PROPOSAL FOR A GENETICS RESEARCH CENTER Submitted to the National Institute of General Medical Sciences June 1, 1973 School of Medicine Stanford University DEPARTMENT OF HEALTM, EDUCATION. AND WELFARE PUBLIC HEALTH SERVICE Farm Approved SECTION I Eudgm Burcw No. 6BA02C9 I LEAVE BLANK TYPE PROGRAM NUMBER GRANT APPLICATION I REVIEW GROUP kOUNCIL (Morth. Ynrr/ FORMERLY DATE RECEIVED I TO BE COMPLETED BY PRINCIPAL INVESTIGATOR f/fwns 1 rhmu@ 78nd 16.4) (1. TITLE OF PROPOSAL (Do not oxcad S3 t~@wdtW8p#~d Genetics Research Center Lederberg, Joshua 28. TITLE OF POSITION Professor and Chairman 2C. MAI LING ADORESS IStrwt. City, SUh, Zip we) 12/31/78 4. TOYAL DIRECT COSTS RE- 5. DIRECT COSTS REQUESTED FTiE;ED FOR PERIOD IN FOR FIRST 12-MONTH PERIOD $2,659,305 $537,800 . FQRMANrrrSlfElSr (Sn /5rn/ctions) Department of Genetics Stanford University School of Medicine Department of Genetics and Stanford, California 94305 Department of Pediatrics Stanford University School of Medicine Stanford, California 94305 N/A 7 . Rrwch lnvolvirq Hums Subjuts fSm ItU~fucDpnS) & lnvomiom IRwmw8l Applreim8 Only - Sm fttstnu204 A.0 NO 6.0 YES Avprwsd: A.0 NO B.0 YES - Not prwiously mponrd ~~~~~-~cnding~cviw6-29-;;) D*t* C. DYES - Pwiowty reoorted TO BE COMPLETED BY RESPONSIBLE AOMINISTRATIVE AUTHORITY hlPS8 thmuph 13and 1581 & APPLICANT ORGANltATIONtSt /Swa IrrrtnutiMs) 11. TYPE OF ORGANlZATlON (Chsk aoplicabh ireml OFEDERAL c] STATE 0 LOCAL m OTW3f IS=ifvJ Stanford University Primtp- non-nrofl -+ Stanford, California 94305 12. NAME, TITLE, ADDRESS, AND TELEPHONE NLlh!EfR OF IRS No. 94-1156365 OFFICIAL IN BUSINESS OFFICE WHO SHOULD ALSO BE Congressional District No. 17 NOTIFIED IF AN AWARD IS MADE K.D. Creighton Deputy Vice President for Business d Finance Stanford University IQ NAME.TITLE. AN0 TELEPHONE NUMBER OF OFFlClALlSI , Stanford, California 94305 lCNlNG FOR APPLltANT ORGANIZATIONISI T.lcphan~Nurn~(415)3~1-3330 :<7,?51 i3 IOEN~ NAL Kathleen C. Butler FOR INSTITUTIONAL GRANT PURPOSES iSea lnsrrucrio~~ Sponsored Projects Officer 01 School of Medicine C/O Sponsored Projects Office 14. ENTITY NUYBER (Fomcrly PHS Account Nunbcr) ToIaphonrNumbw (sI& 5) 32-0 Eyt. ?883_f 458210 IL CERTIFICATION AND ACCEPTANCE Wo, thm undmrrlgne crrriti tht th* nrumena husin uu trur and Compkta to th. bat 0' our kmwlaJp 8nd p m to 8fIy QInt mv.rdmd, tha obll@pn bD c&ply v&h Publk Hwlth Swvlca tonm o nd conditions in 8ff=t n the time of th* SIGNATURES IS-urn rquird on A, SI,GNATLjRE OF PERSON NAMED IN ITEM 2A (See also page i)DATE 107'1 . :, ,- ..z..-- : .* " 1.. ., -`- q MAY 30 IJIJ IN ITEM IO UATC / 5f3Of73 .' klW )yi- (FonmERLr PWS 398 . DEPARTMENT OF HEALTH, EDUCATION, AND WELFARE PUBLIC HEALTH SERVICE RESEARCH OBJECTlVES LEAVE BLANK PROJECT NUMBER NAME AND ADDRESS OF APPLICANT ORGANIZATION Stanford University, Stanford, California 94305 NAME. SOCIAL SECURITY NUMBER. OFFICIAL TITLE, AND DEPARTMENT OF ALL PROFESSIONAL PERSONNEL ENGAGED ON PROJkT, BEGINNING WITH PRlNCl-PAL INVESTlGATbR Joshua Lederberg, Professor and Chairman, Howard Cann, Associate Professor of Pedia Norman Kretchmer, Professor of Pediatrics Leonard Herzenberg, Professor of Genetics Luca Cavalli-Sforza, Professor of Genetic Luigi Luzzatti, Professor ofA Department of Pediatrics ~~1 Clifford R. Earnett, Associ e Department of Pediatrics ;)tp TITLE OF PROJECT Co&unity and Preventive Medicine, GENETICS RESEARCH CENTER USE THIS SPACE TO ABSTRACT YOUR PROPOSED RESEARCH. OUTLINE OBJECTIVES AND METHODS. UNDERSCORE THE KEY WORDS (NOT TO EXCEED 10) IN YOUR ABSTRACI-. .A comprehensive program of basic and clinical research, uniting the efforts of the Department of Genetics a& .Paatz$cs, will apply advances in analytical .P. . . instrumentation to problems of genetic and disease in man. Program areas include: Genetic errors of metabolism identified by computer-managed gas chromatography and mass spectrometry, applied to urine, blood and amniotic fluid; Detection of fetal cells in maternal circulation; Expression of genetic markers in fetal cells, linkage studies; antenatal diagnosis; Polymorphisms involving specific binding to plasma proteins; Psychological impact of genetic disease and counseling practices. * Kenneth Tsuboi, Sr. Scientist, Department of Pediatrics Alan Duffield, Research Associate, Department of Geneti Wilfried Pereira, Jr., Research Associate, Department o PHS-398 RN. 3.70 PAGE 2 The anaersigned agrees to accept responsibility for the scientific dud technical condwt of this project and for provision oE required progress reports if a grant is awarded as the result Bf this application. MAY 3 0 1973 ---------w----- Date 4-J ,! ~~~&.A&-..A~L~~ 4 Y-r---- Joshua Lederberg, '--.' Principal Investigator i TABLE OF CONTFNTS I. introduction and Program Summary .*.*................ P-l Comments on Organization . . . . . . . . . . . . . . . . . . . . . . . . P-3 The Stanford Environment . . . . . . . . . . . . . . . . . . . . . . . . P-6 Clinical Faci 1 i ties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . P-11 Research Program Out1 i ne . . . . . . . . . . . . . . . . . . . . . . . . P-16 il. Screening and Characterization of inborn Errors of Metabolism by Gas Chromatography/Mass Spectrometry Analysis of Body Fluids (Drs. Lederberg, Kretchmer, Cann, and nuffield) .,... . P-17 a-76,~ Budget and Explanation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . P-78 iii. Maternal Blood Stream - Ar.other Source of Fetal +dg Tissue for Pre-Natal Diagnosis of Genetic Disorders (Drs. Herzenberg and Cann) . . . . . . . . . . . . . . . . P-85 Budget and Explanation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . P-97 IV. Polymorphic Genetic Markers in. Amniotic Fluid (Drs. Cann and Tsuboi) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . p-100 Budget and Explanation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . P-119 v. A Search for Genetic Polymorphisms and Variances Of Specific Binding Proteins in Blood (Dr. Cavall i-Sforza) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . P-122 Budget and Explanation . . . . . . . . . . . . . . . . . . . ..*........ P-139 vi. The impact of Genetic Counseling Practices on Family Decisions and Behavior (Drs. Barnett, Cann, and Luzzatti) . . . . . . . . . . . . . . . . . . P-133 Budget and Explanation .............................. P-145 VII. Overall Budgets ..................................... P-148 Program Director's Office Budget ................ P-143 Aggregate Genetics Research Center Budget ....... P-152 VIII. Concluding Remarks (Dr. Lederberg) . . . . . . . . . . . . . . . . . P-157 TABLE OF CONTENTS (cont.) IX. Professional Personnel .............................. P-160 Key Personnel and Backgrounds ................... P-161 Relevant Research Support Summary for Departments of Genetics and Pediatrics .......... P-165 individual Biographies .......................... P-169 SECTION I Introduction and Program Summary INTRODUCTION The Genetics Research Center that would be enabled by this grant will te a new level of cooperative involvement of the Departments of Genetics and Pediatrics of the Stanford University School of Medicine in research in medical genetics and the application of such research to clinical aspects of medical genetics. We are requesting support for an interrelated set of basic and applied research projects involving members of both departments. Besides their importance as basic science, these investigations relate to genetic counselling, pathogenesis and diagnosis of genetic disorders and the mechanisms of human adaptation in genetic variation. 11-l preparing this application, the two departments, labelled as basic science and clinical science respectively, have already enhanced the coordination of their activities for the benefit of patients with genetic disorders and their famiiies. Since 1959, the inception of the School of Medicine on the Stanford University campus, Genetics and Pediatrics each have been deeply involved in various aspects of genetic research. A number of ad hoc cooperative efforts have also evolved. Both departments are together teaching medical genetics to medical students. In- formal communications in training graduate students and postdoctoral fellohs have been excellent. Research projects undertaken in one department have involved consultation or collaboration with various members of the other department, and clinical problems have stimulated basic research activities in both departments, In accordance with its clinical mission Pediatrics has focused on patients with genetic disorders, diagnosis, pathogenesis, and therapy; and the couns2llis~ of such patients and their families. Pediatrics has likewise emphasized perinatal biology, featuring a program of detection and management of high risk pregnancies in which the potential for morbidity or mortality of mother, fetus and/or newborn infant is high. Genetics is well known for its basic research programs in molecular biology, population genetics, and immuriogenetics, whose potential for clinical applications have long been recognized by members o+f 'both departments. As knowledge in molecular biology has increased and the necessary technology has permitted applications to the study of man and his disorders, it has become increasingly evident that relevant research efforts of both departments should converge on problems connected with clinical care. Both departments have begun to cooperatively design and carry out research applicable to genetic medtcine and to plan for the application of the findings to the care of patients encountered on the various clinical services of the Department of Pediatrics. The underlying theme that unifies the range of specific studies outlined below : is genetic polymorphism in man. The pediatrician views this as the source of genetic disease; the basic scientist as an expression of gene mutation and evolu- tionary pressures. These are roles shared within as well as among individual in- !/ a vestigators. To these challenges are brought a combination of clinical insights, 1 experience with several aspects of basic genetics, and new analytical technologies 3 -- the application of instruments like the mass-spectrometer, the computer and the cell sorter. Besides the specific research projects to be funded under the Center grant, we work in an extensive context of genetic and related research -- and indispensable aspect of our own environment, and a set of activities to which the Center organiza- tion may also bring a new focus for developments that should advance both basic scientific knowledge and its application to human problems. P-2 COHtlBNTS ON ORGANIZATXON The Stanford Genetics Research Center is organized as a cooperation of the Departments of Genetics and Pediatrics. Thus, it follows, rather than conflicts with existing departmental authority. This minimizes the need for new, formal arrangements that right be essential in other circumstances. 1t does reflect a deep-seated intention on the part of both departments to improve the application of nev analytical methodology and of basic genetic science to clinical problems, and to relate clinical studies to enhancing knowledge of genetic polymorphism in man. The length of the following comments on organizational detail may lead one to believe that we are xore interested in management than in scientific substance. Exactly the opposite is the case, but we cannot afford the consequences of misunderstanding about how we vi11 work together. Obviously, as at any other university, the actual character of our @@organixationN has a larger compass and bears little resemblance to the forxal hierarchy of imputed power, Research is actually done in individual laboratories under the supervision of an autonomoas faculty member. Best HdirectionM must be in the form of intellectual stimulation and criticism, and in the selection of key people for roles of autonomous responsibility. Professor Joshua Lederbetg, Chairman of the Department of Genetics, vi11 serve as Principal Investigator and Director of the overall Genetics Center Proyraa. Dr. Howard Cann, Associate Professor of Pediatrics, will serve as Associate Program Director with special responsibility for clinical research related to the Center's activities. (If this application is approved, and offers a funding basis for the step, it is anticipate! that Dr. Cann will receive a joint appointment in the Genetics Department as well.) The Director will have executive responsibility for the administration of the program, including the formulation of extended an3 new projects, their budgets, and reports to the NIGHS of progress under the grant, and internal functions of coordination, information and criticism. The internal adainistration of the program is facilitated by the Director's position as Chairman of the Genetics Department, and by the enthusiastic support and participation of Dr. Irving Schulman, Chairma'n of Pediatrics, and of other senior members of both departrents. In practice, the Director will be advised by, and vi11 delegate subproject responsibility to, these colleagues in accordance with their special skills and interests. The Director will also invite an informal visiting committee, principally from among his colleagues involved in similar lines of work at other West Coast institutions, to visit Stanford annually and to advise him of advantageous directions of -2- policy. This group may also help to identify new opportunities for inter-institutional cooperation and coordination, especially to further our ,eaphasis on building complementary rather than competitive capabilities. These annual visits may also advantageously coincide with a local or regional conference on research progress concerning genetic polymorphism and disease. The Director must play both an attractive and a critical role. That is he nust encourage his colleagues to invest in the effort needed to orient their activities towards the conson goals of the Center's research. At various times this attraction may be enhanced by the fiscal support of the Center's budget; on the other hand, it is hard to predict what the level of that support will be, an;3 the level of discretion that will be delegated to the Director. Realistically speaking, then, his role is more inspirational than directive, at least uith respect to the tenured fellow-members of the faculty. If the Center's budget is approved, aad develops some de facto continuity, he may have greater formal authority -- but always with accountability to outside revieu groups and to the independent authority of other professors. The moral and intellectual influence that the Director can exert will obvjously be greatest in relation to people in the same or closely co-functioning departments who already relate in many other ways. His main positive role will be to help bring in new ide&s -- his own and others -- into the progress of the Centeres research. Conversely, he has responsibilities for negative functions -- especially to discourage the growth or even survival of projects that are inherently unsound, or become obsolete. Within a close-knit institution there are often serious personal and political obstacles to the exercise of objective scientific quality judgments. These limitations are both aggravated and compensated for by the limited discretion usually given to a director, i.e., the fact that final decisions about project items are generally made by peer review groups. All of these problems would be much uorse if the Director had the responsibility of coordinating every genetics-relevant activity in the school. He would have neither the budget nor the political mandate to do this well. In fact, the present proposal encompasses a serious and soul-searching effort to initiate a major Center program at a practically achievable and useful level of integration. It will also be the basis for less formal but equally important co-operations with other departments, whose policies and programs can be influenced but not directed in the exercise of our responsibilities. Provision for Succession of Directorship. Day to day deputization will be handled ad hoc in the same fashion as the duties o-f the departmental chairman. The question of possible succession, in the event of incapacity or transfer of the Director, poses a more difficult question. The Associate Director will serve pro-tea, in concert with the (acting) -3- chairaan of each department; and a new director will be proposed by the Dean of the school after consultation with the visiting committee and with the participating faculty of the departments. The Associate Director's role will be to maintain contacts with other clinical departments for patient referals to Center projects, and to assure a high standard of clinical responsibility in all Center research activities that deal with patients. Stanford has long experience with Clinical Research Centers -- e.g. in medicine, in cancer therapy, in cardiovascular disease, and in premature infant research. (These facilities afford further research bed opportunities for protocols involving intensive study of selected cases without burdening the present proposal uith inappropriate ia-patient care charges). In addition, they have led to the institutionalization of formal procedures for the ethical revieu of research involving human subjects. Dr. Cann vi11 be responsible for defending the present proposal before that revieu conmittee, and for assuring that all further research under this grant is properly submitted and reviewed according to established procedures. An Application for renewal of a Genetics Research Training Grant uas approved by Council for the.period 1974-1979. This action was superseded, however, by the executive decision to wind down such programs and we are nou operating on a decreasing budget to cover only the previously enrolled trainees. That training grant reneual had, for the first time, provided for clinical research in genetics as part of our program. Lacking these funds, it is, of course, imperative that the research activities of the Center also double as training opportunities for graduate research assistants and postdoctoral fellows (research associates.) We vi11 raake every effort to fit each fellou into employment in the support roles of the Center. We cannot pretend, however, to be able to support the training functions at the level previously assumed without a compensatory expansion of the funding for these research programs. This is not reflected in the budgets submitted at this time. The department faculties having agreed to the programs of the Center, there is much to.do in substance, but little in organizational process, to coordinate our activities and insure meeting the Center's objectives. We are already in frequent, almost daily, comvtunication in the pursuit of our other duties and socially. These contacts ace promoted by the fact that our laboratories and offices ace immediately adjacent in the nedical Center Duilding. The Genetics Center Budget is intended to fund programs of new substance and scope or those for which other support (e.g. NASA) is no longer available. Lt goes into new territory beyond many on-going research projects which are identified in the material on each affiliated investigator. However, the coordinated funding and administration of the Center allows more careful planning and resource-sharing than would be possible for -4- a disparate set of individual proposals. We intend .to minimize the indefinite prolongation of autonomous projects merely to keep a research group intact. Instead we vi.11 promote an innovative search for new opportunities ana on-going mutaal scrutiny of projects. For the continuity of a program as complex as the Center, we are requesting an award for 5 years. Yet it is obvious that many unforeseen obstacles and opportunities will arise in that interval to warrant changes of tactical direction and evolution of major strategies. The responsibility of the Director to manage this progression of emphasis is his most important executive role. It vi.11 be discussed with the visiting committee and reported to BIGHS from ,year to year, to assure the consistency of his decisions with the approved mandate of the Center grant. This is not an assertion of unwarranted latitude. To the contrary, the political structure of a university ensures, if anything, a great deal of conservatism in changes of direction and support for individual programs, once approved. The present proposU will also be a model for further developments to be initiated uith the Departments of Gyn-Ob., fledicine, Psychiatry and .Dermatology -- to mention only those with which we have had tangible discussions. For various reasons, potential projects uith them are not yet ripe for formal subnissious; these will be t&e subject of future requests, depending in part on the climate for funding expansion of genetic research. Heanuhile, we have working arrangements to assure their awareness of our activities and the provision of patient material for mutual advantage. A brief description of the role of each participant in this prograa and of his research and/or clinical interests are presented here: THE STANFORD ENVIRONW3NT Besides the Frograa Director (Professor Lederberg) and Associate Director (Professor Cann) the faculty and professional personnel associated with the Center, their roles and biographical detail are spelled out in Section IX. In addition, we have included biographical sketches of a number of key members of the environment of the Department of Pediatrics and Genetics, although their research projects dre not included in funding under the Center at this time. Within one or two years after the activation of this prograa. we anticipate the direct participation of one or more colleagues from the Department of Obstetrics and Gynecology. A search for a chairman of this department is presently under Way, and we expect that this individual will reaeu active research P-L -5- here in fetal physiology and fetal monitoring. Ue look forward to interacting in this program with our obstetrical colleagues in various projects pertaining to antenatal detection of genetic disorders and selective abortion. Ye hare also consulted uith the chairmen of the Departments of Dediciae and of Dermatology, and have encountered great interest in the development of the Center, and assurances of cooperation in the referal of patients who would be pertinent to our screening technologies. Their position is best witness8d by the attached correspondence. At some future date, we may expect to formulate more specific proposals, as a consequence of recruiting now under way. The Department of Psychiatry will be chaired by Albert Stunkard (now at University of Pennsylvania) beginning September 1973. He is personally well known to the Director (who served on the search committee for this appointsent, which ua,s chaired by Professor Schulman) and aany other participants and we have strong assurances that the Psychiatry Department's interest in genetic etiologies of psychiatric disease will continue under his leadership. Dr. Stunkard has also voiced his concern that psychiatrists have not hitherto been more directly involved in problems of genetic counseling, like those reflected in Dr. Barnett@s proposal hem; and we are looking forward to closer cooperation on such issues after his assumption of his duties. For some time, Professor Cavalli-Sforza of the Genetics Department has been cooperating with Professor Darchas of Psychiatry on polymorphisms of biogenic amine metabolism. Genetic counseling, per se, is a responsibility mainly of the Pediatrics Department -- diseases of adults only occasionally present serious problems of reproductive policy of the farily. Genetic disease, of course, presents itself to all of the medical and surgical specialities, In recent years, these departments have not seen any requirement for a special organization to deal with genetic aspects of internal rmedicine or surgery -- and indeed these are fully and competently integrated into their overall teaching and practice. Rhen special problems do arise, there are no iapediaents to consultation with pediatricians and with basic geneticists. Plany, nembers of those departments are already involved in immunogenetic and other genetic research. DATE: May 25, 1973 To : Joshua Lederberg, Ph.D. FROM : Daniel D. Fe&man, M.D. (Chairman, Department of Medicine) SUBJECT: Genetics Research Center Dear Josh: I am not really clear&out your timetable for submission of the proposal for a Genetics Research Center, but I thought I would make explicit several areas of potential involvement of the Department of Medicine. 1. Pharmacogenetics - We have a slot in Clinical Pharmacology for a new Assistant Professor. Stan Cohen and I have discussed finding someone with a particular interest in the area of pharmacogenetics, but at the present time, no particular individual has been chosen. 2. Endocrinology - A. Hormonal control of gene expression. We will have at least one new person in the Endocrine Division in the next year and have placed as our first interest finding someone working in the area of the effective hormone on the expres- sion of genetic information. link to a genetic center. This would provide an obvious 3. Early detection of hereditary endocrine disease. There are at least 80 endocrinopathies which show Mendelian segregation and a number more which probably have a major genetic control. Early de- tection of assymptomatic patients can be achieved by suitable pro- vocative testing. This opens the way to family studies in which a) the hereditary mechanism can be defined, b) preventive or therapeutic intervention can be tried before irreversible damage has occurred, and C) the effects that intervention can be compared to the natural history of the disorder. The latter approach has important application to the management of "patients" discovered by multiphasic screening techniques. 4. Imunogenetics - This area of course is strongly represented in our Department and we could, I believe, contribute significantly to the activities of the genetic center in this area. I should have liked to provide more specific information, but until particular people have been identified, it would be difficult to do so. I hope this is of some help -- laboration. 1 am certainly anxious for an active col- Sincerely, d?!f 3-f CLINIC FOFj Ctt[LDf$!`i WPTK ~SO~I.fWS AND OTHEFj ~NH.&kT/-j~bE S,KI;N DISEASES Directors: Eugene M. Farber, M.D. Profe'ssor and Chairman, Department of Dermatology Alvin H. Jacobs, M.D. Professor of Dermatology and Pediatrics In the clinics of the Department of Dermatology there are about 12,000 total patient visits annually,of these approximately 3,000 are children seen in the Pediatric Dermatology Clinic, Over half of these children have either c psoriasis or atopic dermatitis, 60th conditions with important heritable factors in their etiology. In addition many patients are seen with the less common genodermatoses, such as, the vat lous types of ichthyosis, epidermolysis bullosa, and the many types of heritable neurocutaneous disorders. At the present time a special clinic is being established for the care and study of children with psoriasis and other inheritable skin diseases. The purpose of this clinic is not only to offer more complete care for these types of patients, but to study the genetic aspects of these conditions and offer genetic councelling to the patients and their families. The Department of Dermatology 6lready has an unmatched reservoir of material for genetic study in its Psoriasis Life History Research files; a data-bank of approximately 8000 patient histories. Not only is this computerized information a source of information .on the epidemiology of psoriasis, but has served as the basis for several published follow-up studies on the genetics of psoriasis including family and twin studies. At present several genetic studies are under way. For example, Dr. Farber and associates are studying the association between H-LA antigens and psoriasis. Drs. Jacobs and Chan are studying and developing techniques for accurate counting of melanocytes in pigmented and depigmented macules in order to predict the development of certain genodermatoses, such as neurofibromatosis, tuberous P-9 -2- sclerosis etc. Studies are also under way to properly classify the various genetic types of ichthyosis and in cooperatton with Dr. Howard Cann we are planning to investigate the x-linked blood type ichthyosis. in the families with x-linked It is hoped that tn the future, w ith the vast resources availab le in Genetics, Pediatrics, and Dermatology at Stanford, an investigation could be launched into the genetlc aspects of atoptc dermatitis, one of the commonest skin problems of childhood, I CLINICAL FkXLITIES - The Stanford Yedical Center, located midway between the cities of Scn Francisco and SalI Jose on the campus of Stanford University, co;,siats of tk.2 School of Xedicine , the University !lospital and the Stanford Clinics. Staforrl is the only university hospital in the ikgional liedical Program (XZ?) .Arca I11 and functions as a tertiary care center. R`iP Area III consists of eleven coti.i:ie-j in mid California consisting of 2.6 million people, There are 60 acute h@%pital facilities, and 4000 physicians practice in RX ? Area III. Approximately $0 PArcent of patients admitted to Stanford University Hospital live in this area.Xdi'cticnall)`, patients for the clinics and university hospital are also dra5.n from other areas i:, and out of California. ElP Area III and its:population form a base from wbic:l is drawn numerous patients with diseases resulting from major genes or chromosomal abnormalities or with a significant genetic component. bloat of these patients are seen primarily on one of t!le services of the Department of Pediatrics or arc referred for diagnostic evaluation and/or genetic counsclling frcm other clinical services at the medical center. Clinical teaching services at other hospitals;: affiliated with the Department of Fediatrics add to the source of patients k.ith medical genetic problems. As the Genetics Center activities evolve and research developrdnts become available for clinical application, we anticipate that more patients t.511 be referred to this medical canter for genetic counsclling and evaluation and management of genetic disease. The number of patients seen annuall;: by ttc combii:zi clinical facilities of the ikpartment of Pediatrics for genetic disorders 5s estimated to be about 200. Ve predict that this number will double during t;r-r: cctirs;i of the Genetics Center grant. Other clinical divisions of the Medical Center, of course, also see man;: adult patients with genetic disorders. iiistorically, Genetics has had weaker tiss with them than with Tediatrics, although several joint projects connect ::it:? Medi tine (H. !!cDevitt) and vsith Psychiatry (J. Sarchas; S. Kessler). T:?e ap:cint:?nr (effective February 1) of a "genetic endocrinoiogist", Dr. Daniel Federman, as net; head of Medicine should remove administrative obstacles to extensions of Center- related programs. Patients with genetic disorders are seen in the fo_llowing pediatric sub- specialty clinics at the Stanford Xedical Center. *The Children's hospital at Stanford and the pediatric services at the Santa Clara County Hospital (San Jcse) and a,t the Kaiser-Permanente Medical Center in Santa Clara. PHS 2495.1 1245 P-N Confinuot~- n page Genetic Counselling Clinic (Director, Dr. Howard Cann, Associate Professor of Pediatrics) Approximately fifty patients and their families are referred annually for genetic counselling . These include patients from outside of the Stanford ?!cdical Center and from various clinical services at the Piedical Center, About jO% of fami.lies seen are counselled for disorders determined by major genes at singie loci. The remaining 50% of patients represent more complex counselling problems, e.g. possible sporadic cases and phenocopies, multifactorial threshold traits, undiagnosed familial disorders, and excessive exposure to radiation. Counselling i.ncludes confirmation of the diagnosis of the disorder and identification of heterozygotes among asymptomatic relatives at risk (autosomal recessive and X-linked recessive disorders). Families are always screened for the possibility of applying ante-natal diagnostic techniques. The motivation for and expectations from genetic counselling are explored by Dr. Cann and, for selected families, by a social worker, in order to plan for effective communication with each ianily. Birth Defects Clinics (Director, Dr. Luigi Luzzatti, Professor of Pediatrics) Approximately 100 new patients are seen in the Birth Defects Clinics annually for diagnostic evaluaticn and comprehensive and interdisciplinary management of congenital defects. There are about 400 return visits per annun in the clinics. In addition, about 50 newborn infants are seen annually for evaluation of congsnital defects in tlie Stanford Hospital Surseries. >lost ChiiLren seen have multiple congenital dt2fects of unclear etiology, about 507; of thzse are scrt?eneci for c1lronosonal aSnormalities, aSout 25X are patients with dlsor (based on fairly ridgid criteria, e.g. full time neonatologists and basic irlfant monitoring equipment available at all times), and this is evident by referr&ls of I fetuses and their mothers and newborn infants. Approsimat2ly 700 infants arr admitted per year to either the intensive care nursery of the Premature Rosearc Center at Stanford and about 30;; of these are born at other facilitks. +proxlzatc3> 551 of newborn infants receiving intensive care at Stanford are ill because ::?y - are hetero-ygous or homozygous for a major mutant gene, because of a chromosJna1 i abnormality or because of a malformation with a significant genetic contribution. We anticipate a major expansion of pcrinatal activities involving detecticn and I management of high risk coth2rs and fetuses with the appointm&nt in the czar ! future of a Chairman of the Department of Obstetrics and Gynecology. The scar& j is presently underway, and one of the criteria for selection is comT,stsnoe in the areas of fetal monitoring and evaluation and fetal and maternal physiologic Ict2r- i actions. I Pediatric Neurology Clinic (Clinic Director, Dr. Judith Koehler, Assistant Professor I of Pediatrics and N2urology) Approximately 500-600 patients with various neurologic disorders are se2n annually in this clinic. Xany of these patients `nave genetically det2rmined or inherited disease, e.g. learning disorders, familial myopathies, spinoc2r2bellar degeneration, familial spastic paraparesis and other metabolic or degen2racive disorders of the central and peripheral nervous system. The Division of PedlatrLc I Neurology has special interest in clinical treatments of and research into rile bzs%z . mechanisms of epilepsy. Some patients w!t.. h seizure disorders have heredofsmilLa1 i determinants underlying their basic seizure problems, Additionally, response to medication and drug metabolism may be genetically determined. The Divisicn of I Pediatric Neurology also has special clinical and research interest in neuro- muscular disease, much of which is hereditofamilial. Specifically for this purpose, a neuromuscular clinic is being established in conjunction with the Yuscuiar Dystrophy Association. This clinic will see 200-300 patients, mostly children, annually. A diagnostic neuromuscular histochemistry laboratory is already in I function to process nerve and muscle biopsies from these patients. Xctivz ressarzl; I in neuromuscular disease is being carried out by several workers in the 2n;!rt;"-?:::: ,F_ which incorporates ultrastructurz and basic physiologic studies. 3 :iSurOi cfgy , I ! t I --2 h ,-a Hemophilia Program (Clinic Director, Dr. John Gribble) The Hemophilia Program provides diagnostic evaluation and comprehensive management for patients with disorders of blood coagulation. Approximately 109 new patients are seen annually; these patients represent the clinical spectrum of disorders of blood coagulation, the most frequent being hemophilia A, heno?hilia !3 and van Willebrand's disease. The families of all patients with inherited disorders are provided with genetic counselling. A comprehensive care clinic is located at the Children's Hospital at Stanford in order to provide continuing care for patients with hemophilia and includes prophylactic treatment of patients with hemophilia A with Factor VIII preparations. Pediatric Metabolic and Endocrine Clinic (Clinic Director, Dr. R.O. Christiansen, Assistant Professor of Pediatrics) Seventy to eighty new patients are seen annually in this clinic which logs about 703 patient visits each year. Xany of the patients may be classified as inborn metabolic errors and these include phenylketonuria, galactosemia, the various syndromes associated with adrenal hyperplasia, alcaptc>uria, the various glycogenoses and defects in thyroxine oiosynthesis. Diet therapy of patients with phenylketonuria and galactosenia is an ongoing activity of thzse clinics. Furthermore, a number of patients with hypophosphatemia (X-linked) are enrolled in this clinic. Genetic counselling is provided for the families of all patients with hereditary metabolic and endocrine diseases, and this includes attempts to designate heterozygotes among unaffected siblings of probands with inborn errors of metabolism. Growth and Development Clinic (Director, Dr. Norman Kretchmer, Professor of Pediatrics) This clinic deals with genetic and environmental problems in infants and children which affect their growth and development. Usually children are referred to this clinic because of failure to grow or abnormally slow growth. Thus children with hereditary conditions which result in significant growth:failure in the phenotype (e.g. isolated growth hormone deficiency, various aminoacidurias and various chondrodystrophies) comprise a proportion of this clinic's patient population. Approximately 150 new patients are seen annually. Conlinual;-..n page The Children's Hospital at Stanford, located approximately l/2 mile from the Medical Center on the Stanford campus, is affiliated with the Department of Pediatrics for teaching and postdoctoral clinical and researc!] trainin?. There are three clinical services relevant to a medical genetics program at thi; hospital: 1) Cystic Fibrosis Service (Director, Dr. Birt Harvey, Clinicai Associate Professor of Pediatrics), 2) Clinical Immunology Service (Director, Dr. Vincent Narinkovich, Assistant Professor of Pediatrics) and 3) Pediatric Oncology Service (Director, Dr. Jordan Wilbur, Clinical Associate Professor of Pediatrics). These services have in-patient and out-patient facilities at tile Children's Hospital at Stanford. Here patients are evaluated, diagnosed and treated for cystic fibrosis, hereditary immune defects and hereditary malignant tumors (e. g. retinoblastoma). Genetic counselling is provided by the staff of thsst services. I Research Facilities Research facilities in the Departments of Genetics and Pediatrics available to this Genetics Center Program include some 15,000 square feet of laboratc*ri2s, research support areas and offices. The research and administrative Ireas for each department are contiguous on the third floor (the Joseph P. Kennedy Jr. Laboratories for Nolecular Xedicine) of the Joseph D. Grant Building of tiln School of Medicine. In addition the Instrumentation Research Laboratory (D?;artr2nt of Genetics), in the basement of this Suilding,conprises about 7,030 square feet of offices, laboratories, and shops. Thus biochemical laboratories, fully devcicy\ed tissue culture areas, heavy equipment areas, cold and warm rooms and an electron microscope facility are available for this program. A cell separator, a gas chromatograph, a mass spectrometer and an electron microscope lead the long list of equipment which is available for ths research projects to be carried out in this program. Other equipment also includes centrifuges, scintillation counters, lyophilizers, spectrophotometers, incubators, microscopes and coulter counters. Rather good computer facilities are available. Continuation page THE RESEARCH PROGRAM The Center program consists of research projects which have direct relevance to medical genetics. Some of these projects will require blood, urine, amniotic fluid and cells from patients and others will require the patients themselves. In none of these research projects are we ready to test methods for use in the actual clinical situation; rather, we are developing methodology. The general areas of research are as follows: I. Screening and Characterization of Inborn Errors of Metabolism by Gas Chromatography/Mass Spectrometry Analysis of Body Fluids. (Drs. Lederberg, Kretchmer, Cann and Duffield). II. Maternal Blood Stream - Another Source of Fetal Tissue for Pre-Natal Diagnosis of Genetic Disorders. (Drs. Herzenberg and Cann). III. Polymorphic Genetic Markers in Amniotic Fluid. (Drs. Cann and Tsuboi). IV. A Search for Genetic Polymorphisms and Variances of Specific Binding Proteins in Blood. (Dr. Cavalli-Sforza). V. The Impact of Genetic Counseling Practices on Family Decisions and Behavior. (Drs. Bamett, Cann and Luzzatti). The arrangement is also reflected in the budget presentation. I PHS-398 Rev. 249 Peg* SECTION II Screening and Characterization of Inborn Errors of Metabolism by Gas Chromatography/Mass Spectrometry Analysis of Body Fluids Drs. Lederberg, Kretchmer, Cann, and Duffield Screening and Characterization of Inborn Errors of Retabolism with Conpaterized Gas Chromatography And Bass Spec troaetry Dr. J. Lederberg, Principal Investigator Drs. 1. Rretchmer, 8. Cann, and A. Duffield, Associate Investigators A. IYTRODUCTIOB A.1 Objectives The objectives of this work are to develop the uses of gas-1iquiU chromatography (GC) and @ass spectrometry (ES) instrurentation, under computer management, for the screening, diagnosis (pre and postnatal), and study of inborn errors of metabolism. The efficacy of these analytical tools has been demonstrated uhen applied to limited populations of urine samples in the research laboratory environment. Ue propose to enlarge the clinical investigative applications of GCfRS technology and to deaonstrate its utility for o ore econorical and routine diagnosis and screening of disease. Specific goals include the application of GC/HS analysis capabilities to larger and more diversified populations to establisb better defined norms, deviations, and control parameters necessary to relate GC/BS analysis results to identifiable disease states. In order to ease the problems in analyzing the prodigious amounts of information expected in this research, we will augment the existing GC/BS data handling system to provide for increased throughput and automation. Two other on-going or penbiag research projects relate to the present application, each with distinctive aims: 1. DBUDRAL (RIB: BE-00612; Principal Investigator, R. A. Peigembaaa) is concerned uitb the advancement of artificial iate$ligence (computer softuare) techniques for the automated interpretation .of sass spectrometry data. These prograwx attempt to emulate human reasoning processes in constructing explanations for mass spectra fron basic principles. 