Meeting Transcript
November 16, 2006

COUNCIL MEMBERS PRESENT

Edmund Pellegrino,M.D.,Chairman
Georgetown University

Floyd E. Bloom,M.D.
Scripps Research Institute

Benjamin S. Carson, Sr., M.D.
Johns Hopkins Medical Institutions

Rebecca S. Dresser, J.D.
Washington University School of Law

Daniel W. Foster, M.D.
University of Texas, Southwestern Medical School

Michael S. Gazzaniga, Ph.D.
University of California, Santa Barbara

Robert P. George, D.Phil., J.D.
Princeton University

Alfonso Gómez-Lobo, Dr.phil.
Georgetown University

William B. Hurlbut, M.D.
Stanford University

Leon R. Kass, M.D.
American Enterprise Institute

Peter A. Lawler, Ph.D.
Berry College

Paul McHugh, M.D.
Johns Hopkins University School of Medicine

Gilbert C. Meilaender, Ph.D.
Valparaiso University

Janet D. Rowley, M.D., D.Sc.
University of Chicago

Diana J. Schaub, Ph.D.
LoyolaCollege

Carl E. Schneider, J.D.
University of Michigan

INDEX

• Session 1: Stem Cell Research Update
  
• Session 2:   Stem Cell Research Update and Alternative Sources of Pluripotent Stem Cells (May 2005)
  
• Session 3: Overview: Genetics Research and Clinical Applications
  
• Session 4: Overview: Genetic Ethics and Public Policy
  

   

WELCOME AND ANNOUNCEMENTS

DR. PELLEGRINO:  Thank you all for being so prompt.  Welcome to the opening of our meeting of the President's Council.

My first act, as always, is to recognize the presence of Dr. Daniel Davis, who is the Executive Director and gives legal and government legitimation to our proceedings, and even to my chairing, which he can remove me from, I'm sure, any time.

(Laughter.)

DR. PELLEGRINO:  Perhaps should, but thank you, Dan.

I would like before we start just to recognize a distinguished visitor who's a friend of mine and a friend of our first speaker, and for that reason particularly, I'd like to introduce Professor Tony Altieri, who is Professor of Theology at University of Münster.

Thank you very much for being here with us, and we invite you to participate as you see fit.

We have a varied agenda for the next day and a half.  This morning we will be beginning with an update on the science relating to stem cells.  This is the result of our survey of the members of the Council, many of whom said they thought it would be useful to be brought up to date on the scientific changes over the past couple of years.

SESSION 1: STEM CELL RESEARCH UPDATE

And so to that end, we have dedicated the first session to that subject.  You have the agenda before you, and as in the past, we have not engaged in extended introductions.  So I hope you'll forgive us for that, but material is available, and obviously many people around the table know our first speaker, a distinguished investigator in the field of cellular biology and related issues on stem cells.

So I would like to ask you to come to the podium and to begin the session.  When Professor Schöler is finished, we have had agreement by a member of our Council, Dr. Floyd Bloom, who will open the discussion.  I want to thank you in advance for your willingness to do so.

Dr. Schöler.

DR. SCHÖLER:  First of all, I would like to thank you very much for this invitation.  It's a big honor for me to be here, and I'm happy to see some friends here in the audience.  I hope I can provide you with an idea of what I think has been interesting with respect to stem cell research over the last, let's say, one or two years since you had the Alternative Sources of Pluripotent Stem Cells published as a white paper.

The way I would like to start this is by raising an important point that you will see again and again.  That is, our body — soma — is something which is not lasting forever.  As you can see in this scheme, our bodies are aging, and if you think about what is maintained from us, that is our germline, that information which is passed from one generation to the next.

And with respect to regenerative medicine, the germ line has turned out to be extremely important, and a couple of publications on that issue did come out in the last few years, and I'm going to emphasize their importance.

To understand the mammalian germline, scientists are mostly using the mouse, and as you can see here, the highlighted germline of mammals, in order to see the parts of the germline, and it's obvious to all of you that the germ cell lineage giving rise to sperm and eggs is part of the germline.  Some people think that is the germline in mammals, but that's not true because you have cells which give rise to the germ cell lineage and will also give rise to the bodies.

You see here three mice.  We're talking about cloning today.  These have been cloned by the computer, by copy and paste not by nuclear transfer.

Now, these cells that give rise to the three germ layers, ectoderm, mesoderm, endoderm, and the germ cell lineage, these are the pluripotential cells that are in the focus of science and also public discussions, and both together, the pluripotential cells and the germ cells, comprise the mammalian germline.

That's different for other model species, like drosophila or C. elegans, where the germ cell image is set aside very, very early.

Here the germ cell image isn't used until a rather late stage.  In the case of a mouse, it's like one-third of development before birth.  So let's say seven days up to 20 days that it takes a mouse to be born.  That's when the germ cell image is induced.  Before that, there are no germ cells or progenitors of germ cells.

And we can look at this not only in a linear way, but in a cyclical way.  You have these cycles giving rise to new individuals after fusion of sperm of oocyte.  You basically can say the germ line lineage is the only lineage of a cyclical nature in development.  All others terminate at some stage.

So you have these two phases here.  The first phase, the phase where you have pluripotential cells; beginning even totipotential cells come to that, and here at the time that the embryo starts to gastrulate, when the three germ layers are formed, that's when the primordial germ cells are distinguishable.

And then they migrate as the embryo and the fetus develop from a posterior position in the embryo to the gonads and then eventually will have sperm and oocytes to start the cycle again.

So you have these two phases, the germ cell phase and this first phase, the phase of pluripotential cells, and it has, you know, been extremely fortunate for scientists that from this early phase, cells can be derived from different stages, pre-implantation stages.  Cells can be derived that can be cultured in the dish.

And the amazing thing is that these cells in development only show up for a very short period of time.  Once the embryo gastrulates, there are no pluripotential cells, these cells that can give rise to the three germ layers and to germ cells.

But you can take these into culture, and you can basically maintain these cells for an extremely long period of time, and ifyou think about the first embryonic stem cell lines that have been derived by Jamie Thompson from human blastocysts, these, the three lines that are mostly used called H1, H7, H9, three embryos that would fit on the tip of a needle have generated embryonic stem cells distributed all over the world, which I think if it would take them all together, you would have in the grams or even higher numbers.  Maybe you can even have in the range of kilograms by now embryonic stem cells that are derived from these three embryos.  So they have an enormous proliferation potential.

Just to at least mention that here — I will not get into that today — one of the focuses of my research is to try to get the germline, the mammalian germline cycle, into the dish, and that's for scientific reasons, but also for practical reasons that we can derive from zygotes eight cell embryos that we can use to derive embryonic stem cell lines from these, derive oocytes from the oocytes, derive metaphase II oocytes that we can use for nuclear transfer.

So if that cycle is completed and the only missing link for us is that from these oocytes we have not derived from embryonic stem cells, we have not succeeded in getting oocytes that are good enough so that we can do nuclear transfer with these oocytes in mouse, and so that is the major focus of the research of my lab in Minster currently, to fill that gap.

All of these others, nuclear transfer with mouse is something which we do routinely.  These steps and these steps here have all been done at the lab, and we'll start next year to try to do this cycle from embryonic stem cells to oocytes here with human embryonic stem cells.  So far we have been only working with mouse embryonic stem cells.

Now, if you look at embryonic stem cells, you have a very simple definition.   You have cells that make themselves again at more different stage of cells, but there are different levels of stem cells, and you can take the first cells, the mother of all stem cells, the oocyte, that after being fertilized forms a zygote, which is totipotent.  You have pluripotent cells, multipotent, and then eventually you have unipotent cells. 

So there's a restriction in potency during development, and that makes sense.  You'd rather not have a totipotent or pluripotent cell in muscles because you might risk to form a tumor.  Potency goes along with potential to form all of these different lineages. 

So at the end you rather have something which is more restricted and specialized, starting from here, this all-rounder as I call it, and in specific, you want to have a specialist at the end which is doing its job and it's not doing everything.  You want to have somebody who can do the job.  So you have these specialists at the very end.

And it makes sense if you just think about how an organism develops.  So you're starting off with the totipotent zygote, which can form an organism, but then you come to a stage where you have cells that potentially can form all different cell types, but they don't have to do that in a concerted way.  You can show today that a pluripotent cell forms ectoderm tomorrow, and mesoderm and endoderm and germ cells.

And in vivo this would be shown by moving the cells around in the embryo, transplant the cells from one position in the embryo to another one.  That's how you can show that they are still pluripotent.  They can still do all of these different things.

And, again, from these stages here, that's where you can derive embryonic stem cell lines.  You can't get them from a later stage.

And as a summary to my introduction, pluripotential cells, if somebody tells me I've found a new pluripotential cell, then I ask him can it form derivatives of the three germ layers and can it form germ cells.

Germ cells are mostly forgotten in that in proving that these cells are pluripotential and the best way to prove that they are pluripotential is to show this both in vivo and in vitro.  That's something that has been done for embryonic stem cells at least for mouse and partially for human embryonic stem cells.

If you just concentrate for a second on adult stem cells, these are extremely useful cells because these are specialists that can be used to restore some tissues, but not all, and also, they might be able to augment survival after damage, like after heart attack.  If you provide them at the right time, they might help so that the heart cells, the cardiomyocytes will survive.

Even if they are not forming cardiomyocytes, they might help other cells to migrate to that area and help them survive.  So you have to take hematopoietic stem cells.  You all know that is the best system, the best stem cell with respect to therapies.  People have since many years been using after chemotherapy or radiation.  Before the chemotherapy, they took the hematopoietic stem cells and brought them back.

But this has not been shown that you can use hematopoietic stem cells or other cells, for example, to form neurons in a way that these cells then can be used for treating, for example, Parkinson's.  So I think that is something which still has to be explored.

But the potential, if you just think about what I said at the very beginning, the potential is very limited.

On the other hand, if you would try to use embryonic stem cells for therapies, uses all around us, you had better know what the specialists can do and what the specialists are so that you can convert these embryonic stem cells to neural stem cells or hematopoietic stem cells and then bring them back.

If you would try to do this right away, then you would risk that these cells form tumors, and that is an outcome of quite a number of experiments that people do not really know what kind of intermediate, what kind of specialists.  They haven't even tried to inject the derivatives of embryonic stem cells and are surprised that tumors are formed.

In that respect, I was very pleased to see this paper published by Austin Smith last year, in September 2005.  The reason why I thought this is a key paper for me, that he succeeded with mouse embryonic stem cells to derive neural stem cells.  So basically he converted an all-rounder to a specialist.

And this is important.  We can't see it down here.  It's a stable intermediate.  He can culture these cells almost like a cell line and can take these cells and inject them into the brains of mice and then can get functional derivatives and does not risk — as far as I know, there was no tumor formed after these injection experiments, transplantation experiments.

So that is something which I think is very crucial if you would like to benefit from embryonic stem cells.  You need a thorough understanding of adult stem cells.

So I think what I would like to stress here is that both adult and embryonic stem cell research has to go side by side.  If you just concentrate on one or the other, you will not be able to unravel the full potential of either.  I think that's a statement, one of the very strong statements I want to make that you have.  If you even want to think about developing therapies, or develop their full potential, you have to study both side by side, and this is something that we can discuss later.

Now, from now on I will concentrate on pluripotential cells.  And the question is:  how can they be obtained?

And for that reason, it was important for me to show you the distinction between soma and germline because these are the two different sources for obtaining pluripotential cells or how people think pluripotential cells can be derived.

One way is deriving pluripotential cells from germline cells.  The other one is reprogramming of somatic cells.  That means non-germline cells.

This is a picture, which might remind one or the other here about Waddington schemes.  Here you have at the very beginning of this mountain, you have the zygote, which then will form an embryo which contains this inner cell mass, and then this totipotent cell is kind of rolling downhill to eventually form a germ cell.  That will be down here.

And on its way, it's forming all of these different lineages, which leave the mammalian germline.  So you see here the trophectoderm.  You see your hypoblast, and then here are the three somatic lineages.  And the primordial germ cells from here on would then normally not form any of these lineages.  That's at day seven, as I said, in mouse.  That's the time point when the germ cell lineage has been allocated.

What happens here, as you concentrate on the germline, you have an inner cell mass of pluripotential cells.  That is cells of the inner cell mass at a different shading to primordial germ cells which are unipotent.  That means primordial germ cells will give rise to germ cells, but not to somatic lineages.

The pioneer of transplantation of germline cells is Ralph Brinster.  This pioneering work started more than ten years ago where he showed that spermatogenesis following male germ cell transplantation can be done with mouse, in mouse, but also with rat spermatogonial stem cells in mouse test.  So we have complete rat spermatogenesis in mouse.

And this work has been proliferating enormously over the years, and one of his post-docs after he started back in Japan, Takashi Shinohara, he actually showed that you can use not only spermatogonial stem cells from testis, from the testis, from the adult testis and from the neonatal, but you can use primordial germ cells, those very early cells that, as I said, around day seven or later, they can be transplanted into postnatal mouse testis and could even go a little bit further back.

So it's not only here spermatogonial stem cells, primordial germ cells, but also epiblast cells, which I would position right here, he could use for transplantation in testes.

But in general you would say that's fine.  That's going the right direction from, you know, soma cells to primordial germ cells.  Still that was a big surprise.

What I want to say here is that along this germline axis, there's some freedom, experimental freedom to move these cells around from what position here straight to such a position, and you can get sperm, and the sperm can give rise to viable offspring without any apparent problems.

Now, that was germline cells and transplantation in this direction.  Are we going uphill?  And that's where it comes to germline cells and pluripotency, the focus of today's talk.

As I told you before, you can derive embryonic stem cells from the inner cell mass of blastocysts, and we know now that can be done even as early as the eight cell stage embryo, that you can derive embryonic stem cells.  I'm going to come to that later again.

Now, at the time, it was a big surprise that you can derive embryonic germ cells from primordial germ cells.  That was a big  surprise because these cells are unipotent, and by culturing these cells, Peter Donovan and co-workers, Brigid Hogan and co-workers have been able to push these cells basically uphill to convert a unipotent germ cell to a pluripotent cell which has many features in common with embryonic stem cells.

And more recently, two years ago, Takashi Shinohara, the one I have just already mentioned, has been working together with Ralph Brinster.  He succeeded in getting neonatal spermatogonial stem cells to be converted to what he calls germline stem cells.

He had to do a trick once he had these spermatogonial stem cells, but before he got them from testes, he could just culture the testis under certain conditions, and then has seen colonies of pluripotential cells in these testes which we think are derived from these neonatal spermatogonial stem cells, but that is something that still has to be explored.

And even more recently, that's the work of Takashi Shinohara, published in Cell, December 2004.

More recently, a German group, Engel in this collaboration with Hasenfuss, who is the cardiologist; he is the germ cell, the reproduction biologist.  He has obtained pluripotent cells from spermatogonial stem cells from adult mouse testes.  That was a big surprise at the time, and this has to be further explored, but here you would also see this is something where these are pluripotent cells derived from germline cells.

There are a couple of points that have to be discussed with both.  I'm going to come to that later.  There's still uncertainty with respect to stability, imprinting, and cancer.  The question, if they are really pluripotent, and I will come to the litmus test later, what a cell also has to do to be considered a pluripotent cell.

So basically, to complete that section you can derive cells which are pluripotent as far as one can tell at this stage from any given stage here up to the adult testis.  I don't think it is possible from any stage.  I would doubt at this stage that spermatocytes can give rise to pluripotential cells, but this is something that will have to be shown.

Definitely you can get pluripotential cells from all the different time points, stages that I just mentioned.

The second part is reprogramming of somatic cells.  These are now non-germline cells, and one reason to do this, besides the scientific interest, the interest that scientists have in this topic, is how to deal with the problems of rejection of transplanted cells.