2. SIJRBX (RIE: RR-00785 pending; Principal Investigator, J. Lederberg) is a conprehensive resource grant to establish a national facility for developing applications of artificial intelligence in medicine. Our own use of this facility will include the integration of DEYDRAL software with BIGR RESOLDTIOR mass spectroietry instrumentation. Genetic screening can be made even more sophisticated by using these techniques for the corroboration of the structures of newly discerned metabolites. Rowever, the present program can also operate stand-alone, if necessary, using low resolution RS on line with the GC, -2- A.2 Background and Rationale The Instrumentation Research Laboratory was established in the Genetics Department under NASA auspices in 1961. Its task was to define and improve nicroanalytical methods for the detection of living processes that might be useful for the biological exploration of the planets. !iany of the concepts that we explored have been embodied in NASA's planetary mission plans. However, we have not undertaken to design and build harduare for such missions, Instead, we have served as experienced advisors to the experiment teams responsrble for scientific studies on the Mariner and Viking Mars programs. Our work on GC/HS is one of several lines of instrumentation effort. Our original mandate from NASA included generous encouragement to seek health-related applications as a spin-off of the development work they were supporting. However, they have not been able to support the full fledged extension of space-related technology to genetic disease research per se. The present application also comes at a time when overall funding for basic research by NASA is declining rapidly and sag disappear within the next year. We have already begun to reduce our W/MS laboratory staff in response t3 these cutbacks. It is therefore appropriate that we seek NIN support to help maintain this existing laboratory to apply its capabilities to problems of characterizing genetic metabolic disease. Our focus on mass spectroaetrp (ref 3c) originally stemmed from the exquisite sensitivity, speed, and specificity of this technique for the identification of organic molecules. We have had some experience with instruments (like the Bendix Time-of-Plight Hass Spectrometer) which can generate a complete low resolution mass spectrum in 100 microseconds and whose sensitivity is limited by the statistics of the number of ionized fragments, and by the data handling problems of averaging repetitive spectra emerging at a rate of 10,000 frames per second. Ue have also been led to look at the computational challenges of fiS from another standpoint - namely the aechanization of the scientific thinking that is entailed by the interpretation of a mass spectrum. This task has been the focus of the research in "artificial intelligence" of the DENDRAL project. The application of these instruments to genetic research requires another dimension, namely the separation of complex mixtures, e.g., from body fluUs, into individual components. These are then available for identification by the mass spectrometer. Gas chromatography has proven to be a useful companion to the mass spectrometer - the output gas stream can be fed directly to the inlet of the spectrometer and much of the carrier gas (helium) selectively deviated by a semi-permeable membrane. (Automated, continuous flow into a mass spectrometer of other chroaatographic streams, e.g., high pressure liquid chromatography, is a speculation that may eventually materialize .-. i., ,' -3- to great advantage but is not yet available for applications like ours). For some time then we have been developing the means to integrate GC vith MS under computer management. The present project represents the systematic application of these skills to the recognition and identification of metabolic variations, viewed both as genetic polymorphisms and as clinical problems of genetic disease in man. The sample populations compr-ise mainly healthy (control) and problematical newborns already under intensive study in the Stanford Pediatrics Department. Other samples will be furnished by collaborative arrangements with physicians elsewhere and vith other Departments at Stanford (e.g., Medicine, Psychiatry, Dermatology, etc.). These inputs will have been prescreened for conditions likely to relate to possible genetic etiologies or otherwise to exercise the analytical utility of GC/MS screening. The Techniques of nS and GC The technique of sass spectrometry gives information about the structure of a molecular species by measuring the characteristic mass abundance pattern of fragments resulting from ionizing the parent molecule, by electron bombardment. Ionization is usually accomplished The compound under analysis must have a measurable vapor pressure at about 200 degrees C. (This temperature and a pressure of 0.01 microbars are the normal operating conditions of a GC-coupled mass spectrometer ion source). The ionizing electron beam (70 eV energy) removes one electron from some of the molecules of the sample vapor to yield excited positive molecalar ions: II + e ----> El (+) + 2e The molecular ion, a(+), is generally unstable (especially if at a high energy of excitation) and may decompose within a few microseconds to yield a series of positively charged fragment ions. Each fragment ion can in turn decompose to ions of lesser ion where they af-6 sepdrated - - ^ spectrometer into the analyzer reg ratio. In sector instruments according to their mass-to-charge in quadrupole instruments by m this is done by a magnetic field, ght instruments by dn electric field, and in time-of-fli Y- The mass spectrum of an adjustable ion detection time dela a table of positive ions of organic compound thus consists of olecular structure (for different Basses and abundances. n teroatoms) determines the instance number and location of he pture subsequent to ionization, frequency uith which bonds will ru rt, a characteristic mass thereby producing, for the most pa rical isomers may shou subtle spectrum for each compound. Geoaet e mass spectra owing to the differences vithin their respectiv -4- influence of the geometry of neighboring groups. Optical enantioaers yield identical spectra. Although the technique of mass spectrometrp was extensively used by petroleum chemists from the 1940's, it was not widely utilized in organic chemistry until the late 1950's. The first extensive monograph on biochemical applications of mass spectroaetry has just appeared ("Biochemical Applications of Mass Spectrometry, " edited by G, R. Wailer, Wiley-Interscience, New York, 1972). Our colleagues and close collaborators in the Stanford Chemistry Department, led by Professor Carl Djerassi, have been among the pioneers in the development of nS for natural product chemistry, especially as applied to steroids (4 books and in excess of 200 papers on various aspects of the theory and application of MS have been published by Prof.. Djerassi's group since 1961). During tbe 1960's, mass spectrometry was applied to many different types of organic compounds. The accumulation of these reference mass spectra was necessary to establish fragmentation rules for the interpretation of unknown mass spectra. The experienced mass spectroscopist becomes adept at recognizing the mass spectral signatures of those types of compounds with which he works but he cannot encompass within his memory all the relevant information contained in the literature. In addition, many reference spectra determined by mass spectrometry laboratories have not been published. To overcome this problem, libraries of mass spectra are being compiled for computer storage and retrieval so that they will eventually be available for matching by computer against the mass spectra of unknown compounds. Progress is now being made toward compiling libraries of mass spectra relevant to general metabolic studies. These will match the accumulation already available for special classes of organic molecules and for some drugs whose spectra are important for emergency toxicological analyses (ref 2). Instrumentation advances in mass spectrometry during the past decade like improved sensitivity, direct coupling with GC and the use of computers for the routine recording and presentation of mass spectra, a11 facilitate the large scale application of mass spectronetcy to biomedical problems. Body fiuids and other materials encountered in biomedical research are complex mixtures, For example, urine is known to contain several hundred organic compounds at levels exceeding on the order of 1 nanogram per milliliter. The gas chromatograph is indispensable for the separation of such mixtures into discrete components. Yith mediua resolution instruments, the mass spectrometer can be scanned repeatedly once every 2-4 seconds. The gas chronatographic separation of a urine mixture may require 40-50 minutes: the result is the accumulation of over 700 mass spectra per analysis. The simplest way to identify these mass spectra is to search a library of known compounds. Even if the mass spectrum of a test compound does not reside in the library, the best match found may be a related compound. This can facilitate the manual interpretation based on the chemist's knowledge of and guesses about the rules of frageentation. The -5- problem of computerizing the identification of compounds whose mass spectra are not in a library is addressed by the DENDRAL project. Computer programs have been developed to interpret mass spectra of unknown coapounds from first principles (i.e., to emulate the reasoning processes of organic chemists). Prequently compounds of biochemical interest occur in small amounts in biological fluids. (By definition, many frontier problems concern compounds at the limit of easy detection by existing techniques). Thus, the effectiveness of GC/HS as a detector of biological materials is directly related to its sensitivity. Current systems routinely operate vith sensitivities such that mixture components with as little as 50-300 nanograms of material can be measured. This limitation is imposed by the following instrument-related factors. In order to record an interpretable mass spectrum, a low resolution mass spectrometer must have input to its source en the order of S nanograms of material per second. Since a gas chromatographic peak lasts for approximately 5 to 30 seconds, in GC/HS operation some 25-150 nanograms per GC peak are required for mass spectral analysis. Inherent in the gas chroaatographic column and in the semi-permeable membrane separator (used to preferentially remove the helium carrier gas from the effluent stream) are losses of up to 50% which increase the input sample requirements in a practical sense to about 50-300 nanograms of material per GC peak analysis. Another limiting factor in the application of the GC/RS technique to biological extracts concerns the volatility of the material to be assayed. before the system can detect many non-volatile components (e.g., carbohydrates, amino acids, etc.), they must be converted into volatile derivatives uhich will pass through the gas chroeatograph at a maxinutP oven temperature of 300 degrees C. Above this temperature, column bleed from the gas chromatographic phase will tend to enter the ion source and complicate the recorded spectra. Thus the GC/YIS technique is restricted to those organic compounds which can be converted to volatile derivatives and is not, in general, applicable to inorganic compounds. A recent report (ref I) describes the analysis by GC/RS of ketose diphosphates (as their trimeth.ylsilyl (TtiS) derivatives) uhile aldose diphosphates (also as their TMS derivatives) proved to be too unstable to analyze. It is safe to assert, however, based on our uun experience and the literature, that a broad spectrum of organic compounds of biological significance will be amenable to analysis by GC/HS methods. There is another mode of operation of a Gc/HS system which enables greater sensitivities to be attained for the yuantitation of KNOWN aetabolites, If the mass spectrum and the gas chromatographic retention time of the compound to be quantitdted are known, the mass spectrometer can be used as a specific detecting system for this compound. This technique is called mass fragmentography. Under these experimental conditions, the mass spectrometer is not scanned over the ENTIRti mass range but is directed to measure one or two SPECIFIC masses known to be -6- characteristic of the compound(s) being quantitated. Consequently, there is an appreciable increase in sensitivity since the mass spectrometer samples only the significant datd points and can integrate the signal longer. In this mode, existing GC/HS instrumentation matches the new fluorescent reagents for aaines (reported to detect approximately 10 picomoles). It also embodies the specificity of the Bass spectrum at individual mass numbers. At greater cost and cumbersomeness, the W can be extended to a quantum-counting range of sensitivity. These methods are therefore likely to be complementary to the special purpose methods, like flnoresciretry, which are often cheaper and more efficient for well defined classes of compounds. On the other hand, the history of pesticide analysis shows how the GC can also be made ultrasensitive at the cost of some loss of specificity. Using deuterated analogs as standards for the test compound, qnantitation can also be achieved at sub-nanogram levels. We have recently exploited certain characteristics of the quadrupole mass spectrometer and its data system to develop a method for the qnantitation of ten amino acids in soil extracts (ref 3a, copy attached) and subsequently for the amino acid content of biological fluids (ref 3b). This represents an advance in the technique of mass fragmentography since the sector mass spectrometers used up to this time, have been severely limited in the number of ions and the mass range they could monitor for any one experiaent. Our technique of quadrupole mass frayaentography was used for the quantitation of the amino acids in the urine of a patient with suspected branch chain amino aciduria. The results are discussed later (see nethods of Procedure and figure 5). The overall management of the system and the reduction and selective presentation of the large volume of data emanating front the analytical instruments is an important task of the control computer. Our experience in instruaentation comprises a good deal of computational software embracing real time instrument aanagenent, automated data reductxon, and artificial intelligence (ref 4, appended to this application), It also requires considerable effort in electronic and vacuum technology for the instrumentation hardware, and a coherent system approach for the overall integration of these components. Present GC/HS systems are designed mainly for laboratory research, incorporating great analysis flexibility but the ability to handle only a small nunber of samples. Such systems are not practical for large volume screening, but they can be adapted for the pilot studies contemplated here. A properly designed automated spstem could reduce these costs by as much as an order of magnitude, as would be essential for cost-effective applications to general health care. The routine screening of normal and abnormal body aetabolites, including drugs, in human body fluids (ref S) is currently the object of several research programs. Various -7- non-specific methods, including thin layer (ref 6,-I), ion exchange (ref S,lO), liquid (ref 9), and gas chromatoyraphy (ref 11-14 and 17t), are used primarily with the goal of separating d large number of unnamed constituent materials. Using these techniques, compound widentifizatlon8* is made by a comparison or the migration, under identical conditions, of the unknown spot with reference compounds. This approach can lead to erroneous identifications, however. This point is illustrated by a recent article (15) which describes the use of mass spectrometry in the identification of a case of isovaleric acideaia. Previously, the same patient was diagnosed as having butyric dnd hexanoic acidemia on the basis of chromatographic evidence alone. This type of error is especially important when analyzing a @@newtr (previously undescribed) metabolic error uhere rigorous identification of relevant metabolites in various body fluids and tissues is essential. For positive identification using mass spectrometry, the separated components must be transferred from the chromatographic medium to the mass spectrometer. The unknoun spot can be leached from paper or thin layer chrowatograms, or in the case of liquiti chromatography, the solvent removed, and a mass spectrum recorded on the residual material. Trapping the various effluent components Erom a gas chromatograph, with subsequent introduction into the mass spectrometer, has been used. This approach requires considerable time and is inefficient when applied to complex separations. It has been superseded by the direct coupling of the gas chroeatograph and mass spectrometer. Gas chromatography is unquestionably the most convenient separation technique to couple to the mass spectrowater because the carrier \jas can be removed efficiently and easily as the analysis proceeds. For recent examples of the use of the GC/MS technique for the analysis of body fluids see refs 15ld. dased on the work cited, as well as our own on-going programs, the ability of the GC/FiS technique for the analysis of body fluids is well established. We have drawn upon the published literature in helping to design our experimental protocols (ref 18). As mentioned earlier, urine dnd other body fluids (e.g., serum, amniotic fluid, etc.) are complex mi.xtures. The separation (by gas chromatography) dud subsequent identification (by mass spectroaetry) of these components can be a difficult task. To simplifg the separation problem, the body fluid under analysis is chemically separated into a number of fractions which permit analysis for acids, am.ino acids, and carbohydrates present in free or in conjugated form. Drugs and hormones, as well as their metabolites, will also appear in the chromatographic seyardtions. The gas chramatographic analysis of each class of compounds; presents a metabolic profile. Abnormal profiles (ContaininLJ either novel peaks or peaks deviating from their expected amplitudes) are then assayed by mass spectrometry. The mass spectra recorded during the elntion of each gas chromdtogf~~phic peak then serve to identify tha constituents present in that peak. -a- The importance of this diagnostic technology may be illustrated by a family seen for a fatal inherited disease at the Stanford fledical Center. Three male infants have been born to this couple and each of the offspring died in a very similar manner by 10 days of age, The first infant was not seen at Stanford, but the second infant was transferred from another hospital, in extremis. Aside from the striking family history, there were no diagnostic claes, and the second child was dead within 6 hours of arrival. During the period of observation, blood and urine were collected from the proband, and analysis of the latter by gas chromatography (at another institution) revealed an excess of erotic acid. This diagnostic clue, coupled with the clinical picture of relatively rapid delsise after protein (breast milk) feedings were well established, resulted in a diagnosis of a hereditary deficiency of hepatic ornithine transcarbaaylase, with resulting hyperaamonemia (ref 19). 'This diagnosis was eventually confirmed when the third mdle infant of this couple behaved in a manner identical to that of his two deceased brothers and, despite attempts at therapeutic intervention which included low protein feedings, he developed documented hyperamconemia and died in this medical center. Study of the liver revealed the complete absence of ornithine transcarbasylase. It must be stressed that clinical observations of the first two infants and post mortem examinations were not helpful diagnostically, and it was the chemical identification of erotic acid which led to the understanding of the familial pattern of neonatal mortality in this family. Ilost medical centers have access to amino acid analyzers in order to screen patients for metabolic abnormalities of the principal amino acids, but unless a special research interest exists, other inborn errors of metabolism cannot easily be studied. At this institution the tiC/EIS system provides us the opportunity to detect a uide variety of inborn errors which show accumulation of amino acids, fatty acids, and many other metabolites in urine, blood, amniotic fluid, and other biOlOJiCdl fluids and tissues. Another application of GC/tiS pertains to pre-natal diagnosis of hereditary inborn errors of metabolism, Fetal urine contributes to the amniotic fluid by the twelfth week of gestation and it should provide information which is diagnostically relevant to the fetus (ref LO). Surprisingly little is known about the origin, fate, and components of normal amniotic fluid, although information is accumulating because of interest in pre-natal diagnosis and evaluation of the fetus (ret 21). The fetal cells of the amniotic fluid, cultivated IN VITRO, are used routinely for pre-natal diagnosis of ,Jenetic disorders (ref 22). The fluid itself has been used infrequently for diagnostic purposes, although there is increasing evidence for the utility of direct assay of this component of amniotic tluir! for pre-natal diagnosis of heritable metabolic errors. Rahoney et al. (ref 23) have recently reported the first successrul pre-natal diagnosis of methylmalonic aciduria (confirmed by stu*:y of the fetus and performed in time to elect abortion). Their -9- observations suggest that at 12 weeks of gestation, methylmalonic acid is undetectable in the amniotic fluid surrounding a normal fetus and, probably, a fetus who is heterozygous for the (recessive] gene (ref 24). Goo3man. et al. (ref 25) have successfullp diagnosed argininosuccinic aciduria in a 16 week fetus. This involved detection of argininosuccinic acid in the amniotic fluid plus enzymatic studies on cultured amniotic fluid cells. Normally this compound is not detected in amniotic fluid at 16 ueeks of gestation. Thera are reports of other conditions being diagnosed in the fetus by direct asstiy of amniotic fluid (ref 26) as uell. The accuracy of diagnostic procedures probiny amniotic rluid for soluble constituents has been guestioncd because of the possibility of contamination with maternal blood and because of lack of information on the normal state at various times of gestation. If the fluid component could be used for pre-natal diagnostic purposes, the phenotype of the fetus could he detected relatively rapidly as compared to the time required to culture sufficient amniotic cells. Hereditary diseases which are potentially amenable to diagnosis by analysis of the soluble constituents of amniotic fluid are those in which the accumulating metabolite is not cleared by the placenta but is expected to appear in fetal uri-ne. Defects in epithelial transport, e.g., cystinuria an3 Hartnup disease, are examples of such conditions. However, it is clear that this class of metabolic errors is not the only one which might be detectable by direct assay of amniotic fluid. The examples provided above suggest. that fetuses with overflow type metabolic errors may also be detected. The development of diagnostic and screening techniques suitable for various inborn errors of metabolism will require a suitable computer based methodology for screening a large selected sample of subjects with the subsequent resolution of data into classifications describing normal states and ranges as well as specific correlations of lX/HS analysis abnormalities uith disease states. With a modest augmentation of existing instrumentation facilities, we can accomplish these analytical tasks on increasing nuabers of patients. B. SPECIFIC AIM a) We plan to use GC/NS to screen urine and pldsma from normal individuals ok various ages, including premature and newborn infants, in order to establish adequate control clatd and to anderstand variations encountered. b) We plan to use GC/MS in th o diagnosis of inherited metabolic abnormalities an3 in the detection and study of previously unrecognized metabolic disorders. -Y- observations suggest that at 12 weeks of gestation, methylmalonic acid is undetectable in the amuiotic fluid surroundiny a normal fetus and, probably, a fetus who is heterosygous for the (recessive) gene (ref 24). Goodaaa, et al. (ref 25) have successfully diagnosed argininosucciaic aciduria in a 16 week fetus. This involved detection of aryininosuccinic acid in the amniotic fluid plus enzymatic studies on cultured amniotic fluid cells. Nocrally this compound is not detected in amniotic fluid at 16 weeks of gestation. Thera are reports of other conditions being diagnosed in the fetus by direct assttp of amniotic fluid (ref 26) as well. The accuracy of diagnostic procedures probing amniotic fluid for soluble constituents has been questioned because of the possibility of contaaination with maternal blood and because of lack of information on the normal state at various times of gestation. If the fluid component could be used for yre-natal diagnostic purposes, the phenotype of the fetus could be detected relatively rapidly as compared to the time required to culture sufficient arniotic cells. Hereditary diseases uhich are potentially amenable to diagnosis by analysis of the soluble constituents of amniotic fluid are those in which the accumulating netabolite is not cleared by the placenta but is expected to appear i.n fetal urine. Defects in epithelial transport, e.g., cystinuria an3 Hartnup disease, are examples of such conditions. Hoverer, it is clear that this class of metabolic errors is not the only one which might be detectable by direct assay of amniotic fluid. The examples provided above suggest that fetuses with overflow type metabolic errors may also be detected. The dewelopment of diagnostic and screening technigues suitable for various inborn errors of metabolism will require a suitable computer basea methodology for screening a large selected sample of subjects with the subsequent resolution of data into classifications describing normal states and ranges as well as specific correlations of GC/NS analysis abnormalities with disease states. With a modest augmentation of existing instrumentation facilities, ue can accomplish these analytical tasks on increasing numbers of patients. B. SPECXPIC rras a) Ye plan to use GC/HS to screen urine ana plasma from normal individuals of various ages, including premature and newborn infants, in order to establish adequate control datn and to understand variations encountered. b) Ho plan to use GC/HS in th o diagnosis of inherited metabolic abnormalities aad in the detection and study of previously unrecognized metabolic disorder:;. c) Ye plan to use GC/MS to study normal variation of clinically significant aetabolites in amniotic fluid and then to apply these techniques to pre-natal diagnosis of hereditary inborn errors of metabolism. d) In support of the above goals, we plan to augment our existing GC/PlS system and to more fully automate it for screening larger numbers of patients through improved computer management. This would fit with current concepts ot regionalization of diagnostic facilities for genetic disorders and would'& appropriate for a Genetics Research Center. -ll- c. METHODS OF PROCEDURE We use the following procadures to fractionate mine, blood, and amniotic fluid in preparation for their analysis by GC/fiS. In the case of plasma, the protein is first precipitated (by the addition of ethanol) and the supernatant liquid dried IN YACUO and processed as for urine. Amniotic fluid is treated in the same way as urine. URINE (pH = 1, internal standards added) I I 1 ethyl acetate extraction I -------------------------------- I I organic phase aqueous phase (free'acids) L -------------------------------- A (carboh:drates) I I (amino acids) I HC1 hydrolysis c B I and ethyl acetate 1 extraction --------------------------------- I I organic phase aqueous phase (hydrolyz:d acids) (aeino'&cids) D E The experimental procedurs used for uorkiug with a fluid sample is as follows. To an aliquot (2.5 ml.) of fluid is added 6N hydrochloric acid until the pH is 1. Two internal standards, n-tetracosane and 2-amino octanoic acid are then added. Ethyl acetate extraction isolates the free acids (fraction A) which are then methglated and analyzed by W/HS. An aliquot of the aqueous phase (G.5 al.) is concentrate3 to dryness, reacted with n-butanol/hydrochloric acid followed by methylene chloride containing trifluoroacetic anhydride. This procedure derivatizes any amino acids (or uater soluble amines) which are then subjected to GC/BS analysis (fraction B). Another aliquot (0.5 ml) of the aqueous phase can ba derivatized (T8lS) for the detection of carbohydrates (Fraction C). Concentrated hydrochloric acid (0.15 ml) is added to the urine (1.5 al) after ethyl acetate extraction dnd the mixture hydrolyzed for 4 hours under raflux. Ethyl acetate extraction then separates the hydrolyzed acid fraction (D) which is methglated and analyzed by GC/MS. A portion of the aqueous phasca (0.5 ml), from hydrolysis of the urine, is concentrated to -12- dryness and derivatized and analyzed for amino acids (Fraction ?o o The above scheme represents a general method for preparing body fluids for analysis. The conditions required for gas chromatoyraphic separation vary with different classes of compounds. For instance, using our method for chromatographing derivatized free acids, amino acids, and carbohydrates, steroids will not be detected. A different gas chromatography column and derivatization procedure would be required. Large molecular weight compounds (e.g., tripeptides and mucopolysaccharide fragments) cannot be analyzed by this system without their degradation to smaller unit molecules. A suitable computer data system can significantly ease the tedious burden of analyzing the large amounts of data emanating from GC/fiS instrumentation. The computer assists in instrument set-up and calibration, data collection and filing, and data reduction dnd analysis. We have developed considerable experience with such data systems, both for low and high resolution instruments, since our fxrst design uas described in 1966 (ref 27, copy attached). Our most recent efforts have focussed on facilitating user interaction uith the data system and on automating various aspects of data analysis (ref 3~). 'The research proposed in this application entails the analysis of increased numbers of body fluid samples, beyond the capacity of the present system. We therefore propose to augment the current system to handle the larger sample volume and at the same time to extend the capabilities of the system to reduce X/AS data. Our present system is built around the NLH-subsidized ACME tine-shared computer facility (Iflh 360/50) with the real tine data interface being through an Itrtl 1800 computer. These machines vi11 disappear at the termination of the ACHE grant in July 1973 and will be replaced by a fee-for-service IBM 370/158 system with more limited real time support capabilities. With the transition implied by the machine changeover, we have reevaluated the approach to be taken in implementing our GC/HS supFort needs relative to the new requirements presently proposed, the projected costs of alternative approaches, and the evolving computer technology. Mini-computer capabilities have advanced significantl.y sinct? the previous decision was made in 1965 to implement the existing system on the ACPiE .facility. After examining the options of implementing a data system on the new time-shared facility or on a stand-alone mini system, we feel that a mini-system is the more advantageous for our needs. Such a system is the cheaper of the two based on projected costs and is more responsive to real time needs in view of a smaller set of demands on the mini-machine. There are limitations inherent in mini-machines, however, in terms of memory size and software/language support. The large central system (370/158 or SUMEX if approved) will dct as a backup for the mini-system in the relatively small num1;er of instances where these considerations are important in low resolution GC/hS I, - /-- ,... `I -13- applications, In preparation for the transition this July, we are iaplementiug a minimal data system on a PDP-11/20 computer. This machine is well suited for "front endUi data collection and filing activities as vell as simple analysis tasks. It cannot simultaneously support data acquisition and the more sophisticated analysis activities conte0plated in this proJran, however, because of capacity limitations. In addition, it lacks the arithmetic speed desirable for these analysis procedures. Since the proposed research program will entail a heavy duty cycle of data collection throughout the day to analyze the increased number of body fluid samples, data analysis and program development would have ,to take place during off hours. We propose to solve this problem by acquiring a second machine to support the PDP-1 l/20. This machine, tentatively selected as a PDP-11/43, would provide for data analysis and program development support and would be well suited to anticipated data analysis functions because of its speed and extended arithmetic capabilities. We have also considered a PDF-11/40 machine but feel the PDP-II/45 to be a better choice because of the relatively small cost differential (approximately $10,000) in return for a factor of 2-3 in performance. The proposed system configuration is shovn in Pigure 1, Our choice of Digital Equipment Corporation (DEC) maChiAeS is based on oar existing hardvare and software expertise with this equipment. Our existing high resolution PlS system utilizes a similar PDP-ll/20 machine as the data acquisition computer a5 vell. The PDP-11/45 vould have 24K words of memory, floating point hardware and a programmable clock. The memory size is based on projected needs for PORTPAN-based analysis programs. The two disk drives (an existing fixed head drive, 262K vords and a proposed moving head drive, 1.2fi words) provide needed space for system software, programs (source and object riles), and for spectral data. It should be noted that a single GC/rSS run contains some 700 spectra, each containing 500 12-bit spectral amplitude measurements. Each such file therefore requires 350,000 words (uncompressed) so that our proposed disk space would be quickly consumed by several such files in residence. We can increase the effective space available by compressing the data files to eliminate insignificant measurements. Note that this requites at least one data analysis pass over the full file, however. we propose to augment this disk capacity in the second year by adding an additional drive. In general, data files vi11 be stored on luagnetic tape. Both DEC tape and industry compatible tape drives are pravided. The former is required for system maintenance and is ideal for relatively small private program files, data files, etc. The industr,y compatible tape provides for large volume storage of K~LV data during data acquisition and for archival storage. This augmented data system will allow increased sophistication in the analysis of GC/MS runs and in prescreenin\j -14- GC profiles. In process.iny body fluid samples to establish Norma; and to investigate specific clinical abnormalities, full flS analyses of the GC effluent will be required. This involves extracting from the approxieataly 700 spectra collected during each run, the 100 or so representing the components of the body fluid sample for identification. The raw spectra are contaminated with background *gcolu~n bleed" and some are composited with adjacent constituent spectra unresolved by the GC, We have begun to develop a solution to the problem of effectively increasing the resolution of the GC by computer analysis of the data. These programs will allow us to autoaaticaly locate the body fluid constituent spectra and remove the distortions caused by background and poor resolution. These "cleaned-up" spectra can then be analyzed by library search techniques 3r first principles as necessary. By using a disk-oriented matrix transposition algorithm developed for image processing applications, we can rotate the entire array of 700 spectra by 500 mass samples for each run, Tn this way ve can gain convenient access to the "mass chroeatograan form of the data. This form of the data, displayed at a few selected masses, is used in mass fragsentography described elsewhere in this proposal. fYass chrosatograas have the important property of displaying much higher resolution in localizing GC effluent constituents. The automatic analysis techniques currently being developed for mass fragnentograms can be extended to this more general case. Thus by transposing the raw data to the mass ChrOQatOgCam domain, we can systematically analyze these data for baselines, peak positions, and correct amplitudes thereby deriving idealized mass spectra for the constituent materials. These spectra are free from background contamination and influences of adjacent unresolved GC peaks. The results of this work zan also lead to reliable prescreening analyses of GC traces alone by having available a detailed list of expecteu GC effluent positions and amplitudes for the particular body fluid fraction under consideration. By dynamically determining peak shape parameters for detected GC singlet peaks, interpretation of more complex peaks can be made to determine if unexpected constituents or abnormal amounts of expected constituents are present. If so, d more thorough K/M analysis can be trade. This typz ot prescreening is valuable in processing body fluid samples which may or may not be abnormal and saves by committing the expensive mass spectrometer instrumentation to analyzing only suspect samples. This, of course, assumes that norms have been previously established by processing body fluid samples in detail from a large population of normal subjects. The problem of spectrum identification is addressed by using rapid library search techniques for the identification of previously encountered compoun3s. Those not in the library will be identified by the manual interpretation of the spectrum usinq other information as available, These results will be incorporated into the library to extend its domain of usefulness. P-3/ -1% This progressive compilation of a library of bioaedically relevant compounds will speed the throughput of the system. .This library w-ill be freely shared with other investigators. Eventually the extension of the library domain will be assisted by adapting the computer programs under development in the DENDRAL project (NIB RR-UO612). These seek to emulate the reasoning processes organic chemists employ in interpreting spectra using fragmentation rules and other knowledge to infer the correct molecular structure from among those possible. The most appropriate target material for this developmental effort is surely the metabolic output of NORBAL subjects under controlled conditions of diet and other intakes. The eventual application of this kind of analytical methodology to the diagnosis of disease obviously depends on the establishment of normal baselines, and much experience already tells us hou important the influence of nutrient and medication intake can be in influencing the composition of urine, body fluids, and