And one major issue is that scientists try to derive cell lines, stem cell lines that would allow them to study disease in the dish or at least certain aspects of disease in the dish.  Patients with the known genetic disease would provide genetic information for reprogramming of somatic cells, regardless if it's done by nuclear transfer or reprogramming by fusion as I will tell you in a minute.

That's something which I think will lead to a broadening of an understanding of disease, which then eventually can lead, of course to therapy.  But this first is like the basic understanding of disease in the tissue culture dish.

And then there's of course a huge interest in generating allogenic stem cell banks, as I'll mention later, and the major question here is not only with germline cells, but also with somatic cells, can you convert these specialists, these tissue specific specialists or their derivatives to all-rounders.  Can you go uphill with respect to the potency of a cell?  Can you unravel that?

And my personal view with respect to here when it comes to somatic cells, just somatic cells, it's my personal view of what is in the pipeline, what scientists are doing and trying, is highlighted in this picture, and we start off with oocytes and tissue culture oocytes and then come to the other topics.

And as you've seen probably many times, nuclear transfer is so far the only other way to derive embryonic stem cells, to derive embryonic stem cells with the genetic information of a certain mouse in this case, not possible in humans so far.  People are trying hard to do this, but to replace the genetic information of an oocyte by that of another organism, another mouse is working out very, very well in the lab.

And if it comes to human, this search for alternative oocyte sources, people right now, there's a lot of discussion based on what groups in Newcastle have been asking for and applying for, using oocytes from other species. Then there are ways that oocytes may be derived from the ovaries of corpses and biopsies and so on and also egg donations have been discussed.

But one thing that we are concentrating on is in vitro, deriving oocytes from embryonic stem cells in the dish.  This is something that might work out one day, but we can't say that this will work out in the near future.  It's something we are trying hard, but we don't know and others are trying as well.

And if you look at this scheme where I've shown you that from pluripotential cells down to Petri season, down to germ cells, of course, that works very well in vivo and has been shown that you can push cells uphill.

So for us it was not a big surprise that we can use embryonic stem cells to let the cells basically roll downhill to obtain follicle-like structures, and out of these follicle-like structures, structures which resemble preimplantation embryos.

And of course, there's a huge interest in deriving such structures from human embryonic stem cells, and the only thing basically that they ought to do is to be able to reprogram an incoming nucleus. 

I think at the end this will be easier than fertilizing an artificial oocyte, but that's something we really have to see.  The outcome is at this stage completely unknown. 

And as I've mentioned, using embryonic stem cells to develop therapies which understand disease and identifying drugs is something that a lot of scientists are dreaming of.  There are a lot of attempts, as you know, I guess much better even than I, what is happening currently in the States and other countries, Singapore, England, that you derive, for example, neurons from patients with a certain specific disease, and then use them, for example, for small chemical compound screens to see if that disease can be changed to the better.

I have been using now the white paper and also what I have been provided with as kind of a frame to mention a couple of recent publications which fit into that frame, and here in that scheme that has been provided to all of you, there are cells that are obtained from the adult body and which have markers of pluripotential cells, like Oct4.

And in that respect, I would like to mention some interesting papers that these cells or possibly these cells or related cells have been shown to give rise even to male gametes, and here you see this is actually the same group that had published pluripotency  of spermatogonial stem cells from adult testes.  The same person, Karim Nayernia, had three major papers.  Here he was co-first.  He was first here and here.  In three major publications he could show the derivation of male germ cells from bone marrow stem cells.

So I would assume that these cells have been positive for markers of pluripotential cells or due to the culturing of these have developed features of pluripotential cells.  And these are the first to succeed in using embryonic stem cells to give rise to male gametes to fertilize an oocyte, to then generate offspring mice, which were not viable for a long time, but this is as a proof of principle, that you can obtain sperm from embryonic stem cells in the dish.

These publications are complemented by others, one by that of Paul Dyce's lab where he has shown that there is in vitro germline potential of stem cells derived from fetal porcine skin. 

Here he has obtained structures which are very, very similar to oocytes from skin and you certainly have heard about Jonathan Tilly's work where he claims that oocyte generation in adult mammalian ovaries, might occur by putative germ cells in bone marrow and peripheral blood.

It looks like from what I've heard from him at a recent meeting that these cells at this stage are not capable of forming a functional follicle, functional oocyte, but they can kind of develop in a way that the program of oogenesis is developed.

And so the question really is if you have these cells which are Oct4 positive, stage specific antigens, positive, have these been originating from the skin or are these, for example, cells like PGCs that came to certain niches in the adult, then eventually showed up there in the adult body, and then originally were germ cells, were derivatives of the germline, and that is something that has to be studied.

It's not sure at this stage if these are really adult stem cells that we're talking about, but it could be, again, germline stem cells.

So the second part, embryonic stem cell-soma fusion and then segregation, is something that is, again, taken from your overview here, is something that has been studied for quite a number of years in the mouse and then eventually Kevin Eggan's lab has reproduced what has been shown in the mouse also for human embryonic stem cells, and that is that embryonic stem cells can reprogram adult cells and don't have to be adult stem cells.  They can reprogram them after fusion because the embryonic stem cells are dominant.  They take over the program and by all means can convert the adult program to a pluripotential program.

The problem here is that we will still have the chromosome of embryonic stem cells.  So that is something that people are trying to get rid of, and I'll show you one way how people are succeeding and doing that at least to some extent.

So what we have been doing, for example, is to study that process by using cells, different cells from the mouse, fusing them with embryonic stem cells, and we are just looking at the green color being turned on, and by doing this we could actually show that this activity is found in the nuclei of embryonic stem cells.

And a method that has been published by the group of Paul Verma in cooperation with Alan Trounson is that they have been using embryonic stem cells to reprogram adult cells by not allowing the nuclei to fuse.  So you have one that is the adult cell, the other one, the embryonic stem cell.  That's 4N.  So it's twice the number of normal chromosomes, and before the nuclei fuse, they centrifuge these cells.  So the 4N nucleus would be lost during the centrifugation process.

And apparently that appears to be enough to reprogram these adult chromosomes in a way that they acquire features of pluripotential cells.  It's an extremely, from what I can tell from the publication, an extremely inefficient way and has to be optimized to see if, indeed, these are pluripotential cells that are of therapeutic value.

But that would be a way how the nucleus here of the embryonic stem cell can kind of force the adult cell to be reprogrammed.  And here the recent publication which just came out just a week ago or two.  That is that people are trying to get rid of the chromosomes of the embryonic stem cells, and Azim Surani and Takashi Tada have developed a chromosome elimination cassette that would eliminate certain chromosomes of the embryonic stem cells.

So you could use this, for example, to eliminate those chromosomes which would result in host rejection.  Still you would have all of these other chromosomes.  So at that stage, that's an interesting proof of principle study, but it has to be shown if this can actually lead to pluripotential cells that are of therapeutic value.

But I just want to mention these publications, that there are major attempts to have the embryonic stem cell reprogram adult cells and then try to get rid of the chromosomes afterwards.  Of course, that would be something wonderful if this approach would work.

Now, the last group of procedures are here, the cellular vesicles or artificial vesicles or, at the end — I'm not going to talk about this — in situ reprogramming where people will aim to try to bring certain factors to certain organs to reprogram cells to become stem cells in a certain organ.  But this still too (speculative) at this stage.

So pluripotential cells via somatic cell differentiation, you have mentioned this paper, which in my eyes is a key paper, but before I come to this, basically the ideas — a lot of you know this picture better from Kronau— is that you use not human as here, but cells, by buffering cells in a certain cocktail of factors to turn back the program so it would become an umbrella program just by the factors.

And the first paper on that topic has been published by Phillippe Collas, and what he was doing is to use extracts of carcinoma and embryonic stem cells and use this to put adult cells in this cocktail made pores in the adult cells so that the factors of these cells could enter the cells, and he succeeded induction of de-differentiation genome-wide transcription of programming and epigenetic reprogramming by these extracts.