breath (ref 18). Among the most attractive subjects for such a baseline investigation are newborn infants already under close scrutiny in the Premature Research Center and the newborn nurseries of the Department of Pediatrics at this institution. Such patients are, for valid medical reasons, under a degree of dietary control difficult to match under any other circumstance. Many other features of their physiological condition are being carefully monitored for other purposes as well. The examination of their urine and other effluents is therefore accompanied by the most economical context of other information and requires the least disturbance of these subjects. Two obvious factors which could profoundly influence the excretion of eetabolites detected by GC/#S are maturity and diet. Be have already initiated a program for serial screening of urinary metabolite excretion in premature infants of various gestational ages and a determination of changes in the pattern of excretion of various netabolites as a function of age following birth. A synopsis of this research is presented later in this section. These studies are being performed on infants admitted to the Center for Premature Infants and the Intensive Care Nursery at Stanford, a source of some 500 premature infants per year, In addition, in conjunction uith an independent study on the effects of both quality and quantity of oral protein intake on the incidence and pathogenesis of late metabolic acidosis of prematurity, ue plan to measure the urinary excretion patterns of various metabolites and thereby partially assess the effect of diet on this screening method. We shall use the analyses on blood and urine specimens from normal individuals in the final development of rapid, automated identification of compounds described by mass spectrometry. ay compiling records o-f the gas chromatographrc profiles dnd associated computer-identified constituentx of the body fluids of normal individuals, we will establish the statistical norms and expected variations for the component and levels present. Quantitation is achieved by introducing internal stdnddrds into -lb the body fluid fractions prior to analysis. We will seek to minimize or at least stabilize the variations in measurement results by assessing the effects of various aspects of diet and medication. lornal blood and urine specimeos will also allou us to test the system's operational capacity for rapid and accurate aetabolite identification. Given our abilitg to identify various constituents of urine and plasma and to understand normal variation, we shall apply the GC/hS system to patholo$y, making use of patients with already identified aetabolic defects for control purposes. The aain application uill, of course, be diagnostic and patients with suggestive clinical manifestations, such as psychomotor retardation and progressive neurologic disease, as uell as suggestive pedigrees (e.y. affected offspring of consanguineous parents or multiplex sibships) vi11 be investigated. Actually, we have been studying limited numbers of such patients already. These patients are seen relatively frequently at any university hospital, and their presence in the various in-patient and out-patient services of the Stanford Department of Pediatrics is well documented. The GC/M system will be invaluable in diagnosing various inborn errors of metabolism, especially those involviog excretion of various fatty and other organic acids. Some of these are isovaleric acidemia (ref IS), methylaalonic acidemia (ref 28), the recently reported inherited disorder of isoleucine metabolism causing accumulation of alpha-aethylacetoacetic acid and alpha-aethyl-beta-hydroxybutyric acid ,(ref 29), lactic acideaia (ref 20), Refsum's disease (a defect in the oxidation of phytanic acid - ref 31), erotic dciduris (ref 32), dnd as illustrated above, ornithine transcarbamylase deficiency (ref 19). lie recognize the potential of this methodology for defining previously undescribed (*nevcc) inborn errors of metabolism (e.g., ref 15). In considering the strategy for applying tiC/MS techniques to problems of screening and characterizing inborn errors of metabolism, particular liaitations in our present system in terms of detection capabilities or throughput capacity must be accounted for. For example, our present system is somewhat limited for screening and diagnosing those conditions whose phenotypes include the accumulation of amino acids in urine and/or blood. He can detect all of the naturally occurring alpha amino acids except hoaocystine, cystine, and tryptophan. In addition, the derivatization procedure being used, converts asparagioe and glutamine respectively to aspartic acid and glutamic acid. Thus the presence and quantity of asparagine and glutamine cannot be: separately measured from that of aspartic acid and glutanic dcid by current procedures. This situation could be remedied by the use of a different GC column in order to detect homocystine, cystina, and tryptophan. By using an additional derivatization procedure, asparagine and ylutamine could be measured. This approach, while rigorous in its ability to detect and quantitate amino acids, complicates the higher -1'7- volume screening throughput. In effect, it would be necessary to install and operate an additional gas chromatograph just for this purpose because,the temperature programming conditions for the new column differ from those required for the column presently used for acid and carbohydrate fractions. Ye are auare that an amino acid analyzer, separate from the GC/nS system, offers a practical solution to larger szale screening for metabolic errors involving the accumulation of amino acids. This approach also has the advantage of providing increased assurance that the clinical analysis of amino acid accumulations can be performed at times when the GC/LIS system may be tied-up, under repair, or undergoing engineering change. Such an analyzer, using ion exchange chromatography, does not require derivatization of the specimen and can detect all of the naturally occurrinq amino acids. We therefore propose to acquire an amino acid analyzer to assist in the screening of the increased number of samples expected from our work at Stanford and from collaborating researchers elsewhere. In selected cases where the amino acid analyzer alone cannot provide sufficient resolution and quantitative accuracy in characterizing amino acid accumulations, the more lengthy, augmented X/MS approach vi11 be available. Another area in which our existing equipment would limit throughput is in the capacity 3f the single low resolution mdss spectometer. The present GC/MS system can process 1-2 specimens per day (including all five derivatized fractions). This throughput has sufficed for small sample research applications but we anticipate that this load will at least be tripled when we are actively studying normal variations and are screening patients for metabolic errors. We could, of course, eliminate the bottleneck by buying more mass spectrometers. I'his would be quite expensive. An alternative approach appears feasible. By passing the samples submitted for screening through a preliminary GC profile analysis (without HS), we feel it will be possible to identify those deviating from established normal linits and warranting a more rigorous GC/HS analysis. This will allow store efficient use to be made of the HS instrument time. For abnormal samples, the deviation, under IIS analysis, may be readily identified by library search techniques or may require more lengthy analysis procedures. These could require different chemical derivatization techniques or additiondl information such as high resolution mass spectrometry, This additional analysis could consume days or veeks of effort. Thus it is important to make efficient use of these analysis capabilities. The number of: samples eliminated in GC prescreening depends, of course, on the nature of the samples submitted. Prescreening will be less useful for the processing of samples which have been carefully selected as clinically suspect than on a general samFling of "normal" individuals. It is in this latter case that we see a benefit to developing the prescreening capability. Fat: this purpose, we propose to add to our laboratory a 4 column gas ChrOmdtOgrdph. The Department of Pediatrics has affiliations with the Kaiser-Pernanente bediCd1 Center in Santa Clara, the Santa Clar9). 27) Reynolds, W.E., Bacon, V.A., Bridges, J-C., Coburn, T.C., Halpern, 8.. Lederberg, J., Levinthal, E.C., Steed, $., and Tucker, R-B., #'A Computer Operated Hass Spectrometer System,** Anal. Chem., 42, Page 1122, (1970). 28) norrow, G., Schuartz, R.H., Hallock, J-8., and Barness, L.A., *Prenatal Detection of Kethylmalonic Acidemia," J. Pediatrics, 77, p. 12b, (1970). 29) Daum, R, S., Seriver, C, A., namer, 0. A., Delvin, E., Lamm, P ., and Goldman, H., nAo Inherited Disorder of Isoleucine Catabolism Causing Accumulation of Alpha-methylacetoacetate and Alpha-methyl-beta-hydroxybutycate and Intersittant tietabolic Acidosis, Pediat. Res,, 7, 149 (1973). 30) Haworth, J.C., Pord, J.D., and Pounoszai, K.K., "Familial Chronic Acidosis due to an Error in Lactate and Pyruvate Hetaboliss," Canad. fled. Ass. J., 97, p. 773, (1967). 31) Herndon, J.H., Steinberg, D., and Uhlendorf, B.U., nBefsutn's Disease: Defective Oxidation of Phptanic Acid in Tissue Cultures Derived from Homozygotes and Heterozygotes," New England J. of Iled., 281, p. 1034, (1969). ,h _,*, / i, -2b- 32) Fallon, J.H., Smith, L.H., Graham, J.El., and Burnett, C.H.,, nA Genetic Study of liereditary Orotic Aciduria," New England J. of Bed., 270, p. 878, (1964). 32a) Lawless, J-G, and Chadha, E.S., @#Bass Spectral Anallyisis or C(3) and C(4) Aliyhatic Amino Acid Derivatives," Anal. Biochem., 44, p. 473, (1971). 33) Sutton, H. E., "BAIBuria in Inherited Biochemical Disease,*' New York, McGraw-Hill, 1960, pp.792-806, 34) Auapara, J., "The Leukemias, H International Symposium (Detroit) View York, Academic Press, 1956, p. 353. 35) Rubiai, J. R., Cronkite, E. P., Bond, V., and Fleednir, T. n P& "Urinary Excretion of BAIl3 in Irradiated Human Beings.@g . Sot. Exper, Viol. and Bed., 100, Page 130 (lYS9). 36) wright, S. W. and Fink, K., "The Excretion of BAIB in Normal, fiongoloid and Non-Mongoloid Bentally Defective Children," Am. J. Bent. Deficiency, 61, Pnge 530 (1953). 37) Berry, H. K., "Individual betabolism Patterns: .II. Excretion of BAIB." Retabolism, 9, Page 373 (1960). 38) Harris, Ii., "Family Studies on the Urinary Excretion of BAIB," Ann. Eugenics, 18, Page 43 (1953).. 39) Calchi-Novati, C., Cepellini, R., Biancho, I., Silvestroni, E. and Harris, H., "BA'IE) Excretion in Urine. A Family Study in an Italian Population,w Ann. Eugenics, 18, Page 335 (1954). 40) DeGrouchy, J. and Sutton, H, E., "A Genetic Stud.y of BAIB Excretion," Am, J. Human Genetics, 9, Page 76 (1957). 41) Yanai, J., Kakimoto, Y., Tsu jio, T., and sane, I., Am. J. Human Genetics, 21, Page 115 (1969). 42) Gartler, S. M., "A Family Study of BAIB Excretion," Am. J. Human Genetics 8, Page 120 (1956). 43) Gartler, S. B., Pirschein, I. L., and Kraus, R. S., @*An Investigation into the Genetics and Racial Variation of BAIE Excretion," Am. J. Huaan Genetics, 9, Paye 200 (1957). 44) Gactler, S. fi., "A Betabolic Investigation of Urinary RAIU Excretion in Man, Arch. Biochem. 80, 400 (19SY). 45) Pink, K., Henderson, R. B., and Fink, R. ai., "BAIB: A Possible Factor in Pyrimidine fietabolism,ti Proc. Sot. Exptl. Biol. Bed., 78, Page 135 (1951). 46) Crumpler, H.R., Dent, C.E., tiarris, H., and Westall, R.G., "BAIB, A Xew Amino Acid Obtained from Human Urine," ddture, lb7, Page 307, (1951). -29- 47) Goedde, H.W. and Brnnschede, H., 18frAIB: A TLC Plethod for the Quantitative Estimation in Elnman Urine," Clin. Chim Acta, 11, page 485, (1965). 48) Pessas, P.R., Koniavitis, A., and Zeis, P.R., J. Clin, Path., 22, Page 154, (1969). 49) Killmann, S.A., Rubini, J.R., Cronkite, B.P., and Bond, V.P., *Urinary Excretion of Beta-Amino Isobutyric Acid (BAIBA) in Leukemia and Polycytheaia Vera," Acta Haemat., 25, Page til, (1961). 50) Lee, K.B., Reill, D-W., and Bridges, J.B., "Urinary Amino Acid Excretion in Subjects with Leukemia," 3. Clin. Path., 20, Page 70, (196'7). 51) Schreiec, "The 'Behavior of Amino Acids in Body Fluids During Development and Growth: Physiology and Pathology in Amino Acid Pools," J.T. Holden, Editor, Klsevier, blew York, Paye 2'76, (1962). 52) Ranlov, P. and Siygaard-Aadersen, 0.. "Late Betabolic Acidosis in Premature Infants; Prevalence and Significancc,n Acta Ped. Stand., SU, Page 531, (1965). 53) Leviae, S.Z., Bacyles, E., and Gordon. H.H., nA Defect in the Metabolism of Tyrosine and Phenylalanine in Premature Infants, I: Identification and Assay of Intermediary Products." J. Clin. Invest., 20, Page 199, (1941). 54) Dancis, J. and Levitz, M, "Abnormalities of Branched-Chain Amino acid Betabolisr: The Metabolic Basis of Inherited Disease ,I) Stanbury, J-t)., Wpngaarden, J-B., and Predrickson, D.S.. Editors, McGraw-Hill, Raw York, 3rd Edition, Page 426. 55) Bartter, F.C., *Hypophosphatasia: The Retabolic Basis of Inherited Disease," Stanbury, J.b., Wyngaarden, J. B., and Predrickson, D.S., Editors, BcGcau-Rill, Neu York, 3rd Edition, Page 1295. 56) Rasmussen, K., WPhosphocylethanolamine and Rypophosphatasia, Danish fled. Bull,, 15 (Suppl. II), 1 (196d). 57) Bevis, D.C.A., %omyositi~n of Liquor Amnii in Haemolytic Disease of lewborn," Lancet, ii, Page 443, (1950). 58) Deflarca, C., WAminoacidie Chetoacidi Presenti nel Liquid0 Axniotico,N G. Biochem., 3, Page 296, (1954). 59) Thomas, J.A. and Thiery, J.P., "The Use of Deproteinized and Purified Amniotic Fluid. Culture and Prolonged Conservation of the Tissues; Bicrobiological Cultures," Bull. Sot. Chim. Biol., 35, Page 841, (1953). -3O- 60) Orlandi, C., Torsello, ft.!!., and Bottiqlioni, P., *'Analisi Qualitativa e Dosaggio Semiquantitativo degli Aminoacidi Contenuti nel Liquid0 Amniotico,** Attual, Ostet. Ginec., 4, Pacje 871, (1958) * 61) Wirtschafter, Z.L., "Free Amino Acids in Human Amniotic Fluid, Fetal and Maternal Serum,*' Amer. J. Obutet, Gynec., `76, Page 1219, (1958). 62) Sassi, D., *#Sulla Presenzs degli Aminoacidi nel tiquido Amniotico," Honogr, Ostet, Ginec., 33, Page 683, (1962), 63) Warren, C., Allen, J.T., and Holton, J.8.6 "Assessment of Foetal Lung Maturity by Amniotic Fluid Fatty Acid AnaLps&," Clin. Chin, Acta, 44, Page 457, (1973). 64) Reiner, E. and Hicks, J.J., "Pyrolysis-GLC Studies on Amniotic Fluid: A Prospective nethod for Confirming Inborn Errors of Fetuses," Chromatographia, S, Page 529, (1972), 65) Hagenfeldt, L. and Kagenfeldt, K., "Gas Chromatographic-Mass Spectroaetric Analysis of Organic Acids in Amniotic Fluid," Cllin. Chia. Acta, 42, Page 219, (1972). 66) Neu, ff. I., "Antenatal Diagnosis of the Adrenogeaital Syndrome," Lancet, 1, 83 (1970). L I I Existing Equipmentm FIGURE 1. PROPOSED DATA SYSTEM HARDWARE CONFIGURATION FIGURE 3 80 60 40 20 r53 40 60 `6'0 100 120 140 160 180 200 220 240 260 FIGURE 3 Malrs spectrum of P -amino isobutflic acid I- . FIGUW 4 GAS CIIROMATO(;fiAPiI OF URINARY ACID PROPILE A. PATIKIT ACIDOTIC (Z-17-73) I\" !`A'I'T)`yr' vfq."; 1, ( 2,. 1 (l-71 1 l.fIf! n.52 3.51 f-l.23 Q.4P -!I.51 ').7c, 2.7') n.74 I , :: 1 3 "I, Areas A r-as Arr-as Areas Arwis hrfvs Areas Arcas At-pas Art-as Areas 11349.2/ 21382.5 l&LX/ 109h7.3 6272.4/ 6054.7 39;. 21 I;!i53.7 R35,1/ 67621.3 7cn3.1/-330es.r 116?.7/ 2712.q 3293.4/ 31f?4.7 2920.1/ 5971.8 22El.E/ 5751.0 lcZE3.1;/ 19353.8 PATIC! = 0.5308 Locattan Error - 9 ?????? ?? o 0.1771 Locatifh rrrar =`-2 F4T PAT PAT PAT I 1.0277 0.107G 0.1235 -0.2238 0.4270 FIGURE 5 ANALYSIS OF 12 AMINO ACIDS IN URINE USING MASS FRAGMXNTOGRAPHY Location Erroi = 2 Lccntfnn Error - -1 loc?tinn Frrnr = 2 Locatfon Crror = 2 LocatIon Error = 0 Location Error - 0 Lor?&an 'rror = -2 Locattnn rrror = 0 Loci+tlan Frror - -1 THE SIMULTANEOUS QUANTITATIPN OF TEN AMINO ACIDS IN SOIL EXTRACTS BY MA& FRAGMENTOGRAPHY W. E. Pereira, Y. Hoyano, W. E. Reynolds, R. E. Summons and A. M. Duffield Department of Genetics, Stanford University Medical Center Stanford, California Running Title: Mass Fragmentography of Amino Acids Address for Proofs: Dr. A. M. Duffield Department of Genetics Stanford University School of Medicine Stanford, California 94305 Received P- 5-z The analysis of amino acids from terrestrial and extraterrestrial sources is becoming increasingly, important (l-5) , The need for a specific, I sensitive and rapid method of quantitation is desirable. The methods currentiy employed for amino acid analysis involve ion exchange procedures (6,7) or gas chromatography (8-10). These techniques, although of immense value, are limited by their non-specificity for the absolute identification of any substance responsible for a gas chromatographic peak. In the present communication we report an absolute, unambiguous method for the positive identification rnd quantitation of ten amino acids present in soil extracts using GLC-mass fragmentography. In mass fragmentography the mass spectrometer is used only to detect certain preselected ions known to be characteristic for each compound being quantitated, and the internal standard. The technique of mass fragmentography . . using sector mass spectrometers is usually restricted to the simultaneous monitoring of up to three integer mass values (11, 12), although with one instrument five ions were used spectrometer up to eight ions have analog signals monitored (14). We (13) o Using a.quadrupole mass been selected and their respective now wish to report the modification of the gas previously conqo~ of chromatography-quadrupole mass spectrometer-computer system described (15) for the simultaneous monitoring under computer ., . . . , the ion currents from 2.5 cc\nje br*w-- WIICS C Ld 750 i- con+tst 2 r;sc.l;Eetdcnteger mass _ values. -pjcsc via us cc- ;yL UVIIlCbIL 4or n`$l &8y--io+j ;I:;*(j SC' P*r.tr'XIf required this number could be increased by suitable alteration of the computer control programs. Specifically we wish to report the application . of thii system to the quanti'tation of ten of the amino acids present in soil . ___. --. _- ., extracts . Reagents: A deuterated amino acid mixture was supplied by Merck Laboratory Chemicals (New Jersey). 1.25N HCl in n-butanol, 25% (v/v) trifluoroacetic anhydride in methylene chloride and Tabsor column packing were obtained from Regis Chemical Co., Illinois. A standard amino acid solution was purchased from Pierce Chemical Co., Illinois. Equipment: A Varian model 1200 gas chromatograph was coupled by an all glass membrane separator (16) to a Finnigan 1015 Quadrupole mass spe-trometer which was interfaced to the ACME computer system of the Stanford University Medical School (15). CLC separations were conducted using a 6 foot by 4 mm. (I.D.) coiled glass column packed with Tabsorb (Regis Chemical CO.). The flow rate of the carrier gas (helium) was 60 ml/minute. . . The uniqueness of the mass spectrometer instrumentation lies in the -_- modified computer software (program) used. The hardware is the system previously described (15) and as:umes an operating cycle of: (a) transmission of a control number, N, from the computer to an interface controller which sets the quadrupole mass analyser to a particular mas-s point in the m/e continuum. -- (b) an integration of the ion.signal for a pre-set period, T (integration time = 8 milliseconds in our work), and (c) computer reading of the integration value with a twelve bit A + D conversion. . * ' For the recording of normal mass spectra N is selected such that successive cycles result in m/e values of 1,2, . . ...750. At the beginning of each -- day the instrument is calibrated using a reference compound. Idiosyncracies of the IBM 360,/50 to IBM 1800 computer data paths dictate that the mass s values be buffered into groups of 250.% For normal g.c.-m.s. procedures the operator is allowed to select a mass range of 1 to n x 250 (n = 1,2, or 3 buffers). For mass fragmento- graphy n is set to zero and instead,a "precision collect" buffer of 250 control-data acquisition cycles is employed. The operator must then enter the pre-selected m/e values he wishes to scan. When the precision collect -- buffer is constructed, 10 cycles are allocated to each m/e value selected. -- The first of the 10 cycles sets N to K -4. In The returned integrated ion measurement is discarded; this cycle serves only to slew the quadrupole electronics from anywhere in the m/e continuum to the mass region of -- InterFst. The additional 9.cycles are used with N = N,-4....Nm.....Nm+4. The returned values represent a set of readings about the m/e value of -- Interest + 0.5 amu. The center three points are then smoothed with a five point qudratic function (17). The highest value of these three smoothed points is then selected,as the precision collect value. Thus , small drifts in calibration are corrected and a signal average obtained. Finally ,i the abbreviated "spectrum" of 25 precision intensities for each m/e are filed on disc. a- Such a "spectrum" is recorded every 2 seconds and a summation of all the ion intensities is used to cou$truct the ion chromatogram shown in Fig. 2. Inditidual ion chromatograms can also be constructed if required (Fig. 3). A threshhold is established from the ion currents before and aftet each gas chromatographic peak and a computer program performs integration of the ion currents under each peak. Procedure 1 g of sieved, air-dried soil (Stanford University garden soil) was refluxed with 6N HCl (10 ml) for 20 hrs. The mixture was filtered and the residue washed kth 1N HCl (5 ml). The combined filtrate and washings were extracted with chloroform (4 x 10 ml) and the aqueous phase evaporated to dryness. The residue is dissolved in water (5 ml) and passed through a column of "Ion Retardation Resin" AG 11-A8 (So-100 mesh, 1 x 21 cm). The amino acids were eluted with water (50 ml) and the eluate evaporated in vacua to dryness. The residue is dissolved in water (5 ml) and placed on a column of p-57 cation exchange resin (AC SOW-X12, SO-100 mesh, 1 x 21 cm) and washed with water (50 ml) to remove neutral and anion contaminants. The amino acids were eluted with.4N NH4?H (80 ml) and the eluate evaporated to dryness. The residue was disiolved in water and made up to a volume of 4 ml. A portion of this solution (1 ml) was used for the amino acid analysis using an amino acid ,analyser. To another 2 ml of the processed solution was added 2 ml of the deuterated amino acid standard solution (100 mg in 100 ml of O.lN HCl) and the mixture evaporated to dryness. The residue was refluxed with 1.2 N HCl in n-butanol (1 ml) for 30 min. and evaporated to dryness in vacua. To the residue trifluoroacetic anhydride in methylene chloride (0.7: ml) was added and refluxed for 10 min. The solution was evaporated to dryness at room temperature and the residue dissolved in ethyl acetate (100 ~1). An aliquot (1 ~1) was injected into the injector port of the gas chromatograph and the oven kept at 100" for 1 min. when it was programmed at 4"/min. to 220'. To each of 4 tubes containing 2 ml of the deuterated amino acid standard solution (100 mg in 100 ml of O.lN HCl) was added 150, 200, 250 and 300 ~1 respectively of a standard amino acid solution (2.5 ,,moles -e of each amino acid per ml). The solutions were mixed and evaporated 'to dryness. Each residue was derivatized by the above method and an aliquot of each (1 l.J) injected into the gas chromatograph which was operated under the conditions described above. This procedure . . . . was used to construct a standard curve for the quantitation of each am&o acid. A typical standard curve is shown (Figure 1) for glutamic. acid. p- 5-z RESULTS The N-TFA, 0-n-butyl derivative was chosen for the derivatization s of amino acids for two reasons.. Firstly, these derivatives have 14 excellent glc separation characteristics @$j and secondly the selected . characteristic fragment ions of the deuterated and non-deuterated derivatives do not interfere with each other, nor with other a-amino acids. Table I records the individual ions monitored for quantitation in the mass spectra of each of the deuterated and non-deuterated amino acids. Thezomput~tegrates-the-intensity of the deuterated and Be-ratio-of-their-respective-peak-areas7 Our results of a typical soil analysis are compared with those from an amino acid analyser in Table II. The higher value obtained with lysine by the amino acid analyser is due to a ninhydrin positive substance in soil interfering with the quantitation of lysine. In this respect mass fragmentography is superior to the amino acid analyser in that using a mass spectrometer as detector only characteristic pre-selected ions are detected and quantitated and any impurity present' under the same gas chromatographic peak is not measured* A summation of 20 such characteristic ions was plotted as an ion chromatogram of a derivatized soil sample and is shown in Fig. 2. Preliminary experiments showed that when the deuterated amino acid mixture was added directly to the soil sample extensive hydrogen- deuterik exchange occurred during acid hydrolysis of the soil extract. The removal of the isotopic label was catalysed by the hot mineral acid in presence of excess mineral used in the soil hydrolysis step. Fox and collaborators have reported (4) a similar finding concerning the _ - decomposition of amino acids in soil upon direct acid hydrolysis. In the present work the deuterated amino acid mixture was added just before derivatization (i.e. after hydrolytic extraction of the soil) in order to avoid this problem. However, in cases where it is necessary to quantitate the free amino acid content of complex mixtures, such as In serum or urine samples, the deuterated amino acid mixture may be added sample before processing without any deleterious Although only ten amino acids present in soil were quantitated the method can be extended to all the normal amino acids found in protein. The deuterated analogs of arginine, histidine, serine, threonine and tyrosine are commercially available. Appropriate deuterated analogs of methionine, tryptophane, cysteine and cystine would have to be chemically synthesized from the appropriate precursors. In these instances at least two deuterium atoms should be incorporated in non-exchangeable positions so that for the characteristic ion chosen the P + 2 peak is separate from the 13 C isotope contribution of the unlabeled amino acid. Furthermore, the deuterium substitution need not be quantitative (>90%) provided the same characteristic ion of that deuterated analog is used for the construction of a standard curve such as Figure 1. -Lx Instrument analysis time is approximately one hour and with our system we have been able to achieve accurate quantitation with samples containing as little as 10 nano'grams of an amino acid, SUMMARY A specific and sensitive method for the identification and simultaneous quantitation by mass fragmentography of ten of the amino acids present in soil has been developed. The technique uses a computer driven quadrupole mass spectrometer and a commercial preparation of deuterated amino acids is used as internal standards for purposes of quantitation. The results obtained are comparable with those from an amino acid analyser. In the quadrupole mass spectrometer-computer system used up to 25 pre-selected ions may be monitored sequentially. This allows a maximum of 12 different amino acids (one specific ion in each of the undeuterated and deuterated amino acid spectra) to be quantitated. The method is relatively rapid (analysis time of approximately one hour) and is capable of the quantitation of nanogram quantities of amino acids. . . . . . ACXNOWLEDGMEXTS This research was funded by the Planetology Program Office, Office of Space Science, NASA Headquarters under grant NGR-05-020-004. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. Il. 12. '13. 14. POCKLINGTON, R., Anal. Biochem. 45, 403 (1972). TAYLOR, R. and DAVIES, M. G., Anal. Biochem. 51, 180 (1973). - HALPERN, B., WESTLEY, J. W., LEVINTHAL, E. C. and LEDERBERG, J., Life Sciences and Space Research - North Holland, Amsterdam, 239 (1967). HARE, P. E., HARADA, K. and FOX, S. W., Proc. Apollo 11 Lunar Science Conf. 2, 1799 (1970). = KVENVOLDEN, K., LAWLESS, J. G., PERING, K., PETERSON, E., FLORES, J., PONNAMPERUMA, C., KAPLAN, I. R. and MOORE, C., Nature, 228, 923 (1970). Z SPACKMAN, D. H., STEIN, W. H. and MOORE, S., Anal. Chem. 3&, 1190 (1958). HAMiLTON, P. B., Anal. Chem. 35, 2055 (1963). GEHRKE, C. W., KUO, K. and ZUMWALT, R. W., J. Chromatog. 57, = 193 (1971). GEHRKE, C. W., ZUMWALT, R. W. and WALL, L. L., J. Chromatog. 37, 398 (1968). . . . ._ JijNSSON, J., EYEM, J. and SJGQUIST, J., Anal. Biochem. 51, 204 (1973). - HAMMA!, C. G., HOLMSTEDT, B. and RYHAGE, R., Anal. Biochem. 2, 532 (1968). _- HAMMAR, C. G. and HESSLING, R., Anal. Chem. 43, 298 (1971). -- - SNEDDEN, W., PARKER, R. B. and WATTS, R. E., Int. Conf. on Mass -- Spectrometry Brussels, 31&. i 4 Sept. 1970, Vol. 5, Institute - of Petroleum, London, and Elsevier, Amsterdam, 1971. p. 742. . . KNIGHT, J. B., Finnigin Spectra, &, No. 1 (1971). .lS.. REYNOLDS, W.: E.; BACON;& A;, BRIDGES, J.. C., COBURN;T. C.,. HALPEPN, B., . LEDERBERG, J., LEVINTHAL, E. C., STEED, E. and TUCKER, R. B., Anal. .`, . Chem. 42, 1122 (1970). = 16. HAWES, J. E., MALLABY, R. and WILLIAMS, V. P., 2. Chromatog. Sci. 2, 690 (1969). (%H. GEHRKE, C. W. and STALLING, i. L., Separation g. 2, 101 (1967). 1q.6 PEREIRA, W. E., BACON, V. A., HOYANO, Y., SUMMONS, R. and DUFFIELD, A. k., Clin. Biochem. (In Press). Table I. CHARACTERISTIC FRAGMENT IONS SELECTED FOR MASS FRAGMENTOGRAPHY OF UNDEUTERATED AND-DEUTERATED . N-TFA-0-IJ-BUTYL-AMINOrACIDS. Amino Acids i Fragment Ion VAL GLY ILEU LEU PRO PHE ASP GLU LYS CH3CH=kICOCF3 (m/e 140) CD3CD(NH2)COOH I-C3H7CH=zHCOCF3 (m/e 168) I-C3D7CD(NH2)COOH CH2&COCF3 (m/e 126) NH2CD2COOH C2H5CH(CH3)CH=zHCOCF3 (m/e 182) C2D5CD(CD3)CD(NH2)COOH i-C3H7~2CH=kXOCF3 (m/e 182) I-C3D7CD2CD(NU2)COOH kCOCF3 (m/e 166) C6H5CH=CHCODHl + (m/e 148) BuOOCCH2CH&XOCF3 (m/.e 240) HOOCCH2CH2CH=kKOCF3 (m/e 198) CX2-CHCH2CH2CH-&COCF3 (m/e 180) Deuterated Amino Acids D C6D5CD2CD(NH2)COOH HOOCCD2CD(NH2)COOH HOOCCD2CD2CD(NH2)COOH Fragment Ion CD3CD&COCF3 (m/e. 144) . . I-C3D7CD=hCOCF3 (m/e 176) CD2=iHCOCF3 (m/e li8) C2D5CD(CD3)CD=zHCOCf+3 x+/e 1%) i-C3D7CD2CD=hCOCF~~~(m/e 192) D D K2 hi -COG3 (m/e 173) DD ; D C6D5CD=CDC001q+ (m/0.155) , BuOOCCD2CD=hCOCF3 (m/e 243) HOOCCD2CD2CD=$HCOCFj, (m/e 203) CD2-CDCD2CD2CD&O~~3 (m/e 188) LEGENDS TO FIGURES Fig. 1. Standard curve for the quantitation of Glutamic acid. Fig. 2: Typical ion chromatogram of soil amino acids. Ftg.3. fv\',sr Fc=p e-2 Q+ub& -i--*u- O- f . Table II. ANALYSIS OF AMINO ACIDS IN SOIL (pg/g SOIL) Mass Fragmentography Amino Acid Ala Val GUY Ileu LeU Pro Phe Amino Acid Analysis 206.5 148.3 215.4 95.4 154.2 143.4 80.3 218.3 227.0 129.7 Bl 198.7 151.0 ASP Glu LYS S96.8 100.4 152.1 141.4 80.5 217.1 217.2 115.3 #2 202.7 150.5 201.6 100.2 149.7 142.8 80.7 219.8 215.6 113.5 83 198.3 149.9 201.3 92.3 154.2 141.2 80.0 219.9 214.1 114.9 P-Lb Fig. 1. GLU - 4dded D5^GLU - 3 J 3 E i, I IIC. 3 A Lomputer Uperated Mass Spectrometer System W. E. Reynolds, V. A. Bacon, J. C. Bridges, T. C. Cohurn, Berthold Halpern, Joshua Lederbcrg, E. C. Levinthal, Ernest Steed, and R. B. Tucker Department o$ Genetics, Stan$ord University School of Medicine, Stanford, Calif. 94305 An integer resolution mass spectrometer-computer system has been developed in which the computer controls the "scan" of a mass spectrometer. In this system, the computer queries the user for operating parameters which are then translated into control functions which operate the mass analyzer. The spectral information acquired from the mass spec- trometer is made available to the chemist within min- utes in an on-line graphic system. processing of GLC effluent are given. Examples of the THE USE OF htAss SPECTROMETRY has been hampered by the lagging development of a fast and convenient method of re- ducing the spectral output of the mass spectrometer (MS) to numerical data. Usually the operator must convert a MS chart recording, which is an analog plot of intensity cs. time, to a digitized plot of intensity cs. mass number. Because of instrument instabilities, wide range of signal amplitudes, large amounts of data, and other operational ditficulties, (I; 2), it is often dificult and time-consuming to establish all the correct mass peak identifications. One aid is to use a reference compound (3) tither prior to the run or as an in- ternal standard with the unknown sample. By counting from known mass peaks, unknown spectral peaks can bc idcntificd. However, the processing of data by this tczhnique is still a formidable task and it may take several days to accumulate all the information from a gas chromatograph-mass spec- trometer (GLC-hIS) run. Several workers have demonstrated MS-computer systems in which the computer monitors and records digital data from a MS. In most of these applications the mass spcc- trometer has operated indepcndcntly oi the computer, scannmg in some time dcpcndcnt mode. mcnsuring ion intensities at all points within the range of (500 to 5000 samples per second) and afterward performs the computations required to reduce the large amounts of digital data to useful information (4-7). Much instrument time and sampling effort is ex- pended in the intervals between integer peak positions where there is little or no information. One system that improved (1) K. Biemann, "Mass Spectrometry." McGraw-Hill, New York, 1962, p 10. (2) J. Lederberg, E. Levinthal, and Staff. "Cytochemical Studies of Planetary Microorganisms Explorarlons in Exobiology," IRL Report No. 105-1, Instrumenration Research Laboratory, Department of Genetics, Stanford University School of Medi- tine, April 1966 to October 1966. NASA Accession No. N66- 34195. - (3) J. H. Beynon. "Mass Spcctrometry and Its Applications to Organic Chemistry," Elsevicr Publishers. Amsterdam, 1960, p 44. (4) R. A. Hitcs an K. Biemann. ANAL. CHEhq., 39,965 (1967). (5) ILGd., 40, 1217 (196s). (6) R. A. Hitcs, S. Markey, R. C. Murphy, and K. Biemnnn, 16th Ann. CO/I./. ,lfrw Spcc/rarwrr.v Aliid To,Cs, ASTM E-I J, Pittsburgh, Pa., hla>, 196s. (7) R. B. Tucker, "A hlass Spcctromater Data Acquisition and Analysis System." IRL Report No. IOG?, Instrumcntntion Research Laboratory. Depnrtmcnt of Gcncticb. St:tniorcl Um- versity School of Medicine, NASA Acc:ss~on No. NOS-25743, 1968. upon this latter ineficiency used step switches to step the scan from position to position (8). We now describe a MS-computer system, suitable for routine laboratory use, in which the computer controls the operation of a quadrupole mass spectrometer (9, 10). In this system the "scan" is calibrated by relating known mass positions of a reference compound to a computer g~ncxtcd control voltage (V,). R, is gencratcd as the result of a number N, serit from the computer to a Digital-to-Analog (D-to-A) converter in a MS-computer interface. The parameters of this V,, or the N for each integer mass position, arc de- termined by a computer program and stored in memory. The subsequent use of this information allows the computcr- directed MS output to be recorded directly as mass,chnrgz (nz/r) cs. intensity. On request, this data is then made al,ail- able to the operator in an on-line system. The use of this computer-.MS interaction, combined with the decision-making ability of the opcra+?r, permits n sig- nificant saving in data processing costs. Furthermore, a much larger duty cycle of analyzer "on peak" time is obtain- able, resulting in the detection of more ions for a given rnxs position than is possible in conventional time based scanning. The new MS-computer system has at least thrcs unique features. There is a hardware control intcrfncc to connt':t the MS intimately with the computer; there is an improv:d etlicicncy of information acquisition from spectral psahs that are limited in ion production rates; and there is a uscr- oriented control and data presentation system that csnc.xls the foregoing details from the operator, but prssen:s 1i7c gxr with prompt and concise data which include normalirsd mass spectral plots. The described system has evolved through three mass spectrometers, three computers, and two basic computer pro- grams (I I, 12). The later systems have greater range. scnsi- tivity, and convenience, but they all have a common concept. Therefore the description that follows will be concrprunl rather than specific to any one configuration. (8) H. L. Friedman, H. W. Goldstein. and G. A. Griffith. "\lnjs Spectrometric Thermal Annllsis of Polymrr DCCOnlpOhiIiJn Products," 15th d~vr. CU/I~: .\fcrss S~cctronre!r.v rlllrc*ti 7'0,~jcs. ASTM E-Id, Denver, Colo., hfay 1967. (9) W. E. Reynolds, "A Small Computer Approach to Lou Resolution hlnss Spectrometry." Pacitic Conference on Chc:l:ls:r> and Spectroscopy, Anaheim. Cnlif.. November 1967. (IO) W. E. Reynolds. T. B. Coburn. J. Bridges. and R. Tuc`ker. "A Computer Operated ,Ilass Spectrometer S~stcm." IRL Report No. 1062. lnstrumcnration Research Laboratorv [)c- partment of Genetics, Stanford Univcwty School of \lc::c!zc. NASA Accession No. N68-l ! S69. Nov. 1967. (11) W. E. Reynolds. R. B. Tucker, R. A. Stillman. and J. C. Bridges, "hlass Spcctromcrers In a Time Shxcd C:xn:~u!~r Environment," I7 t h rlrur. Co,rjI iIl:,ss Sprcrror,~c*tr~ .~li!;,x Topics, ASThl E-14. 1969. (I?) J. Lectcrberg. E. Lcventh:ll. and StatT. "C! tochmxnl Stt~d~c; ot Planetary hlicroorgcanisms Esplorshons in t\;olxoioc~ ." II;L Report No. 1076. Instrumcntntion Rcscarch Labornror). I):- partmcnt of Gcnctizs. Stnnf'urd C'mvcrsity School of X1:, I;,!::. October 1967 to r\prll IYM. Appcndiu A IS a ~LI!~~I~IICLI IC;':.IIL of the abovc "hlass Spcctrometcrs in 2 Tuw Shurcd t. II\ iron- menl," NASA Accession No. N6S-ZYS-IG. Reprinted from ANALYTICAL CHEMISTRY, Vol. 42. Papc 1112, Scptentbcr 1970 Copyright 1970 by the Amcricm Chemical Society and rcprintcd by permission of the copyright owner P-7 1 , The present system is operating with a Finnigan 1015 quadrupole MS and a Varian Aerograph GOOD chromato- graph. The same computer programs and a similar interface were also operated successfully with a Bendix Time-of-Flight (T-o-F) MS (13) and an EAI qundrupole (14) MS. In all cases the GLC-MS, the teletypew/riter, and the digital plotter were situated in a wet chemical laboratory. A schematic diagram of the GLC-MS combination is shown in Figure 1. The emuent from the gas chromato- graph, equipped with a flame ionization detector, first passes through a variable splitter that diverts between Ii3 and *I? of the flow through a Biemnnn separator (15) and into the MS. A solenoid-actuated valve in this line helps to keep the large initial solvent peak from entering the MS system. A reference gas reservoir containing a fluorine compound at a vapor pressure of approximately 3 X IO--' Torr is also in- corporated in the system and is connected to the MS by another solenoid valve. The computer, tin the interfacing electronics, has direct control of gas valves, and can valve in or shut off the reference gas whenever it is needed for the calibration routine. These valves were constructed in our shops in such a way that the back side is open to the vacuum system when the valve is closed. This avoids the common pressure burst when conventional valves are opened to a vacuum. The right side of Figure 1 illustrates the major components and functions of the interface. This computer-MS interface was built in our Instrumentation Research Laboratory (16) and contains all the electronics not normally supplied with a standard configuration IMS or computer. Al1 of thcl operating parameters of the hlS are, or may be. controlled by a digital word (binary number) sent from the computer. The principal control is ricl fhe "N" register to the D-to-A converter. The analog signal, V,, from the D-to-A sets and holds the mass analyzer to pass ions of a predetermined ))I e. Alternately the digital output may be coded to operate auxiliary control functions, such as actuate valves. set amplifier gains, the low speed multiplexer, or enable the digital plotter. STlEV'3 ONll33NN03 UOHS 1 The characteristic method of controlling the nl:e passband and taking measurements while the mass analyzer dwells upon a m/e value converts what is normally measured as a time dependent parameter, to a stationary signal. This statistically stationary property of the signal enables the em- ployment of full integration to enhance signal to noise. Both the electrometer and the integrator are standard commercial FET operational amplifiers of the 550.00 class. The time allowed for integration and the operation of the integrator reset are controlled by signals (numbers) from the computer to the "T" register. The output of the integrator is sampled, held, and read ciu the Analog-to-Digital (A-to-D) converter. Auxiliary signal sensing is provided by the low speed multiplexer. This is useful to determine the automatic settings for self calibration,or may be used to record tempcra- ture, pressure, etc. These sense functions, plus some valve (13) D. B. Harrington and R. S. Gohlkc. "High Resolution Time of Flight Mass Spectrometers." 10th .Irw. Coqj: ,Iltrss Sp~wrum- erry A//id Topics, ASTM E-14. New Orlcnns. La.. 1962. (14) W. I'& H. I'. Keinhard, and U. van Znhn, Z. Plrys., 152, 143 (1958). Figure 1. The GLC-AIS instrumentation and the electronics interface to enable computer systems integration control and checkout functions, are controlled by the "C' register. (IS) J. T. Watson and K. Bicmnnn. ANAL. CIIEM., 37, WI (1965). (16) W. E. Kqnolds, J. C. Hric!gcs. R. B. Tucker, and T. U. Co- burn, "Computer Control of Xfass AnalyLcrs," 16th .&III. Co\!/: Mass Sprclrowe~ry d//M Topics. ASTXl E-14, Pittsburgh. Pa.. 1968. Thcrr are no manual opcrntor control functions in :lny o' the above steps. The control is nccompli>hcd iIt tl~ I 11 writer keyboard. This keeps the system llcsiblc ant! IILI,L,. it indcpendcnt of the idiosyncrasies of indi\,idual con~put~ I P-1' ANALYTICAL CHEMISTRY, VOL. 42, NO. 11, SEPTEMBER 1970 . 1123 COMPUTER DIALOGUE (The rcser's respome is underlined) OPTION = collect EXPERIMENT; = test - T=g MODE = single PLOT, TYPE, or FILE = plot - BY MASS, AMP. or ENTIRE = entire FROM MASS = 1 g,;g n PLOT, TYPE, or FILE = 1 T=I EXPExIMENT,+' = MS26x15 --~ CONTINUATION = yes T=6 - MODE = continuous # OF SPECTRA = 130 FILED IN POSITION 930 to 1070 T=/ EXPE`i%IMENT; = / OPTION = sum - EXPn = MS26x15 _~--- FROM SPECTRU,M = 9-U) TO = 1020 - FROMXSS = ilo TO MASS = 500 PLOT = yes - EXP,+ =, 1 OPTION = plot EXPERIMEtii+ = XlS26x15 _- - FRO&M SPECTRU.M = 957 TO = 957 - &OM%iASS = 1 TO MASS = 340 - - interrupt lines and/or individual computer characteristics. This straightforward system definition makes the software design much like conventional computer programming rather than encouraging intricate techniques highly dependent upon the specific hardware. Thus the system is not oriented specifically to any given computer. It has operated on an early model LINC (17) com- puter with 2K words of 12 bits. memory, and on a time-shared, locally programmed, IBM 360;50, butTered with an IBhl 1800 (18). In all cases the computer was somewhat remote, separated by some 500 ft of cable from the rest of the in- strumentation. The system is very economical of computer resources. Most of todak's small general purpose computers would be able to operate the described functions if it were desired to avoid time-shared computer dependency. Some (17) R. W. Stacy and B. Waxman. "Computers in Biomedical Re- search." Vol. II. Academic Press. New York. N. Y.. 1965. DD 35-66.. . . (18) W. J. Sanders, G. Breitbard. G. Wledcrhold. er (I/.. "An Ad- vanced Computer for hlcdicnl Research." Full Joiut Cortrpwr Colt/. Proc., ACM, Anaheim, Calif., 1967, p 497. COhlMEm The user reauests the data collection chase. A catch-all name, "test". is given; the spectrum will be used simply for a systems check. An integration time of 35 milliseconds per peak is requested. Only one spectrum will be taken (single mode). The data is acquired after this answer. The user can plot, type. or file the data col- lected; here a plot is requested. The user can plot selected masses, the highest intensities, or the entire spectrum within requested limits. The user indicates the limits. A QUICK plot omits annotation, etc. Note that "y" and "n" mean "yes" and "no". The user completed the checkout and now wishes to proceed with the experiment. The "/" is used to backup through the conversation. An existing experiment name is given here. The user confirms that the spectra are to be added to the eslsting experiment file. The user requests that spectra be collected continuously until 130 are taken. Data collection is complete. The user then wishes to sum the elements of each spectrum to produce a "total ion" plot, analogous to a GLC trace. Mass position 40 to 500 are summed for each of the collected spectra. The "total ion" curve is now plotted. (Figures 6 and 7 are illustrative of this &tiple dialogue.) Guided by the total ion plot. the user will plot interesting m3ss spectra. Only the spectrum filed in position 957 is chosen. The plot (Figure 17) is drawn and normalized to the base peak, nlje = 31. Figure 2. An example of the user-computer dialogue during operation sort of magnetic storage for object code programs and data storage is most desirable. DEC-t)-pe tapes ha;-c be:n uzd on the LINC system and disc packs on the IB\l s\-stem. THE SOFIV.IRE STRUCTURE The objectives of the software are to operate and conrrol the MS, acquire data from the MS, process and prcsznr rhis data in a manner useful to the chemist, and provide ctrtn:n control and information to aid in maintaining and ser\-l:ing the instrument. With the program loaded into the computer, the Csfr requests any one of several functions (see Table I) by t!Fing the name of that function. The computer responds \x::h a series of prompts (see Figure 2) to elicit user m;lcrocomm:!ntis. The computer then generates the detailed conrrol funz!ons to perform the assigned task. At the comp:.!- tion technique makes the system both tlesiblrt an;l rl) self-instructing. 1124 . ANALYTICAL CHEMISTRY, VOL. 42, NO. 11, SEPTEMBER 1970 P- 72 Table I. A List of Program Options (1) CALIBRATE: Creates an accurate N Table. The .V "urn- hers which correspond to the peaks in the reference gas are used as the end points of a piecewise linear interpolation pro- cedure for calculating a complete .V Table. (2) COLLECT: Is the primary data collection step. It is here that the 750 N Table values are sent to the MS and the 750 m/e intensities rccordcd. This operation can be repeated at five-second intervals as the data are tiled on disk under an e.y- periment name. (3) TYPE: Allows the user to print out spectral data by indicating what spectra in a given file are to be reviewed. The user can request that the amplitudes of particular rtr/e positions be typed ; that a given number of the highest amplitudes be typed, or that a consecutive number ot' them over a given range be typed. (4) PLOT: Enables the user to have bar graphs produced by the computer controlled digitai plotter. The amplitudes to be plotted can be selected with the same flexibility as described in TYPE. (5) SUM: Produces a plot of the total ion current over a series of gathered spectra. All responses of a spectrum are summed to produce one datum point on the plot. This plot corre- spondsclosely with the GLC output when runrung with theGLC. (6) TRACE: Produces a record of a spectrum similar to the normal chart recording output. The analyzer is sampled at all N values (about 10 per amu) over a given range and the result is plotted as a "broken line." (Used for system check out) (7) MONITOR: Provides for inspecting the peak profiles by sampling the ,$>ctrum around a given or position. The gathered dara are then typed out. (Normally used for system service or service log) (8) DISPLAY: Enables the user to display a given mass position (or N numbcrj in the center of the console oscilloscope. (Used in the adjustment of the mass spectrometer) (9) GAS: Allows the user to remot+ turn the reference gas on or off. This is helpful when operating the system from a remote position. n 31 69 :! 1'" 1212 .0600 2060 .7600 & 2507 .260I .il. 169 : 3327 1 D 1.300 # PRES Jr lo'7/orr T FACTOR 40 rlICA ' 40,&U. 123351 L/he I IONV 20 r&,;r's 691218 dafe Figure 3. A monitor plot indication of instrument scr\iccability The example of a user-computer conversation gi*:cn in Figure 2 represents the day-to-day conlput-r-r~sc:!r~her dialogue given to direct the system's olxrarion. D:?per level programming may be done at the terminal to rcdctine these functions or to add new modes. AddiIionnl b!`;tcm development may be done by the chemist-user, or his program- mer in a manner typical of general purpcse ccrnptitphilia, phenylketonuria and ornithine transcarbaaylase are some of these prenatal diagnostic *Dorphansa. For other hereditary diseases, sufficient information on the underlying biochemical 3efect is not available, and numerous autosonal recessive, autosomal dominant and X-linked pathologic characters can be cited for this category. Cystic fibrosis (although this disorder may soon be liberated from this category), X-linked ichthyosis, neurofibromatosis and huntington*s Chorea are just a few. while we can optimistically look forward to increasing progress in the elucidation of basic mechanisms of these hereditary disorders, a waiting period before application to prenatal diagnosis of these conditions is not necessarily implied. In other Yards, even though a biochemical or other suitable cellular phenotype may not be available, it may still be possible to diagnose with considerable accuracy some of these conditions in the fetus. Genetic linkage may offer this possibility. Human linkage (syntenic) groups are relevant to prenatal diagnosis of inherited disorders because the detection of the phenotype of a genetic marker which is closely linked to the locus of an allele determining disease provides the possibility of genotyping the fetus. This is already possible for myotonic dystrophy, a disease expressed in heterozygotes for an allele at a locus which is linked to the ABli secretor locus, the recombination fraction being 0.04 (9). ABh substances are secreted early in gestation by the fetus into amniotic fluid, forming the basis of determining its secretor status (5). Thus, if the coupling phase is known for an individual who is heterozygous at both loci and married to a non-secretor, detection of the secretor status of the fetus will predict presence or absence of the allele determining mpotonic dystrophy. The wagnituae of error in this prediction, 8X, is slightly greater than the recombination fraction (9). The genetic map of man is growing. More and more liakeci loci are being found, autosoaal linkage groups containing more thdn two loci are recognized and loci are being assigned to visible -3- autosonal chromosomes (IO). The majority of the newer linkage groups involve polymorphic biochemical or serological markers rather than inherited disease markers. This is understandable because ascertainment of infrequent diseases in families is by-passed and accumulation of data is facilitated. Furthermore, linkage analysis by in vitro, Sendai virus mediated, interspecific somatic cell hybridization (11) does not require polymorphic gene markers so long as differences between interspecific allozyaes or other homologous markers can be detected. Still, for prenatal diagnosis, spnteny involviny a locus uith a disease determining allele is essential. At present, a small number of autosomal syntenic groups involving clinically significant loci are known. The loci of five autososal dDainant disorders have been shown by family studies to be linked to polymorphic loci (revieued in reference 10): ,,c. , (' = MAIN LOCUS TEST LOCUS DISTAPCE 95% PROBABILITY COFMENTS CENTIHORGAIS LKHITS OP I'lAP DISTANCE CENTIMORGANS Congenital total Duffy 0 o-17 Probably syntenic nuclear cataract with the two loci for pancreatic auylase. Assigned to chromosoae #l, Elliptocytosis Bh 3 2-7 Nail-Patella b Sclerotylosis 2 e Hyotonic Dystrophy ABO hdenylate Kinase 13 0 HkiS 4 Secretor 4 8-21 O-6 0.3-19 Syntenic with loci f Or 6-phDSphDglUCO- natedehydrogenase E phOSphDglUCDmUtaSe (first locus), and peptidase C. If these loci are correctly assigned to chromosome #l, they are syntenic with loci for the Duffy system, con- genital total nuclear cataract and pancreatic amylase. Linkage studies with Rh have detected genetic hetero- geneity for ellipto- cytosis. -s- The biochemical basis of almost all autosomal dominant disorders, including those listed above, is unknown, denying us a rational fOUndatiD8k for prenatal diagnostic testing. Genetic linkage affords a potentially useful strategy for antenatal detection of autosomal dominant disorders. This also applies at present to some clinically significant loci on the human X chromosome: P-/c 5 MAIN LOCUS TEST LOCUS DISTANCE CENTIHORGAI'IS Hemophilia A Glucose-ti- (Factor VIII phosphate deficiency) dehldroyenase 4 Lchthyosis Ocular Albinism XY 18 xg 18 95% PRUBABILITY COLIBEITS LIHIT OF HAP DISTANCE CENTIWORGANS O-16 Polymorphism at the test locus in black, tlediterranean E some Asiatic populations ll-31(*) 7-34(*) (references 12,13 and 14, respectively) (+I - 90% probability limits -7- With respect to the above autosonal syntenic groups it should be pointed out that the ABU types of secretor + fetuses and secretor types have been detected from AF (5). and that Gphosyhogluconate dehpdrogenase (6-PGD), of the Rh syntenic group, has been detected ia cultured AP cells (7). The electrophoretic phenotype of glucose-6-phosphate dehydrogenase (G-6-PD) has also been detected in cultivated AP cells (7). Thus, the potential exists for diagnosing the nail patella syndrome, myotonic dystrophy, hemophilia A and perhaps elliptocytosis in fetuses at risk by virtue of slntenic relationships. It is clear that, at present, there are limitations in the use of syatenic groups for prenatal diagnosis of inherited disorders. The method will be useful only for informative families, i.e. those with a doubly heterozygoos (at test and main loci) parent for autosomal dominant traits, a doubly heterozygous mother for X-linked recessive characters and both parents doubly heterozygous for autosomal recessive disorders. lleiotic crossing over between the marker (test) and main loci vi11 always provide the possibility of error in predicting the fetal genotype, and the magnitude of this error is provided by the recombination frequency. The smaller the distance between these loci, the lover this error vi11 be. Still this error will be, in general, much less than the 50% "error" that nou attends genetic counseling based on detecting a male fetus at risk for an X-linked disorder. Another limitation of utilizing linkage information for prenatal diagnosis is the small number of syntenic groups involving clinically significant loci. At present, there is good reason to be optimistic about mapping the human chromosomes, Huddle has indicated that each of the chromosomes wiJ.1 have a known linkage assigament within "the next several years" (15). Family studies and interspecific somatic cell hybridization are providing data for new s,yntenic groups, assignment to recognized groups and to visible chromosomes, and these in vivo and in vitro techniques for linkage analysis are also confirming each other's findings (e.g. reference 16). Ye should inject a slightly less optimistic coraent concerning the application of genetic linkaye groups detected by somatic cell hybridization to prenatal diagnosis of inherited disorders. The genetic markers used in these studies with interspecific hybrids are usually enzymes vhich are not necessarily polymorphic characters, as classified by electroyhoresis. This map bn especially true for autosomal recessive genes determining various metabolic errors. For prenatal detection of autosomal recessive disorders, both carrier parents must also be heterozygous, preferably for codominant alleles, at the linked test locus, an unlikely expectation if the test locus Ls not polymorphic. Thus, this restricts the number of marker loci and the frequency of couples at risk which are informative for application of linkage for the prenatal diagnostic test. The situation is only slightly improved for X-linked loci. because of the paucity of X-linked polymorphic systems (Xg, Xm and in some populations, glucose-6-phosphate dehydrogenaso; color blindness is a recessive trait). -B- Another problem with linkage data collected from analysis of somatic cell hybrids resides in the inability to estimate the frequency of recombination betueen test and main loci. For purposes of prenatal detection, the recombination frequency is dn estimate of the error in the prediction of the fetal genotype and phenotype. The limitation in observing polymorphism at a number of the (enzyme) loci uhich will be linked by this technique suggests that for some linkages, confirming family studies will not be forthcoming because informative families will be rarely encountered. Despite these reservations, the more linkages determined between loci and between loci and identifiable chromosomes, the more likely this knowledge can be applied to prenatal diagnosis of various inherited disorders. Certainly we can expect assignment of polymorphic loci to linkage groups and visible chromosomes, and detection of polymorphism in a number of presently monomorphic or idioaorphic systems is more than a reasonable prediction, The amount of genetic variability in terms of polymorphism, has surpassed estimates of a mere decade ago, and, indeed, it is not unreasonable to consider the possibility that almost all loci are polymorphic in man (17). Thus, we can expect assignment of a number of clinically significant loci to syntenic groups. A ,final limitation to the use of synteny for prenatal diagnosis stems from the relatively small amount of information available on the expression of polymorphic genetic markers in AF. Should additional, clinically relevant, syntenic groups be detected, ue still must be able to test for the expression of the marker loci in AF in order fo infer the fetal genotypes. In addition to providing information pertaining to the biology of the fetus and BP, a systematic study of polyaorphisas in AP can provide the information necessary to use syntenic relationships for the prenatal diagnosis of inherited disorders. B. SPECIFIC AIHS 1. We propose to seek in AF and cultured AF cells the expression of known polymorphisms. Among the polyaorphisrs to be studied will be erythrocyte antigenic systems, the hL-A system and systems of various intracellular enzymes, 2. Ye vi11 delineate variation in expression of allelic markers in AF and attempt to understand the basis of the variation. 3, We will delineate expression of polymorphic markers in Af in terms of gestational age. 4. Me will compare the expression of polymorphic markers in AP with their expression in other tissues (e.g. blood, cultured fibroblasts, etc.). -9- 5. As ue come to understand the variation encountered in the expression of polymorphic markars in AF and the limitations of the tests ue use, we shall begin to employ or help others employ those markers involved in a syntenic relationship with clinically significant loci for prenatal detection of affected fetuses. C. HETHODS AND PROCEDURE 1. AP Samples AP samples for these studies are being obtained from appropriate patients by staff physicians with full-time or clinical faculty appointments to the Department of Obstetrics and Gynecology. These physicians provide lo-15 AF samples each year for prenatal diagnosis of chromosonal disorders (primarily trisomy 21); these specimens are usually obtained at 12-16 gestational ueeks. Ye expect the numbers of Al? samples sent for prenatal diagnosis will gradually increase as more obstetricians take advantage of the service provided by the Cptogenetics Laboratory at Stanford. One problem in this source of Al? samples which concerns us pertains to maternal age. Host of the samples obtained for prenatal diagnosis are taken from women over 35 years of age. The effect of maternal age on expression of polymorphic markers in AP is a variable about which there is no inf ormatioa. The obstetricians have been cooperative in providing us with AF samples collected at the time of therapeutic abortion; these specimens are usually obtained at 18-20 weeks of gestation. About 2-3 therapeutic abortions are performed each week at Stanford University Hospital. 2. Cultivation of AF CELiS We shall work with cultivated AF cells in order to assure ourselves that we are working uith only fetal cells. After amniocentesis AP usually contains a mixture of maternal blood cells (probably resulting from the procedure) and fetal cells. Although it is possible to remove most of the maternal erythrocytes by lysis (I&), other nucleated cells from maternal blood may remain. Cultivation results in dilution of maternal blood cells by the replicating fetal cells. The use of polymorphic markers and of markers of fetal sex will enable us to decide on the origin of the cells growing in culture (fetal or maternal), Obviously cultured cells carrying a fluorescent interphase Y marker (19) or showing no sex chroadtin (Barr) bodies are of fetal origin. For cells which do show evidence of the XX karyotgpe, ue vi11 compare tha phenotypes of the polymorphic markers we employ with the phenotypes in the mother and father. In most instances these precautionary procedures vi11 help us avoid mistaking maternal for fetal cells in AF. -lO- We have been cultivating AF cells for the past year. Our culture technique is similar to that used by other investigators in the field. Gsually 10 ml of Af (more, if obtained before instilling a hypertonic saline solution for therapeutic abortion) are centrifuged at 100 g for T-10 minutes. The supernatant Ah' is removed and saved (see below) and the sedimented cells are suspended in 0.5-1.0 ml of fetal calf serum. We carefully place drops of the serum-cell suspension on the surface of a small plastic Petri dish or on a cover slip in a Petri dish and then incubate the cells at 37 degrees C in a 5% CO;! and air environment. After 6-18 hours, during which time cells have attached to the surface of the dish (or coverslip), we add tissue culture medium (FlO-Grand Island Uioloyical Co,) with 30% fetal calf serum. This medium is changed every other day. with this procedure ve can see cell growth within one week. There are sufficient cells in a dish or on a coverslip for the first subculture (0.05% trppsinsolution) within 2-3 ueeks. After the first subculture, we find that we can perform the second subculture uithin a ueek. At this time the cultivated BP cells are groving well. The cel.ls with which ue are dealing at this time usually are epithelioid cells. It is our experience that these cultivated AY epithelioid cells will grow vell for about 4-S subculture passages after which their grouth will cease. Hithin these limits we have been able to cultivate large qnantities of these epithelioid cells in roller culture bottles. This will be important for the study of polymorphic enzyme markers in cultivated AP cells. Less frequently we have noted fibroblasts growing in cultures. These cells grou for longer periods than the epithelioid cells. For instance, one AE' fibroblast culture which we are propagating and using for various investigations (7B2B) is now in its 14th subculture passage. We know this line is of fetal origin because it does not shou Barr bodies and possesses the XY karyotype; this is a diploid culture. It is our impression that more time is required for fibroblastic growth to be evident initially than for epithclioid cell replication. We will use either morphological type for our studies of cultured BP cells, although for purposes of prenatal diagnosis epithelioid cells may be more relevant. Aliquots of replicating cells from each AF culture are frozen ia 10X DHSO (lo**6 cells per ml per vial) and storect in liquid nitrogen for future studies with live cells. Aliquots of lo**7 cells vhich have been washed, sedimented and drained of supernatant liquid are stored in liquid nitrogen for future electrophoretic and enzpme activity studies. 3. The Supernatant AP Fluid After AP cells are separated from the supernatant AF by centrifugation, we again centrifuge the latter (2,900 r.p.m. fee 10 minutes in an angle head International Clinical Centrifuge) to p-//t -1 I- remove any remaining cells or large pieces of cell debris. The supernatant AP is then transferred to another container and stored at -20 degrees C. This fraction of A.F is used for ABE hemagglutination inhibition studies and will be used for studies on fetal Lewis substances (6). 4. Controls for Expression of Polymorphic lYarkers Whenever possible blood samples will be obtained on both parents of the fetus under study. These samples will be typed for the various polymorphic markers being studied in AF. In addition umbilical card blood specimens will be collected at birth of those fetuses whose AP was studied and who were not aborted. These specimens will also be typed for the various polymorphisms which were studied in the AF. In those instances in uhich fetuses are aborted, ve shall atteapt to use appropriate material from the abortuses to type for polymorphisms. The collections of the various specimens just listed are meant to provide us with material which will serve as controls for the observations we make on Al?, Working uith polymorphic systems permits us to predict the phenotype of the fetus, provided we know maternal and biological paternal phenotypes. A high frequency of discordance between observed (from BP) and expected phenotypes will signal us to examine our testing procedures, to consider that changes are occurring in cell culture or that interesting variations in interallelic expression are occurring. The direct controls of studies of expression of polyrorphisms in AF vi11 be the results of testing cord bloods or aborted material. 5. The A&O Secretor and Lewis Polymorphisms Fetuses which are heterozygous or homozygons for the gene which determines secretion of ABH substances (Se) secrete these soluble blood group antigens which appear in AP (5,6). The secretor type of the fetus and the ABO type of secretor + fetuses can be detected by studying the AF in early gestation free of cells, for hemagglutination inhibition of appropriate detector systems. Inhibition by AP of A,B, or H hemagglutinins indicates that one (or tuo) of these blood group substances have been secreted by the fetus. In this case the fetus is secretor t, and his ABO type is determined directly by specific inhibition of the A,B or ti heragglutinia. If the AF does not inhibit agglutination the fetus is a non-secretor and the ABO type cannot be determined. About 25% of Caucasian fetuses will be non-secretors (20) o Wet are already determining in our laboratory secretor status and ABO types of ABH secretor + fetuses. Al? is serially diluted for nine or ten doubling dilutions in three series of tubes. In one series of tubes the AF dilutions (including undiluted AF) due incubated at room temperatures for 1 hour with an egual volume of single donor, non-commercial, anti-A hemagglutinin. In the second series of tubes tht? AF dilutions are irlcubated with single donor, non-commercial, anti-B and in the third with anti-h (Ulex europaeus), A single dilution of each hemagglutinin is used throughout the determination, this being determined from the anti-A or anti-B titer of the serun; we do not dilute the anti-H reagent which has a very low titer (commercial preparations or our own preparation). After the incubation of A8 and heeagglutinins, ue add A, B and R erythrocytes to the first, second and third series of tubes, respectively. Following thorough mixing of cells and reagents (and AP), the tubes are centrifuged and are then observed for gross hemagglutination. As a control for each AF determination, agglutination inhibition is carried out in a similar manner using salivas containing A, B and H substance. As of the date of preparation of this application, we hdVe standardized (for our laboratory) the conditions of the hemagglutination inhibition procedure (e.g. titer of hemagglutinins, volumes of reagents, incubation times, centrifugation tines and speeds) and tested five amniotic fluids: YBEK OF GISTATION PBTAL TYPE BECIPBOCAL BEDAGGLUTINATION ABO SECRETOR IRHIBITTOB TITER II AF A B H 19 weeks B + 0 64 0 17 l/2 weeks ? 0 u 0 16 weeks A + 8 0 0 14 weeks + 16 16 weeks + 0 We believe that we vi.11 be able to distinguish between Al and AZ secretor fetuses on the basis of inhibition of anti-R; A2 individuals secrete nore H substance than do Al individuals (20). We are continuing to type AF specimens for ABH henagglutination inhibition and to compare the inhibition titer with that of the saliva controls. He plan to check the ABO types of secretor + fetuses by typing cord blood specimens and ABH secretor status with saliva collected in the newborn period. In a manner analogous to the methodology just described, secretion of Lewis a, Le(a), and Lewis b, Le(b), substances will be studied (6). Non-secretor fetuses do secrete Le(a) substance. We are planning to decrease the amount of serological reagents and AF used in the ABH and Le typing procedures. He vi.11 attempt to adapt these hemagglutination inhibition tdsts to tissue typing piates (21) which require only 1 nicroliter each of AP dilution, antiserum and erythrocytes. These plates can be "centrifuged n on a serological rotator and hemagglutination abserved microscopically. Direct reacting (complete) antibodies must be used in this nicrotechnique. Fortunately, ABH hemagglutinins and some dntibo3ies to Le(a) and Le(b) do not require anti-human globulin (Coomb's reagent) to agglutinate erythrocytcs, 6, Other Polymorphic Systems of Erythrocyte Antigens -13- Recently, the expression of the blood group P has been detected on cultured cells and interspecific cell hybrids by complement fixation (22). If this finding can be confirmed, a technique might be available for detecting the expression of various blood group polyaorphisas on cultured AF cells. ABO, Rh, HtlS, Duffy, P and Xg specificities will be sought on cultured Al cells by complement fixation. Other serological methods which we will explore for the detection of these substances on AF cells include absorption of specific antisera and mixed agglutination. Fuchs et al. (23) used the latter technique to detect ABO types of uncultured AP cells. Although there is no indication whatsoever of secretion of blood group antigens other than ABH and Le substances, (except for Sd(a), see reference 201, we shall examine AF for hemagglutinin inhibition activity for the above mentioned erythrocyte antigens. 7, The EL-A System Ue have been able to type for HL-A antigens cells in the fifth passage of the fibroblastic euploid culture (7R2R) derived from AP obtained at 19 l/2 weeks of gestation. Cultured AP cells grown to confluence in a plastic tissue culture flask (75 square centimeter growing area) were released from the surface of the flask with 3 ml of 0.05% trypsin solution. The cells were exposed to trypsin for approximately five minutes, They were washed and resuspended in medium PI0 without fetal calf serum; the cell. concentration of this suspension was lo**6 cells per ml, Yh:,se cells mete prepared and typed for HL-A antigens in Dr, Rose Payne's laboratory, Department of Hedicine, Stanford, as follows: Aliquots of the cell suspension were incubated with fluorasr:ein diacetate (PIiA 2 micrograms in O-1 ml tissue culture medium; added to each P ml aliquot of cell suspension) for 30 minutes an6 the cells were separated from the FDA by centrifugation. Examination of the FDA-treated, cultured AF ccl Is under the fluorescertc:,? microscope revealed masses of fluorescent cells. Aliquots of l,OOO-2,900 of these cells were then added to approximately 60 HL-A antisera, representing 25 antigenic specificities, on tissue plates and incubated in the dark at room temperature for 30 minutes. Rabbit serum, absorbed with cultured human fibrohhasts, was used as a source of complement; an excess of complement uas added to each well of the tissue typing plate containing cells and antibodies. Following an incubation of 1-4 hours at room temperature in the dark, the cells were observed with a fluorescence microscope for evidence of cytotoxicity. Cells which remained fluorescent failed to react with antibodies and thus did not carry the HL-A antigen which the antibodies detect. Peu+r or absent fluorescent cell& indicated cytotoxicity mediated by the antigens recognized by the antibodies with which the cells had been mixed. Cytotoxicity was graded as 4+ when no fluorescent cells were seen, 3+ few fluorescent cells, 2+ more fluorescent cells and lt no decrease in fluorescent cells. This method has been used by Dr. Payne and her associates to detect HL-A antigens on cultured cells (24). P-113 -14- Typing of the 7B2B cells by this method revealed HL-A first series antigens, I&-A9 and R29, and the second series antigen RL-A3. Ue ace especially confident of the detection of HL-A9; most of the anti HL-A9 reagents in Dr. Payne's sublist of antisera reacted with these cells. Although more than one of the antisera for W29 and for HL-A7 reacted with the 7B28 cells, there were some uhich did not. Pe are about to type this AF cell culture again, now at passage 14. We are especially interested in the gain or loss of BL-A specificities, especially involving Y29 and HL-All. The genetic structure of the HL-A spstea ( 2 closely linked loci with a lou frequency of recombination between them, reference 25) serves as a control for our typing results. If we are correctly detecting the HL-A phenotype of the fetus, we should find no aore than two first series and two second series antigens on the cultured AF cells. Unfortunately, this fetus was aborted (by hypertonic saline injection), so that we cannot colapare the UL-A antigenic type of the cultured BP cells with that of (aborted) fetal tissue or of lymphocytes in cord blood. He look forward to studying variation in expression of HL-A antigens in cultured AF cells. The genetic structure of the system will permit us to look for variation within sub-series as well as betueen series (i.e. do the antigens of one sub-series tend to be expressed more frequently in cultured AF cells?) Furthermore, we will study the evolution of expression of RL-A antigens on AP cells throughout the life of the cell culture as well as at different weeks of gestation. Finally, shifts in expression of cross-reacting antigens (26) will be sought. 8. Polymorphic Enzyme Karkers of Cultured AF Cells The demonstration of the electrophoretic phenotypes of G-6-PD and 6-PGD in cultured AP cells by Eadler (7) raises the possibility of detecting other polymorphic enzyme markers. It is not clear from ladler's report that he considered polymorphism for G-b-PD as the source of the variation he observed in the cultured cells frotu different AF samples. blor is it clear that he studied sufficient saaples to note variation for 6-PGD in cultured AF cells which he reported as failing to show variation. Three to eight percent of Caucasian individuals shov variant 6-PGD electcophoretic phenotypas (27). Harris and Hopkinson (28) have listed some 20 loci determining enzyme structure which have been found bg electrophoretic studies to be polymorphic in Europeans, and G-6-PD can be added to this list for Black populations. Of these enzyme polymorphisms ue can test extracts of cultured amniotic fluid cells for seven by starch gel electrophocesis: phosphoqlucomatase locus 1 (PGRl), phosphoglucoautase locus 3 (PGM3), adenylate kinase (AK), adenosine deaminase (ADA), 6-PGD, hexokinase and G-6-PD. Furthermore, we believe we will be able to develop procedures for five additional systems: Peptidase A, C and D, glutamate-pyruvate transaainase (soluble - SGPT) .and galactose-l-phosphate uridyl transferase (gal-I-P-tfase). -lS- Cultured AF cells will be tested for total enzyme activity (per unit weight of cell protein) and for the electrophoretic phenotypes of the systems listed above (many of the procedures which we will use are provided in references in 28). Quantitative determinations and qualitative observations will be made throughout the lifetime of the same AP cell cultures and on cultivated cells from AF specimens taken at different times in early gestation. We will attempt to ascertain all common phenotypes of each polgmorphisa. Over a three year period we will probably have had the opportunity to encounter the expressions of comu~oa alleles for most if not all of these phenotypes, Ye shall use standard cell cultures uith reference phenotypes to control our electrophoretic procedures. These cultures will be obtained from cell culture repositories (e.g. The klaemalian Genetic Wutant Cell Repository, Institute for Hedical Besearch) or from biopsies (after informed consent procedures) from individuals who have been typed for various enzyme electrophoretic phenotypes. 9. Applications to Prenatal Diagnosis As this study progresses we will become familiar with the behavior and the limits of variation of expression of various polymorphic markers in Au. This information will serve as a base for using syntenic relationships for prenatal diagnosis. WC envision a cooperative effort uith a number of other centers involved in genetic counseling and prenatal diagnostic activities in finding families who are at risk for having affected fetuses and to which the methodology discussed herein can be applied for prenatal diagnosis of those fetuses. Such families will be typed for the test marker in order t3 determine that one or both (for autosomal -recessive traits) parents are heterozygons and to work out the coupling phase. We will then seek the expression of the syntenic marker in the AF from those informative and suitable pregnancies. Certainly, familias uith hereditary disorders seen at Stanford vi11 be screened for the possibility of applying the methodology presented in this application, but we believe we will need access to a larger number of families to apply this prevatal diagnostic technique. D. SIGNIFICANCE 1. This project will enable us to infer information about well knoun polymorphic markers in the developing fetus. This includes the development of the expression of these markers in early antenatal life, The project gives us the opportunity to find fetal forms of these markers. By understanding differential expression of polymorphic alleles in fetal life, we may be able to make inferences about the nature of selective forces which mRay be acting on these polpmorphisms. 2. This project could ultimately result in expanding the number of hereditary conditions a&enable to prenatal diagnostic techniques. This is especially true for autosomal dominant -16- disorders for which little information on basic biochemical mechanisms is available to fashion appropriate techniques for prenatal detection of the fetal phenotype. -17- REFE BENCES 1. Bosnes, R.W., Clin. Obstet, Gynec. 9:440, 1966. 2. Plentl, B.A., Clin. Obstet. Gynec. 9:427, 1966. 3. Huisjes, H.J., Amer. J. Obstet. Gynec. 106:1222, 1970. 4. Nadler, H-L., Adv. Hum, Genet. 3:1, 1972. 5. Harper, P. et al., J. fled, Genet. &:438, 1971. 6. Arcilla, K.B., Sturgeon, P., Pediat. Nes. 6:853, 1972. 7. Nadler, H.L., Biochem. Genet. 2:119, 1968. 8. Horton, R.E., Amer. J. Phys, Anthrop. 28: 191, 1968. 9. Renuick, J.H. et al., J. Med. Genet. 8:407, 1971. IO, Renuick, J.H., Ann. Rev. Genet. 5:81, 1971. 11. Ruddle, P.H., Adv. Hue. Genet. 3:173, 1972. 12, Bayer, S-R., Graham, J.B., Amer. J. Hum. Genet. 17:320, 1965. 13. Adam, A. et al,, Lancet i:877, 1966. 14. Fialkou, P.J., Giblett, E.R., Hotulsky, A.G., Amer. J. Hum. Genet. 19:63, 1967. 15. Ruddle, F.H., Birth Defects: Original Article Series 3:188, 1973. 16. Westerveld, A., Kahn, P.R., Nature 236:30, 19'12. 17. Cavalli-Sforza, L.L., Amer. J. Hue. Genet. 25382, 1973. 18, Lee, C.L.Y., Gregson, N.H., iJalker, S., Lancet ii:31b, 197$i. 19. Pearson, P.L., Bobrou, H,, Vosa, C.G., Nature 226:78, 1970. 20, Race, R.R., Sanger, R., Blood Groups in tian (5th ea.), Blackwell, i)xtford, 1968. 21. Bodmec, W.F., Tripp, M., Bodmer, J.G., Uistocompatibility Testing, Runksgaard, Copenhagen, 1967. 22. Felloos, !I. et al., 4th Internat. Congress Hum. Genet., Abstracts, Excerpta Redica Intern. Congr. Ser. No. 233, page bb, 1971. -It)- 23. Fuchs, F. et al., Lancet i:996, 1956. 24. Brautbar, C., Payne, R., Hayflick, L., Exper. Cell Res. 75:31, 1972. 25. Bodaer, W.F., Bodaer, J-G., Tripp, R!., Histocompatibility Testing, tinnksgaard, Copenhagen, 1970. 26. Mittal, K.K., Terasaki, P.I., Tissue Antigens 2:94, 1971. 27. Giblett, E.R., Genetic )larkers in Blood, Davis, Philadelphia, 1969. 28. Harris, f-f., Hopkinson, D.A., Ann. Hum. Genet., London 36:Y, 1972. EGED COMMUNICATION SECTlClN It SUBSTlfUTE TWIS PAtF FOR DFTAILFO Allnt.ET Pdr.1 ,__. _----- -- ---..-.. . . --- -... -. - . . ..- _.-- -.. --.-.--- _--__, _", SUBSTITUTE PERIOD COVERED GRANT NUMBER FROM )ETAILED BUDGET FOR FIRST 12.MONTH PERIOD THROUGH l/1/74 12/31/74 PERSONNEL (List all perso~el endaged 0" project) TIME OR EFFORT AMOUNT RECUESTEO fOrnit cents) NAME (Lad. first, initial) TITLE OF POSITION A/HRS. I tOTAL Cann, H. Principal Investigator or Program Director 20% Tsuboi, K. Senior Scientist 20% PoL'fbfORPHIC GENETIC MARKBRS '..' ,, : . . Van West, B. Research Assistant 100% '- '." Makk, G. Research Assistant 100% ./ " Open - Pediatrics Cusumano, M. Murray, R. Research Assistant 50% ;.I i, : Laboratory Diener 50% .I.; . . . Secretary 25x : .j: f .: :.: 1. ,' I:..: 6'. . . . : : . . I ',::.;. TOTAL R ' 52,330 , CONSULTANT COSTS (Include Feoa and Travel) . EQUIPMENT (Itemize) Eppendorf Microfuge and accessories $500 Buchler Power Supply (constant voltage/current) $600 * SUPPLIES Chemicals, antisera, tissue culture supplies, glassware, etc. s S 1,100 * 5,000 S . STAFF a. DDMESTIC 1 East Coast Meeting TRAVEL t sea xn8tNsrions~ b. FORE, GN . PATIENT COSTS (Separate Inpatienf md Outprtlenf) S 500 S S . ALTERATIONS AND RENOVATIONS S . OTHER EXPENSES (Xlwni;e per Inrt~crlon~ Office supplies, telephone, repro., postage, publication costs, etc.