These cells look very promising, but there are still so many tests to be done to see if these indeed will fulfill these hopes that one would have if you look at this publication, which I think is worth reading.

The only problem with this publication, I think, is that they didn't have the rigid biological tests.  Otherwise it would have been published  in Cell and not in Molecular Biology of the Cell.

This is the paper that you have here  and the paper that you have distributed, and I, indeed, consider this one one of the key papers of the last years: "Induction of Pluripotent Stem Cells for Mouse Embryonic and Adult Fibroblast Cautions by Defined Factors."

So in contrast to what I've mentioned before, nuclear transfer or fusion, in this case he has been using defined factors which have been provided to these cells by viruses and has succeeded by using four different factors, c-Myc, Klf4, Oct4 and hidden here is Sox2, to convert a differentiated cell to an undifferentiated.

So of course, he would see that there are many problems with providing viruses and so on, but just the idea by having to have a defined set of factors and converting one stage to another stage is, I think, a major step in understanding, and now people will say, "Okay.  I don't want to have c-Myc.  I think this factor is better than c-Myc or Klf4, which have oncogenic potential.  So you might not want to have this if you want to think about therapies.

And you don't want to have viruses in them, and you don't want them to have them consistently be expressed.  You basically want to bring them as proteins to a cell and then convert it, just like Collas did it with the extract, with defined factors converting one cell type to another.

And then it has to be stable and pluripotent.  There are a little bit of techs who can, because I think this key paper has a couple of features which have to be really understood because based on this paper, there might be too many hopes at this stage, and there are so many things that you still have to understand before something like this can lead to something that can be used with respect to therapies or at least to obtain pluripotential cells.

So I think that the results suggest that Takahashi and Yamanaka, the two authors of that paper have successfully reprogrammed terminally differentiated cells to a state that has features in common with those of pluripotent cells.  I would not call them pluripotent.  I would say that they have features in common with those of pluripotent cells.

However, several observations indicate that as they call them, induced pluripotent stem cells are similar but not identical to embryonic stem cells, and there are three major differences that I want to go through.

One is the absence of any contribution of these cells, these induced pluripotent cells to postnatal animals following blastocyst injection suggests that the cells have a limited capacity to stably integrate into normal tissue in vivo.  That is something that has to be studied more thoroughly and at this stage is a problem.

Although rare induced pluripotential cell clones showed expression patterns of known embryonic specific genes that were very similar to the controls, embryonic stem cells as controls, a substantial degree of clone-to-clone variation was observed, and some clones failed to reactivate a number of the genes assayed and notably none were found to express embryonic stem cell-associated Transcript 1, Ecat1, which apparently is an important player.

Transcription profiling experiments revealed that although these cells cluster more closely to embryonic stem cells than they did to their parental fibroblasts, they still present a distinct gene expression signature.

And the third point is that DNA methylation of the Oct4 promoter as one marker and the post-translational modification of histones positioned there suggested that these cells are caught in an epigenetic state that is intermediate between their somatic origins and fully reprogrammed embryonic stem cells.

So there are things missing, and I think the next months and years will have to be used to find what is missing, but I think these guys are on the right way.  They are on the right way to become, as I trust, to become pluripotential cells.

And so in summary — that's the last slide with a lot of text — in summary, the nuclear reprogramming observed by introduction of these four transcription factors into somatic cells is substantial, but it differs from the more complete reprogramming that is observed after transfer of nuclei from somatic cells into oocytes or after fusion of somatic cells with embryonic stem cells.

By all means, this here, these two ways are resulting in a complete reprogramming.

Several important questions remain. Are these cells trapped in an intermediate state between somatic cells and embryonic stem cells or are they actually some other pluripotent cell type, for example, those that correspond to cells of the epiblast?

And one possibility is that they are, instead of being embryonic stem cells, they could have more features that come with embryonic carcinoma cells.  These are still questions that have to be solved before we can even think about using such cells in organisms.

Now, basically what this type of research is trying to do is to convert the unipotent somatic cell to a pluripotent IPS, induced pluripotent cell, and this at the end might not lead to therapies, but I think it is right now one of the most exciting fields in biology, to try to use this system as a way to understand how a cell is converted from one stage to the other, and you have to do this by using defined factor to understand the molecular biology behind that.

And I guess many groups are going to concentrate on this work based on what Shinohara has published in that key paper.

Now, we have here this scheme, but there's a big "but" here, and the reason for this big "but" is that for some reason a lot of people think that their face is getting older and older, but the DNA is staying young.  This is a major problem.  We are aging, and with us our DNA is aging, and if you think about cloning of an aged person, just by knowing a little bit of biology, it is ridiculous.

But even therapies might be very problematic if you would like to use the genetic material of an aged person, and here is one scheme that I took from a review article, and that is the increase as you see here of mutations in the human population based on what people have outlined in that paper.

So you see that from the very beginning of our life we are accumulating as a human population, accumulating mutations, and statistically seen that is resulting in an increase of tumors in the population, and statistically seen a young person has less risk of getting a tumor than an aged person.  We all know that.

But what we sometimes forget is that there is a time point where there is almost like an exponential increase, and this is called in the literature — it's not my terminology — the end of warranty.

(Laughter.)

DR. SCHÖLER:  Well, I'm beyond this end of warranty because that's 45 years.

So if you think about this, what that means, if you would like to use that genetic material for reprogramming studies, I would say you either have an extremely good screening procedure or you're risking that you're causing problems by therapies and that there are genetic problems.

I just show you one example that we can actually show by cloning.  These are two clones from the same mother, two mouse clones.  It's pretty obvious, and that's why mouse geneticists love this kind of phenotype.  It has a short tail.

And this is interesting because you don't have to open the mouse to see that there's a genetic problem.  There is a genetic problem.  They both originate in the same genetic material, and the offspring, as you can see here, some of them actually have a short tail.  They have a normal length.  They have a short tail.  So this is genetic because it's passed from one generation to the next.

At birth the mice are naked, don't havefur, and they develop this like after two weeks or so, that they get fur.  So that's why they look like small pigs instead of mice in that picture.

So that is genetic, and if you just look at the chromosomes, either this way or by chromosome painting, you won't see that they have a genetic aberration.  It's not obvious from this.  So if you would like to use genetic material of an aged person, you would, I think, run into many more problems than this one I've been showing you, and you still wouldn't be able to pinpoint before you do this that there is a problem or there's not a problem.

And the same note of caution I would raise if it comes to such procedures, which is using — and this is also nicely described in the white paper — I think there might be a reason why these embryos arrest; that if you're not sure that the arrested embryos that are obtained here, like Miodrag Stojkovic has succeeded in deriving embryonic stem cells; if the embryonic stem cells that you have are as perfect as the ones that you derive from a nonarrested embryo, you might be risking that at the end this attempt is a failure.

It's important to follow this, I think, but there are a couple of question marks that you have to be aware of.

And that brings me to my vision, how I think what should be used as genetic material, and that is umbilical cord blood, and not because these cells are pluripotential.  Umbilical cord blood cells are limited in their potential.  They are not like embryonic stem cells.  They might have a bigger potential than originally thought based on the publications that have been out there since during the last few years, two or three years, but I would find them extremely interesting because the DNA is very young, and you would not risk to the same extent that you introduce problems by genetic mutations if you take one of these procedures to reprogram these cells so that they will be pluripotent.

And so umbilical cord blood or another way to use nuclei of HLA-compatible donors, to use any of these procedures to convert these cells into banks of pluripotent and/or multipotent stem cells.  I think that is something that at least in Germany I'm trying to get that established in a network with other researchers working on umbilical cord blood.  Peter Wernet in Düsseldorf is the one person who has these banks and should be with whom we collaborate, and I'd like to see if we can get from these, let's say, at best multipotential or alipotential cells to pluripotential cells, but we'll see if that works out.

Now, the point here that I would like to make is if you ask if you can go back from these unipotential cells to pluripotential cells, I stressed enough that this, I think, is one of the most exciting topics in biology, and the therapeutic potential needs to be explored.