$400 Central computer usage and terminal rental $1600 S 2,000 . Subtotal - Items 1 thru 8 c ,I 60,930 10. TRAINEE EXPENSES f&e Instnrctions) PREDOCTORAL No. Proposed s FOR . a STIPENDS P0ST00CT0RAL No. Proposed 5 OTHER (Specify) No. Proposed S TRAINING DEPENDENCV ALLOWANCE S . GRANTS TOTAL STIPEND EXPENSES b. TUITION AND FEES ONLY , e. TRAINEE TRAVE', (Deari&) 11. Subtotal - Trainee Exponrer Ii. TOTAL DIRECT COST (Add Subrorala, Irema 9 uld II. and enter on Pale 1) c s S S +S * s 60,930 h . . . S&rtitutp Budget Pop. %72 c-m c--, PUC 20s --A 0oC uoo.1 f--/l ;r GPO 933.:i' SECTION II -PRIVILEGED COMMUNICATION POLYMORPHIC GENETIC MARKERS BUDGET ESTIMATES FOR ALL YEARS OF SUPPORT REQUESTED FROM PUBLIC HEALTH SERVICE DIRECT COSTS ONLY (Omit Cents) DESCRIPTION PERSONNEL COSTS ST PERIOD (SAME AS DE. ADDITIONAL YEARS SUPPORT REQUESTED (Thisapplication on/y/ TAILED BUDGET) 2ND YEAR 3RD YEAR 4TH YEAR 5TH YEAR 6TH YEAR 7TH YEAR 52,330 56,035 59,897 64,022 68,424 CONSULTANT COSTS (Include fees, travel, etc.) EOUIPMENT 1,100* SUPPLIES 5,000 5,300 5,700 6,000 6,400 DOMESTIC 500 600 600 600 700 TRAVEL FOREIGN PATIENT COSTS ALTERATIONS AND RENOVATIONS OTHER EXPENSES 2,000 2,100 2,300 2,500 2,600 TOTAL DIRECT COSTS 60,930 64,035 68,497 73,122 78,124 TOTAL FOR ENTIRE PROPOSED PROJECT PERIOD (Enteron Pege 1, Item 41 - s 344,708 I REMARKS: Justify all costs for the first year for which the need may nor be obvious For future years. justify equipment costs, JS well JS any significant increases in any other CJtJgOfy. If e recurring annual increase in personnel coets is requested, give percentage (Use conbnuarion page if needed. I Budge't explanation attached. BUDGET EXPLAYATIOI' Professor Cam's and Dr. Tsuboi's time are each budgetted dt 20% in support of this project. The project also requires two full time and one part time research assistants. The part time research assistant will assist in typing cultured cells from amniotic fluid for W-A antigens. One full time assistant (firs Van West) will assist in the development of serologic tests on amniotic fluid and amniotic fluid cells. The other research assistant (6, tlakk) will assist in culturing amniotic fluid cells and in performing electrophoresis on cultured cell extracts. The laboratory diener uill provide the needed support for cell culture work ( washing, sterilization of equipment, etc) and also for general laboratory uork. We are also requesting 25% of secretarial time to support the activities of both professionals in this project. Salaries are increased at a rate of 6% per year to cover merit and cost of living increases. Staff benefits are applied based on the following University projections: 17X, g/73-8/74; 18.3%. 9/n-e/75; 19.3%. g/75-8/76; 20.3%. 9/76-a/77; 21.3X, 9/77-a/78; and 22.31, Y/78-8/79. The microfuge will be use3 to centrifuge small dBOUntS of cultured cells. The power pack will be used for electrophoresis. Mecessarp supplies are budgetted to support laboratory work including chemicals, glassware, antisera, etc. We are requesting $100 per month for computer time and data file storage and $400 per year for our share of the rental of a computer terminal. Access to c3aputation is necessary for cataloging of cultured cells from AF specimens, automatic filing and storage of test results on various cell cultures and amniotic fluid samples, filing and storage of data on phenotypes of parents, rela,ting parental data to fetal data, monitoring lists of expected dates of delivery of fetuses ue have studied and data analysis. P-/l/ SECTION V A Search for Genetic Polymorphisms and Variances Of Specific Binding Proteins in Blood Dr. Cavalli-Sforza Genetic Polyaorphisns and Variants of Specific Binding Proteins in Blood Dr. L. L. Cavalli-Sforza, Principal Investigator A. IRTRODUCTIOR A.1 Objectire To search for neu medically significant genetic variants and genetic polymorphisms of specific binding proteins in blood. A. 2 Background and Bationale The recognition of yenetil" differences by electrophoretlc studies of proteins is by now a widely applied procedure, nethods employed for detectiny proteins after electrophoresis range from unspecific Protein staining to the identification of specific proteins by a great variety of techniques. Aarris and Hopkinson (1972) have recently summarized the evidence collected in electrophoretic studies of specific enzymes in Caucasians. Out of 7 enzymes analyzed, about l/3 were found to be polymorphic. Certain other categories of enzymes may be subject to a lower rate of polymorphism (Oaenn, Cohen and Rotnlsky, 1971). One method of labeling a variety of proteins is already establ,ished but has not been tested systematically for its capacity to identify polymorphisms or rare variants. It consxts of adding to serum (or other biological fluids) a radioactive ligand molecule and following its binding to a specific protein (or proteins) by electrophoresis followed by autoradiography. This method was used (Giblett, Rickman and Smithies, 1959) to amplify the classical notion that transferrin binds iron and that electroyhorntic variants of this molecule retain the capacity to bind iron. The physiological function of this protein is fairly well known and its important role in the organism uell ascertained {for details see Giblett, 1969). Polymorphism is well documented and many rare variants are also known. Other proteins known to specifically bind metals (e.g. copper: ceruloplasmin, see Giblett for details) and other substances (e.g. thyroxine; see Heinonen et al., fialkow et al., and Penfold et al.) have also been studied. The sugyestion advanced in the present application is to test systematically samples of human blood for proteins binding specific substances, taking advantage of the fact that many physiologically important ligands are available in a radioactive form. Two aims could be thus obtained: 1) increase the present uealth of polymorphisms, a very desirable aim (see Cavalli-Sforza, 1973); 2) a functional basis for each specific polymorphism or rare variant conld be sought, given that the nature of the substance would usually suggest possible advantages or handicaps of the variants. p -/`;z : -2- B. SPECIFIC AIdS (1) Search for polymorphisms in proteins binding specific substances available in radioactive form. This may enable us to look for a large number of new polymorphisms, allowing us to considerably increase the genetic map and to find new linkages. (2) In all cases in which polymorphisms are .detected, test for developmental behavior of the proteins in question, and for the chemical specificity of the ligand. (3) In cases of toxicity or idiosyncratic drug reaction, test for variation or absence of proteins binding the specific poison or drug. C. KETLiODS AllD PROCRDURE The experimental methodology has already been developed on the basis of experience accumulated in another research line. In the analysis of platelet proteins binding certain neurotransmftters (in particular, serotonin and norepinephrine) a method of electrophoresis followed by autoradiography was set op. In order to test the method for its capacity to reveal serum proteins binding specific substances, it was applied to lead-binding proteins. A short description of the method follows: Pb(210) at an appropriate concentration is incubated at 37 degrees C. for 30 minutes with human serum. 0.1 ml of the incubated serum is electrophoresed on acrylanide gel. Bromophenol blue is used as a tracking dye and electrophoresis stopped when the dye is at the bottom of the column. (The procedure can be used both on columns and on s13b gels). At the end of electrophotesis, the gel column is cut longitudinally and the flat surface applied on an X-ray Kodak film for 4-10 days in conditions guaranteeing close adhesion and maximum efficiency of arrival to the plate of the electrons emitted by the decaying radioactive atoms. A diagram of a developed autoradiogram is shovn in Figure 1. Four different individuals are shown. There is considerable binding of l?b to albumin which forms the large thick band. Albumin is known to have a high affinity for many substances. In addition, there are other lighter bands of Pb-binding proteins in the globulin region. Some of these correspond to thin protein bands visible by staining. Tuo proteins in the beta globulin region have been comstantlp found in all individuals tested so far. In the gel at the extreme left, two bands are visible near the origin (the top of the figure) while there is only one band at the same location in the other three individuals. The double band may be due to heterozygosis but so far only one individual with a double band has been found on the limited number of individuals tested. It is planned to continue the testing on at least a hundred normal individuals, to establish if a polymorphism really exists, to test the families of individuals showing electrophoretic differences, and to test all cases of plumbism which may come to our attention by the cooperation of -3- the hospital staff. Some of these individuals may be idiosyncratically sensitive to lead; or, alternatively, may show physiological responses to lead intoxication. Other disease states suspected of affecting plasma protein distributions will also be examined. The extension of this uork to substances other than lead is the subject of the present grant application, The substances to be tested for binding to serum proteins can be many, the main limitation being that the organic substances must be labelled with C(14) or otherwise produce beta radiation of similar energy. Pb emits also gaana-radiation, so that exposed plates must be shielded; this, however, is no serious liaitation since contact of the experimenter with the radioactive material is very short and his or her exposure controlled by the supervision ordinarily carried out in the laboratory (film badges, routine checks of benches and equipment). Results with h(3) labelled material have so far been unsatisfactory due to the short range of the weak electrons eritted in E(3) decay. C(14) labelled material is eatirely satisfactory, as shoun by the previous experience with platelets. A shortened list of the substances to be tested includes the following: (1) Elements which hava radioactive isotopes suitable for the test. (2) Amino acids, peptides (3) Uucleotides, nucleosides (0) Sugars (5) Lipids (6) tiormones (7) Vitamins W Qruw, including antibiotics, addiction drugs, pesticides, poisons, etc. The analysis should be carried out initially on a sample of at least one hundred random iniividuals. Over 100 substances can be tested, but each test requires only 0.1 ml of serum and thus 20-30 ml of blood obtained from an individual will be enough for all tests. On the assumption that one or more proteins binding specifically a substance are found for most of these substances, this experiment should give a good sample of prospective polynorphisrs from which the frequency of polymorphism and average heterozygosity for each can be computed and compared with that observed for enzymes (see Introduction). -Y- Substances found to hdVe polymorphic binding proteins cdn then be subject to the following series of observations: 1) Tests on families scored for other markers uhicb have already been collected in other laboratories. Prospective collaborations are being considered. It is expected (but should be first tested) that in serum stored in freezers the specific binding activity is stable. The existence of a number of projects in which blood samples have been collected from families, examined and stored makes it easier and more efficient to test on such material inheritance of the protein differences (i.e. segregation analysis) and linkage of the corresponding genes to standard markers. Several such collections of samples are already available. 2) Ye plan to examine newborn infants born at Stanford Hospital of matings in which the mother is honozygous for d polymorphic protein of the type described, and the father heterozygous (or hosozygous for another allele). The paternal protein would be searched in cord blood and if not present, the child uould be followed further to establish the age of appearance of the paternal protein. This would give us a chance to seek regulatory genes for the developmental pattern of these proteins. P3r instance, we will seek variation among individuals of age of appearance of the protein and analyze the variation with family studies. 3) For every specific substance, patients with diseases that may be explained by a variation or absence of a binding protein, the specific substance should be examined. . D, SIGNIPICAYCE It is difficult to anticipate the total number of proteins that can be identified by this procedure, hut existing information would suggest that it can be as high as several hundred. The method suggested then supplies a very economical procedure for testing a great number of potential polymorphisms. The frequency of polymorphic genes is one of the quantities which is of interest to estimate for comparison with the existing enzyme data. This result has obvious evolutionary significance in view of the present discussion on neutrality of polymorphic genes. If the proportion is the same as is known to be among enzymes, then this investigation may generate enough markers to more than double the existing genetic map of man, with all consequent advantages of increased precision in genetic counseling and research. The interest offered by such new polymorphisms would be greatly enhanced by the possibility of detecting variation for regulatory genes in the manner explained before. This is one of the most d.ifficult fields in human general genetics today, the development of which may be most fruitful. -4- Substances found to have polymorphic binding proteins can then be subject to the following series of observations: 1) Tests on families scored for other markers uhicb have already been collected in other laboratories. Prospective collaborations are being considered. It is expected (but should be first tested) that in serum stored in freezers the specific binding activity is stable. The existence of a number of projects in which blood samples have been collected from families, examined an3 stored makes it easier and more efficient to test on such material inheritance of the protein differences (i.e. segregation analysis) and linkage of the corresponding genes to standard markers. Several such collections of samples are already available. 2) Ye plan to examine newborn infants born at Stanford Hospital of matings in uhich the mother is homozygous for a polymorphic protein of the type described, and the father heterozygous (or homozygous for another allele). The paternal protein uould be searched in cord blood and if not present, the child uould be followed further: to establish the age of appearance of the paternal protein. This uould give us a chance to seek regulatory genes for the developaental pattern of these proteins. F9r instance, we will seek variation among individuals of age of appearance of the protein and analyze the variation with family studies. 3) For every specific substance, patients with diseases that ray be explained by a variation or absence of a binding protein, the specific substance should be examined. . D. SfGhIFXCIYCE It is difficult to anticipate the total number of proteins that can be identified b,y this procedure, but existing information uould suggest that it can be as high as several hundred. The method suggested then supplies a very economical procedure for testing a great number of potential polymorphisms. The frequency of polymorphic genes is one of the guantities uhich is of interest to estimate for comparison with the existing enzyme data. This result has obvious evolutionary significance in view of the present discussion on neutrality of polymorphic genes. If the proportion is tha same as is known to be among enzymes, then this investigation may generate enough markers to more than double the existing genetic map of man. with all consequent advantages of increased precision in genetic counseling and research. The interest offered by such new polymorphisms would be greatly enhanced by the possibility of detecting variation for regulatory genes in the manner explained before. This is one of the most difficult fields in human general genetics today, the development of which may be most fruitful. P-126 -5- Finally, each and every one of the proteins thus detected and identified may offer unique possibilities of further research and therapeutic developments. Taking again the model of transferrin, there is one well known case of congenital absence of this protein which was lethal (Reilmeyer et al., 1961). III similar cases, substitutional therapy by transfusion or plasma infusions may prove life saving. Several dangerous rare drug idiosyncrasies are kooun to exist, e.g. to chloraaphenicol. Should they prove to be connected to the lack of a specific binding protein, transfusion or plasma infusions may again prove useful or at least these,patients could be identified before becoming the victims of the administration of a drug potentially lethal for them. Cases of vitamin or hormone resistance might find similarly an unexpected explanation and therapeutic benefit, -6- REFEREYC ES Caralli-Sfnrza, L.L. 1973, Some current problems of human population genetics. Am. J. Hum. Gen. 25:82-104. Giblet, E.R. 1969. Genetic Markers in Huaan Blood. Blackwell Scientific Publications, Oxford. Giblett, E-B., Bickran, C.G. and Smithies, 0. 1959. Serum transferrins. Rature 183:1589. Harris, II. and Hopkinson, D.A. 1372. Average heterozygosity per locus in man: an estimate based on the incidence of enzyae polymorphisms. Ann. Hua. Genet. 36:9-20. Omenn, G.S., Cohen, P-T, and Hotulsky, A-G. 1971. Genetic variation in glycolytic enzymes in human brain (abstr.). Excerpta Bedica Int. Cong. Ser. Yo. 233:135. Xeinonen, 0-P.. Lanberg, B.A., Virtaao, J, 19'10. Inherited decrease of the binding capacity of thyroxine-binding globulin (TBG). Acta Endocrinologica 64:171-180. Pialkow, P.J., Giblett, E.R. nnd Husa, B. 1970. Increased serum thyroxine-binding globulin capacity: inheritance and linkage relationships. J. Clin. Endo. E hetab. 30:66-75. Penfold, J.L., Kneebone, G.B., Welby, tl., Oldfield, B-K, 1971. Growth retardation and thyroxine-binding globulin deficiency. Arch. Dis. in Childhood 46:115-117. I j .: , I i I ! ~"".,.."I..~-. I",. *Lb I I",. II avua 1 I I" I L I r1,a I ClVL r "R YL I mmLLY ""VVC t 13-v: SUBSTITUTE PERIOD COVERED GRANT NUMBER IETAILED BUDGET FOR FIRST 12.MONTH PERIOD -O;,l,,4 THROUGH 12131174 PERSONNEL (List all personnel en9eged on project) NAME (Last, first, initfal) TITLE OF POSITION CavalUrSforza, L. Principal Investigator or Program Director TIME OR EFFORT AMOUNT REQUESTED (Omit cents) %/HRS. I TOTAL , >.; 10% .'. open - Genetics Research Associate 100% ';;.~PCLYMCRPRISMSS OF.SPRCIFIC ,.: Open - Genetics Research Tech. 100% .'.j,' : ~smimc PRCTRINS : +;,,:. ..:I ,..: j :,.,. :.: . . .:. . . I. 1: i. .: "".S :;:.: . .., ; . . '.. .:.::, . . .::., .: 1 . . . ; .:: .: :,.., . . .I... ::.. ,. ::. I ..: . . .:.:.... " ::.. / .1..:: : .." .::: :. :. :. . . .j "'. : :... :':' :.: / .;.: . . . '.' :.. '..'. ., : .:: :. : . . : TOTAL *s 30,489 CONSULTANT COSTS (Include Fees and iravel) EQUIPMENT (Itemize) Constant current power supply $1,000 Slab gel electrophoresis 300 Column acrylamide gel electrophor. 200 Destainer 200 SUPPLIES Radioactive tracers $5,000 Chemicals, glassware, lab app. 1,000 Expendable lab supplies, 1,500 (photographic sunplies. etc.1 I S 1.700* s 7.500 STAFF 0. DOMESTIC 2 East coast meetings TRAVEL . :ee rn*tJuctfons) b. FORE,GN PATIENT COSTS (Separate Inpetient md Outpatient) Venapuncture for blood samples ALTERATIONS AND RENOVATIONS OTHER EXPENSES (Itemize per in&uctiond S 1,000 S S 500 S Office supplies, telephone, repro., postage, publications costs, etc. 1000 Central computer usage 1000 S 2,000 Subtotal - Items 1 thru 8 * s 43,189 10. TRAINEE EXPENSES (See Instnrctions) PREDOCTORAL No. Proposed s FOR . o sT,pENDS POSTDOCTORAL No. Proposed S OTHER (Specify) No. Proposed 5 rRAlNlNG DEPENDENCY ALLOWANCE S GRANTS . TOTAL JTIPEND EXPENSES - S b. TUITION AND FEES S ONLY . C. TRAINEE TRAVE'- (Dercrlbe) S 11. Subtotal - Trainee Erpmses *S 2. TOTAL DIRECT COST (Add Subtotala, Items 9 md 11, and enter on Pa&?= 1) c ' 43,189 ,. _.__ ,a. .I - -- n r.- kP~rmm rruagor rag* ~--II For Forms PHJ 398 and PHS 2499-l r-/Y v GPO 930.731 SECTION 11- PRlVlLEGED COMMUNICATION POLYMORPHISMS OF SPECIFIC BINDING PROTEINS I BUDGET ESTIMATES FOR ALL YEARS OF SUPPORT REQUESTEO FROM PUBLIC HEALTH SERVICE DIRECT COSTS ONLY (Omit Cents) CONSULTANT COSTS (Include fees, travel, etc.) EQUIPMENT I 1,700* I 500* SUPPLIES I I 7,500 8,000 8,600 1 9,000 1 9,600 1 DOMESTIC 1,000 1,100 TRAVEL FOREIGN PATIENT COSTS I I 500 500 ALTERATIONS AND RENOVATIONS OTHER EXPENSES I 2,000 I 2,300 TOTAL DIRECT COSTS 1 43,189 1 45,048. TOTAL FOR ENTIRE PROPOSED PROJECT PERIOD (Enter{ REMARKS: Justify all costs for the first year for which the nea sbnifimnt increak in any other category. If a recurring annual page if needed.) Budget explanation attached YEARS SUPPORT REQUESTED (Thisapplication only) 3RDYEAR 1 4THYEAR 1 5TH YEAR 1 6THYEAR 1 7Tt-i YEAR 34,898 37,301 39,867 500* I I 1,200 1,200 1,300 (,-I--- -2,400 / 2,400 1 2,700 ) may not be obvious For future years, justify equipment costs, as well as any ncrease in personnel costs is requested, give percentage. (Use continuation I PHS398 Rw. &70 L P-/.2/ BUDGET EXPLAHATION 10% of Professor Cavalli-Sforza's time along with a full-time Research Associate and a full-time Research Technician are budgetted in support of this project. The Research Associate, a biochemical geneticist, will be responsible for the electrophoretic analysis of plasma proteins and will be assisted by the Research Technician. Salaries are increased at a rate of 6% per year to cover aerit and cost of living increases. Staff benefits are applied based on the following University projections: 17X, Y/73-8/74; 18.3%. g/74-0/75; 19.3X, g/75-8/76; 20.31, 9/76-8/77; 21.3X, g/77-8/78; and 22.3X, g/78-8/79, The budget includes slab qel and column gel electroyhoresis equipment, and associated pouer supply, etc., as well as supporting supplies. These supplies include radioactive tracers, chemicals and laboratory apparatus, glassware, and expendable supplies such as photographic plates, etc. Travel funds are requested for attending two professional meetings on the east coast. Patient costs covering venepuncture to obtain blood samples, are estimated at $500 per year. P-/3;? SECTION VI The Impact of Genetic Counseling Practices on Family Decisions and Behavior Drs. Barnett, Cann, and Luzzatti The Impact of Genetic Counseling Practices on Paaily Decisions and Behavior Dr. C.R. Barnett, Principal Investigator Drs, R. Cann and L, Lozzstti, Associate Investigators A. INTRODDCTION A.1 Objectives The overall objective of this study is to provide systematic answers to some of the basic, unanswered questions in the practice of genetic counseling. (1) Yhat is the impact of genetic counseling, that is, do families not receiving genetic coanseling make decisions different from those who do? (2) What is the difference in counseling effectiveness betueen a physician trained in genetic counseling and a social worker trained in genetics? (3) What is the difference in effectiveness between a counselor who is directive in his counseling and one who maintains a neutral stance? (4) Uhat is the relationship between the structure and content of a genetic counseling session and the pre-counseling training and attitude of the counselor'? (5) What is the difference in effectiveness between a counselor who receives social and psychological information about the family before counseling and one uho 3oes not have such information? (6) What are the expectations of farilies seeking counseling and how do they use the information they obtain in making decisions? A.2 Background A recent revieu of the social aspects of human genetics (1) and an editorial on genetic counseling in the Neu England Journal of Medicine (2) have consisted largely of lists of questions regarding genetic counseling for uhich there are as yet no answers. While much has been learned over the years regarding the genetic basis for many diseases, their mode of inheritance and the probability of occurrence in a given population, little research attention has been paid to how this information is transmitted to patients and the use they make of it. Typical of the state of the art and the still prevailing emphasis on agenetic prognosis", rather than "genetic counseling", is a recent textbook on genetic counseling (3) which devotes only 3 of its 355 pages to the counselor-patient relationship. The major issues in the field may be subsumed under three basic questions: 1) What is or should be the impact of counseling ? 2) What should be the counselor*s role? 3) Who should do the counseling? The first question is most difficult to answer at this time since there are little data available on the impact of counseling. A few studies have looked at impact by measuring the: number of children families have had post-counseling, or by learning of their post-counseling decision to practice or change p-134 -2- their aethois of contraception. With regard to the findings from such studies, Hecht and Holmes (2) have noted: @*What is the objective of genetic counseling ? If it is to lessen the chance of subsequent affected sibs being born, the available data are discouraging .I) One of the major problems with studies which have reported somewhat favorable results (4) is that they have not utilized control groups. The only study that used a control group (made up of faailies uith children affected with a non-genetic, chronic condition) reported that SO% of the control group decided to limit the size of their families, in the absence of any genetic counseling to do so (5). Studies to measure the effects of counseling have also used a number of other outcome criteria, such as knouledge of probability or risk and information about hereditary transmission of traits that was retained by the family. There are two major deficiencies in these studies, First, with one exception (6) none of the studies have made an assessment of knowledge before the counseling took place. Indeed, in some cases, the follow-up of knowledge retained by the families was as long as 4 to 10 years after counseling (4,7), uith no control or assessment of the effect of other sources of information on the families. A second deficiency with the studies which have tested post-counseling risk and genetic knowledge of families, is that there is no indication, from the point of view of families, that biological knowledge and inforaation regarding risk is used by them in making decisions about reproduction, The actual decision-making process in the family has remained an unopened Rblack boxa. The second question regarding the counselor*s role involves sharp differences of opinion on two issues: the question of whether the counselor should be neutral or directive; and whether the counseling should be narrowly focused or broadly comprehensive. The traditional neutral stance is most often associated with the focused role prescribed for the counselor: *It can be argued that a counselor's job is simply to estimate.... risk as uell as possible and try to ensure that this is understood. This is, of course, true. It is entirely a matter for parents to decide whether to avoid having further children, or to seek sterilization or termination of pregnancyn (3). Yhen a genetic counselor feels called upon to violate this principle of neutrality, he makes a point of explaining the deviation, as does Carter (4), in order to reassure low risk parents. There are two untested assumptions in the argument presented by experts on both sides of this controversy. The first assumption is that a neutral counselor will not communicate unconsciously by his tone of voice, mode of presentation or non-verbal cues, his true feelings about what decision a family should make. Secondly, it is assumed that presentation of his feelings of "what he would do if he were in their shoes" will have a marked effect on the faiPily*s decision. These assumptions can and should be tested, since findings nay suggest that the argument for either position is irrelevant to the outcome of counseling. P-135 -3- The issue of whether genetic counseling should be narrowly focused or broadly comprehensive has been linked to the question of who should do the counseling. Thus those who have taken the position that genetic counseling should be considered part of family guidance, have argued that the family physician can best play this role (8). Those vho argue that the priaary purpose of genetic counseling is to answer questions the patient may have about risk, feel that counseling should be left to the clinical geneticist. Franz Kallman (9), uho favors a comprehensive approach has phrased the question most realistically by suggesting the type of training needed by the counselor: "There can be no qaestion .,.-that the constructive management of genetic family problems requires either geneticists uho are experienced in counseling techniques or family guidance workers who have adequate training in genetics*'. The issue of whether to take a narrow or a brodd approach to counseling could be settled if information were available regarding what problems they may acquire as a result of the counseling. At present, genetic counseling has been not subject to the same types of analysis that have been brought to bear on the physician-patient relationship in other situations (lo), Thus, what happens during genetic counseling has been described only in anecdotal form (1). The question of whether the geneticist, the family counselor, the family physician or any other type of professional or lay couaselor can best meet the needs of the family seeking genetic counseling can be determined by systematic evaluation of what these people actually do in counseling and what impact they have on the families. Uhen new roles have been established in other areas of care delivery, the behavior and effectiveness of people taking the neu roles have been evaluated (11). There is no reason uhy the same approach cannot be taken with regard to this issue in genetic counseling, A.3 Rationale Genetic counseling involves at least two parties, the counselor and the family, and both parties to the event must be studied, as well as the event (counseling) itself to determine the effectiveness of genetic counseling under varying conditions. The counselor vi11 bring to counseling his expectations about the nature of the coanselin9 situation, and a predetermined view of the risk and burden a defect may represent for the family. He may decide, beforehand, to communicate an optimistic, pessimistic or neutral point of view to the family. His upublicH position map vary from his "privately'* held view. Ke may, if he is supplies uith additional information about the family, (their state of knowledge, their values regarding having children, differences between husband an3 uife on basic issues, other decisions they are in the procsss of making, and their expectations regarding counseling), tailor the information and counseling he provides to the specific needs of the family. -4- The family, as noted above, may have expectations regarding counseling that are at variance with those of the counselor. They also come to counseling with a state of knowledge about the risk of having a child with a defect, the burden it represents, the genetic and biological principles underlying the defect and the basis fot computing risk. Further, in their own fanily decision-making experience they may make great or little use of probabilities in coning to decisions. The fattily also comes with a set of values or attitudes regarding what they want as individuals and as members of a family unit, and these values also determine the kind of information they seek and how they use the information. Families will vary even with regard to the number of sources of information they use, so that for some, the genetic counselor may be the principal source, while for others, the genetic counselor may be one among many. The counseling session represents an interaction between these two parties and no matter what the prior expectations on either side, the event say differ from what the two parties believe will happen, and after the event, what they think happened. Thus, the event itself aust be studied and compared both witb prior expectations and with post-counseling recollections. Did the counselor consciously or unconsciously break his stance of neutrality, and uas it noted by the family? Yas the family so immersed in absorbing the information about burden and prognosis that they recollected little about the risk information given by the counselor? The expectations of both parties in genetic counseling provides two measures of effectiveness of counseling, rather than the single measure (goals of the counselor) which has been used up to nou. Information obtained prior to counseling about the values, knouledge, and decision states of the family, as well as their expectations may enable the counselor to satisfy both his and the family's expectations. An ideal design for answering the basic questions regarding genetic counseling should satisfy the canons of experimental design even though the issues are basically behavioral and social. The study would be prospective in that it measures the status of the parties before the counseling takes place and then measures changes following counseling, against the pre-counseling base (6). It should randomly assign counselees to varying types of counselors (such as a physician or a social worker), and to counselors uho have di-fferent types of information available to them about the family before counseling. Finally, control groups should be utilized to control for both the effects of the research contacts on the family, ds vell as a control group which does not receive genetic counseling, but say also make decisions about having children. B. SPECIFIC AIHS 1. To test the hypothesis that genetic counseling can be done at least as effectively by a social worker with some ?-i37 -5- training in clinical genetics as by an fl,D. trained in clinical genetics. 2. To test the hypothesis that genetic counselors, even when holding consciously to the principle of "neutrality," will divulge their ntrueM feelings to their counselees. 3. To test the hypothesis that counselors who are informed prior to counseling regarding the values, knouledge, decision status and counseling expectations of the counselees will be more effective than counselors who are not so informed. 4, To develop measures for determining the effectiveness of genetic counseling which utilize the goals of the counselees, as veil as the objectives of the counselors. 5. To learn how families utilize information provided in genetic counseling (such as risk and burden) in reaching decisions about child bearing. C. STUD? DESIGB Four experimental groups and 3 control groups will be established in order to test the significance of the major variables in the study. all four of the experimental groups will be subject to the following procedures. 1. 48-72 hours, pre-counseling. Family receives pre-counseling interview by 2 members of research team and fills out inventory instruments to assess their values relating to child-bearing, faeily relationships and life expectations; their knowledge of probabilities, genetics and the disease or condition about which they are seeking counseling; the family decisions they have recently made or are in the process of making; and their expectations regarding the counseling they are to receive. 2. 24-48 hours pre-counseling. Genetic counselor writes a summary of his understanding of the case; his expectations regarding the session; the position he expects to take with the family (neutral, optimistic, pessimistic), and his personal feelings about the decision the family ought to make. .3. Family receives genetic counseling. The entire intorviev is audio-taped for analysis of the structure and content of the interaction. 4. 24-48 hours post-counseling. Summary and evaluation of the counseling session by the genetic counselor including his prediction about the decision the family vi11 make and differences between his expectations recorded at point 112 and what actually occurred during the counseling at point 83. 5. 48-72 hours post-counseling. Interview and administration of instruments to the family, similar to point Xl. Probes on: their view of the counseling sassion; what they learned; were P-138 -6- expectations net; what position did they feel the counselor took. 6. 1 month post-counseling interview with family. Information obtained as at tl; probes on other information obtained by family sources of information, new experiences which have led to value changes and decisions. 7, 6 month post-counseling interview with family. Information obtained as in 16. 8. 1 pear post-counseling interview with family. Information obtained as in #6, The 4 experimental groups will vary according to vhether they receive counseling by an H.D. trained in 8edical genetics or by a social worker trained in genetic counseling. They vi11 also vary according to whether the counselor receives or does not receive information about the family obtained from the pre-counseling contact (point 1c1, above). The families in all 4 of the experimental groups defined belov will be subject to the procedures outlined above (#l-R). Families seeking or referred for genetic counseling will be assigned randonly to one of the folloving treatment groups: Group E-l, Group B-2, Group E-3. Groay B-4. Receives counseling from M.D. trained in medical genetics, Counselor receives no information obtained from pm-counseling interview. Receives counseling from M.D. trained in medical genetics, Counselor receives information aboat the family obtained in pre-counseling research interview. Receives counseling from social worker trained in genetic counseling. Counselor receives no information obtained from pre-counseling research interview. Receives counseling from social vorker trained in genetic counseling. Counselor receives information about the family obtained in pre-counseling research interview. It has been our experience with other longitudinal studies (12) that oaltiple interviews with families in order to obtain research data actually provide considerable psychological and social support for the family. In the case of the proposed study, it could ev8n influence the decision made by the family by helping them to focus on the problems they face and to make more explicit the alternatives they may have. In order to control for the effects of the interviews and instruments on the decisions that may be made by the faeilias, the following 2 control 'droups will be established by random assignment of families: -7- Group C-l. Pamily does not receive pre-counseling research interview (#I above). Receives counseling from H. D. trained in,medical genetics (as does Group R-1). Counselor completes pre- and post-counseling summary (points t2 and Y4). Family does not receive post-counseling follow-up (points #S,tb, and t7) until 1 year post-counseling (point #8). Group C-2. Family does not receive pre-counseling research interview (#l above). Receives counseling from social uorker trained in genetic counseling (as does Group E-3). Counselor completes pre- and post-counseling summary (points 12 and BY). Family does not receive post-counseling follow-up (points 15, f6, and t7) until 1 year post-counseling (point t8) . A third control group (C-3) will consist of parents who have a child with a chronic, non-genetic condition and who have not received genetic counseling. This group vi11 provide an overall control on the effect of genetic counseling on family decisions, particularly with regard to knowledge and limitation of family skze, Like control groups C-l and C-2 they vi11 be interviewed one year after receiving information from a physician (in this case, inforration about the diagnosis and prognosis for their child). MTRAYCE CRITERIA FOB THE STUDY For families in the 4 experiwental groups and families in control groaps 1 and 2: 1. Family must seek or be referred for and receive genetic counseling at Stanford University Medical Center. 2. Family must be intact, i.e. there must be a couple in an already-established marriage or common-law relationship. 