However, we currently only have as a source for useful pluripotential cells and embryonic stem cells those cells which are derived from embryos, and these cells are the gold standard.  And any other cell that you obtain by reprogramming, you have to be able to compare it with these embryonic stem cells.  If they are as good — I doubt they would be better, but they have to be as good, and we don't know if such cells once available can actually replace embryonic stem cells.

There might be genetic/epigenetic problems, cause tumors, and you can see this down here.  So we'll skip right to the next slide.

The crucial litmus test at least in mouse is that these cells have to be able to give rise to a mouse in this tetraploid aggregation experiment.  I took this scheme from Janet Rossant's technical report here.  So basically what has to be done to show that these cells are pluripotent is that you use a clump of embryonic stem cells that you have obtained or embryonic stem cells or pluripotential cells obtained after reprogramming and combine them with tetraploid host embryos, and the host embryo would then form trophoblast and establish the yolk sac, and the rest here, this diploid part, would then give rise to the embryo proper, the mesoderm of the yoke sac, but the embryo proper then has to be born.

If that's not working, these cells are not as good as embryonic stem cells.  That's a standard procedure with embryonic stem cells, and even the report which I think is extremely well done, the one from Takashi Shinohara where he showed generation of pluripotential cells from neonatal mouse testes, he hasn't done this experiment.  Many people who are doing the studies, they either do not report them or don't even do them, these complementation studies.

What he has done here, after deriving these pluripotential or these induced pluripotential cells, a total of 92 tetraploid embryos were created by electrofusion.  So they went ahead with that procedure, and aggregated with these AS cell-like cells and transferred to pseudopregnant ICR females.

When some of the recipient animals were sacrificed at day ten and a half, we found one normal looking fetus and several resorptions with normal placentas.  The normal placenta, of course, is coming from the tetraploid part.  It has nothing to do necessarily with this part.

The fetus showed some growth retardation, but clearly expressed this gene, and none of these were born.  So if you even have this problem with cells of the germline, I am not surprised that people who have been trying to do these experiments with reprogrammed cells are not reporting their failures.

This is something which has to be really worked out, and this, as I mentioned here, has to be the test.  If you have pluripotential cells and claim you have them, you don't only show that the three germ layers and germ cells are formed, but you have to go through this test in mouse and then you know that the procedure is I would say very good or even perfect.

And that brings me to the only way I think one can go ahead at this stage, and that is by pluripotent stem cells derived from biological artifacts, and I would like to provide you with some data from our lab, which I think is making a good case that the proposal, the ANT proposal, is a procedure that at this stage in my eyes is the best way of going ahead if it comes to trying to provide an embryo stage.

And I'm going to show you this data, and it's something we can discuss.  It's what Guangming Wu in the lab has done with the help of a couple of other people in the lab, is to use Cdx2.  That's the gene that has been widely discussed in this group, to knock Cdx2 down, not out.  This is the knock-down approach, and he has done it by siRNA, not like Rudolf Jaenisch has published it, by a viral infection of the nuclei that are transplanted into the oocyte, but in this case, we have been using fertilized oocytes.  See here?  That would be the female pronucleus and that would be the male pronucleus, and injected siRNA against Cdx2.  That's like small, 23 base pair RNAs are scrambled.  The same nucleotides were used, but scrambled.

And then you look at what happens when the zygote is formed and the embryo is developed, and this is a very efficient way of knocking down a gene.  You can see here in this scheme this is quantitative realtime PCR.  That means you can really look at levels.

Cdx2 in normal development with scrambled RNA would increase more and more.  As you see here, this is the eight cell embryo.  The early morula, the morula, the early blastocyst and blastocyst.

You see here that the Cdx2 knock-down experiment reduced levels more than 95 percent.  There's just a little bit left here after that knock-down experiment just by injecting this RNA once at that early stage.

And what you can see here if you look at the development of stages, you see here pictures of early blastocysts and late blastocysts, and these are the ones that have been control treated with the scrambled RNA.

Now, you look here at the Cdx2 treated and you see these stages look very similar.  The eight cell, the early blastocyst and this, the late blastocyst, as we can see here — I hope you can see it from the back — all of these embryos here failed.  They are all intact in the zona pellucida.  They have not hatched in comparison to the late blastocyst that you have here in the controlled treated one, and that's something that none of these in any of the embryos that we have obtained did hatch.

And I've said embryos, but I rather would not even call these embryos.  These stages which correspond to late blastocyst I should say.

Now, if you look here for the protein, this is now by immunocytochemistry.  So you can actually look at the Cdx2 protein here.  You see there is, of course, protein in the control treated one, and you see that there's no protein here in the knock-down experiment.

Now, we look taking a marker of pluripotency.  That is Oct4, and you will see here in the control group Oct4 is where it's lying.  It's in the inner  cell mass, in that area which will give rise to the embryo proper.

In this case, it's all over the place, and if you have an overlay, you'll see here Oct4s all over the place, and there is an Oct4 restriction here in the control treated embryos.

It's important to stress at this point that these look very similar to blastocysts.  If you would look at these here, you would say these are blastocysts, but they aren't.  They look like blastocysts because the oocytes already have RNA and protein which would pump in fluid into these structures.

But this is just a pumping activity which are depending on proteins and RNA laid down in the oocyte.  That you would get regardless if this is an embryo or not.

And as I mentioned none of these embryos — you've been using large numbers — none of these embryos actually hatched out of the zona pellucida.

Now, when we tried to understand, if these embryos at an earlier stage are any different from the control embryos, we looked at the whole genome by RNA profiling.  So we used eight cell mouse embryos that were obtained from eight-cell stage embryos by the control and here compared them with what happens if Cdx2 is knocked down.

And this was done with Kuniya at the RIKEN Institute in Japan.

And if you look here just at this scheme, this is just a comparison.  You will see that even at the eight cell stage, there are differences between the two types of eight cell stages.  You see here even at that early stage, you have like 300 which are higher and 300 which are lower than normal, supporting the idea that the development programs of the two are different, and this is based on the fact — and this has been published by others in the meantime, Dr. Roberts — that there is an early expression of Cdx2.

Here we show this again by quantitative view on PCR, and I should mention, stress that this is a logarithmic scale.  So these are always jumps of ten.  So that actually means that there are very low levels in the metaphase II oocyte.  That's what is used for nuclear transfer, then the two-cell, even lower in the four-cell.  It is really so low that it's a base level and you need a couple of embryos to really be sure about the numbers here.

But that's the nice thing about the siRNA, that you can use large numbers.  You know that you have a group of embryos which behave the same way.

So this goes down and then you have an increase, and you see that there is expression of Cdx2 RNA, and we have been looking also for the protein because that's what's actually important if you want to express genes to see your Cdx2 protein at the zygote stage, and it's very, very difficult to really prove that this is not unspecific.  It's much easier than if you can do the knock-down, if you can look at the result of the knock-down experiment.

Here we see the eight cell stage, and now we have to help you.  I can convince you that if you treat these zygotes with siRNA and compare this to the control which shows weak expression of this protein in the nucleus, you see that there is no expression in the nucleus in the case of the Cdx2 knock-down.

So RNA protein and the profiling data are all in agreement with the fact that at this stage the embryos, the control embryos are different from these knock-down stages.  And since this is a transcription factor, you don't need a lot of transcription factor to turn on these 300 genes and turn off other genes, other 300 genes.

And we wanted to know what's happening here at later stages to see quite nicely when it's strongly expressed, and that's what people have been mainly looking at, Janet Rossant and others.

You see quite nicely that the expression in the nucleus is much stronger, and you see here that there is no expression or there's basically no signal detectable in the knock-down.  That's now the morula stage.

And this is the first time that we see something like asymmetry. This is for Bill Hurlbut.  We had a discussion on that yesterday.  That's the first time that we see something, and it's actually not always like they're fore it in one correct.

At the four cell stage, we don't have any evidence that one nucleus has more protein than the others, what we see at a later stage.