3. Family must be willing to participate in the number of sessions involved for data collection. Counseling costs and transportation for research interviews will be borne by the project to encourage participation. Paorilies in control group 3 will meet the same criteria, except that they vi11 have a child with a non-genetic, chronic condition diagnosed at Stanfor University fledical Center or the Children's Efospital at stanforj. The purpose of the entrance criteria is to control for some of the background variables which must be considered in data analysis. Patients receiving genetic counseling outside of the medical center must be presumed to be a population with somewhat different characteristics than the population seen at the medical center, and the counseling they receive must also be assumed to be somevhat different. A population outside of the aedical center -0- could be studied only b,y increasing the size of the study population by 100%. Use of a medical center population, combined with the requirement that families be intact, vi11 serve to provide a population uith some homogeneity with regard to income, education, occupation and family situation (13). This requirement, for example, rules out from the study couples seeking genetic counseling before marriage, unmarried teen-age mothers, etc. Hhile the impact of counseling on these groups is deserving of study, given the umber of variables in the study, control of some of the population characteristics is necessary. These criteria will also allow for random assignment of families to treatment and control groups thus obviating the difficulties and possible bias of selective matching. IISTRUMITS ABD SCHEDULIW The first year of the study will be devoted to the developnent and validation of the instruments to be utilized, the training of personnel to do the coding of the transcripts of the counseling and interview sessions, and a pilot test of the study design. Approximately 50 families will be utilized during the first year. Wring the second and third year of study, approxisately 125 families will be taken in and followed each year. The 4th year will be devoted to continued one year post-counseling follow-up of the families and data analysis, The 5th year vi11 be exclusively data analysis and write-up of the study. Among the instruments to be developed are those to assess the attitudes, decision state, knowledge and expectations of families relevant to genetic counseling. These are the instruments to be utilized at point 81 in the study design and at future follow-up points. These instruments will be pre-tested with a variety of patients to determine their ability to distinguish significant differences among families, their ease of administration and numerical szoring. Pace validity vi.11 be determined through use of standard pre-test procedures (14). Particular attention will be given to the development of instruments which vi11 deternine the ability of the families to apply probability figures to every-day life situations. During the development period, genetic counseling sessions will be tape recorded and a scoring system developed for analysis of the sessions. Coders, who will have no knowledge of the pre-counseling data obtained from the families or the counselors, will apply the scoring system. Using an adaptation of the interaction methods developed by Bales (15), both the structure and the content of the sessions will be analyzed. These data will be tested against the pre-counseling data obtained from both tht! counselors and families and against the recall, post-counseling, of counselors and families. Pre-coded and pre- and post-counseling forms to be used by the counselors will be developed, Counselors will record their understanding of the case, the stance they propose to take and -9- their own personal feelings about the decision the family should make. The standardized post-counseling report vi11 include their evaluation of the session, any changes from the pre-counseling stance and their estimate of the decision the family might make as a result of the counseling. The expectations of the counselor regarding the session vi11 be compared with the pre-counseling expectations elicited independently from the couple. Similarly, the post-counseling summary from the counselor vi.11 be compared with the post-counseling wiev of the session obtained from the counselees. Post-counseliny interviews will also be conducted with the families (points 115-e in the study design). Some of the same pre-counseling instruments vi11 be used along with a standard intervieu format combining general and specific probe questions similar in form to the type developed by the study director for a study of family response to the birth of premature infants (12, 13). Included in the post-counseling interviews vi11 be questions to elicit family reactions to the counseling, their assessment of the point of view taken by the counselor, decisions they may have reached and the reasons for making the decisions they have arrived at. On the basis of our previous family studies, the husband and uife will be interviewed separately to prevent contamination of the decision-making process by forcing consensus or facilitating husband-wife communication. Since the pre-counseling assessment will also be obtained independently, one form of data analysis will be to see to what extent the values and information of the husband and wife coincide after counseling, The timing of data collection for the post-counseling period, beyond the first post-counseling interviews, is not rigidly established. One purpose of the first year of developmental work is to determine the best timing that will take us closest to the point at which families do make decisions. LIflITATfOffS There may be some loss of subjects to follow-up, but this uill be minimized by paying transportation and counseling costs. The number of families who refuse to participate in the study will be kept to a miaimum through the same devices, but background data will be obtainad in any case to see whether refusing families differ in important respects from the study population. The findings of the study will not apply, of course, to couples who seek pre-martial counseling, to individuals who do not constitate a faai1.y unit, and to those who do not seek or are referred foe counseling. Further, it is anticipated that because of the nature of the entrance criteria, the population Will have fairly homogeneous middle class ChardCteriStiCS (as definer! by income, occupation and education). A number of genetic coonsaling studies have attempted to determine the relationship between the decisions families make about reproduction and the risk and burden they face, As noted previously, the meaning of risk from the fnsilycs point of view has not been determined. Further, there appear to be significant differences among counselors regarding the nature of the burden for the same disease. Therefore, we have not chosen to classify families on the basis of risk and burden before assigning them to the experimental or control groups. Risk and burden will be analytic variables in the study and random assiynlaent of families should provide an appropriate mix of these variables in each group, SIGNIPXCAilCE The study will provide the first systematic test of the significant questions relating to the practice and inpact of genetic counseling. The study is unique in the experimental nature of the design. The instruments to be developed in the course of the study should be useful to counselors in guiding their practices and in evaluation of their effectiveness. Conceptually, the study places genetic counseling within the general framework of family decisions, so t.hat the effect of variables other than counseling on decision-making can be assessed. -11- REPEHEPiCES 1. Sorenson, J.R.: Social Aspects of Applied Human Genetics. Social Science Frontiers, No.3, Hussell Sage poundation, N.Y., 1971. 2. Hecht, F., Holaes, L.B.: New England J. fied. 237:464, 1972. 3. Stevenson, A.C., Davidson, B.C.: Genetic Counseling. J.P. Lipincott Co., Phila., 1970. 4, Carter, C.O., tvans, K.A., Fraser Roberts, J.A., Buck, A.R.: Lancet 1:281, 1971. 5. Leonard, C.O., Chase, G.A., Childs, B.: Neu England J. bed. 2873433, 1972. 6. Reiss, J-A., henashe, V.: J. Peds. 80:655, 1972. 7. Carter, C.O.: Proc. of the Third Xntl. Congress of Human Genetics (J. Crow E J. Reel, eds.), Baltimore, 1966, p. 97. 8. Gordon, H.: JARA 21.7:1215, 1971. 9. Kallmann, F-J.: Genetics S the Epidemiology of Chronic Diseases (J. Neel, LI. Shau G W. Schull, eds.). Public health Service Publication 1163. Washington, b.C., 1965. 10. FreeaDn, B., legrete, V-F., Davis, Pl., Korsch, B.H.: Pediatric Research 53298, 1971. 11. Adair, J., Deuschle, K., Rabin, D.: The People's Health: Medicine and Anthropology in a Navajo Community. Appleton-Century-Crofts, N.Y., 1'370. 12. Barnett, C.R., Leiderman, P.H., Grobstein, R., Klaus, R.: Pediatrics 45:197, 1970. 13. Seashore, R., Leifer, A., Barn&t, C., Leiderman. P.ki., Williams, J.: Personality and Social Psychology (in press). 14. Festinper, L., Katz, D.: Research Methods in thz Behavioral Sciences. Bolt, Rinehart E Winston, N.Y., 1953. 15. Bales, B.F.: Interaction Process Analysis: A tlethod for the Study of small Groups. Addison h'esley, Reading, Mass., 1950. P-/`/r/ "b.b 1 ,"I. I, ~"YJIIIVIL IrllJr N"L rvn YLI~IiL'J~`J"`J=I rr. SUBSTITUTE PERIOD COVERED GRANT NUMBER DETAILED BUDGET FOR FIRST 12.MONTH PERIOD FAo;,l,74 THROUGH 12/31/74 --- . PERSONNEL (Lisf ail perso~ef enfagcd on project) TIME OR EFFORT AMOUNT REQUESTED (Omit cenrs) NAME (Last. first, initial) TITLE OF POSITION wnus. I TOTAL Principal Investigator or ~ Barnett, C. Program Director 20% . . Cann, H., Assoc. Prof./Peds. 10% IMPACT OP'GBNBTIC COUNSELIXG Luzzatti, L. Professor of Peds. 10% PRACTICES Open Research Assoc.-Stat 20% 1 Open-Pediatrics Res. Assoc.-Sot. wk. 50% :: Open-Pediatrics Interviewer 50% '. Open-Pediatrics Interviewer 25% .' :. Open-Pediatrics Statistical Clerk ,50x Open-Pediatrics Data Coder '65% ' Open-Pediatrics Typist 100% .;: I ".-e--*,--- :. CONSULTANT COSTS (Include Fees and Travel) _ "_-.I_. ..!- . -I~- Tape Recorder -...-_.---_--- -.-.- -._ --- --- ----.-. .-_--~- _I_-- 1. OTHER EXPENSES (itemize per inatmctions) Office supplies, telephone, repro., postage, publicatti costs, etc. 1200 Central computer usage \- a. STIPENDS kkstituh Budast Paae S-72 SECTION II -PRIVILEGED COMMUNICATION IMPACT OF GENETIC COUNSELING PRACTICES BUDGET ESTIMATES FOR ALL YEARS OF SUPPORT REQUESTED FROM PUBLIC HEALTH SERVICE DIRECT COSTS ONLY (Omit Cents) DESCRIPTION PERSONNEL COSTS 1ST PER100 ISAME AS DE. ADDITIONAL YEARSSUPPORT REQUESTED lThisapp/ication only1 TAILEO BUDGET) 47,377 SUPPLIES DOMESTIC TRAVEL 500 600 600 600 700 FOREIGN PATIENT COSTS 2,500 RENOVATIONS ---I-- 5,000 5,000 ALTERATIONS AND OTHER EXPENSES 2,400 2,600 2,800 3,000 3,200 TOTAL DIRECT COSTS 53,277 58,932 62,629 61,564 65,850 TOTAL FOR ENTIRE PROPOSED PROJECT PERIOD (Enter on Page 1. item 4) _ s 302,252 REMARKS: Justify all costs for the first year for which the need may nor be obvious For future years, justify equipment costs, as well as any significant increases in any other category. If a recurring annual increase in personnel casts is requested, give percentage. (Use continuation page if needed) Budget explanation attached BUDGET SXPLAhATIOU Barnett has a 9 month academic appointment supported joint!;.by the Department of Pediatrics and the Department of Anthropology, His project salary 1s computed on the basis of 1Oi time during the 9 month academic year and 70% time during July and August for an average of approximately 20% during each year. The social worker (50% time), a research associate (23% time), two interviewers (50% and 25% time respectively), a statistical clerk (50% time), a data coder (65% time) and a typist (100% time) are require3 for the project. Two interviewers are required because husband and wife will be seen separately. The research associate is a biomathematician experienced in design ot and data analysis for behaviorial research projects. Computer time will be used for data analysis. Salaries are increased at a rate of 6X per year to cover 8erit and cost of living increases. Staff benefits are applied based on the following University projections: 17X, Y/73-8/74; 18.3%. 9/74-a/75; 19.3X, 9/75-8/7b; 20.3%. 9/76-h/77; 21.3X, g/77-8/78; and 22.3%. g/78-8/79. The tape recorder vi11 be used by the typist to transcribe tape recordings of genetic counseling sessions and pre- and post-counseling interviews in the project on qenetic counseling impacts on the family. To insure that ve obtain adequate patient material for the project on genetic counseling, ue propose to waive the fee for this service to any participating family and therefore include these costs in our budget. The cost of genetic counseling to d family is $50. This does not include laboratory tests and amniocentesis. We anticipate doubling the number of patients in the second and third years of the project. Xn years 4 and 5 no new patients will be studied although follou-up interviews will be carried out for those studied in year 3. p- i'r 7 SECTION VII Overall Budgets DIRECTOR'S OFFICE BUDGET EXPLANATION Salary support for the Program Director (Profassor Lederhery) has been included entirely under the subproject budget for Screening and Characterizatiou of Inborn Errors of Hetaboli~m Using GC/HS. The 2UX of his time budgetted there, includes support for his role in overall program direction as well ds his direct involvement in that research project, This 20% allocation has not been subdivided between that budget and the present Program Director's Office budget. Such a suballocation would be difficult to make realistically since the apportionment of Professor Lederberg's time vi11 vary from time to time, depending on program needs. This budget does include support for 30% of the Program Director's secretary. She will support the Director in overall program management as veil as in liaison vork with the Visiting Committee and in implementing the planned annual symposium on aspects of genetic disease. An important responsibility of the Director is maintaining current awareness of the relevant literature which spaas a number of fields. Us. Aedse will spend considerable time in assisting at this task with the help of modern information services an?L devices. She will also undertake to disseminate notices to the appropriate colldboratiny investigators. ns. ilease's salary is increased at a rate of 6% per year to cover merit and cost of living increases. Staff benefits are applied based on the folloving University projections: 17%, 9/7 3-8/74; -16.3X, Y/74-8/75; 19.3%, Y/75-8/76; 20.3%` Y/76-8/77; 21.3%, Y/77-8/78; and 22.3%. Y/78-8/79. Secretarial support for the individual Principal Investigators is provided in those respective subproject budgets. The bulget also covers estimated expenses for Visiting Committee honoraria and travel and expenses related to the planned annual symposia. 1, , PERSONNEL (Lisf all personnel enlale m omiect) TIME OR - EFFORT AMOUNT REQUESTED fOrnit cen,s) NAME (Lest, lirsl. hiHal) TITLE OF POSITION OIHRS. I TOTaiL Principal Investigator or Program Director 1 Redse, R. Program Director's 30 :. PROGRAM DIRECTOR'S OFFICE Secretary '. :. i 2 , CONSULTANT COSTS (Incfude Fees and TravelJ 3 I . SUPPLIES 5 . STAFF Q. DOMESTIC TRAVEL I. Sea rnducrfond b FORE,GN 6 . PATIENT COSTS (Sepsrs~e InpsUenl md Oulpalienl) 7 . ALTERATIONS ANO RENOVATIONS 6 -. 6 . OTHER EXPENSES (ILemire per insOucUona) Visiting Committee honoraria and travel and expenses for annual symposium. Communications: information services (e.g., abstracts, ASCA; MEDLINE). $5,000 9 Subtotal - Items 1 thru 8 t $8,414 10. TRAINEE EXPENSES (See Ins,mctions) PREOOCTORAL No. Proposed s - FOR o. ST,PENDS POSTDOCTORAL No. Proposed s OTHER (Specrfy) No. Proposed s TRAINING DEPENDENCY ALLOWANCE s GRANTS ., TOTAL STIPEND EXPENSES - s s ONLY b. TUITION AND FEES c. TRAINEE TRAVEL (Describe) s 11. Subtotal - Trainee Expenses c s I 1 I& TOTAL DIRECT COST (Add Sublolals. Ilsma 9 and 11, and enter on Page I) c $8,414 s ~PRIVILEGED COMMUNICATION SECTION It SUBSTITUTE THIS PAGE FOR DETAILED BUDGET PAGE . SUBSTITUTE PERIOD COVERED GRANT NUMBER DETAILED BUDGET FOR FIRST 12.MONTH PERIOD FRoM THROUGH :;j.; :.,.:>. :.:. .J' :.. .:.. ..:.:.. ( ..:. . . . . . ..:: :. . . . ,. s - s - s - s - Substltut. Budget Page S-72 For Forms PHS 398 and PtlS 2499-1 P cio GPO 933.751 SECTION II -PRIVILEGED COMMUNICATION PROGRAM DIRECTOR'S OFFICE BUDGET ESTIMATES FOR ALL DESCRIPTION 1ST PERIOD ISAME AS DE- TAILED BUDGET) PERSONNEL COSTS CONSULTANT COSTS (Include fees, travel, etc.1 3,414 EOUIPMENT I - SUPPLIES DOMESTIC TRAVEL FOREIGN PATIENT COSTS ALTERATIONS AND RENOVATIONS OTHER EXPENSES 5,000 TOTAL DIRECT COSTS 8,414 TOTAL FOR ENTIRE PROPOSED PROJECT I REMARKS: Justify all costs for the first year fc significant increases in any other category. If a I pege if needed.) fEARS OF SUPPORT REQUESTED FROM PUBLIC HEALTH SERVICE DIRECT COSTS ONLY (Omit Cents) ADDITIONAL YEARS SUPPORT REQUESTED llhis application only) 2ND YEAR 3RD YEAR 4TH YEAR 5Tti YEAR 6TH YEAR 7TH YEAR L I 3,656 3,656 3,908 3,908 4,177 4,177 4,464 4,464 ZNDYEAR 1 3RD YEAR t 4TH YEAR 1 5Tti YEAR 1 6TH YEAR 1 7TH YEAR 5,300 5,300 5,700 5,700 6,000 6,000 6,400 6,400 8,956 8,956 9,608 9,608 -0,177 -0,177 10,864 10,864 I ERIOD (Enter on Page f, Item 4) - s 48,019 ERIOD (Enter on Page f, Item 4) - I s 48,019 I r which the need may not be obvious. For future years, justify equipment costs, as well as any Cuffing annual increase in personnel caHs is requested. give percentage. Use continuation Budget explanation attached MS-398 Rev. 3-70 p-/y/ BGGREGATP BUDGET EYPLAUATIOR The following budget is the aggregate of the five individual subprogram budgets and the Program Director's Office budget. Individual budget items such as personnel appearing in more than one budget and supply items like Vhemicals, glassware, etc." have been combined to show only one such item each in this overall budget. In considerinq the percentages of personnel time committed to this project, it should be noted that.facnlty personnel, as distinct from staff, also receive a portion of their salary from the institution. This institutional salary support assists in covering fasulty efforts along research lines overlapping those in the present application. Therefore the faculty time percentages reflect contractual lower bounds on faculty commitment to this program, Salaries are uniforrlp increased at a rate of 6% per year to cover expected merit and cost of living increases. Staff benefits are applied based on the following University projections: 17%, 9/J3-0/74; 18.3%, Y/74-&/75; 19.35, g/75-8/76; 20.3%. 9/J&0/77; 21.3%. 9/77-b/78; 22.3X. 9/30-B/19. Budget items other than %quipmentH and "Patient Costs" are in general increased by 6% per year to cover inflation, except in specific instances noted in the individual budgets. n,. ,LL"LY JELIIUN II DETAILED BUDGET FOR Cann, H. Kretchmer, N. Herzenberg, L. of Genetics Cavalli-Sforza, L. of Genetics - :AkGREGATE PROGRAM BUDGET Bamett, C. Luzzatti, L. Tsuboi, K. nior Scientist Duffield, A. search Associate Pereira, W. search Associate Rindfleisch, T. search Associate Hulett, H. search Associate Open - Genetics search Associate I Open - Pediatrics Research Associate . CONSULTANT COSTS (Include Fees and Travel) see attached emtzs per matruct see attached s 21,500 Subtotal - Items 1 thru 8 . s 537,800 IO. TRAINEE EXPENSES (See instructions) PREDOCTORAL No. Proposed s FOR Q. STIPENDS POSTDOCTORAL No. Proposed s 0 TH ER (Specity) No. Proposed - s rRAlNlNG DEPENDENCY ALLOWANCE s GRANTS TOTAL STIPEND EXPENSES c s b. TUITION AND FEES ONLY S C. TRAINEE TRAVEL (Describ.,) S 11. Subtotal - Trainee Expenses *s 2. TOTAL DIRECT COST (Add Subtotala, Item= 9 and II. and enter on Page I) t s 537,800 Substltuto Budget Peg. S-72 P-63 For Forms PM 398 and PHS 2499-1 GPO 930.751 continued 1. PERSONNEL Open Open - Pediatrics Veizades, N. Steed, E. Tucker, R. Wegman, A. Waters, R. Sakaguchi, S. Van West, B. Makk, G. Open - Pediatrics Summons, R. Open - Genetics Open - Genetics Pearson, D. Wyche, M. Boswell, M. Open - Genetics Open- Genetics Open - Pediatrics Open - Pediatrics Open - Pediatrics Open - Pediatrics Jamtgaard, R. Redse, R. Allen, M. Murray, R. Meyering, P. Open - Pediatrics Harlow, W. Cusumano, M. Open - Genetics TITLE Research Assoc.-Stat. 20 Research Assoc. - Sot. Worker 50 Research Engineer 33 Research Engineer 33 Computer Programmer 75 Senior Res. Assist. 10 Research Assist. 100 Research Assist. 50 Research Assist. 100 Research Assist. 100 Research Assist. 50 Post.Doc. Fellow 100 Grad. Res. Assist. 50 Grad. Res. Assist. 50 Electronics Tech. 40 Lab. Tech. 100 Lab. Tech. 100 Engin. Tech. 50 Research Tech. 100 Interviewer 50 Interviewer 25 Statist. Clerk 50 Data Coder 65 Administrator 15 Secretary-Prog.Dir. 30 Secretary - IRL 25 Secretary - Peds. 50 Secretary - Gen. 10 Typist 100 Machinist 10 Laboratory Diener 50 Lab Glasswasher 25 X TIME continued 3. EQUIPMENT 4 Column Gas Chromatograph $14,400* Mini-computer System & Inst. Interface 59,900* Amino Acid Analyzer 32,500* Event Counter 2,700* Digital Voltmeter 900* Carbon Dioxide Incubator 2,500* Laminar Flow Hood 1,000* Fluorescence Microscope 9 ,ooo* Eppendorf Microfuge 500* Buchler Power Supply 600* Constant Current Power Supply 1,000* Slab Gel Electrophoresis 300* Column Acrylamide Gel Electrophoresis 200* Destainer 200* Tape Recorder 500* Total Equipment $126,200* 4. SUPPLIES Chemicals, glassware & lab. apparatus GC Supplies (Columns, Phases, etc.) Dry ice & liquid nitrogen Data recording media (GC/MS, Calcomp, etc.) Mini-computer supplies (start-up 6, continuing) Mass spectrometer repair parts & supplies Electronic parts & supplies Amino acid analyzer supplies Radioactive tracers Expendable supplies (photo. plates, etc.) $18,000 1,100 500 1,800 1,600 2,300 1,900 2,000 5,000 1,500 Total Supplies $35,700 8. OTHER Visiting committee honoraria & expenses for annual symposium Office supplies, telephone, repor.,postage, publication costs, etc. Mini-computer maintenance Freight on capital equipment Central computer usage & terminal rental $5,000 6,200 6,000 500 3,800 Total Other $21,500 II - AGGREGATE BUDGET .",I",. I, -, lll"lLL"LV Y"I.II.I"I.IVr.lIV.. BUDGET ESTIMATES FOR ALL YEARS OF SUPPORT REQUESTED FROM PUBLIC HEALTH SERVICE DIRECT COSTS ONLY (Omit Cents) OESCRIPTION PERSONNEL COSTS CONSULTANT COSTS llnclude fees, travel, etc.) 1ST PERIOD ISAME AS DE- ADDITIONAL YEARS SUPPORT REQUESTED (Thisapplication only1 TAILED BUOGET) 2ND YEAR 3RD YEAR 47-H YEAR 5TH YEAR 6TH YEAR 7TH YEAR $345,600 370,068 431,528 461,243 492,966 EOUIPMENT 126,200* 23,000* 18,100* 5,300* 6,000* SUPPLIES 35,700 37,700 42,400 44,800 47,700 DOMESTIC 4,000 4,700 4,900 4,900 5,400 TRAVEL FOREIGN 3,000 5,500 5,500 500 500 PATIENT COSTS ALTERATIONS AN0 RENOVATIONS 1,800 OTHER EXPENSES 21,500 24,900 26,400 27,800 29,700 TOTAL DIRECT COSTS 537,800 465,868 528,828 544,543 582,266 TOTAL FOR ENTIRE PROPOSED PROJECT PERIOD (Enteron Page 1, item 4) - t 2,659,305 REMARKS: Justify ail costs for the first year for which the need may nor be obvious For future years, justify equipmenr costs, as well as any significant increases in any other category. If e recurring annual increase in personnel msts is requested, give percentage. (Use continuation page if needed, I See individual subprojects for budget explanations SECTION VIII Concluding Remarks Dr. Lederberg p-k-7 Continuation page CONCLUDING REMARKS (Joshua Lederberg) The overt justification for the proposed Center is, of course, the collective value of the well-defined research projects that will make up its day to day work. However, its most important utility in the long run may be a new institutional arrangement that will generate now unforeseen lines of investigation. Molecular biology as a basic science has leaped far beyond its practical application to human problems. The more intimate intellectual association of gifted scientists and clinicians envisaged here, the development of an active interface among workers who spend much of their time still either in basic laboratory work or in the clinic, is the only possible way of advancing these applications. Speaking more personally, I can point to a number of anticipations from my own research career that might have been explored more aggressively had I then pressed them in an interdisciplinary context. My early work (with Tatum, Zinder and others) on genetic recombination and on viral transduction in bacteria was foreseen years ago as laying a groundwork for the development of a genetics of somatic cells and of means of importing reparative genetic information ("genetic engineering"). Subsequently, I tried to stress that the new molecular genetics would overtake other lines of concern about genetic impairment, and improvement, and that ethical issues would loom as large as technological opportunLties in public policy debates. While public reactions to these potentialities are in some respect overdrawn, geneticists obviously must inculcate and exhibit great sensitivit! to the ethical issues of genetic intervention and how they are perceived by the public (1). Although much of my own laboratory research might go under the heading of genetic engineering of bacteria (2) I have been persuaded that prenatal diagnosis represents the most important practical avenue of genetic therapy or rather pre- emption of disease. For this reason, the application of automated technologies (growing in large measure out of NASA-supported work) is emphasized in this application. What is more difficult to foresee is the intensive development of the whole repertoire of tools of molecular genetics. Somatic cell genetics is well launched; but we will surely also have to learn how to use specific DNA replication, hybridization, transcription, and translation, i.e. the full range of gene action vitro, in to solve pressing diagnostic problems. Laboratory research shares a place with other interests. I can summarize these under the heading of a concern for the overall process of science, and its application for human benefit. These meta-scientific interests have been expressed in the public arena, as in efforts to enhance public understanding of science and provision for its support. More recently, I have been more involved in efforts to enhance the infrastructure of science, with work on new instrumentation coupled with feeble steps towards the use of computers for scientific "intelligence" (3). The Genetics Research Center is a plan to bring all of these themes to a focus 'on an area most likely to advance genetic knowledge -- my fundamental scientific base -- and to generate tangible health benefits for the mutual advantage of both disciplines, and to satisfy the public's motives for continued investment in the health sciences. PM-398 Rev. 2-69 Page Continuation page Bibliography 1. Lederberg, J.: in Challenging Biological Problems, Oxford LJ. Press, N.Y. 1972, p. 7 (J.A. Behnke, ed.) "Biological Innovation and Genetic Intervention". 2. Sgaramella, V.: Proc. Nat. Acad. Sci. 69:3389, 1972. "Enzymic Oligomerization of BacterGphage P22 DNA and of Linear Simian Virus 40 DNA." 3. Lederberg, J.: in Biochemical Applications of Mass Spectrometry, John Wiley & Sons, N.Y. 1972, p. 193 (G.R. Waller, ed.) "Use of a Computer to Identify Unknown Compounds: The Automation of Scientific Inference." HS-398 BY. 2.49 Page 0Po : ,889 0 - 350-360 P-/Q SECTION IX Professional Personnel P-/60 KEY PERSONNEL AND BIOCRAPBICAL MATERIAL Joshua Lederberg, Ph.D. - Principal Investigator, Professor and Chairman, Department of Genetics. Professor Lederberg will serve as the Principal Investigator for the overall program, and for part 1, automated screening methods (SC/RS) for inborn errors of metabolism. In addition, Professor Lederbery aaintains active research programs in genetic recombination in bacteria; mechanisms of D#A replication and reunion; interactions of environmental mutagens with DYA; and computer emulation of human cognitive processes. Reward Cann, M.D. - Associata Program Director, Associate Professor of Pediatrics. Dr. Cann vi11 assuae responsibility for coordinating laboratory research with clinical applications within the program. This will involve screening of patients for their suitability of participation in the relevant reseach projects, obtaining appropriate patients for projects, informing patients about the research projects and obtaining their consent for participation, insuring that proper evaluation, care and counseling are provided to patients and ianilies involved in these projects and planning for the application of the research findings to patieat care. Dr. Cann will be directly involved with research projects on genetic markers in amniotic fluid, separation of fetal cells from the maternal circulation, the impact of genetic counseling on family decisions and screening and detection of inborn errors of metabolisn. Dr. Cann's research activities pertain to human polymorphisms and to somatic cell genetics and his clinical work involves him with hereditary illness and genetic counseling. He has set up and directs a laboratory for typing for various polymorphisms of erythrocgte antigens and enzymes and serum proteins. He has initiated an investigative program for applying somatic cell hybridization to prenatal diagnosis of hereditary disorders. L.L. Cavalli-Sforza, H.D. - Professor of Genetics 'The investigations of Professor Cavalli-Sforza have long contributed and continue to contribute important information on human population genetics. He has pioneered in applying demography to studies of population and genetic structure of man. Particularly pertinent to this program is his interest and work on variation among populations in the frequency of inherited disorders and their determining genes, Professor Gaoalli-Sforza als3 directs a laboratory program oriented toward the detection of specific binding proteins in P-/6/ -2- blood. The detection of genetic polymorphisms of these proteins will be carried out by Professor Cavalli-Sforza as a research activity of this Genetics Center. Norman Itcetchmer, H-D., Ph.D. - Professor of Pediatrics. Professor Sretchmec will participate in the research project dealing with screening and detection of inborn errors of metabolism. De is well known for his research and clinical uork in various inborn errors of metabolism. One of Professor Kretchmer's main research interests presently deals uith lactase intolerance in man. His research program also involves the developmental biochemistry of the urea cycle and the pyriaidine biosynthetic pathuay in eukaryote cells and tissues. He has recently relinquished the chairmanship of the faculty committee for teaching the human biology curriculum at Stanford University. Prom 3959-3969 Professor Kretchner was Chairman of the Department of Pediatrics, Leonard Herzenberg, Ph.D. - Professor of Genetics. Professor Heczenberg directs an active research program in immunogenetics with special emphasis on genetic and structural studies of mouse immunoglobulins and on the mechanism and control of antibody synthesis. The development under his direction of instrumentation for separating cells by means of fluorescent sensing has provided potential methodology for separating fetal cells from the maternal bloodstream. Professor Herzenberg vi11 participate in the project which will apply this methodology to antenatal diagnosis of genetic disorders. Clifford Barnett, Ph.D. - Professor of Anthropology and Associate Professor of Pediatrics. Professor Barnett has worked in medical anthropology for 10 years. His research activities have dealt with cultural and genetic aspects of congenital dislocation of the hips, fertility and birth spacing of partially acculturated Guatenalan Indians, and mother and infant interactions in reference to premature infants. He has designed and will direct the research project on the impact of genetic counseling on family decisions and behavior. Alan Duffield, Ph.D. - Research Associate in Genetics. Dr. Duffield is an organic chemist who has carried out fundamental studies in mass spectrometry of organic compounds. He is presently directing research involving the development of a multicomponent, automated system for analysis of biolorjic substances. He shall apply this system, which involves d gas liquid chromatograph, a mass spectrometer and computer -3- facilities to screening, detecting, and studying inborn metabolic errors in various body fluids and tissues. Kenneth Tsuboi, Ph.D. - Senior Scientist, Department of Pediatrics. Dr. Tsuboi*s research interests have included the physiological chemistry and enzymology of erythrocyte interaediate metabolism and more recently, the biochemical genetics of various erythrocyte enzymes. He is also working oti the enzyaological characteristics of various priaate cell culture lines. In this prograa he vi11 participate in the project on linkage and prenatal diagnosis of inherited disorders. In particular, he will uork in the detection of polyaorphic enzyme markers in cultured amniotic fluid cells. Luigi Luzzatti, M.D. - Professor of Pediatrics. Professor Luzzatti is the director of the clinical cytogenetics laboratory and of the Birth Defects Clinics in the Department of Pediatrics. His clinical activities which are pertinent to this program, include comprehensive care for patients (and their families) with birth defects, genetic counseling and screening amniotic fluid samples {usually from women over 35 years of age) for chroaosomal abnormalities. He will participate in the project which investigates the impact of genetic counseling on family decisions. Herbert Schwartz, tl,D. - Professor of Pediatrics. Professor Schwartz's research interests in hemoglobin synthesis, structures and function in health and disease, and their application to the prenatal detection of hereditary disorders involving hemoglobin, e.g. sickle cell anemia crnd thalassemia, will be incorporated into the program on exploring the maternal bloodstream for fetal cells. Dr. Schwartz will COlldbOCdte with Drs. Herzenherg and Cann in preliminary studies of fetal erythrocvtes separated from maternal blood. Dr. Schwartz directs the hematology service of the Department of Pediatrics. Other members of the Departaent of Genetics who will not participate directly in this program but with whom ve interact daily are Dr. Eric Shooter (Professor) dnd Dr. A.T. cidnesan (Associate Professor). Frofessor Shooter directs research into the structure and mechanism of action of the nerve growth factor protein. Dr. Ganesan is investigating the genetic control of chromosome replication in B. subtilis. the chemical basis of DEiA replication, the mechanism of genetic recombination during Dt~b mediated transformation and the role of the nuclear membrane in chromosome replication in mammalian cells. -4- The clinical and research interests of Professor Irving Schulman, Chaiman of the Department of Pediatrics, are devoted to various defects of coagulation, including inherited disorders. Other members of the Departaent of Pediatrics whose clinical and research activities relate to this prograta are Dr. Phillip Sunshine (Associate Professor of Pediatrics) and Dr. John Johnson (Assistant Professor of Pediatrics), Director and Associate Director of the Revborn and Premature Infant Nurseries, respectively. Dr. Sunshine and Dr. Johnson are together investigating the metabolic consequences of neonatal ornithine transcarbamylase deficiency and detection of heterozygotes for the gene which determines this disorder. The research activities of Dr. Merton Bernfield, Associate Professor of Pediatrics, are designed to assess the aorphogenetic behavior and cell surface properties of human cells and to utilize these assessments in studies of cells derived from individuals with birth defects, Dr. John Gribble, Assistant Qrofessor of Pediatrics, is a pediatric hematologist whose research activities have dealt with in vitro biosynthesis of hetaoglobin and biochemical and physiological aspects of pinniped hemoglobins. Dr. Gribble's clinic& activities involve evaluation and treatment of patients with hereditary disorders of blood coagulation, including genetic counseling ot their faailies. The clinical and research activities of Dr. R.O. Christiansen (Assistant Professor of Pediatrics and Director of the Pediatric Pletabolic and Endocrine Service) an3 Dr. Judith Koehler (Assistant Professor of Pediatrics and Director of Pediatric Neurology) are quite relevant to the Genetics Center program. Even though the research activities of all of these individuals will not be supported by this grant, we are including bibliographic sketches on each of them because of their relevance to the overall Genetics Center program. Within one or-tro years after the activation of this program we anticipate the ditect participation of one or @ore colleagues from the Department of Obstetrics and Gynecology. A search for chairnan of this department is presently under uay, and ue expect that this individual will bring competence to Stanford in fetal physiology and fetal monitoring. We look forward to interacting in this program with our obstetrical colleagues in various projects pertaining to antenatal detection of genetic disorders and selective abortion. Principal Investigator: (unless otherwise noted) Research Support Summary for Departments of Genetics and Pediatrics Relevant to Genetics Research Center Program Director: J. Lederberg Associate Program Director: H. Cann HOWARD CANN Associate Professor of Pediatrics Department of Pediatrics Grant Title Current Year Total Award Grant Term Budgeted % time 1) NIH:HL-15008 Pre-Natal Detection of Sickle Cell Anemia $ 38,717 LUCA CAVALLI-SFORZA Professor of Genetics Department of Genetics 1) AEC:AT-04-3-326 Mutation Rates and Mutational Loads in 32,000 Man 2) NIH:NS-10711 Genetic Study of Metabolism of Neural Transmitters 65,000 3) NIHiGM-20467 Gene Diffusion, Natural Selection and Drift in Man 47,587 ADAYAPALAM GANESAN Associate Professor of Genetics Department of Genetics 1) NIH:GM50,199 Development Award-Research Career Program 21,433 Chromosome Replication, Recombination and Cell Division 2) NIH:GM14108 DNA Synthesis and Genetic Recombination 34,902 $118,000 g/72-8/75 32,000 10/72-g/73 10% (Renewal pending) 207,000 g/72-8/75 20% 147,351 5/73-4/76 20% Support recommended for 2 additional years ending 12/31/75, the amounts to be determined annually. 211,996 6/72-5177 50% LEONARD HERZENBERG Professor of Genetics Department of Genetics 1) NIH:GM-17367 2) NIH:AI-08917 3) NIH:CA-04681 4) NIH:HD-01287 Automated Cell Sorting - Clinical and Biological Uses Genetics of Immunoglobins Genetic Studies of Mammalian Cells Fetal-Maternal Immunological Inter- actions JOHN D. JOHNSON Assistant Professor Department of Pediatrics 1) United Cerebral Developmental Aspects of Heme Palsy Protein Catabolism NORMAN KRETCHMER Professor of Pediatrics Department of Pediatrics 1) NIH:RR-00081 Clinical Research Center for Premature Infants 2) NIH:HD-02147 Biochemical Studies of Development 3) National Found. CRBS-252 Growth and Differentiation of the Placenta 4) NIH:HD-CC047 Human Development and Pediatrics Train- ing Grant 5) NllH:HD-00391 b *; P Regulatim of Enzyme Action During Development 160,802 585,977 l/73-12/75 15% 52,774 246,386 5/69-4/ 74 20% 80,994 424,981 9172-8177 30% 38,198 222,897 5/73-4178 10% 28,772 418,532 206,593 23,293 90,588 42,289 53,322 7/71-6/73 1,974,008 10/69-9/74 5% (Renewal pending) 1,309,278 6/66-5/74 15% (Renewal pending) 23,293 7172-16174 15% 448,936 7170-6 J74 10% 183,822 g/68-8/73 15% (Renewal pending) NORMAN KRETCHMER (continued) 5) NIH:CAl4917 Pyrimidine Synthesis and Cellular Proliferation in Colon 7) Educational Found. Epidemiology, Etiology, and of America Physiology of Diarrhea in the American Indian JOSHUA LEDERBERG Professor and Chairman Department of Genetics 42,689 45,156 1) NASA:NGR-05-020 Cytochemical Studies of Planetary 180,000 Micro-organisms 2) NIH:AI-05160 Genetics of Bacteria 60,000 3) NIH:RR-00612 Resource Related Research - Computers 194,408 and Chemistry (E. Feigenbaum, Principal Investigator; J. Lederberg and C. Djerassi, Co-Investigators) 4) NIH:RR-00785 Stanford University Medical Experimental Computer Facility (SUMEX) 765,573 5) NIH:GM0295 Training Grant in Genetics i43,964 GILDA LOEW Research Associate Department of Genetics 1) NSF:GB17980 Quantum Chemical Investigations of Heme Proteins and Ferredoxins 2 2) NIH:DAOO770 w Quantum Chemical Szudfec ef Opiate Narcotics 130,737 6/73/5/76 20% 92,915 7/73-6/75 5% (Renewal pending) 3,800,OOO g/60-8/73 (Future support dubious) 11% 280,000 g/68-8/73 15% (Renewal pending) 6i5,OOO 5/71-4174 0% (Renewal pending) 4,246,621 8/73-7J78 (pending) 20% 755,550 7/69-6J74 15% 34,799 34,799 2/72-l/74 50% (Renewal pending) 57,557 184 * 139 g/73-8/76 (Pending) 50% ERIC M. SHOOTER Professor of Genetics Department of Genetics 1) NIH:NS04270 Molecular Neurobiology - Proteins in the Nervous System 2) NSF:GB31982 Structure and Mechanism of Action of the Nerve Growth Factor KENNETH TSUBOI Senior Scientist Department of Pediatrics 1) NIH: Contract- Biochemical Parameters of Primate Div. of Biol. Cell Cultures Standards 84,558 404,979 12/70-11/75 40% 60,000 60,000 l/72-12/73 20% 23,598 47,196 6/72-5 J74 ROT. j. lo*:?+g'r: . , SECTION II - PRI\`ILZ.;ED CO'.".:`J:!!CATIO'J ppnt+Y.`.' -1 ,!-..,A a. -- BIOGF:RPl-IICAL SKETCH lGivc the following information for allprcfcGcw~l personnel listed on page 3, beginning with the Principal /nvA-tigator. Use continwtion p.?gc' a~:d fol!o;v rhe same general format for each pe,son.j --- NAME TITLE BIRTHDATE IYo., Dab/, Yr.) Clifford R. Barnett Associate Professor of Pediatrics Professor of Anthropologv 8/17/29 PLACE OF BIRTH (City, State, Country] --- FCiESENT NATIONALITY I/f non-U.S cifizen, in6'icate kind of visa end expiration date) SEX New York, New York U.S. Citizen m Male l-J Fenlol;: EDUCAl ION (Begin with baccalaureate training and include postdoctoral) INSTITUTION AND LOCATION DEGREE YEA? SCIENTIFIC CONFERRED FIELD The City College, New York, New York B.S.S. Cornell University, Ithaca, New York M.A. Cornell University, Ithaca, New York Ph.D. 1950 Anthropology-Psychol$;: 1951 Anthropology 1960 Anthropology HONORS Executive Board, Society for Applied Anthropology 1967-70, Vice-President, 1971-72, President 1972-73, Past President 1973-74; Executive Board, Society for Medical Anthropology 1971-73; Chairman, Committee on Ethics, American Anthropological Association, -.- Cultural Anthropology RESEARCH SUPPORT I.% instructions) I Investigator Research Grant: Genetic Studies in the Lake Atitlan Basin, Guatemala, GM 15593; $13,133 for the current year; $300,000 for the six year period 1967 - 1973; National Institutes of Health. Howard Pi. Cann, M.D. - principal investigator. RESEARCH kr:D/OR PROFESSIONAL EXPERIEF!CE IStar:im v/;thpresentposition, list traininqandexperiencerelcvant toarea Sfprojjcct Lasta:; Or mat representative publications Do not exceed 3 pages for each individL:al. ) 1964- Associate to Full Professor, Anthropology; Associate Professor, Pediatrics, Stan. :Jr,;1 1964-1970 Assistant Director to Acting Director, Program in Medicine and the Behavioral Sciences, Stanford University. 1962-1964 Research Associate to Associate Project Director, Navajo-Cornell Field Health Research Project, Dept. of Public Health, Cornell Univ. Xedical College. 1961-1963 Resident Anthropologist, Navajo-Cornell Field Health. Research Project at ?!z-q Fzrzs, Arizona; Resident in ?rofessional Practice, Russell Sage Foundation. 1955-1961 Senior Research Associate to Team Chairman, Foreign Areas Studies Division of Specid Oper.ations Research Office of the American University, Washington, D.C. Publications (selected) "Untreated Congenital Hip Disease: A Study of the Epidemiology, Natural History and Social Aspects of the Disease in a Navajo Population". With D. Rabin, W. Arnold, R. Freiberger and G. Brooks. Supplement, @erican Journal of Public Health 55, 2. 1965. "Genetics of Diego Blood Groups in Guatemalan Indians: Use of Antiserums to Diego a and Diego b Antigens". With Howard M:Cann and Betty Van West. Science, 162:1391, 1968. "Neonatal Separation: The Maternal Side of Interactional Deprivation". With P.H. Leiderman, R. Grobstein, M. Klaus. Pediatrics 45:197, 1970. - (publications continued) --- RI-IS-398 Rev. 370 P-q Publications (continued) "Child Spacing in a Highland Guatemala Community". With Jean Jackson and Howard M. Cann. In Culture and Population: A Collection of Current Studies, Polgar, (Ed.). Monograph 9, Carolina Population Center, Chapel Hill, 1971. pp. 139-148. "Health Care Experiment at Many Farms". With W. McDermott and K. Deuschle. Science 175:4017, January 7, 1972, pp. 23-31. "The Effects of Denial of Early Mother-Infant Interaction on Natemal Self-Confidence". With M. Seashore, A. Leifer and P. Leiderman. Child Development 43:1203, 1972. - "Effects of Mother-Infant Separation on Maternal Attachment Behavior". With A. Leifer, P. Leiderman and J. Williams. In Press, Personality and Social Psychology. . . . .*<. a._\"-" "C. - 4I\I"`-.I ICS,l -- _* . BIOGRAPi-!ICAL S1:E-l ~4 (Give the folkwing infurmSiou fgr al/prof~~;rion;rlp~rsonn~/ listed on pqe 3, bsinning with the Princ!~al I,qvc f c.- f: t. Clltnic, Dcpartmeili: of Yediatri.cs, Stanford University ScliGol of 1.;ediclx; Stanford, California 1967-1970 Assistact Professor, Department of Pediatrics, Stanford Ur;iversity StkxP,. p!edicine; Stanford, California J 96-i-1367 Chief ksident, T~e~~arixir,~~t of Pcdbtrics, Stanford Medixal Center, Sta:;f>:- f96S-1966 Research Invcsjgaxr, Nstional 1Ixtitut.e of Child llcalth and 1k:;l:x Dr~;.~~:z: with Dr. Cliffo?:d Grcbstcin, i~i the D~p2rttncrit of fiiology, University 0; 19634965 California, San Diego; La Jolla, California Research Associate, btional Heart Institute, with Dr. brshall Kire?bcr:;:; Bethesda, 'Xarylznc? 1962-1963 Assistilnt Resident, 1'!e~?:~rtx.:rznt cf Padiatrlcs, Xew York ~losp~tril-Corni~3.!. I'.. Center; Ilcw York, 33: i'urk 1961-1962 Rotating Internship, Research anh Education Hospitals, Unj.versify of I1El: Chicago, Illinois P-171 i7iTxx-- -u___--" . . -o--e- --_-.-_ . . . -. - . llev. 3-m 51 BioBrhical. Sketch of Dr. Merton Bernfield -- 1'8;~ Two, continuation of page one PLblications -... -- --- Bernfield, M.R. and Nirenberg, M.W.: RUA Codewords and Protein Synthesis. Iv, The nuclcotide sequences of multiple codewords for phcnylaianine, serine, leucine, and proline. Science 147, 479-484, 1965. Bernfield, M.R. and Rottmzn, F.M.: Ribonuclcase and Oligoribonucleotide Synthesis. III. Oligonucleotide synthesis with 5'-substituted uridine 2', 3'-cyclic phosphates, J. Biol. Chem. 242 4134-4143, ..-' 1967. Haenpaa, P.R., and Eernfield, N.R.: Quantitative Variation in Serine Trcnsfc;l Ribonuclcic Acid durir;SEstrogen-Induced Phosphoprotein Synthesis in Rooster Liver. Biochemistry g, 4926-4935, 1969. Bernfield', K. R. : Collagen Synthesis during Epitheliomesenchymal Inter- .actions. Develop. Biol. 22, 213-231, 1970. Eernfield, >I.R., and Vessells, N.K.: Intra: and Extracellular Control of Epithelial Korphogenesis. Develop. Biol. Supplentnt 4-, 195-249, 1970. Bernfield, M.R., Banerjee, S.D., and Cohn, R.H.: Dependence of ,Salivnry Epithelial 1lorphology and Eranching Morphogencsis upon Acid Huco- polysaccharide:Protein (Proteoglycan) at the Epithelial Surface. J. Cell. Biol., 2, 674-689, 1972. 52 P-/7& -_.____ ------ ----- - ----------_-_-_ _ .__ ._-__ -.-. -_--. NAME -:. - -.I Howard M. Cann, M.D. PLACE OF BIRTH (City, State, Country] `Associate Professor of Pediatri s March 3i, 12;: PRESENT NATIONALITY (If non-US citizen, SEX indicj te kind of visa and expiration da tel Chicago, Illinois U.S. Citizen I D hlzie 0 Female EDUCATION (Zqin cvi:h baccalaurcste training and include poztdoarorall INSTITUTION AND LOCATION I DEGREE I YEAR CONFERRED I SCIENTIFIC FIELD University of Colorado, Colorado B.A. 1950 Chemistry, Gun Laude (General Studies) University of Colorado School of Medicine, M.D. Colorado. 1954 . , HoNDRS 1971 - 1972 National Institutes of Health Special Research Fellowship, Genetics Laboratory, Department of Biochemistry, University of Oxford, Englad. 1966 - 1971 Scholar in Academia Medicine, The John and Mary R. Markle Foundation. I MAJOR RESEARCH INTEREST ROLE IN PROPOSED PROJECT k-M "`I - b +---+ Human Somatic Cell Genetics ASSOCIATE INVESTIGATOR RESEARCH SUPPORT (See instructions) Research Grant: `Genetic Studies in the Lake Atitlan Basin, Guatemala, GM 15593; $28,260. for the current year; $300,000. for the six year period 1967 - 1973; 35% effort; National Institutes of Health. National Institutes of Health Special Research Fello\Iship for sabbatical leave at the University of Oxford (Professor-Walter F. Bodmer), Genetic Control of ?ucan Transa?2x:?:i.: Antigens, l-F03, HD51401-01; $12,371. for one year, September 1, 1971 - August 31, 1'172,- 100% effort. RESEARCH AND,`GR PROFESSIONAL EXPERIENCE /S:ar:irrg v.:ithp;esentposition, /i:t trainingandexperiancer~;rvant toarc's cf,?rl;,-!l; i.;: c : Orfmxt representative publrcationr Do not ex cecd 3 pqes for each ino. didual.) September 1, 1971 - 1970 - 1964 - 1970 1963 -` 1964 1962 - 1963 1960 - 1962 1957 - 1960 1956 - 1957 1955 - 195G 1954 - 1955 August 31, 1972 National Institutes of Health Special Research Fellowship, Gene tics Laboratory , Department of 3iochenistry, ..H+?gr,si ty of Osf ord , England. Associate Professor, Department-of ,Pediafrics ti,Stanford Un,iv.ergity School of Medicine. .W' ) Assistant Professor of Pediatrics, Stanford University School of Medicine Instructor, Department of Pediatrics, Stanford University School cf . Medicine. National Science .Foundation Senior Postdoctoral Fellow, institute of Genetics, University of Pavia, Pavia, Italy. Postdoctoral Fellosr, Department of Genetics, Stanford University School of Medicine, Palo Alto, California. United States Pub1j.c Health Service, Washington, D.C., Director, National Clearinghouse for Poison Control Centers. Draft Classif ication: 4A. Assistant Resident and Senior Resident in Pediatrics, Stanford University Hospital, San Francisco, California. Assistant Resident in Pediatrics, Cniversity of Colorado >Iedica1 Center, Denver, Colorado. Rotating Intern at San Francisco Hospital, University of California Service, San Francisco, California. RHS-393 Rev. 3-m Page 17 -- b/7.3 'Committees and Consultant 1968 - Committee on Drugs, American Academy of Pediatrics. 1965 - 1968 Genetic Consultant, Hereditary Defects Unit, California State Department of Public Health. 1965 - Genetic Consultant, Congenital Malformations Branch, Epideniolcgy Section, Dental Health Center, U.S. Public Health Service. Societies and Organizations American Academy of Pediatrics American Association of Poison Control Centers American Federation for Clinical ,Research American Public Health Association, Epidemiology Section American Society of Human Genetics Western Society for Pediatric Research Specialty Board Certification 19tio American Board of Pediatrics State and National Certification L__- Diplomate of National Board of Medical Examiners Licensed to practice medicine in California and Colorado. _ BIBLIOGR!?HY 1. Cann, H.Ff. and Herzenberg, L.A.: In Vitro Studies of Mammalian Somatic Cell -- Variation. I. Detection of H-2 i?henotype in Cultured Mouse Cell Lines, J. 2. Cann, H.hf. and Herzenberg, L.A.: In Vitro Studies of Xaznalian Somatic Call -- Variation. II. Isoimmune Cytotoxicity !*Jith a Cultured Xouse Lycphoma and Selection of'Resistant Variants.'. J;,, Expi-.Med. .2..=:267,- i963...:. . . ~... ., ,-,. ,_,. :' 3. Segregation Analysis o) Juvenile Diabetes I - Berrai, I. and Cann, H.M.: J. Med. Genet. 2:8, 1965. Xellitus, / I 4. Cann, H.M.: Computer Analysis in Human Genetics: *Segregation Anal::sis and 1 Demographic Genetics, Ann. New York Acad. Sci. 126:728, 1965. 5. Barrai, I., Cann, H.M., Cavalli-Sforza, L.L. and DeNicola, P.: The Effect of 4 Parental Age on Rates of Mutation for Hemophilia and Evidence for Differing Mutation Rates for Hemophilia A and B, American J. Hum. Genet. g:175, 1968. 6. Cann, H.M. and Cavalli-Sforza, L.L.: Effects of Grandparental and Parental Age, Birth Order and Geographic Variation on the Sex Ratio of Liveborn and Stillborn Infants, American J. Hum. &net., 20:381, 19G8. - 7. Cann, H.Ef. : Principlks of HLnan Inl-eritanca, in Human Genetics, Birth D~fectd, Original Article Series, The National Foundation, Vol. IV, November, 1958. Qo9e 18 ?-/7f/ __ - *u.s.cPo. 1910 - l/J-cqi 8. 9. 10. 11. 12. 13. 14. BIBLIOGRAPHY (Continued) Cann, H.&l., Van West, B., and .3arnctt, C.R.: Genetics of Diego Blood Grox?s in. Guatemalan Indians: Use of Antiserums to Dia and Dib Antigens, Science, 162: 1391, 1968. Greenstein, R.M., Harris, D.J., Luzzatti, L.L. and Cann, H.M.: Cytogenetic Analysis of a Boy with the XXXY Syndrome: Origin of the X-Chromosomes; Pediatrics, 45:677, 1970. - Dungy, C.I., Aptekar, R.G. and Cann, H.M.: Herediatry Hydrometrocolijos with Polydactyly in Infancy. Pediatrics, 47:138, 1971. Barnett, C.R., Jackson, J. ind Cann, H.M.: Childspacing in a Highland Guatemala Community, in Culture and Population: A Collection of Current Studies, Polgar, S. (ea.), Carolina Population Center, Univ. North Carolina, Chapel Hill, 1971. Watson, W. and Cann, H.M.: Genetic Counseling in Dermatology. Pediat. Clin. N. Ame,rica 5:757, 1971. Watson, W., Cann, H.M., Farber, E. and Nall, I.,.: The Genetics of Psoriasis. Arch. Dermatol., In Press. -- - Bodmer, W.F., Bodmer, J.G., Coukell, A.,.Cann, H.M. and Van Widest, B.: SO"2 Further Data on the Joint Segregation of HL-A and Haptoglobin. Ann. Hum. Gene:. (London), In Press. Book Review Carter, C.O.: Human Heredity, Cytogenetics 1:55, 1963. HONORS (Continued from Page 17) 1964 Society for Pediatric Research 1963 - 1968 Research Career Development Award, National Institutes of Health, U.S. Public Health Service 1962 - 1963 National Science Foundation Senior Postdoctoral Fellowship. CURRICULUM VITAE NAME: L. L. Cavalli-Sforza BIRTHDATE: January 25, 1922 NATIOXALITY: Xtalian Married, four children ACADEXIC HISTORY: University of Pavia, Italy Cambridge University, U.K. Universities of Pavia and Parma, Itily EMPLOYXENT RECORD: 1943-44 Intern of Genetics Department, University of Pavia (then located at Istituto Italian0 di Idrobilogia, fc.rSar-ia, Italy) 1944-45 1945-47 Intern and resident at Vatbania Hospital, Italy Assistant in research, Istituto, Sieroterapico Xilanese, Milan 1948 Scholarship in genetic statistics, John Innes Horticultural Institution, London (U.K.) 1948-50 Research Fellow and Assistant in Research, Genetics De- partment, Cambridge University (U.K.) 1950-57 Director of Research in Microbiology, Istituto Siero- terapico Mlanese (Milan, Italy) 1951-54 Lecturer in Genetics and in Statistics, Science Faculty (part-time), University of Parma and L'niversity of Pavia, Italy 1958-62 1962-70 Professor of Genetics, University of Parma, Italy Profeisor of Genetics, and Director of Istituto di Genetica, University of Pavia (Italy) 1970- Professor of Genetics, Stanford University, Palo Alto, Present California TITLE: Professor of Genetics PLACE OF BIRTH: Genoa, Italy SEX: Male M.D., 1944 iA ., 1950 Libera Docenza, 1952 (!Ecrobiolog:;1 1952 (Genetics) 1960 (Statistics) P-/76 Curriculum Vitae - 2 L. L. Cavalli-Sforza Earlier Sojourns in the United States: 1954 Rockefeller Fellow (six months) at Genetics Department, University of Wisconsin, Madison, Wise. 1958 Research Associate tit University of Wisconsin 1960 1962 Visiting Professor at Genetics Department, Stanford University (three months) * Visiting Professor at Genetics Department and Statistics Department, Stanford University (one month) 1964-65 Visiting Professor at Department of Biology, Harvard University (three months) 1968-69 VisitTng Professor at Genetics Department, Stanford University (one year) Expeditions to Africa in the Fall-Winter periods of 1965-66, 1966-67, 1967- 68, 1968-69, 1969-70, 1970-71, conducting research in the following countries: Central African Republic, CaEerocn, Zaire and Ethiopia ACADEMIC HONORS: President, Biometric Society, 1967-68 Vice President, International Congress of Genetics, Tokyo, 1968 Foreign Honorary Member of American Academy of Arts and Sciences, 1969 ' Royal Anthropological Institute of Great Britain and Ireland, Huxley Award in Anthropology, 1972 MILITARY SERVICE: Medical Officer in the Italian Army, 1947-48 MAJOR RESEARCH INTERESTS: Bacterial' genetics; presently, human population genetics and allied fields. RESEARCH ACTIVITY: Apart from early work in the fields of immunology and Drosophila popu- lation genetics, research activity during the first fifteen years was concen- trated mostly on bacterial genetics. Among results of the analysis of sex and recombination in E. coli strains: the finding of the first coli mutant with a high recombination frequency (Hfr), of Hfr linkage with chromosome markers, and of the infective transmission of mating capacity (F episomes). Among results on the analysis of drug resistance in bacteria: a quantitative p-177 Curriculum Vitae - 3 L. L. Cavalli-Sforza technique for sib selection, demonstrating the spontaneous origin of drug resistant mutants; the analysis by crossing of polygenic inheritance for chlor- amphenicol resistance; streptomycin resistance as a genetic modifier. Some of this,work was in collaboration with the Lederbergs. From 1954, activity was concentrated increasingly on human population genetics -- the analysis of a population in the Parma Valley showed that microgeographic variation can largely be explained by drift alone. This required the development of special techniques, including the computer simu- lation of human populations, methods for the reconstruction of differentiation of racial groups, and the characterization of selective and random causes of variation. Several archives, from consanguinity records to parish books, have been the subject of study to extract genetic information. A full description of the history of consanguinity in Italy has resulted. Demographic analysis of pari,h book records by computer is still in progress. An analysis, during six winters, of an African Pygmy population with the help of a large team of collaborators allowed to accumulate information on the population structure of hunters-gatherers, to detect new genetic types, to account for the physiology of 10:~ stature in Pygmies and other problems of this population. Yost recent work has centered on the impact of technolcgical developments and cultural change, essentially the domestication of plants and animals, on human biological evolution. AUTHOR OF BOOKS: "The Genetics of Human Populations" in collaboration with W. Bodmer (now Professor of Genetics at Oxford, U.K.), and a short handbook of biostatistics. /3-/7F 100. A. ?lORONI, 1960. Sources, reliability and usefulness of consanguincity data with special reference to Catholic records. International Sy:;n- osium of Demography and Genetics, Geneva. 101. L.L. CAVALLI-SFOEZA, 1960. Indagine'speciale sulla consanguineita dei matrimoni. Istituto Centrale di Statistica, Roma. 102. A. FlORONI, 1960. Analisi metodologica de1 rilievo della consanguineita. Parte Prima - Legislazione e materiale religioso e civile italiano per il rilievo della consanguineita. fast. 3: 199-247. Folia Hereditaria et Patholcgica, 2, 103. L.L. CAVALLI-SFORZA, 1961. Lczioni di metodologia statistica per ricercatori - 11 numero di osscrvazioni necessaris in esperinenti semplici e l'efficienza dell'esperimento. Istituto di Calcolo della Probabilita, Istituto di Statistis, 4: Articolo Terzo. - 1 104. L.L. CAVALLI-SFORZX, 1961. Un netodo per la stima della frequenza di ,nutazione dell'uomo; risultati preliminari. Atti X.G.I., r1: 151-162. - 105. L.L. CAVALLI-SFORZA, J.A. ROPER, G. SERXO!ITI, S.I. ALIKIXXAS, D.X. HOPKOOD and J.F. STAL'FFER, 1YGl. Panel discussion: microbial genetics and its application to fermentations. Sci. Repts., 1st Super. San%l-.a, 1: 441-483. 106. L.L. CAVALLI-SFORZA, 1961. Biologia e genetica. L'Xteneo Parr.ens;l: -.,.y- --' -\-:-IT, fast. 6: l-20. 107. PI. ?fAINX-RDI, I. BAR?&, L.L. CXVALLI-SFORZX, 1962. The distribution of t:le number of collateral relatives. Xtti X.G.I., VII: 123-130. 108. L`I>. CAVALLI-SFORZX, 1960. !,fetodi e problemi nella analisi degli eff2tt.i biologici delle radiazioni sull'uomo. VII Basser.na I~.tsrnazi~n~l~ - Elettronica e Sucleare, ;\tti Ufficiali de1 Congresso Scitnrific?. %=a. -- 109. L.L. CAVALLI-SFORZA, 1962. Studying human evolution by census and computer. New Scientist, 13: 92-93. 110. I. BARRAI, L.L. CAVALLI-SFORZ.! and A. XORONI, 1962. Frequencies of pedi- grees of consanguineous marriages and mating structure of the populaticn. Ann. Hum. Genet., 25: 347. - 111. L.L.CAVALLI-SFORZA, 1961. I fattori letali nell'Uom0. Prim0 Simposio di Statistica !ledica, Roma, 11-12 giugno 1961. 112. L.L. CAVALLI-SFORZA, 1962. Risposta ad alcune critiche sul neodamrinisco. Rendiconti della Classe di Scienze Fisiche, ?fatematic!le e ?jaturali; Serie VIII, Vol. SXXI, fasc.1. 113. L.L. CAVALLI-SFORZA, 1962'. Indagine speciale su alcune caratteristiche genetiche della popolazione italiana. Note e Relazioni N. 17, Istltuto Centrale di Statistica. 114. L.L. CAVALLI-SFORZA, 1963. Sir Ronald Fisher. Patti A.G.I., VIII: l-8. 115. G. MODIANO, A.S. BENERECETTI-SANTACHIAPA, F. GONANO, A. CAPALDO e L.L. CAVALLI-SFORZA, 1963. Analisis della popolazione leccese per gruppi sanguigni G6PD, aptoglobine, e transferrine. Atti A.G.I., S: 64-79. 116. L.L. CAVALLI-SFORZA, 1963. Nuclear threat and human genetics. Exposure of man to radiation in nuclear warfare. Elsevier Publishing Company, Amsterdam. 117. L.L. CAVALLI-SFORZA, 1963.. The distribution of. migration distances, models and application to genetics. Entretiens de Monaco en Science Humaines, Preniere Session, 24-29 Xai, 1962. Human Displacements, 139-15s. 118. E.M. LEDERBERG, L.L. CAVALLI-SFOBZA and J. LEDERBERG, 1964. Interaction of streptomycin and a suppressor for galactose fermentation in E. coli P-12. P.N.X.S., 51: 678-682. - 119. I. BARRAI, L.L. CXVALLI-SFORZA and >I. YAINAPDI, 1964. Testing a model of dominant inheritance for metric traits in man. Heredity, 19: 651-668. - 120. A.W.F. EDIJARDS and L.L. CXYXLLI-SFORZA, 1964. Reconstruction of c-solution- ary trees. Svstematic Association Publication X.6, ?henetic and rl:..-1c- Benetic Classification, 67-56. 121. L.L. CAVALLI-SFOBZA and A.!d.F. EX~ARDS, 1964. Analysis of human evolution. Genetics Todav (Proceelinos of the XI International Congress of Genetics, The Hague, Netherlands, September, 1963. ;/oL.;r: y&z-7&w 122. L.L. CAVALLI-SFORZX, I. BARRAI and A.W.F. EDb!ARDS, 1964. Analysis of huinan evolution under random genetic drift. Cold Soring Harbor Symposia on Quantitative Biolc!athematics and Comouter Science in Siologv and Y?edicine, Medical Research Council, 51-60. 124. A.W.F. EDXARDS and L.L. CAVALEI-SFORZA, 1965. A method for cluster analysis. Biometrics, 21: 362-375. - 125. G. MODIANO, A.S. BENERECETTI-SANTACHIAR4, F. GONANO, G. ZEI, A. CAPALDO and L.L. CAVALLI-SFORZA, 1965. An analysis of ABO, XX, Rh, Hp, Tf and G6PD types in a sample from the human population of the Lecce Province. Ann. Hum. Genet., London, 29: 19-31. - 126. L.L. CAVALLI-SFORZA, 1965. Interesse genetic0 dei matrimoni consanguinei. Atti III Corso di Acgiornamento Professionale "Ereditarieta nella Patologia e nella Clinica" dell'ordine dei Xedici di Rona e Provincia, 95-101. 127. CAVALLI-SFORZA, L.L. and A.W.F. EDWARDS, 1965. Estimation procedures for evolutionary branching processes. Bulletin of the Internaticnel Statistical Institute, Proceedings of the 35th Session, Beograd. 128. CAVALLI-SFORZA, L.L., 1966. Genetic drift in popolazioni umane. Atti A.G.I., 11: 3-50. -- 129. CAVALLI-SFORZA, L.L., 1966. Population structure and human evolution. Proceedings, Royal Societv of London, 194: 362-379. 130. CAVALLI-SFORZA, L.L., :4: Kimura and I. Barrai, 1966. The probability of consanguineous marriages. Genetics, 54: 37-6Q. - * 130. DE NICOLA, P., I. Barrai, H.M. Cann and L.L. Cavalli-Sforza, 1966. bis Mutation rate in hemophilia A and B, and effect of parental age. Proc. 3rd Congr. Wld. Fed. Hemophilia, Paris 1965; Eibl. haenat., fast. 26: 91-93, Sew York. r I 131. C.LVALLI-SFORZA, L;L., 1967. Human populations, Heritage from Xendei. -- Ed. Alexander Brink. University of Wisconsin Press, >iadison, 309-331. 132. CAVALLI-SFORZA, L.L. and A.W.F. Edwards, 1967. Phylogenetic analysis. Models and estimation procedures. Am. J. Human Genetics, 19: 233- 257; - and Evolution, 21: 550-570. - 133. CAVALLI-SFORZA, L.L., 1967. Some old and new data on the genetics of human populations. T'ne Alabama Journal of :,!edical Sciences, 2: 376-381. 134. CAVALLI-SFORZA, L.L., 1967. Da ?lendel alla Genetica Umana ?Ioderna. "Cosi Comincio la Genetica", Acta Geneticae ?!edicae 2: Gemel- lologiae, Roma, 103-130. 135. RIMOIN, D.L., T.J. Xerimee, D. Rabinowitz, V.X. XcKusick and L.L. CAVALLI-SFORZA, 1967. Growth hormone in African Pygmies. The Lancet, 523-526. 136. CAVALLI-SFORZA, L.L. and G. Zei, 1967. Experiments with an artificial population. Proc. III Intern. Congr. Hum. Genet., Johns Hopkins Press, Baltimore, 473-478. 137. CAVALLI-SFORZA, L.L., 1968. Introduzione all'Impiego dei Calcolatori in Genetica Umana. Quaderni de "La Ricerca Scientifica," 47: 13-16. - 138. BODMER, W. and L.L. CAVALLI-SFORZA, 1968. A migration matrix model for the study of random genetic drift. Genetics, 59: 565-592. 139.. BARRAI, I., H.M. Cann, L.L. CAVALLI-SFORZA, P. De Nicola, 1968. The effect of parental age on rates of mutation for hemophilia, and evidence for differing mutation rates for hemophilia A and B. Amer. J. Hum. Genet., 20: 175-196. - 140. CAVALLI-SFORZA, L.L. L.A. Zonta, F. NUSSO, L. Bernini, W.W.W. De Jong, P. Meera Khan, A.K. Ray, L.N. Went, M. Siniscalco, L.E. Nijenhuis, E. Van Loghem and G. Nodinao, 1969. Slitdies on African Pygmies. I. A pilot.investigation of Babinga Pygmies in the Central African Republic (with an analysis of genetic distances). F+.J~-/u.&~;L/: 34-2 -279, 141. PAOLUCCI, A.M., M.A. Spadoni,. V. Pennetti and L.L. CAVALLI-SFORZX, 1968. Elevated serum phenylalanine-tyrosine ratios in African Pygmy and non-Pygmy subjects. Unpublished. 142. RIVA, S., I. Barrai,+ L.L. CAVALLI-SFORZA and A. Falaschi, 1969. Dependence on the buoyant density of single-stranded DNA on base composition. J. Yol. Biol., 45: 367. - . 143. SANTACHIAII-BESERECETTI, A.S., G. Modiano e L.L. CAVALLI-SFORZX, 1968. Studio de Alcuni Pol!imorfismi Enzimatici dei Globuli Rossi nei Pigmei Babinga. Atti A.G.E., 12: 99-101. - 144. CAVALLI-SFORZA; L.L., 1968. Teaching of biometry in secondary schools. Biometrics, 24(3): 736-740. - 145. CAVALLI-SFORZA, L.L., 1968. Recherches Genetiques sur les Pygnees Babingas de la Republique Centrafricaine. Cahiers de La I.:abol-e, 5(l): 19-25. 146. CANN, H.?l. and L.L. CXVXLLI-SFCRZA, 1968. Effects of grandp&rental and parental age, birth order, and geographic variation on the sex ratio of live-born and stillborn infants. Genet., ZO(4): 391-391. - 147, CAVALLI-SFORZA, L.L., 1968. Research on African national Biological Programme. Biology of Xan .tier. 3. Hum. Pygmies. Inter- in Africa, Warsaw meeting, 1968. N.78 Yaterialv I Prace Antrooologiczne Wroclaw (Poland), 1970. -- 148. CAVALLI-SFORZX, L.L., 1968. Population structure. Proceedinss of the Conference on Computer Xnnlications in Genetics, Honolulu, Hawaii. 8 149. CAVALLI-SFORZA, L.L., 1969. Human diversity. Proc. XII Intern. Congr. Genet., Tokyo, Japan, 1968, 2: 405-416. 150. RIMOIN, D.L., T.J. Merimee, D. Rabinowitz, L.L. CXVXLLI-SFORZh and V.A. McKusick, 1969. Peripheral subresponsiveness to human growth hormone in the African Pygmies. New England Journal of ?!edicine, 281: 1383-1388. (December, 1969). 151. CAVALLI-SFORZA,, L.L., 1968. Studi sulla struttura genetica di una popolazione italiana. Le Scicnze, L(4): 7-19. 152. RIIIOIN, D.L., T.J. Merimee, D. Rabinowitz, L.L. CAVALLI-SFORZX and V.A. McKusick, 1969. Genetic aspects of isolated growth hormone deficiency. Proc. Int. Symp. on Grcwth Hormone, !4ilan, 11-13 September, 1968, 418-432. 153. MERIXEE, T.J., D.L; Rimoin, D. Rabinowitz, L.L. CAVALLI-SFORZA and V.A. McKusick, 1969. Metabolic studies in the African Pygmy. Unpublished. 154. PAOLUCCI, A.!4., M.A. Spadoni, V. Pennetti and L.L. CXVALLI-SFORZX, 1969. Serum free amino acid pattern in a Babinga Pygmy adult population. Amer. J. Clin.'Sutrition, 22(12): 1652-1659. - 155. NALCOLN, L.A., P.B. Booth -.nd L.L. CAVALLI-SFORZA, 1971. Inter- marriage patterns and blood group frequencies in the Bundi people of the New Guinea highlands. Human Biology, 43(2). - 156. BAR&X, I., L.L. CAVALLI-SFORZA and A. Yoroni, 1969. The prediction of consanguineous marriages. Japan Journal of Genetics, G<(l): - 230-233. 157; CAVALLI-SFORZA, L.L., 1969. Genetic drift in an Italian population. Scientific American, 221(2): 30-37. 158. EDWARDS, A.W.F. and L.L. CXVALLI-SFORZA, 1969. Affinity as revealed by differences in gene frequencies. Unpublished. 159. BARRAI, I., L.L. CAVALLI-SF&A and A. Xoroni, 1969. Demography --_ and genealogy. I. Family reconstitution by computer. World Conference on Records and Genealogical Seminar, Salt Lake City, Utah, 5-8 August, 1969.. P-/P3 2i. 160. CAVALLI-SFORZA, L. L;, 1969. Present trends in basic biomedical research. C.I.O.~LS, Table Ronde organisee avcc l'assistance de 1'O.X.S. et de l'LiXESC0, Geneve, S-10 Octobre 1969. 161. CAVALLI-SFORZA, L. L., F. COXTERIO and A. NORON. 1970. Valutazione de1 carico genetic0 in relazione a matrimoni fra consanguinei. Societa Italiana di Ostetricia e Ginecologia. 54' Congress0 Nazionale, Elano, 16-19 * Settembre 1970. 162. BODKER, W., and L. L. CAVALLI-SFORZX, 1970. Intelligence and Race. Scizntif.'-=. -- American, 223: 19-29. 163. CAVALLI-SFORZX, L. L., 1970. Proble'ms and prospects of genetic analysis of inteliigence at the intra and interracial level. Disadvantsned Child. 3: 111-123. Ed., J. Hellcuth, Brunner >!azel, Inc., Xe:J York. 164. CAVALLI-SFOUA, L. L., 1971. Sisiiarities and dissimilarities of socieculzxrz1 and biological evolution. In Xbth.xzntics in t:?e ~~rch~eolc~icsl 2-2 I-`.lr:rl::l ---. Sciences. Eds. F. R. Hodson, D. G. Kendall, and P. Tautu. Ediaburzh ~~<-.-~r~ 1:;: Press, Edinburgh. 535-541. 165. KIDD, I;. K. and L. L. CXVX,LI-SFOXX, 1971. Sumber of characters esamir.zi and error in reconstructicn of evclutionary trees. In vht;~~~.~tf=S ;- -.-z -.. -..- Archaeoloqical and 'Ilsccrical Scf~~ces. Eds. F. R. Hodscn, D. G. Kendall, and P. Tautu. T-,dizhurgh Cniv2rsi.E:; ?ress, Edinburgh. 335-346. 166. sKoLsIcK, ?f. H., A. XORO?z, C. CAhYISZS and L. L.-hYhLLI-SFOXZ-4, 1?71. The reconstruction of genealogies zrom parish books. In >!ethemstics I:: -167. 168. 169. 170. 171. 172. 173. 174. 175. 176. 177. 22. CAVALLI-SFORZA, L, L., and W. BODXER. 1971. THE GEXETICS OF HLXU POPiTLXTIO:;S. W. H. Freeman and Company, San Francisco. A?DEEM, A. , and L. .L. CAVALLI-SFORZA. 1971. Measuring the rate of spread of early farming in Europe. Man, 4-+l&w &b?4- L ?f AYMER?44~~, A. J ., and L. L. CAVALLI-SFORZA, 1971. A population model for the diffusion of early farming in Europe. Proceedings of the Research Sc>inar in Archaeology and Related Subjects, Sheffield, Dec. 13-17, 1971. In press. KIDD, K. K., P. ASTOLFI, L.' L. CAVALLI-SFORZA, 1971. Error in the reconstruc- tion of evolutionary trees: In GEXETIC DISTUCE, Ed., J. F. Crow, in press. CAVALLI-SFORZA, L. L., 1971. Pygmies, an example of hunters-gatherers, and genetic consequences for man of ldomestication of plants and animals. IV Int. Conq. !r?uman Genetics, Paris, Proceedines. In press. RINOIS, D. L., T. J. XkRI?IEE, and L. L. CAVALLI-SFORZA, 1971. Growth h0rzor.e unresponsiveness in the African Pygmies. IV Int. Conpr. lhzan Genetics, Par. CAVALLI-SFORZA, L. L., 1971. I1 &co genetic0 nell'uoirio. Atti A. G. I., 16: ZEI, G., A. XORONI, and L. L. CAVALLI-SFORZA. 1971. Age of consanguineous marriages. In "Genetique et Populations. Hommage a Jean Sutter." Institut National d'Etudes Demographiquei. Travaux et Documents. Cahier n060. Presses Universitaires de France. 147-153. KIDD, K. K., and L. L. CAVALLI-SFORZA, 1971. An analysis of the genetics of schizophrenia. IV Int. Congr. Human Genetics, Paris, ?roCeedinyS. h -, Sde-G&. 178. 179. 180. 181. 182. 183. BOD>IER, W., and L. L. CAVALLI-SFORZX, 1971. Nigration matrices. IV Int. Congress Human Genet., Paris, Proceedings. In press. CAVALLI-SFORZA, L. L., and K. K. KIDD, 1972. Considerations on genetic models of schizophrenia. Xeurosciences Research Program Bulletin. r:5: pws./&Z+a@,/~74 CAVALLI-SFORZA, L. L., 197.2. Book review (David Rosenberg) in Psychiatry. SGARMELLA-ZOSTA, L., and L. CAVALLI-SFORZA, 1972. A method of the detection of a denic cline. Proceedings, Workshop on Population Structure, Hawaii. In press. CAVALLI-SFORZX, L. L.; and !I. W. FELDXAS, 1972. Models for cultural inheritance. I.: Group mean and within group variation. Theoretic?1 Population Biolozv. In press. CAVALLI-SFOPJX, L. L., and C. CASNISGS, 1972. Human Pcnulation Strcct*ire 2 Advances in Human Genetics. Ed. K. Hirschhorn. 184.. CAVALLI-SFORZX, L. L., 1972. Phylogenetic analysis. In Histoconpa:ibilL::* Testing, Nunksgaard, Copenhagen, 1972. 185. CAVALLI-SFORZA, L. L., 1972. Elements of human genetics. An Addison- Wesley Xodule, Addison-Wesley, Reading, >!ass. In press. . 186. CAVALLI-SFO.RZA, L. L., 1972. Evolution. In Enciclopedia de1 Sovecentro. Fd. V. dappelletti. In press. 187. CAVALLI-SFORZA, L. L., 1972. Some current problems of human population genetics. Am. J. Hum. Gen. In press. :* 188. CAVALLI-SFORZA, L.L. and N'. Yasuda, 1972. The evolution of surnames. For submission to Theoretical Fonulation Biology. -- pLj\CE OF` 8111 iti lo:;<, S!src; Courr!ryj S$n Francisco, California U.S.A. INSTlTUi-ION AN0 LOCATION OEGREE Stanford university B.A. Stanford University School of Medicine M.D. YEAR SCIE::TIFIC COfdFEflFiED FIELO 1958 Basic Medical Science: 1961 Medicine MAJOR AESE23CH INrEfiEST flOLE ItJ PROPOSC-0 PROJECT Endocrine Bioshemistry RESEA.HCH SL;i~?O:iT ISA2 ;f/,:fIuc!,o:ls~ 1. Support from September 1969 to September 1972:HD02147 (principal investigator: Norman Kretchmer, M.D.,Ph.D.) Program Grant in Human Development, from which I was allocated $25,000 per year, which supported ongoing projects herein described. 2. Current.Research Support: HDU6&68-01, Biochemical Studies of Male Sexual Maturation, from 9/l/72 to 8/31/75. Budget for 9/1/72,to 8/31/73 is $26,342. 3. Patient care costs for ongoing projects are derived from RR-70, General Clinica. Research Centers Branch, and RR-00-81-11, Premature Infant Clinic Research Centers Branch, both of NIH. RESEARCH AxC.Oi? P3OiE2SIOX;IL E XPE ZIEiJCE (StJrtrr:g v,,tn pfescnt position, list trainhp andexpcricncc fcfCvJftt :O area Of prO,'?C: LL: d. ormost rcprcsenratwc puohc~tions.~ 00 not cxcccd 2 pqcs for each individual.) Assistant Professor of Pediatrics, Division of Metabolism, Stanford Medical School, "1969-present . Post-doctoral Fellow, Department of Biochemistry & Molecular--Biology, Cornell University, Ithaca, New York, 1967-1969 .'. Post"doctora1 Fellow in Metabolism, Department of Pediatrics, Stanford Medical School, 1967 Chief Resident in Pediatrics,Sta.nford Medical School, 1966 Post-doctoral Fellow in Metabolism, Department of Pediatrics, Stanford Medical School, 1965-1966 -. Resident in Pediatrics, Stanford Medical School, 1964-1965 Pediatrician, USAH, La Chapelle Saint Mesmin, France, 1962-1964 Intern, University of Utah Medical School, Salt Lake City, Utah, 1961-1962 Societies: American Association of University'Professors American Association for the Advancement of Science Society for Pediatric Research Western Society for Pediatric Research The Endocrine Society Society for Developmental Biology Licensure: National Poard of Medical Examiners, 1952 State of California, 1964 Diplomate, American Board of Pediatrics, 1967 'Awards: Special Fellotiship, American Cancer Society, 1967-1969 P-/87 Publications: 1. 2. 3. 4. 5 ` 6. 7. 8. 9. !O. Ll. L2. b3. L#c,>:,.: `. ., .?' -. - Loyter, Aor R.O. Christiansen, and E. Racker. Respiratory control in s*&tito- chondrial particles and Ca++ transport. Biochem.Biophys,?.es,Com?, 29:450, 1957. Christiansen, R.O., L.A. Page, and R.E. Greenberg. Glycogen storagein a hepatoma: Dephosphophosphorylase kinase defect. Pediatrics 42:694, 1968. Christiansen, R.O., A. Loyter, and E. Racker. Effect of anions on oxidztive phosphorylation in submitochondrial particles. Biochem.Biophys.Acta lEg:207, 1969. Loyter, A.; R.O. Christiansen, H.. Steensland, J. Saltzgaber, and E. Racker. Energy-linked ion translocation in submitochondrial particles. I. ~a++ accumulation in submitochondrial particles. J.Biol.Cnem. 242:4422, 1969. Christiansen, R.O., A. Loyter, H. Steensland, J. Saltzgaber, and E. Racker. Energy-linked ion translocation in submitochondrial particles. II. Prnrnrties -v-d- of submitochondrial particles capable-of Ca++ translocation. J.Biol.Chea. 244: -- 4428, 1969. Loyter, A., C. Burstein, R.O. Christiansen, and E. Racker. The polarity of the mitochondrial membrane, Proceedings of the Bari Symaosium, B.B.A. Library, Adriatica Editrice Bari, p. 235, 1970. Greenberg, R.E., and R.O. Christiansen. The critically ill child. XV. Hypoglycemia. Pediatrics 46:915, 1970. -- Loyter, A., R.O. Christiazen, and E. Racker. The two sides of the mitochon,d:ial membrane in B. Chance (ed.), Johnson Foundation Symposia: "Probes of structx-2 and function of macromolecules and membranes", p. 407, Acader:c Press, :;mr York, 1971. Mann, E. and R.O. Christiansen. Adenosine 3',5'-monophosphate phosphodiesteraser Multiple molecular forms. Science 173:540, 1971. Mann, E. and R.O. Christiansen. "GGylate kinase in man: Multiple molecular forms", Human Heredity 22:X-27, 1972. Johnson, J.D., X0. Chrztiansen, and N. Rretchmer. "Lactose synthetase in mammary gland of the California sea lion". Biochem.Bioz:hys.Res.Csx?., 47:393-397, 1972. Enn, E., M. Desautel and R.O. Christiansen. Highly specific testicular adenosine 3' ,5'-monophosphate phosphodiesterase associated with sexual maturation, Endocrinolcgy 91:716-720, 1972. Johnson, J.D., Hansen, R.CR Albritton, W.L., Werthemann, V. and R.O. Christiansen. Hypoplasia of the anterior pituitary and neonatal hypoglycemia., J.Ped., in press. Abstracts: 1: Christiansen, R.O., L.A. Page, and R.E. Greenberg. Analysis of glycogen storage in hepatoma: dephosphophosphorylase kinase defect. J.Pcdiat. g:?'83, 1966. 2. Christiansen, R.O., and R.E. Greenberg. Hypoglycemia due to diminished hePatic gluconeogenesis. Proc.Xest.Soc.Pediat.Rcs. 14:79, 1967.. 3; ChriStianSen, R.O.,and R.E. Greenberg. Reguihon of gluconeogenic enzymes in the newborn period. Soc.Pediat.Rcs.Abs. 37:158, 1967. --- I 4. Greenberg,R.E., R.O. Christiansen, R.X. Rcmcn, A.?. iCohr;na, and G. Re~~~nn. Physiologic mechanisms in infantile hypoglycemia. lediat.Rcs. 1:292, 1?67. --- 5. Christiansen, R.O., and A. Loyter. Respiratory control and calcium binding in submitochondrial particles. Fcd.Proc. 27:527, 1968. 6. DiJeso, F., R.O. Christiansen, H. Steensland, and A. Loyter, Localizatio:] of inner mitochondrial meniorane components. Fed.Proc. 2G:663, 1369. 7. Christiansen, R.O., pf.J.W. Fan, D.A. Eclenky, and Lx. Page. Effect of chlorpropamide in p;:: - - .?i , . central and nephrogenic.diabetes insipidus. Amer.Iedi,z:..C:::z.; Soc.Pediat.Res.Abs., p. 192, 197Q. --- Christiansen, R.O., J.D. Johnson, J. Kellema, and G. Reaven. Severe prolonged neonatal hypoglycemia, nodular hyperplasia of the pancreas and hypcrinsulinism. Amer.Pediat.Soc.&Soc.Pediat.Res.Abs., p. 193, 1971. Christiansen, R.O., Z. Monn and M. Desautel. Highly specific isozyne of cyclic nucleotide phosphodiesterase associated with sexual maturation. Cli.n.Res., 20: 252, 1972.. - Hansen, R., J. Johnson, W. Albritton, R.O. Christiansen and V. Werthemann. Absent anterior pituitary and neonatal hypoglycemia, Clin.Res., 20:254, 1972. - Christiansen, R.O. and W. Desautel. Highly specific testicular isozyce of cyclic nucleotide phosphodieterase associated wtih sexual maturation., Ped.Res. c&349, 1972. Christiansen, R.0 ., E. Monn and M. Desautel.'Highly specific isozyme of phosphodiesterase associated with male sexual maturation. Abstracts, IV int. Cong.Endo., 4~536, 1972. lNSTtTUTlON A?:i) LOCATION University oI e Uestern Australia University of Western Xustrnlia SCIELJTIFIC Orcanic C:-,e=istry . Organic Che=si",:y MAJOH RiSiAFcCH iN:EiiEST A0L.r I:4 ?FiO?CSED i'iO;f CT Applicatiocs of tzss s?ectrc=etr/ to Organic Chesist/zass s;ectroscor?st B~olory/ c,d =~~r.c:c'l =ZCo'~le-s ..- - e... .- "Zb... 5ESEAFICH SU??Oa i iSr* `nstrcCLonsJ N/A PUBLTCATIO::S SIKCE 1971 ,I ILlUl ,II,L11 t:,Lti,b.. ..s . ..- Inrerpreto!isn of b',sss CZCc!:::. I . R.W.G. Milne, Ed., John \Viicy ono kns, 1. o ? ????oo????*??? ??? ????? . .,* . An Application of Arti"-.- Mass Spcctromct? , New York, 1971, ps, &;i- - i~~~~,,d By B . G 3:Jchoncn, A, M. Dufficld and A. V. Robertson `m. 5 70 P-150 2. 3. 6. 7. 8. 9. 10. 11, 12, Mass Spectromeiri in Structural a,r.l Stereochemical Hrsble~s. CCI'I. Sc=--c- ---a 1 of Hydantoins.11. Electron In?sct Induced Fracectaticn of sme S*>stirzrei Hydantoins. Org. Mass Spectr. , 5_, 551 (1971) By R. A. Corral, 0. 0. Orazi, A. M. Duffield and C. Djerassi Electron Impact Induced Hydrogen Scradling in Cyclohexanol and Isczeric Methylcyclohexanols. Org. Mass Spectr'., 2, 383 (1971) By R. H. Shapiro, S. P. Levine and A. M. Duffield Derivatives of 2-Biphenylcarboxylic Acid. Rev. Roumain. Chen., 16, 1095 (1971) By A. T. Balaban and A.!4. Duffield Alkaloide aus Evonymus europaea L. Helv. Chin. Acta, 54, 2144 (1971) By A. Kldsek, T. Rxchstein, A. M. Duffield and F. Santa@ I Studies cn Indian Medicinal Plarts. XXVIII. Sesquiterpene Lactones cf Enhy Fluctczns Lou-. Strccturc. of Enhidrin, Fluctuanin and Flu~~'~=~; c ,,,,n , Tetrahedrcn, 23 32SS (13'7.1). By E. Ali, P.3: Ghosh Dastiizr, S. C. Pakrashi, L. J. Durham ar,d A. L1. Cuffield The Electrsn Impact Prcmoted Frazmentaticn of Aurone Epoxiies. Or-z. Mass SZr'C? By B. A. Brady, _. 6, 139 (lc72j VI r. G'Sullivan.and A. M. Duffield The Detezzicaticn of Cy clshex~~lazine in Aqueous Solutions cf Scdi*:n Cyclz.t:e by Electron Ca;ture Zas Ch~zarcgrzphy. Anal. Letters, 5, 331 (17-71) .By !!. D. Solman, ii. E. ?erei-a and A. ti. Duffield . Computer Zeccznition of Ilctastable Ions. Nineteenth Anr.l;al Ccnference cr: Mass Spectrcmetry, ;stlanra, 1971, 2. 63 =, By A. $1. Duffield, :l. E. Reynolds,.D. A. Anderzon, R. A. Stillzn, u'r. and C. E. Carroll Spectrometrie de E&se. VI. Frasentation de Dimethyl-2,2-dicxolanes-1,3- Insatures. Org. Mass Spectr., 5, 1409 (1971) By J. Kossanyi, J. Chuche and A. M. Duffield Chlorprccazlne !.:et&olism in Sheep. II. In vitro Ketzbolisn and Preparaticz of 3H-7-Hydroxychicrprcnlzine. _ --. Journees D'Qressolcgie, 12 333 cl9711 By L. G. Brooks, Iy. A. Hold., I. S. Forrest, V. A. Bacon, A, M. Duffield and M. D. Solcmon . . Mass Spectronetry in S&c&al and Stereocheaical Problems. CCXVII. Electron Impact sromoted Frac--? ,.,,,ntaticn of 0-Yethyl Oximes of Some a&Unsatwa+ed Ketcnes and Ijethyl Substituted Cyclohexanones. 'Canadia;; J. Chem., 50, 2776 (1972) By Y. M. Sheikh, R.?. Licdtke, A. M', Duffield`afid t. `Djerassi ADAYAPhLA?l T. GXSESXN Born: Madras State, India. May 15, 1932. Citizenship: U. S. Citizen Married Ann K. Cook, Ph.D. (Stanford University, Department of Genetics), Research Associate, Department of Biology, Stanford University. Eddcation: 1947-1951 Annamalai University, Madras State, India. Physics, Chemistry, English. B. S. (1951) Botany. 1951-1953 Annamalai University, Madras State, India. M. A. (1953) Plant Physiology and Genetics 1959-1963 Stanford University, Department of Genetics, Palo Alto, California ("laticnal Institutes of Health Trainee under Professor Joshua Lederberg). Ph. D. .