And to get an idea of why the embryos fail, why do the embryos degenerate at a later stage?We again did a profiling experiment with Kuniya, and now at the early blastocyst stage, and there you can see that there are tremendous differences.  You see that about here more than 2,000 probes, more than 2,000 proves are below this level to indicate that there is differential gene expression.  This because there is no trophoblast being formed.  These embryos don't have a trophoblast.  These mainly are trophoblast genes or genes which are expressed  in the trophoblast.

And since there are a couple more pluripotential cells or cells which have features in common with pluripotential cells.  You have a couple more genes about this level here, but this is indicating that there is lineages missing, that these cells, that there's structures here that you can see here by using two different pluri Tarticipation.  This group of cells is now over all the place with Oct4 expression that is present for all cells, and you have the same for a non-knock.

And the reason why they are failing, we think, one reason for that is that the cells don't have tight junctions as they should have. The cells are not linked together as they should, and that's indicated by ZO-1 you see is missing to quite some extent in comparison to the control, and another one, E-Cadherin, which he had nicely distributed in the embryo — see the green color, quite nicely distributed here.  You see that it is a problem with respect to E-Cadherin, and this with Cdx2 knock-down, the phenotype is even stronger than with the knockout that was been published by Janet Rossant.

And now this is really, I think — when we started doing electron microscopy, this was for me an eye opener of what's happening.  We wanted to look at the tight junctions, and you can nicely see here that the way the cells in  trophoblasts are linked together, see here?  These are really tightly knit together here.  Here you can see them and here.

Now, look at the knock-down.  You see that basically they are kind of sticking together, but they're not really tightly linked as you have them here and here.  Here you actually see that this is opening, and that's why it's no surprise that such embryos would pump, but they would collapse because they don't have these tight junctions.

But look at something which is even more exciting, which I did not expect.  Look at the mitochondria.  These are the energy departments in the cells.  You see here these are mitochondria, as they should look like in trophoblasts.  They are long, longitudinal, and have a lot of what is called crista, these structures inside which are providing energy, which are generating ATP.

And look at those here in the knockdown.  These are round mitochondria, which have an embryo appearance here, and you can see them here.  These are not energy producers.  They have more of a resemblance to those of pluripotential cells.

This is quite nice.  I just found this publication, "Energy Metabolism of the Inner Cell Mass and Trophectoderm of the Mouse Blastocyst."  The trophectoderm consumes significantly more oxygen producing more ATP and contained a greater number of mitochondria than the inner cell mass.  These data suggest that trophectoderm produces about 80 percent of the ATP generated, and responsible for 90 percent of the amino acid, not as a turnover compared with inner cell mass. In conclusion, the pluripotent cells of the inner cell mass displays a relatively quiescent metabolism in comparison to the trophectoderm.

So sine you don't have any power houses in these embryos, as you can see here, the control, this is an assay.  It's called JC1 assay, which is kind of showing where the active mitochondria are.  So these red dots there indicate there are active mitochondria.

In this case, the knock-down, even if you have a longer exposure, you at best see a very, very weak signal.  So what I think happens here is that these are pluripotential cells or cells which have a lot of features in common with pluripotential cells, but they  need energy to further develop, and the trophoblast is providing this since this is basically one lineage instead of two.  This is not, to my understanding, an embryo, but is something which is just a number of pluripotential cells.

And now the way that we're trying to show that these are one lineage, just one lineage of pluripotential cells that comes out of this Cdx2 approach is here by visualizing pluripotency, and that is by using the green color, the green fluorescence protein, which has been integrated into the gene of Oct4.

And if you now look at these three stages here, it's an early blastocyst and late blastocyst, and you want to derive embryonic stem cells, you see that these early blastocysts from the control treated ones, in mouse you get about 90 percent embryonic stem cell lines.

In this case, since you know that these are degenerating structures, you can get one out of — you get one line out of 50.  That means two percent which is a tremendous drop, which means that at this stage they degenerate.

Now, if you then ask what you get out of the eight cell stage, here you see that the green color is distributed like a lost egg.  There's some green cells here, but there are a lot of other green cells.  Of course they are because they are two different lineages, one which will give rise to the trophoblast and one which will give rise to the inner cell mass.

And if you take these embryos in culture, that's that you get, derivatives of the outer cells and the inner cells.

Now, look here if you take these ones, which is where I've been claiming that this is just one lineage.  Here you see that this is one glowing green ball of cells.

And if you look at numbers now, you have 22 percent embryonic stem cell lines, which is the same range as you have here with the eight cell embryo, and here you're going up to 34 percent.  So it's not only much better than the two percent, but it's even better than the control treated.  That means if you use Cdx2 in that type of experiment, you get more cells, I think, that have features of pluripotent cells, and my interpretation is that's why you have a higher efficiency of deriving embryonic stem cell lines, and that these are by all means as good as normal cell lines.

Now, the last two slides.  Here, first of all, is section through here.  You see that.  Just look at it.  These are different cells.  If you look here, all of these nuclei, they look over similar.  So this is a more uniform type of cell that you have here, which I think if you do it this way, derive cells at the eight cell stage embryo, you have basically a group of pluripotential cells.

And here, this is the embryonic stem cell line that has been derived from one of these that can give rise to germ cells, that you can form chimeras, and they have even long lasting effects on these chimeras.  And as you can see here, these are stem cell niches where if that would be in a transient, that would not exist.

So in the end, I would just like to highlight again that this here coming from here to here by the Cdx2 knock-down is a very, very efficient process.  So we have now going forward been using them to derive oocytes.  We're trying to get them useful for nuclear transfer so that we can do all of this in the tissue culture dish, but at this stage, I think if you would like to derive embryonic stem cell lines without generating embryos, I think we have to go through a procedure where a gene like Cdx2 is affected.

I think I'll leave this for the discussion.  This is the procedure that Rob Lanza has published.  I had a lot of problems with that procedure because he has been, as a proof of principle, has been destroying so many embryos to show that the procedure is working and selling this as something of high ethical standards that I had a major problem with that.  But that's something we also can discuss.

At the end by reprogramming and by looking at embryonic stem cells, we're always thinking about therapies, but this work and the work that was from Stewart Arc and Rudolf Jaenisch, Doug Melton and quite a number of people will at the end show us what pluripotency is, and that is very important, that we don't forget the basic science behind all of these approaches, that we understand actually what a pluripotential cell is.

And I think that many excellent groups are now working on that topic, and I think once we understood that, we also have a better way of developing therapies.  My credo is that good basic science is an important step towards applied science, and along these lines something has been published by Peter Donovan that neutrophins mediate human embryonic stem cell survival.  By understanding this here, he, for example, was able to show why or giving one reason why  trisomies happen when embryonic stem cells are cultured, because something like this is missing, and we hope that this something that we have just published with collaboration with Sheng Ding and Peter Schultz, that we can obtain substances that can maintain cells in the pluripotent state by repressing differentiation.

All of these approaches I think are required if we at the end would have a pluripotential cell in hand, and maybe a substance like this which is freezing in the pluripotent state might also help us to derive embryonic stem cell lines from other species.

So this is my international group of people.  You can see here all of the different countries, a lot of European countries, but also we have no problem of Iranians in my lab working next to Americans and Chinese and South Koreans, Indians, Greek and so on.

Sine this is 13 and 13 is not a lucky number, we have the Kingdom of Bavaria as number 14, and finally we moved into a new institute.  Whoever come close to Minster, please come visit me.  It would be a pleasure for me to host any of you at the new institute.  We just moved in there three weeks ago. 

That is the Max Planck Institute for Molecular Biomedicine, which brings me to this slide, Rembrandt, where I think some people have the feeling they know everything.  That's like this person, but I'm one of these guys.  I'm still looking, and I'm completely confused with what's going on.  I try to get a better understanding.

Thanks for your attention.

(Applause.)

DR. PELLEGRINO:  Thank you very much for a very complete overview.

I think we'll have a break of about 15 minutes before we ask Dr. Bloom to open the discussion, if that's okay with you, Dr. Bloom.  So let's take a break and be back in 15 minutes and a little shorter if you can make it that way, please.

(Whereupon, the foregoing matter went off the record at 10:27 a.m. and went back on the record at 10:43 a.m.)

SESSION 2: STEM CELL RESEARCH UPDATE AND ALTERNATIVE SOURCES OF PLURIPOTENT STEM CELLS (MAY 2005)

DR. PELLEGRINO:  Floyd, may we turn the meeting over to you?  Would you like to comment from there or up at the  podium?

DR. BLOOM:  I can do it from here just fine.

DR. PELLEGRINO:  Okay.  Thank you.

DR. BLOOM:  I want to start by thanking Dan and Ed for giving me the chance to relive the last three years of the President's Council and go through the enormous literature that you've produced on the controversies in embryonic stem cell research.

And I was reminded in so doing that in 1997 the Thompson paper appeared in Science while I was editor, and we wrote an editorial on publishing controversial research, not realizing at the time how really controversial the entire topic area would be.

I want to congratulate you, Dr. Schöler, on such an intellectual inspiring and graphically advanced presentation.  I had thought from the papers that were sent to us on your work that you were going to emphasize cell fusion.  So in a minute I'll ask you about that, but in fact, what you've done is give us a great introduction to the next hour's worth of work of reexamining the progress that's been made across the field.

My questions for you really start with your own opening slide where you said that you were trying to develop the oocytes to the point where you could do somatic cell nuclear transfer into them.

But then you explored all of the range of options from cell fusion to embryonic cancer cell extracts, to small molecules that can cause de-differentiation.  I find the whole concept that you can de-differentiate a  somatic cell into a pluripotent cell such an astonishing biological result that it is really hard to imagine how that can take place scientifically and under control.

What you've shown us though is that the research is advancing very, very broadly across a wide range of mouse embryonic stem cell opportunities.  And my first question for you is regardless of whether you take fusion or somatic cell nuclear transfer, how much of what you've talked about in the mouse can we imagine in the near future taking place with any of the human embryonic cell lines that exist for which there is the opportunity to do research?

And if so, which are the ones that are most likely to be successful?

DR. SCHÖLER:  I think that the papers that have been published so far show that things that have been developed in the mouse system to a large extent can be transferred, can be also repeated in the human system.

I think Kevin Eggan's paper is a wonderful example for that.  By using human embryonic stem cells, you can reprogram adult somatic cells.

The nuclear transfer that is working now very efficiently in mouse might be a much bigger hurdle in human, and I think there may be at the end intelligent ways of reprogramming by fusion or by using a cocktail of factors, will be faster.

We have to see because there's not a lot of things I learned from Woo-Suk Hwang's paper, but one thing I learned:  that he used 2,221 or so oocytes, and (unintelligible).  So it's kind of an (unintelligible), but tells us a lot.  So we have to improve the procedure, and I think he had some points that he made that will help future researchers how to in an intelligent way go on with that type of research.

Personally I'm always saying that if he would have worked together with Jamie Thompson to derive embryonic stem cells from clones, person that knows what he's doing, not repeating something that others have, I think he had the wrong collaborators to derive it.  That's my personal understanding.  He might have had embryonic stem cells at the end.  I might be wrong.

But nuclear transfer at the end might turn out to be much more difficult.  Maybe Jerry Schatten with his (unintelligible) science at the time was more right than he afterwards believed himself.

But I don't see why the procedure published by Shinya Yamanaka should not work, and maybe you will exchange one or the other players, but in principle, that procedure I think will work.

From what we and colleagues are doing, we're actually thinking in a different direction that maybe even factors that you get from Drosophila, from Planaria, and so on can do a lot of the job with respect to reprogramming.

This is still something which I think there are still some parts missing in the picture.  I think that's what Orkin with his beautiful paper in Nature is showing, that how complex interactions, protein-protein interactions and so on.

So if you just take the middle player and put it into a somatic cell, you might have problems with efficiency.  You might have problems with really reprogramming the cell, but this was such an important step into the right direction, I think, that others will — if he's not doing it, others will fill the gaps to get to a more pluripotent state.

And I don't see why this should not be possible with embryonic stem cells, and I don't see why this should not be possible with the existing, using the existing human embryonic stem cells.

From my perspective, with respect to basic science, understanding the general principles, I think you can get very far with the existing embryonic stem cell lines.  There are certainly problems if you would like to think about therapies, if you want to go into that direction, but —

DR. BLOOM:  Maybe we just focus on that because we're going to spend this next hour talking about all of the areas of advances, and the science is clearly advancing.  There's no question about that, but for this Council's purposes, the really important questions have to do with if you have to start with a human embryonic stem cell, we're back where we were.  If you have to start with a human oocyte, we're back in the supply business where we were.

So we're not going to debate the science with you because the science is going to do what it is, but if you had to put your resources into the most likely place of advancing to achieve regenerative medicine potential without the destruction of a human embryo, where today would you put your resources?

DR. SCHÖLER:  Basically, I would try to go along with the three major areas that I've described.  I think at this stage we just don't know which is going to be the most successful.

I think that if you are reprogramming a somatic cell and you have to go back to an intermediate stage, a pluripotential cell is an intermediate stage, it's not going to be as perfect as if you go all the way back and then go again to that intermediate stage because here you would have a full erasure, and then you would come to a stage and the difference layers of gene regulation are then established.

If you go back to reprogram a somatic cell to become a pluripotential cell, I think we will find out more and more problems.

So in respect to therapies, if I would bet, I think you have at this stage to use oocytes, and we can discuss the sources for the oocytes, but going back and then to that stage is something that is the only way so far based on the mouse work that is giving pluripotential cells which are the same quality abembryonic stem cells derived from IVF embryos.

And so all of the other things, this exciting basic science, but in terms of if you ask me where I would fit, I would use oocytes, and that would mean you would need to derive new cell lines.

DR. PELLEGRINO:  Thank you very much.

We are now open to questions from the Council.  Dr. Gazzaniga.

DR. GAZZANIGA:  In your Cdx2 example, was it not the case that you were fusing an oocyte with a male sperm, that first slide you showed?

DR. SCHÖLER:  Yes.

DR. GAZZANIGA:  And then you introduced the micro RNA to stop the processes and develop the two classes.  The reason to be addressing these problems is to try to get around certain moral questions that people have.  Why wouldn't the people that have those concerns not be happy with that approach either?  Because you're basically taking an entity that could be developed into a human, an animal, a mouse in your case, and therefore, it has all of the problems of somatic cell nuclear transfer and all of the rests.

DR. SCHÖLER:  So first of all, I should like to stress that we have been using this specific stage and there's no reason for us to believe that we won't be able to use this approach also as an earlier stage, like the metaphase II stage for a nuclear transfer.

It's a different approach than what we have done in comparison to the one that has been propose in the ANT procedure where the nucleus has been changed.  We are introducing this to the oocyte.  So the genetic material has not changed.

We have not done this experiment for the sake of developing an ethically sound way, but we have to be doing this for the science and found that there is some parts there that might be interesting with respect to the ethical problems that we have or many people have with that type of research with embryos.

So, first of all, that's something which I don't think is fixed to that specific stage.  The reason why I think this isn't part of the solution is because I don't see it as an embryo.  This is like cell division.  It's like a cell that is dividing.  It's not giving rise to an embryo, and I've tried to make the point that even if you look at the whole genome profile, and you have to do this if you really want to compare one to the other, you see tremendous differences, and they get bigger and bigger because just not having this second lineage.

And if you agree with the fact that an embryo at that stage requires a trophoblast and inner cell mass, from the viewpoint of a development biologist, this is not an embryo.

So if you say that you're manipulating something that ought to be like a fertilized oocyte, ought to be an embryo and you're changing that from what you're doing, you won't convince somebody who has a problem with doing that.

But if you extend this really to the other direction, combine this, for example, with ANT, I mention this type of research because I think it's giving you some ideas about what's happening with the embryo, regardless if you're doing nuclear transfer according to the ANT or if you inject this in the oocyte or in later stages.