(1963) Genetics. Thesis title: Phyeical and Biological Studies on Transforming D:SX from B. subtilis. Profession,1 Experience: Research Fellow (1953-55), Department of Biochemistry, Indian Institute of Science, Bangalore, India. Awarded Institute Fellowship. Research Associate (1955-57), 2otar.y Department, Indian Agricultural Research Institute, Xe:.r Delhi, India. Responsible for plant tissue cu1turs, teaching of genetics course (laboratory and lectures). Fellowship (1957-59). ATlarded by Rask @rsted Foundation of Denmark for study at: Department of Physiology, Carlsberg Laboratory, Copenhagen, Denmark. Worked under Professor 0. !:'inge, F.S.S. (fermentation genetics and some aspects of cytology). Department of Physiological Chemistry, Carlsberg Laboratory, Copenhagen. Xorked under i?rofessor Halter (physioiogy of sporu- lation in yeasts).. Department of Gei?etics, University of Copenhagen. Seurospora genetics and methods. Research Associate (1963-65), Department of Genetics, Stanford University, ?alc Alto, California. Xssistant Professor (1965-70); Xssociate Professor (1970-present). Special Fields of Research Interest: D>?A Replication and Recombination Mechanisms in B. subtills; Repair of DX in vitro Synthesis of Biologically Active DXX. -- 1Iolecules and its genetics. Phage DSX Biosynthesis. Cytogenetics of Eukaryotic Cells. Recent Publications: Studies on in vitro Replication of g, subtilis DNA. Cold Spring Harbor Syap. Quant. Biol. 33:45 (1968). _ . ATP Dependent Synthesis of Biologically Active DNA by Azide Poisoned Bacteria. Proc. Natl. .'.zad. Scie, 6831236 (1971). DNA Synthesis in Bacteriophage SPO-1 Infected Bacillus subtilis. J, Virology 9:263 (19721, with C. 0. Yehle. . A Deoxyribonucleic Acid Polymerase I deficient Xutant of B. subtilis. J. Biol. Chem. 247:5867 (19721, with P. J. Laipis. C' In vitro Repair of X-irradiated DNA Extrac'ted from a Polymerase I Deficient Bacillus subtilis. Proc. Natl. Acad. Sci.(i+~~~ber$-9?2), with P. J. Laipis. I .,T : t In vitro DSA Synthesis and Function of DNA Polymerasds&i6 Bacillus subtills -- in "DKA Synthesis in vitro".' Ed. R..D. \!ells and R. B. Inman, University Park Press, Baltimore, Maryland (1972), with C. 0. Yehle and P. J. Laipis. Source of Funds: NIH, General Pledical Sciences NIH Research Career Development Award (G?l-50199) Title of Project: DNA Replication and Recombination in Bacillus subtilis Principle Investigator: A. T. Ganesan Grant Number: GM 14108, G?l-50199 (AwardTthrough November 1975) P-133 SECTION II - PRiVILEC-ED CO'ZX!~IFJIC,~TIC)N nil. j. -.:-. : :q co?;c,Te-: pl ? rt, . . BIOGI7APHiCAL SKETCH (Give the fOhv~~rtg fnformarion for allprof~:;,;riop.?/ p,?r~o,,~c~ listed on o z;e 3, bqinning w.th tho Principal Invcst;g3ror, Use COfJfifWafiOn P3tfs Jfld follo:v the ume general former for each person.) NAME TITLE ' . ' BIRTHDATE (20 GJY Y/ I .., I. Terence John Gribble Assistant Professor April 6, 1937 . PLACE OF BIRTH ICity, Stare, Country) PRESENT NATIONALITY f/f non-US fndica te kind of visa and cxpira tion date) citizen, SEX Cardiff, Wales U.S.A. CT h4a!e EDUCAl ION Iikgin vitlr bscch/eurtare training and ir~/ude portdoctora/) r3 Ftira!c! INSTITUTION AND LOCATION DEGREE YEAR SCIENTIFIC CONFERRED FIELD - University of the South, Scwanee, Tenn. B.S. 1959 Stanford University, Stanford, California XD. _ Chemistry 1964 HONORS Sigma Pi Sigtm,,Physics Uonor Society (1958); Phi Beta Kappa (1959); Alpha @mea Alpha; Faber Pediatric,kard (1964); Eorden Student Research Award (1964); Dernhan Senior Fellow (1970). SOCIETIES: Western Sot. for Pcdiat. Resaarch (1968):Pzer. See. EeL-.atolc MAJOR RESEARCH INi ERESI ROLE IN PROPOSED PROJECT *Pediatric Hematology - Hemoglobin Investigator Dernham Senior Fellow, Amricsn Cancer Society, July 1, 1970 - June 30, 1973 Current year $20,690. Total $60,989.' = e -_ -- RESEARCH AtdD;gR PHOFES%l'OS,\L EXPERIEt~!CE (S:~:i;l~~ ~,,;r;lhprc"cc,~t'po:if;on, /;sr tr~i~inoand~~pericnccrc/er,cnt toarc o:crckecf LJT ormost reprcscfmrivc publ;c,?tion~. DO 83 t FxcPCd J" ,E;eS for uch ino'ivid::si.) 1969 to present - Assistant Professor of Pediatrics, Stanford University 1969 - Chief Residc~t in Pcdiatr.ics, Stanford University 1968 - Research Fcllor~~ in Fcdiatric Rezatology, Stanford University . 1966 --1968 - U.S. Public Health-Service (NIH) Rational Eeart Irktitute, Eethezda, Naryland, Publi&ions (Selected) Walters, T.R., Gribble, T.J., and Sch::artz, U.C.: Heme S$nthesis in Norm1 and Lcukezi Leukocytes. Xaturc 197:,,1213, 1963. 5 Gribblc, 'T.J. and Scht?artz, 1I.C.: Effect of Yrotoporphyrin on Hemoglobin Synthesis, Biochcn. Riophys. Acts 1@3:333, 1965. Gribblc, T.J., Cornstock, T.J., and Udcnfrimd, S.: Collagen Chain Pm-nation 2-d Per\:: Proline Uydroxylati.on in PIonolaycr Tissue Cultures of L-929 Fibroblasts. Arch.' Biochcm. Biophys. 129:3OS, 1569. -- Cornstock, J.P., Gribblc, T.J,, aild Udenfricnd, S.: Further Studies on the Act?\-atioil of Collagen Prol.kc i!yci,jrox~l.st:i.on in Cultures of L.-929 Pitrob3asts. rQ: c :i f.>\' (J r .I Biochcx. cr.d Eiq~I:ys* 1.37:!.!..5, 1!?20. -we Lincoln, D.R., Edmunds, D.J., Gribblc, T.J.', and Schvart-z, H.C.: Studies on the Ucmoglobins of Pinniped. Ill pre!;s, Jclnunry 1373, Clood. pj-:r. J, 1:-. ._ : Q#y+~;t ;f 1:: ' :" .-; wai w-c . . . --i .,-. I ;-;5 -1.; :..c,:' :e 2.7-i j.%.`.-,, ";,-ra: `3.. *jc,y;.T,-;,,:., -. -- ____ -. NANE TITLE BIRTHDATE ia;:=., 2ji, `:.j Leonard A. Herzenberg Professor of Genetics Nov. 5, 1931 PLACE OF BIRTH (City, Stare, COUnw/ PRESENT NATIONALITY l/f n0n-U.S c/r;zen, SEX indicate kind of visa and expiration date) Brooklyn, New York . U.S. XHhlzle c Fr?jl: EDUCATION (Begin with bacca/aurea :e training dnd include pcrstdx:orJ/j INSTITUTION AND LOCATION OiGREE YEAR SClEIvTlFlC CONFERRED FIELD Brooklyn College, Brooklyn, Net:/ York A.B. California Institute of Technology Pasadena, Calif. Ph.D. Pasteur Institue, Paris, Postdoctoral Fellos 1952 Biology, Chemistry 1955 Biochemistry, Irxunclc;, 1957 HONORS Phi Ekta Kappa, Sigma Xi Distinguished Alumnus blard, Brooklyn College, 1970 Genetics Study Section, Eational Institues of Health MAJOR RES'ARCH.PJTEREST FiOLE I." PR.O?GSED PROJECT Immunogenetics, somatic cell genetics Prlnclpal Investigator RESEARCH SUPPORT (See instmcdons) I N.I.H. G:+17367, Automated Cell--Sorting-Clinical & Biological Uses, S160,&02 current year direct costs, total funds for nro.ject S585,977 (l/1/73-12/31/75). N.I.tl/ AI-02917, Genetics of !mmuno9loSulins, 552,774 current year direct costs, total funds for project 5246,386 (5/l/69-4/31/74). N.I.H. CA 04681, Genetic Studies xizn ;!amma~ian Cellsj-SE0,993, current year cirect costs, total funds for project 5424,981 (9/l/72-8/31/77) N.I.H. HD 01287, Fetal-maternal Immunological interactions. S35,GOO current year dirzit costs, total funds for project $101,572 (5/l/70-4/30/73) RESEARCH AS3 OR ?=CFESSiCNAL EXPEF'.lE:~-7? (:' Hulet:, II. i:., Bonncr, :I. A., E.~rr,?tt, J. and L. A. Uerzcnbcrg. "Ccl1 sor;ir.;: automated scpnration of mammalian cells as a function of intracellular fluorcsccncfz:" Science 166: 747-749 (1969) _---__ A rcl B A:::'& me P-20& - .__ _`.._m._ __ -_ _..._. - .--..-a. a.- - Hulett, H. R. "Optima world population." Bioscience., g:160 (1970) Hulett, II. Ii. "Non-cnzynatic hydrolysis of adcnosine phosphates." K'attire 225:1248 (1970 HuleK H.-R., Coukcll, A., and W. Bodmcr. 'Tissue typing instrumcnta:ion using the fluorochtonatic cytotoxicity assay.' Transplantation lo:135 (1970). Hulett, H. R, and L. A. Herzenberg: "Approaches to prcscrecniG with special consideration of flow systems'." Acta Cytologica (in press) Hulett, H. R. and L. A. Herzenberg, W. Bonner and R. L. Wolf. "Rapid cell sorter -- a new tool for cell study with clinical appliations." Laboratory Investigations 22:501 (19.70). Hulett, H. R. "Shock.synthesirof amino acids in simulated primitive atmosphere." Science 17091000 (1970). Letter to the Editor. SzSen patents on electro-optical devices and instrumentation Publications after 1970: , Merrill, .I. T., N. 'Veizades, H. R. Hulett, P. L. Wolf and L. A. Herzenberg. "An Improved Cell Volume Analyzer" Rev. Sci. Instr. 42, No. 8, 1157-1163 (1971). Bonner, W. A., H. R. Hulett, R. G. Sweet, and L. A. Herzenberg. . "Fluorescent Cell Sorting,, Rev. Sci. 'Instr. 43, 404 (1972). Hulett, H. R., R. G. Sweet, L. A. Heryenberg. "Development and Application of Rapid Cell Sorter,,, to-be presented at O.R.N.L. symposium on Advanced Analytical Nethods for the Clinical Laboratory, Xar. 15, 1973 (and later published in Clinical Chemistry.) Hulett, H. R. "Formaldehyde and Ammonia as Precursors to Prebiotic Amino Acids. Science 174, 1038, 1971. John D. Johnson PIACE Of BIRTH ICity, Sate, Country/ Palo Alto, California . Assistant Professor September 14, 1935 PHESENT NATIONALITY (If nonUS cirizcn, SEX indicate kind of visa end expiration datel U.S.A. . ah!ale n Fen-i!- EDUCATION (fie@ v,,ifh hxcalaurate trainmg and include postdocrorai) INSTITUTION AND LOCATION DEGREE YEAR SCIENTIFIC CONFERRED FlELq Wabash College, Crawfordsville, Ind. B;A. 1960 _2oology'~~ e Stanford Medical School, Stznford, Calif. M.D. .. 1965 Medicine Johns Hopkins Hospital, Baltimore, Md. 1965-67 Pediatrics,2 yea : Stanford Medical School, Stanford, Calif. I 1970 Post-Doctorel F;- lSf?~5~~fE~~~ HONORS Phi Beta Kappa, Alpha Omega Alpha, Borden Award for Undergraduate Research (Stanford Medical School) MAJOR RESEARCH INTEREST .- /ROLE IN PRGPOSED ?ROJECT Developmental Biochemistry, bilirubin metabolism RESEARCH SiJ?PORT~ irlstructionsl Investigator United Cerebral Palsy Associations, II-X. Grant R-245-71, Developmental Aspects of Mene Protein' Catabolism - -. Current $28,772. July 1, 1971 to June 30, 1973 Totif $53,322. RESEARCH AIC3jGfI Ph3FESS;GNitL EXfEfilENCE (St.?fi;f,l ?::I -h pr~=cnt ~O:jt;o,), list trG;rlhT sndcxperialce retevent 10 2red Of ;W:Xf L;., i t ormost rcprcsentatire publicirtions. Do nor cxcrcd 3 pqes fsr c:ch ir;dividulual.J .a 1971 to pre'sent - Assistant Professor of Pediatrics, Stanford University 1971 to present - Assistant Director of !'Jl'i. Submitted for publication to Giochim. Xiophys. Acta. .*s+--- -c-w.---- -..---.-- . . .-m.-.. .-.-.-em____ --I --.e.- RHS.:4~ir; . . P-;rw :: P i :: :m .-.. -. SECTIOI"J It - PRIVIl.FGED CO:::I1VNICATION Cpe,<--.?-a ! q-.- . . .- BIOGRAPHICAL SKETCH (Give the following information for all prv/rssion,-I personnel lis:ed on p?yc 3, &inning v;ith the Principal Invcstig&ar. Use continuation p2g7w and follow the wme general forma: for each penonl NAME TITLE Assistant Professor of BIRTHDATE /MC., Lby, Yr.) Judith P. Koehler, M.D. Neurology and Pediatrics April 27, 1939 PUCE OF BIHTH ICjry, State, Country) PRESENT NATIONALITY I/f non-U.S. citizen, SEX New York, New York indicate kind of visa and expiration date) U.S. Citizen 0 Male a Fcmzk EDUCATION Begin with baccalaurca te training and include postdoctoral) INSTITUTION AND LOCATION Mount Holyoke College -. Dartmouth Medical School University of Pennsylvania DEGREE YEAR SCIENTIF:C CONFERRED FIELD ' A.B. 1960 B.M.S. . 1962 M.D. 1966 American Academy of Neurology (Junior Member) MAJOR RESEARCH INTEREST Pediatr&* Neurology RESEARCH SUPPORT (Set instrucrions) ROLE IN PROPOSED PROJECT -- RESEARCH AND,`3R PROFESSIONAL EXPEFtlENCE (Starting with p,.esantposition, list trainicqandexperience relevant toarea of projzc List ,r.i or most rcprasenta tive publications. Do not exceed 3 pages for each individual.) 1966-1967 Intern, Mixed Pediatrics, Xontefiore Hospital and Medical Center, Bronx, N.Y. 1967-1968 Junior Resident, Neurology, Albert Einstein College Hospital and Bronx ?funicipal Eos 1968-1970 Senior Resident, Neurology, Albert Einstein College Hosp. and Bronx >!unicipal Hospital Center, New York. 1969-1971 Postdoctoral Fellow, Anatomy, College of Physicians and Surgeons, Columbia C'niv., X. 1971-1972 Fellow in Pediatric Neurology, Columbia-Presbyterian Medical Center, S.Y. 1972- Assistant Professor of Neurology and Pediatrics, Stanford Medical Center. \ Publications (selected). Koehler, J.P., Lovelace, R.E., Spiro, A.J.: Basement membrane and capillary endothelial altsra- tions in hypokalemic periodic paralysis: an electron microscopic study. (Abstract) Present--c at the American Association of Neuropathologists, 47th Annual Meeting, June 1971. Koehler, J.P., Spiro, A.J., Lovelace, R.E.: I. Thyrotoxic periodic paralysis: light and electrr microscopic observations of nerve and muscle. (In preparation). Koehler, J.P. and Chuzorian, A.: Tissue culture of medulloblastoma cells from spinal fluid, a useful diagnostic technique. (In preparation). Spiro, A.J., Koehler, J.P., Taylor, J.M.: Oculopharyngeal dystrophy: ultrastructural and his:*>- chemical observations of skeletal muscle. (Abstract) Presented at the American Associatiz- of Neuropathologists, June 1972, Koehler, J.P., Duffy, P.E., Carter, S. (1973) Selective type I fiber atrophy in childhood neuropathy: light and electron microscopic observations of peripheral nerve, Schwann ccllj. neuromuscular junction, and muscle. (In preparation). (Ghe the foh'owing information for s:iprofzsion~/ perscnnrl Irsrcd on pqs 3, brj!nrriry ?ji!h t.`w Principal Inrzr;,-3rof. .-.I .. Use conrifwatinn pq3j- ~4 ff2lcw th* 2rne genefit format fcr wctj parson.1 NAME Norman Kretchmer TITLE,' Harold K. Faber professor IClaTHpATE (a!!~, Cdl', Yr.l- of Pediatrics I January 20,-1923 PUCE OF ljlHTH (Ciry, State, Cowtfq4 -. PRESENT NAIIOt2ALITY f/f non-U,S cir;ien, SEX .,.. indicate kind of visa ad expiration date) New York, N.Y. U.S.A. Q M3Ie 0 Fr~:le EDWXTIDN (&yin v.?th Dsccckurtute rrainh? aqd incluti ros;doctoq[j INSTITUTION AND LOCATION : Cornell University University of Ninnesota University of Xinnesota . Downstate >lcdical Center, State Univ. of DEGREE B.S. M.S. Ph.D: YEAR -+ - SCIENiIFlC CONFERRED FIELD 1944 Animal Phycidlc;? 1945 Physiological C!;e;:. 1947 Physiological Cheer.. m& Commonvealth*Fund Fellow, 1952,57,65 1957 President, Int. Organ. Stuc!y i;.uzan i>cy:r!is . .Mead Johnson Award, 1958; ment, 1969; President, Soc.Ped,Res., Y$:c:c. A-/. s_ . Borden Award, 1969 Council, Amer. Sot. Clin. Invest., 1064-~j: MAJOR RESEARCH VJlEfiEST C~ESEARCH n?i'DRi (kr ins:rccrjn,T.c) Alumni Hedallicn SlXS, 1969. -- ROLE IN PROWSED PKOJECT Investigator RR 81 - Clinical Research Center for Premature Infants, 10/l/63-9/30174 -- .1 Amount current year: 418,532 - Total amount: 2,021.300-. (5%;) renewal pence., HD 02147 - Eiochemical Studies of Dcvelopcent, 6/l/66-5/30/73, Amount current year: 206,593 - Total amount: 1,309,278- (15X) rexc$.-al pendil:z. CRBS 252 - sational,Foundation - Cellular and Xolecular Determinants cf X~r~:ic;~nc,ii~ Amount current year: $27,273 - Total amount: 54,546 (7/l/71-6/30/73)- ilS::> HD 00049 -'Human Development and Pediatrics Training Grant* 7/l/70-6/30/74, . Amountrurrent year: $$9,538 - Total amount: 448,936.- (1X)renewal pzr.@ing . (Please see continuation on attached sheet) - RESf1.f.:C!i b?.!D,:>R izI:3PiS.5ib:;i;L :!,r * Of most rcprcxnr2tLe prh5rrcr!icrB.5 ~i:I'El~! E;`:CE'(Str;i;t~~ ,:*/IA F:CSZT~ positio;t , --md!~t,c am c*pcri'en:e fc:ev~nr ta 8x3 c/~:rr,:.-;; L;.;: I Un flcf wccrd3 ps;lcs ior cxh iwYlrid23f.) 1971 to present - Harold El. Faber Professor of Pediatrics 1969.-.1972 .-. Chairman, .Progran.in Ruman Biology (Eaccalaureate Prog., Stanford i'r:-.i 1959 - 1969 - Professor and Executive kad, Pediatrics 1953 - 1959 - Assistant then Associate Professor, Dept. of Peds, Cornell L`Liv. --. New York l!ospital 1950 -:1935 - Lecturer, Dcpartmcnt of Biology, Brooklyn College 1948 - 1952 - Research hssxiate, Dept. of Pathol., State Univ. of Xew Ycrk 1947 - 1948 - Assistant Professor, Departments of Pathology and Biocheris:rg, L'niv. of Vermont,. From 1952 - Various cl+nical appointments from Intern to Pediatrician-in-Chief Publications (Six relevant publications are listed from a total of 101) Rretchmer, N.: Lactose and lactase: .A historical perspective. Castrcenteroiogy 61:&O-, 1971. Levine, R.L., Hoogenraad, N.J., and Kretchmcr, N,: Regulation of activity of cerb z:yl- phosphate synthctase from mouse spleen. Biochemistry 10:3694, 1971. Lebenthal, E., Sunshi.ne, P,, and Kretchner, N,: Dffect of carbo@rate and ccrticostcrc~jds on ac';j.vi:y oi 3.-~~lUCOSLiG2SC!S in ir=ssr:ine Of the . infant rat , J, Clin, Invest. 51.:1244, 1972. -4 Mretchmcr, D., Ransomc-Ruti, O., llur*Jitz, R., Dungy, C., and Xlakija, W.: Intestinal absorption of J.actosc *iii Kigerian ethnic groups. The Lancct 2:392, 1971. p-2Ob ic {CT+<. p --..I-- _A_--- ---f*.-.. __.- - . _ : -- --7 -- Biographical Sketch of Dr. Norm& Kretchmer Page Two Research Support, continued . HII 00391 i Regulation of enzyme'actlon durlng'development, 9/l/68-8/31/73 * 'Amount current year: 42,289 - total amount': 183,822 (15X) Publications, continued Welchsel, M.E., Jr., Moogenraad, N.J., Levine, R.L., and Kretchmer, N. Pyrlmidine biosynthesis during development of rat cerebellum Pediatric Res. 6:682, 1972. Johnson, J.D., Christiansen, R.O., and Kretcber, N. Lactose synthetase in mammary gland of the California sea lion. Biochem. Biophys. Res. Comun. 47:393, 1972. Note: Renewal of HD-02147 is pending. This present renewal application, HII 00391, in part overlaps with some material include4 in ID-02147. a .I,, _I. ,,-r....I.b"." ~~I~I..."...~0..~.. ( =----= -- -_ .--=- .,-, -----.. BIOGRAPHICAL SKETCH APPLICATION NUMiEY (Ir.,, r l~~e,k/ (Give tha followiT infarmatton for o och KEY professional staff member, whether or not solorY is requested. Begin witti tha Program Director.) JAMf (Last. jint, i~likiUlJ fl"fProfessor of Pediatrics and BIRlHDATE I IJo.. 1L1.t. j-r., Community & Preventive Medicine I 916114 Luigl Luzzatti, M.D. SEX `LACE OF BIRTH (City, Slrtre. Cornrr~) PRESENT NATIONALITY (If aorr-L'.S. ririzm, indic&r ?I3 MALE 0 FEMALE ha sprbof J SOC. SEC. NO. Rome, Italy U.S. Citizen 570-42-7539 lElATlONSHlP TO PROPOSED PROGRAIA Investigator . EDUCAUON f Begin -wir/, b.ar&wratc truiving and inchdr pnrtdxtorul) ORGANIZATION AND LOCATION DEGREE YEAR CONFERRED DISCIPLINE University of Minnesota, Mpls, Minn. M.S. 1942 University of Minnesota, Npls, Minn. M.D. 1943 . 40NORS MJOR RESARCH OR PROFESSIONAL INTE IS1 (If apphbk) Congenital Defects and Cytogenetics IS1 RECEM RELEVAM PUBLICATIONS Miles, C.P., Luzzatti, L., Starey, S. and Peterson, C.D.: A male pseudo- hermaphrodite with a probable XO/AXY mosaiclsm. Lancet, Sept. 1, 1962, 455. Luzzattl, L.: Failure to thrive - a diagnostic approach. Postgrad. Med. 35:270-8, March 1964. Peterson, C.D. and Luzzatti, L.: The role of chromosome translocatlon in the recurrence risk of Down's syndrome. Pediat. 35:463-9,-March 1965. Welchsel, XE., Jr., and Luzzatti, L.: Trisomy 17-18 syndrome with congenital extrahe? blliary atresia and congenital amputation of the left foot. J. Ped. 67:32G-7, .-\uz. ' `WFESSIONAL AND/OR RESEARCH EXPERIENCE (S&r: with pr~mt poritim und list recmt sigu&mt r.~$wrimsr rrlrumt lo pwww~) (publications con: . 1972- Professor of Pediatrics & Community and Preventive Medicine 1967- Director, Birth Defects Center, Stanford Univ. School of Xedicine 1963- Director, Cytogenetic Lab., Stanford Univ. School of Medicine 1957-72 Associate Prof. of Pediatrics and Preventive Yedicine, Stanford Univ. Sch. of ? 1955-67 Director, Pediatric Outpatient Dept., Stanford Univ. School of Medicine 1954-57 Assistant Prof. of Pediatrics and Preventive hledicine, Stanford Univ. Sch. of 1. 1953-54 Director of Cerebral Palsy Training Program, Children's Hospital of San Francisco . . P-2Db PHS 2499-l PAGE trv. s-70 U.8. OOt+ENNMSNT PRINTING OFPICE : 1070 OF - 166-466 Publications(continued) Craig, A., and Luzzatti, L.: Translocation in De Lange's syndrome? Lancet 2:445-6, August 1965. Craig, A., and Luzzatti L.: Translocation in trisomy D syndrome. A case of probable D/18 translocation. J. Pediat. 70:264-g, Feb. 1967. Greenstein, R.M., Harris, D.J., Luzzatti, L. and Cann, H,.M.: Cytogenetic analysis of a boy with the XXXY syndrome: origin of the X-chromosomes. Pediatrics, 45:677-686, April 1970. Knight, L., Sakaguchi, S., and Luzzatti, L.: Unusual mechanism of transmission of a chromosome translocation from mother to offspring. American J. Dis. of Children 121:162-167. Feb. 1971. Knight, L., and Luzzatti, L.: Replication patterns of X & Y chromosomes in partially synchronized human lymphocyte cultures. Chromosoma, Vol. 40, 153-166, 1973. NAME TITLE GIFITHD;\TE /::I. ;J,; i'r./ Wilfred E. PSPZI?A PV\CE OF BIRTH fGry, Sirte. Counrryl Research Associate June 23 193.5 PfiESEF;T P'AT,.`;' * 1 . " .C.L ITY l/i xc-US c/rizen. SEX indicate kind of viz and exsirdrlon dare) INSTITUTION AND LOCATION I DEGREE YEa9 I SCIESTIFIC CONFERRED FIELD ' Madras Medical College, EIadras, India B. Pharm Saugar Univ, 2. . ?f2dhva Pradesh,, India 14. Pharm U.C. Med. Center, San Francisco, Calif Ph.D. HONORS 1960 1902 1968 Pharmaceutical Che~ist~~~ Pharm. Chern L Cher. of :;a% harm:Chen b Phzrrscolo- . MAJOR RESEi,RCH lNTE.i;EST Identificetion of !(stabolites t drug metabolites in Bzological fluids RESEARCH SiJiPORT (See:nsmcoonsl ROLr IN PROPOSED PROJECT Organic chemist RESEARCH A:;o,oa tT:C-cS3iOXAC EXiE?,E?:ZE rS:3mr.~ .-.trn orzen. . ~ouo'on, jjs: pj,c,rrq and expene.xe rQ?fdnt :O ared Of Ur:;t'." i:ir j.' e-t3rIve ;~:.'~:3:: oi~ggY~97 0 or! Go no: exceed 3 c3;es for ex.7 mcivraust.1 Fcst 3cctoral Pellcv, Zep5, of Genetics Stanford University :.!ed. School 1970 - present Resezreh kscciate s.ze institution e During these fox years i have been in-solved wizh Feptide syxtkeiis, azino acid esz~~sis and synthetic orgzic chemistry. I hel; ed develop rethods for the segarzzicn of diasterioisocers ky gzs ck.rc~2to~~~~y zzd kzve been involved with t.*.e r0r;tir.e t;se of `gas chrozsto~~a3~9 :zss ssectrczetry for the identification of *xir.zrj- -,etsbolizes i:: xrxi and pathological ~7ir.e and serum sezrples. t?y a??lications of ~255 spectrozezrg k27t included the Ceveclyez 0:' ~12~s fra,zeatoGgagky for the detemicztion of tke amino aci? Contents of soil and *zxx serum. 2~2 present project involves tkc screening of xrice fkom leukenic aatiezts for 2bnorz21 cetabolites and to investigate the metabolic fate of anti-leukemic chemotherapeutic agents in the body, PUBLICATICX 1, Transesterification with an Anion-exchange Resin; W, Pereira, V. Close, W. Pattcn and B. Halpern, J. Org. Chem. &:2032 (1959). . 2. Alcoholysis of the ,. verrifield-type Peptide-polymer Bond with an Anion Exchange Resin; W, Pcrcira, V. A. Close, E. Jellum, W. Patton and 9. Xalpern, Australian J. of Chem. =:1337 (1969). 3r The Action of Xitrosyl CbLoriCe cn Fhenylalanifie Pegtides; W, Patton, 2, Jell=, D, Zitecki, W. Pcreira and B, Halpern, .Australian J. of Chen. 22:2709 (1969). - 4, Abnomal Circular Dichroisn of #.-Amino Acid Esters; J, Cyzermn Craie and $1. Z. Pereira, Tet. Let. M-:1563 (1970). 5, The Use of (+)-2,220Trifluoro-LPhenglethylhydrazine in the Optical Analysis of Asymetric %eto=les by Gas Chrorzatography; W. E, Pereira, Is!. Solocon acd B. Ealpern, Australian J. of Chez,&:1103 (1971). 6, The Kicrosoral Ozq-genation of Ethyl Benzene. Isotopic, Stereocherical, snd Induction Studies; * R, E, Kc3!&on, H. PI. Sullivan, J. Cjr;leman Craig and W. E. Pereira, Arch, Eiochen. Biophys, 132:575 (1969). 7. The Steric Analysis of Aligha tic Azines with 'P-do Asyxzetric Centers by Gas-liquid ChrosatoGra,.. -by of Ciastereoiscceric At&es. W. E, Pereira aad 3. Ralpern, Australian J. Ches. 25:667 (lgn). 8, Optical Rotatory Dispersion and Absolute Configuration -XVII. &-Alkylphesrlacetic Acids; J, Cyizexan Crai<, V. Z. ?ereira, B. Halpern and J. W. Vestley, Tetrahedroa 2:X73 (1971). 9. The Optical Rotary Dispersion and Circ ilar Dichroisn of &-Amino and _ A-Hydroxy Acids; J, Cy-,er=an Crztg, a-,i W. 6. Per&a Tetrahedron sj: $57 (1970) . 10,' The Detemination of Cycl,,.,. *&arJlaziice in Aqueous Solutions of Sodium Cyclari3te by Ziectron-capture Gas Chrocatography; M, D. SO~OLO~, 3. 2. Pereira ar.d A. X. Duffield, __ AntLL Let. 4:301 (1971). u. 12, 13. 14. 15. 16. 17. . 18. 19e 20. Chlorination Studies. I. The Reaction of Aqueous vith Cytosine; lCsn Hypochlorous Acid W. Patton, V, S~bx, A. M. Duffield, B. Halpern, Y. Hoyano, W, Pereira and J, Lederberg, Biochen. Biophys. Res. Cormun. %:880 (1972). The Use of R-(+)-l-Phenylethylisocyanate in the Optical Analysis of Asymmetric Secondary Alcohols by Gas Chroztography; W. Pereira, V. A. Bacon, W. Patton, B, Halpera, and G, E, Pollock, Anal. Let, 2:23 (1970). A Rapid and Quantitative Gas Chronatographic Analysis for Phenylalanine in Serum; B, Halpern, W. E. Pereira, M. D. Solomon and E. Steed, Anal, Biochea, 39:1.56 (1971). Electron-i&pact ?ro;noted Fraq entation of Alkyl-X-(1.Phezjjlethyl)- CarbaLzates .of Primary, Seccr,dary 2nd Tertiary Alcohols; W, E, Pereira, B. Ralpern, b!. D. SoloTon and A. M. Duffield, Org. blass Spectrozetry 2:157 (197?). Peptide Sequerxing by L&f Resolution Xass Spectronetry; V, Bacon, E. Jell=, il. ?ztton, ii. Pereira aad 3. iialpern, Biochen. Eiophys. 3es. Cozr,;~?. x:873 (1969). A Gas Liquid Chrozstographic Xethed for the Detemination of Phenylalasise in Serum; E, Jellun, V. A, Close, V. Patton, W, Pereira and B, !Mpern, Anal, Biochea. 2:227 (1959). Quantitative Deterzication of Riologically Important Thiols acd Disulfides by Gzs 3iy;id C1:roza:o;rzp?~; E, Jellun, 'rl, ?atton, V. A. Zacon, 'ri, 3. Pereira and B, Halpern, Anal. Biochea. z:339 (1~69). e . . M A Study of the Zlectron Icpact-,, --c=:oted Fragmentation of Prczazine Sulfofide a& pro-szi,?e Usiz,- Specifically Deucerat-ed h.&ogUeS; M, D. Soloxn, R. S~xs, 'ri, 3ereira and A. 51. Duffield, Australian J. Chen. (1973, in press). The Detemination of PInenylalenirie in Serum by Mass Frag=ectogapkJ; WI Pereira, V. A. Sacon, Y. Eoyano, R. Surxons ard A. 14. Duffield, Clin, Biochen. (In press). Chlorination Studies II. The Reaction of Aqueous HyFocblorous Acid vith A-Amino Acids and Dipeptides; W, E, Pereira, Y. Eoyano, R. Su==ons, V. A, Bacon and A, fi. Duffield, Biochen, et Biophys. Acta (In press), r 026) L SECTION II -PRIVILEGED COMMUNICATION BIOGRAPHICAL SKETCH (G&e the fol~owhtg infomution for aN professional personnel listad on page 3. boginnlng wfth rho Principel Invatl~tor. Use continuation pages and follow the mm gene& fmet for eech penon) NAME TITLE WRTHQATE (Ma. Day, Yr.) Thomas C. Rindfleisch Research Associate 12-10-41 PLACE Of Elf? TH (City, Stete, Country) PRESENT NATIONALITY (If non-US citizen, SEX indiceh khd of vita end o xpimtlon &to) Oshkosh, Wisconsin, USA USA I3 MaI0 0 Femrlr EDUCATION (Begin with becceleuroete treining end include postdoctor8lj INSTITUTION AN0 LOCATION DEGREE YEAR SCIENTIFIC CONFERRED FIELD Purdue University, Lafayette, Ind. B.S 1962 Physics . California Institute of Technology, M.S 1965 Physics Pasadena, CA Ph.D Thesis to bc completed. All course work and examinations completed. HONORS Purdue University, Graduated wlth.Hlghest Honors, Sigma Xi. MA@R RESEARCH INTEREST Space sciences, computer science and image processing ROLE IN PROPOSED PROJECT Technics1 Support RESEARCH ANDDR PROFESSIONAL EXPERIENCE (Staning rvithpr~ntposidon,~ondoxpe~ecrr~w~nt toana ofprojac Listrtl W mai nprwenmdve publiationr Do not uce& 3 es for mch indfvidrul.) 19710Present Stanford University Medical School, Department of Genetics, Stanford, CA. Research Associate - Mass Spectrometry, Instrumentation research. 1962-1971 Jet Propulsion Laboratory, California Institute of Technology, Pasadena, CA. Relevant Experience: 1969-1971: Supervisor of Image Processing Development and Applications Group. 1968-1969: Mariner Mars 1969 Cognizant Engineer for Image Processing 1962-1968: Engineer - design and Implement image processing computer software. 1. Rindfleisch, T. and Willingham, D., "A Figure of Merit Measuring Picture Resolution," JPL Technical Report 32-666, September 1, 1965. 2. Rindfleisch, T. and Willingham, D., "A Figure of Merit Measuring Picture Resolution," Advances in Electronics and Electron Physics, Volume 22A, Photo-Electronic Image Devices, Academic Prese, 1966. , Thomas C. Rindfleisch PUBLICATIONS (cont'd) 3. 4. 5, 6. 7. a. 9. Rindfleisch, T., "A Photometric Method for Deriving Lunar Topographic Information," JPL Technical Report 32-786, September 15, 1965. Rindfleisch, T., 'Photometric Method for Lunar Topography,' Photo- grammetric Engineering, March 1966. . * . Rindfleisch, T., "Generalizations and Limitations of Photoclinometry," JPL Space Science Summary Volume III, 1967. , Rindfleisch, T., "The Digital Removal of Noise from Imagery,' JPL Space Science Summary 37-62 Volume III, 1970. Rindfleisch, T., 'Digital Image Processing for the Rectification of Television Camera Distortions," Astronomical Use of Television-Type Image Sensors, NASA Special Publication SP-256, 1971. Rindflpiach, T., Dunrie, J., Frieden, H., Stromberg, W., and Rulz, R., "Digital Processing of the Mariner 6 and 7 Pictures," Journal of Geophysical Research, Volume 76, Number 2, January 1971. Rindfleisch, T., "Digital Image Processing," To be published, IEEE Special Issue, July 1972. ' (Give the following informs don for all prof~ssio,rajperson~el liskd on ,oage 3, bginnbg with the Principal I~~vcsti~~lcr. USC continuation pqcs and foilov/ the Simc yencrel formet for each parson.) PRESENT NATIONALITY I/f non-US ritizen, indicate kind of visa end expiration date) New York City, New York I U.S.A. I n VI+ J, . ,.. . . g Fcmr;lc _ ED lJ CA TI ON (Eq!n v;irh b~cc~:aur~~tc trdinirq and include pas tdoc toral) INSTITUTION AND LOCATION , I DEGREE YEA9 SCIENTIFIC COiJFERRFD FIELD New York University, Kcw York . New York University College of Medicine, B.A. 1942 New York M.D. 1945 Phi Zeta Kappa, Alpha Omega Alpha Wead Johnson Aorard for Pediatric Research, 1960 President, Society for Pediatric Research, 1966 _ ----. MP..IOF( OE:;!ll,taI /n~s: L-r:g,: U:e conthua?iu:l pqcc ii!d f.?\lO;Y fhe ShW? gencfJ/ fofn:;lt for e&l per;on.) NAME --.--_---_ TITLE ' BIFtTiiDA't E I,:`?., ,-. Herbert C. Schwartz Professor of Pediatrics May 8, j-926 PLACE OF BIRTH (city, Smte, Country) -- PRESENT NATlOlJALlTY Cf con-U.S indicate kind of vim and cxpirztion date/ citizen, 55x New Haven, Connecticut U.S.A. f?J 5.';!-? EDUCATION licp:`n with bxcil~vrc.~tc trJainin~andinc/u~cpo:cd~:torJ// INSTITUTION AKD LOCATION DEGREE YEAR CONFERfiED Alma College, Alma, :*!ichigan Illinois Institute of Technology, Chicago Yale University, New Haven, Connecticut A.B. - 1948 Psychology-C!:::.:. State University of New York, Brooklyn M.D. 1952 . HCNORS - _ - John and Mary Markle Scholar in Acade mic Meeicine (1962); Visiting &of. State yr:i..- Netherlands (19G7); SOCIETIES: Society for Pediatric Research (1961); &.ericz.n s:z. Clinical Invesgigation (1962); American Pediatric Society (1967), etc. MAJOR FiESEARCH If47 EHEST -. ROLE IN PROPOSED PROJECT Hemoglobin Structure and Synthesis Investigator U. S.P.H.S. Grant P.01 X4 12467-10, ForrAtion of Hemoglobin and Other HezproteLnc June 1, 1968, to Play 31, 1973. Current year $28,866.- Total (Years 6-10) SiSj,TSJ. . RESEARCH kl<&`Zi? PFOFESSIOSAL EV?EEIE>~CE -- fSI~rti,-~ !:?th prmmr posirion, ks t I rain I:?? end exocricnce r2;e.v .? t to c .-r'~ c :. :` _ -7 . . . OInlOSt Cpk5enlJi;VC publ~w;ionr. 00 ncr cxcezd 3 ;.ye s ror P3ilI ind:`viSYa/. ) 1968 - 1971 Professor of Pediatrics, Stanford University 1969 - 1971 Ch.cirTan, &.pnrtcxtt cf E'eGiatrics, Stanford University 1963 - 1968 Associate Proiesscr cf Pediatrics, Stanford Unversity 1960 -.1963 Assistant i?rofcssor'of.Pc:cdLci.nc, University of Utah 1957 - 1358 Research Fellow in Ciochaxistry, University of Utah 1955 - 1957 Research Fcllotr in hfedicine ( Hematoiogy ), `Cnivcrsity of Utah Publications (Selected from a total of 19.) . Schwartz, H.C. , Cert\,right, G.E., Smith, E.L., and Wintrohe, M.M.: Studies on th? : thesis of Hcxc from Iron and Protcporp?iyrin, Blood 14:486, 1959. - Hill, R.L., S>z.nson, R.T., and Scheartz, H.C.: Characterization of a Chcxical. i-t:.:: in Hemoglobin C. J. Biol. Chcm. 2?5:3l.S2, 1960. Gribble, T.J., and Schwartz, H.C.: Effect of Protoporphyrin on Hemoglobin Sy:::::,:: them. Biophys. Acts. 103:333, 1965. Dnllun, P.R. and Sch~zrtz, 1i.C.: :i:,.oy,?o'~ir. and C srOCl:!:C-:C C Cci:OS~sc dl!l-ilt~ ::~ . ...1 DCiiCiCtlCy ir; the !;at. J. Clin, invust. 4<:1631, 1365. - FIalters, T.R. , \.!el.I.at~d, F,H. , Gribble., T.J. ,a and Sch~xrtz, 1I.C. : Bj.osynth~t7~ :` '. LCUkCTTl!.C J8Clll:OC~tCSs CZllCClY ?.0:117# 1367. --- Idj.~~co]p, D, I l:dpunds, 1).,1, , Gr:i.i,i!!.c, T,J.,aDd SCkl;:artZ, fl*C*: pcybfefcd !ic.- '1% : 5,' ~.,S~::i:-;~,~-c'crd-..(~tl-!:~:c~~-,s ,-- Jriu~ls.r:y-,l9-%3).. .__. _-_--__.__--a- ,,-,.,~-,-__-..,~.:_: . rrv CURRICULUM VITAE Eric M. Shooter Born: April 18, 1924 Mansfield, Nottingham, England. Married: Elaine Arnold (Born Dec. 22, 1924) Newhall, Burton-on-Trent. Children: Annette (Born Nov. 18, 1956) Redhill, Surrey, England. Permanent Address: Department of Genetics - 1942-45 1942 1943 1945 1945-46 nd 1946-49 1949 1950 1949-50 1950-53 1953-63 1961-62 1963-68 1964 Senior Scientist in charge of Biochemistry, Brewing Industry Research Foundation, Nutfield (Proteins and enzymes of barley and other brewing materials). Lecturer in Biochemistry, Department of Biochemistry, University College, London with Professor Ernest Baldwin. (Molecular biology of normal and abnormal haemoglobins; protein-ion inter- actions of ribonuclease). : U.S.P.H.S. International Fellow, Department of Biochemistry, Stanford University School of Medicine, with Professor R. L. Baldwin (Replication of DNA). Associate Professor of Genetics, Stanford University School of Medicine (Molecular Neurobiology). Head of Neurobiology Group, Lt. Joseph P. Kennedy, Jr. Laboratories for Molecular Medicine. D.Sc. University of London (awarded for distinguished work in the field of Biochemistry). 1968 Professor of Genetics, Stanford University School of Medicine. Stanford University School of Medicine Stanford, California 94305 Natural Sciences Tripos (Part If in Chemistry) University of Cambridge Exhibitioner of Gonville & Caius.College, Cambridge Minor Scholar of same. B.A. (Cantab.) : Research under Professor Sir 'Eric Rideal in the Department of &lloid Science, Cambridge and the Davy Faraday Laboratory of the Royal Institution, London (Proteins of the ground nut). M.A. (Cantab.) Ph.D. (Cantab.) Postdoctoral Fellowship with Dr. J. W. Williams, Department of Chemistry, University of Wisconsin, *Madison, and partly with Dr. D. E. Green, Enzyme Institute, University of Wisconsin. (Enzymes of the electron transport system). 1968-present Professor of Genetics and Biochemistry, Stanford University School of Medicine. 69. Varon, S., Nomura, J. and E. M. Shooter, 1968. Reversible dissociation of the mouse nerve growth factor protein into different subunits. Biochemistry 1, 1296-1303. 70. Shooter, E. M., Smith, A. P., and S. Varon, 1968. Heterogeneity of the nerve growth factor protein and its subunits. 'Fed. Proc. 27, 464. - 71. Herschkowitz, N., McKhann, G. M., Saxena, S. and E. M. Shooter, 1968. '.. Characterization of sulfatide-containing lipoproteins in rat brain. J. Neurochem. l5, 1181-1183. 72. Smith, A. P., Varon, S. and E. M. Shooter,'1968. Multiple forms of the nerve growth factor protein and its subunits. Biochemistry L, 3259-3268. 73. Greene, L. A., Shooter, E. M. and S. V&on,' 1968. Enzymatic activities of mouse nerve growth factor and its subunits. P.N.A.S. 60, 1383-1388. 74. McKhann, G. M. and E. M. Shooter, 1969. Genetics of Seizure Susceptibility & Basic Mechanisms of the Epilepsies, H. H. Jasper, A. A. Ward, Jr., and A. Pope, Ed., Boston, Little, Brown and Company, Chapter 24. 75. Herschkowitz, N., McKhann, G. M., Saxena, S., Shooter, E. M. and R. Herndon 1969. Synthesis of sulfatide-containing lipoproteins in rat brain. J. Neurochem. l6, 1049-1057. 76. Goodall, P. T. and E. M. Shooter, 1969. Changes in Heme Environment due to subunit interaction in hemoglobin. J. Mol. Biol. 39, 675-678. 77. Smith, A. P., Varon, S. and E. M. Shooter, 1969. Equilibria of the Nerve Growth Factor proteins and their subunits. Fed. Proc. 28, 897. 78. Greene, L. A., Shooter, E. M. and Silvio Varon, 1969. Subunit interaction and enzymatic activity of mouse 7s Nerve Growth Factor. Biochemistry 8, 3735-3741. 79. Shooter, E. M., Smith, A. P., Greene, L. A. and S. Varon, 1969. Aspects of the dissociation equilibria between 7s NGF and its subunits. Abstracts of Second International Meeting of the International Society for Neurochemistry, p. 366. 80. Waehneldt, T. V., Grossfeld, R. M. and E. M. Shooter, 1969. The solubili- zation and electrophoretic analysis of membrane proteins from mouse brain. Abstracts of Second International Meeting of the International Society for Neurochemistry, p. 411. 81'. Swfth, A. P., Greene, L. A., Fisk, H, R,, Varon, S. and E. M. Shwter, 1969. Subunit equilibria of the 7s Nerve Growth Factor protein, Bit?- chemistry 8, 4918-4926. 8& Shooter, E. M, and S. Yawns 1974, Macromolecular aspects of the Nerve Gmwtk Factor proteins a Protefn MetaB&isar of the Nemw $ystem, A. Lajtha, Ed. q New York, N,Y. $ Plenum Pww, 41%438,- SECTION II - FPIVILEGED CC:!!.`U"JIC:iTlOtJ CI ,.,-. ,- ( .) - . -- --- BIOGRAPHICAL S!:ETCH (Give the fofio&ng information for ~IIprofcss.kn~l F?rsonnd lislcd or) pqc 3, bYinning v.5'th the Prin?ipaf Inv=t:gzlor. N&E Philip Sunshine Use continuarion p.?:cs and IO:/OW th? same general format for each person,) TITLE * BIRTHDATE !:.!a, Cd/, Yc.1 . 'As'sociate Professor June 16, 1930 PLACE OF BIRTH Kiry, Sfafc, Country) PRESENT NkTlOrdALITY (If noa-U,S CitiZFn, SEX indicate kind of visa and expiration date) Denver, Colorado . American gQ F.%lls g FPT>:~ EDUCATION (Sqin rvith bzcczlaornfc twining and kcludo pcstdsctoral) INSTITUTION AND LOCdTlON DEGREE YEAR SClElJTlFlC CONFERRED FIELD University of Colorado, Eouldcr, Cola, B.A. 1952 Pre-Medicine . . . University of Colorado,. Denver, Colo. M.D.* '1955 Medicine . HONORS Alpha Omega Alpha Council Western Society for Pediatrics Ross Award for Pediatric Research 1970 Research, 1972 MAJOR RESEAfiCH I;,!TE!?EST ROLE IN PROf'OSEU PfiOJECT Developmental Gastroeneerology and Nutriticn Investigator RESEARCH SUPPORT (Se jnsrrv;tjonsj RR 81 - Clinical Research Center for Premature Infants, lo/%9 - g/30/74 Amt. Current Year: $385,882 - Total Account:- $2,3&0,59$ (Xorman Kretcker - Principal Investigator) . RESEARCH AND,QR PROFESSIONAL E.XPERi EIJCE (Starting r;ith pruent po:ition, list rr,~~nin~~andexpcience rc!&ant 13 arca nf ,?ro,+st L..:- `Of most rr?prcscnt.&c pub//cations. 00 not pxcced 3 pqes for each indrviduaI,I . - Associate Professor of Pediatrics 1968~present Stanford Univ. Sc!;ool of licr`lcl::c Director, Cmter for Frerxature Infants 1968- present Stanford Vniv. Scksl of :-Ictlic1-.: Instructor through Assist. Professor 1963-1968 Stanford Univ. School of :!sdicir% Fellow in Pediatrics . . -'-X961-1963 Stanford Univ. Sch:c)ol of :I;3ici::.z Resideqt in Pediatrics 1959-1961 Stanford Univ. School of tlz.diclz;~ Publications (Selected) . . Sunshine,. P. and Kretchmr, N.: Studies of mall intestine during developezt. III. Infantile diarrhea associated with intolerance to disaccharides. Pediatrics 22:. 38,' 1964. Sunshine, P. and Kretchmcr, N.: Absence of intestinal disaccharidases in two species of se2 lions. Science 144:SSO, 1964. Herbst, J.J., Hurwitz, R., Sunshine, P. and Krctchmer, N,: Effect of colchicir:e on intestinal disaccharidascs: Correlation with biochcnical aspects of ceJ.luler rcneval. J. Clin, Invest. 19:532, 1970. - Sunshinc, P., Ilcrbst, J.J., !:oldwsi:y: 0. md Krctchxr, !3.: hdqtztion of t!.,? gastrointesr.inal tract to extrautcrJ.nc iife. Ann. N.Y. Acad. Sci. lik:iG-7'?, 1971. , * Sunshine, P., ct al.: 1Ipperaemoncmia due to a dcfcct in hcpatic ornithine t!-:rrr-.cnr? '. -- n,ls.~;~r Pediatrics z:lOO, 1972, -- -...-- ---s-L_ 1 , . . - -pz5i- - -. , . . Tsuboi, Kenneth K. Senior'Scicntist - t- February 7, 1922 I'LI.CC CF BIRl H (City, Stole, Country) PRESENT NATIOtJALITY (If rmn4.1.S~ citizen, hdicu te kind of visa and expiration date) Okayama, Japan --- INSTITUTION AND LOCATION St , Thomas College, St. Paul, Minnesota University of Xinncsota, Xinn. , Plinil. . University of Xinnesota, Ninn., Minn. d . tlOI:OHS DEGREE B.S. 1944 F1.S. 1946 Ph.D. 1948 P ,ch.fc ~?,,sfdo:to: YEAR CONFEftfcED - Established Investigator; . American lieart Association; 1960 - 1964. . SCIENTIFIC FIELD Chem, Biochem. Biochem. MAJOR RESEAHCtf I!:TIiliay 31, 1972. Current $12,500. Total $102,880. RESkAfXtl AtiD/OR PHOf`ESSlO!:I..L EXf C'itEt:Cc _ (Sftir!ir~.? with prrsrnt position. fist trnininb and cxpcricnco rolcvcnt tz (IICG ol prc;rc?. Lirt nil 0' rr8.3:t tc,zrar.nt.~tive p;r.l,licoticns. (Ijo net c*csrS ----.i 1966 to prcsc:nt 3 p;;c s for cat.? indiviiu;z:.) - Senior Scientist, Biochemistry in Pediatrics 1460 - 1966 - Associate Professor, Biochzxistry in Pediatrics, Stanford ?!edical Schoo: 1957 - 1960 - Assistant Yroicssor, Siochcmistry in Pediatrics, Cornell _ Xedical '(:::ool 1951 - 1957 - Researcil Associate, Ziocixxiistry, 1948 - 1951 Columbia University ?ledicai Sc:;col - Research Associate, Oncology, University of Kansas Xedicrl Sc!;ool Publicati&x (Five relevant publications are listed frcm a total of 48). Tsuboi, K.K., Greenberg, R.E., and Kretchner, I!.: CarbohydGhte PIetnbolisn, ix Bi.olo:ic,-1 Baais of Pediatric Practice, R.E. Cooke, cd., XcGraw-Hill (1968). ----.A--. ---- Tsuboi, X.K., Fukuncga, K., and Petricciani, J.C.: Properties of Eryt!lrocyte Uridine Purification and Specific Kinetic Diphosphste Glucose Pyrophosphorylase. J.;l.lol. Chxn. 2U, 3.008 (1969). Tsuboi, K.K., i'ukunaga, K.: Relationship of solute permeability to crythrocytc gl~colys::. Biochem. Biophps. Ac.ta, w9(, 215 (197Oj. Uchino, 3. and Tsuboi, K.K.: Actin Accumulation in Developing Rat Muscle. Amer. J. Physiology, 2l9, 154 (1970). Tsuboi, K.K., Fukunn:a, I;., and Chervenka, C.H.: Phosphoglucose Isonwrase from Iluxn Erythroc:;tc. Preparation and Propertxes. J. dial. &cm., 2!!6, 7SSG (1971). -- CII--- l:l:S.?!% . -m-q- .- ---II--- _- pv. 7.69 @-2zs