Just to give you a complete picture because so far people are just saying these embryos fail, and you have no idea what we're talking about.  Now I think if we ask for this, you get aa clearer picture that they're failing because the inner cell mass needs support.  We think ATP is of real importance so this embryo can survive.

And what we have been doing, we have been replacing the trophoblast, which is not present because this is not an embryo, by the fetal layer, and if it only works that you get embryonic stem cells, if you put them on a fetal layer, so the fetal layer is nurturing the pluripotential cells, this then gives this nice ball of green cells, and then you can derive very efficiently embryonic stem cells.

That is my understanding what is going on here.  We're trying to or we might have succeeded in supporting what we think maybe is supporting also this ANT procedure.

Did this answer your question?

DR. GAZZANIGA:  I's kind of fascinating because if I recall correctly, basically human embryonic stem cell research cannot go forward in Germany presently, and so you are taking opportunity to advance stem cell biology in various animal models, and one of the unintended consequences is that maybe German biology is actually advancing the understanding of what's actually going on in these incredible phenomena like de-differentiation, where we have sort of slowed down in that within our own country.

DR. SCHÖLER:  May I disagree on this point?   I think that —

DR. GAZZANIGA:  Am I wrong?

DR. SCHÖLER:  I think that the last papers that I've shown, Stuart Orkin, Rudolf Jaenisch, Ihor Lemishka, they are going to the key points of what a pluripotential cell is, and what I think is very interesting, that Stuart Orkin, Ihor Lemishka, and also if you take Irv Weissman, these are people who have been working all their life with hematopoietic stem cells and now putting a lot of their resources into trying to understand embryonic stem cells and trying to understand what pluripotential cells mean.

And with all of their experience that they have developed with human embryonic stem cells, I know kind of diving into that topic in a way that is very, very astonishing, and this is from this country.  It's not from Europe.  You have such great amount of science here in this country.

So even if they are more than Germany into working with human embryonic stem cells, but still we can do work with human embryonic stem cells in Germany.  I also want to make that point clear.  It's just that it's like four, five months later, the cell lines that we can use and the presidential cell lines I think was the first of generally 2001 or 2002, the ones that we can use in Germany.

And we have a discussion now, a big discussion if that date is going to be shifted or going to be dropped.  I have here this what has been presented last Friday from the DFG, the major German funding agency.  Hans Biniker has had a press conference where this has been presented, (speaking German) "Stem Cell Research in Germany, Possibilities and Perspectives."

But he has been asking for Germany to drop this state at all, not even to push it, and there's a lot of discussion.  I just received this morning E-mail that our chancellor, Angela Merkel, has been looking very positive into that something has to be changed.

So we have a discussion, a big discussion in Germany.  It still won't be possible that we as scientists would be allowed to drive our own stem cell lines.  We only also in the future would be able to import them.  That's for German scientists.  It's a problem when interacting with scientists from other countries, who say, "Why don't you do it yourself?"  This is a problem, but we have to live with that.

DR. PELLEGRINO:  Professor Schöler has kindly agreed to send us a translation of that paper which we'll distribute to the members of the Council.

Thank you very much.

We have three commentators waiting, Dr. George, Dr. Foster, and Dr. Kass, in that order.

PROF.GEORGE:  Dr. Schöler, I just would like to ask a brief follow-up to Dr. Gazzaniga's question.  From your description, it sounds to me — and I'm asking you to correct me if I'm wrong about this — from the description you give, it sounds as though not only would you not have an embryo, something that would qualify as an embryo, but the lack of integration and self-direction or capacity for self-direction along a developmental trajectory in the direction of maturity such that you wouldn't really be able to say you have an organism of any sort here.  It just isn't an organism.

So my question is is it right to say not only is this not an embryo.  It's not an organism.  Do you see so far as to distinguish between an embryo and say a non-embryonic organism?

DR. SCHÖLER:  So first of all, I would like to apologize that I've been using the term "embryo" in a very loose way.  I'm in a way caught in a situation that whenever I give this structure a name, I'm being accused for kind of trying to hide something.  So if I call this "structure" or if I call this "pluripotency ball" (phonetic), somebody stands up and says, "Why don't you call it embryo?"

Actually I always try to say this is a stage which compares to the eight cell embryo, and during a talk I might lose this and not be able to explain this is a stage that corresponds to the blastocyst stage or this corresponds to that.

But I hope that you forgive me if I have not in every case done it that very way during my talk, but that's what I meant.  So whenever I had this comparison, I wanted to point to the embryonic stage.

So I don't consider this an embryo.  I don't consider this an organism anyway.  No, I think this is, for me, this is cleavage.  This is cell division of a structure that starts off as an oocyte, but it would not cleave and divide to give rise to an organism.

PROF.GEORGE:  Am I correct that in thinking about what is and is not an embryo, what is and is not an organism the focus should be on whether we have a self-integrating entity that is developing along a trajectory in a direction?  Is that the correct way to think about it?

So that if we see a lack of integration and a lack of self-directed development along a certain trajectory that we associate with the normal developmental trajectory of the species, if we see a lack in these respects, that would be the ground for judging that we have here something that is not an embryo, that is not an organism.

If, on the other hand, we find that there is a degree of integration, that there is self-development along a trajectory,  we would conclude that we have an embryo although in a particular case the embryo may be damaged, the embryo may have defects that will prevent its full manifestation of its potential.  There may be an impossibility of implantation.  There may be an impossibility of survival so that we might not have viability, but we would still have an embryo as something as distinct from something that's not an embryo.

Am I looking at it in the way that you would advise?

DR. SCHÖLER:   I think one would have to look at it exactly the way you have been describing it because otherwise if you would not be looking at that that way, you could come to the conclusion that an embryo that will fail at some stage, like in mouse there's this one mutation, Lim-1, which gives rise to fetuses without a head, and you could say this doesn't have any potential.  The answer would be why don't we produce these and at the end we can use their organs, which I think would be not in agreement with the view that you have been presenting.

I think the view that you have presented is the view I would see it as well.

PROF.GEORGE:  Thank you.

DR. GAZZANIGA:  But were you here for the presentation?

PROF.GEORGE:  I got here late.

DR. GAZZANIGA:  So he missed the key slide, which is the fusion of the oocyte with the fertilization, and that occurred first and then the intervention of the micro RNA that caused for the two classes of embryo.

So you're starting out with something that is on that trajectory, and you are interfering with it to produce this artifact that can be used in the way that has been demonstrated.

So the question is that I thought would offer you a problem in the final analysis.

PROF.GEORGE:  So then the question would be does Dr. Schöler agree that what he's beginning with is an embryo that is transformed into a non-embryonic condition.

DR. SCHÖLER:  Nucleated zygote to my understanding is not yet an embryo.

PARTICIPANT:  But it is an oocyte.

DR. HURLBUT: When you gave your presentation, you were talking about the use of pronuclear stage, but in answer to Mike Gazzaniga's previous question, I understood you to say that the same thing would almost certainly work if you did the silencing in the oocyte —

DR. SCHÖLER:  Yes.

DR. HURLBUT: — before nuclear transfer or before whatever procedure, the point being that that would satisfy the full concern about ever creating an embryo.

DR. SCHÖLER:  Yes.

DR. HURLBUT: And some people might argue that a pronuclear stage entity is a embryo.  I know German law says differently, but the point is in America that criterion probably wouldn't be the same.

So to satisfy the American concern, one would have to do the silencing in the oocyte, and you say that you think that's feasible.

DR. SCHÖLER:  Yeah, that's why for this we're trying to see if nuclear transfer into oocytes can be the (unintelligible) derivation of the nuclear transfer into nucleated oocytes can be also improved.  As I tried to explain, we have at this time not done the experiments for getting out of ethical problems, but for scientific reason, and if we would have planned them differently, we would have started at an earlier stage.

DR. PELLEGRINO:  On this point?  I have a list, Bill, of people who are waiting.  You're on this point?

DR. HURLBUT: Yes, on this point.

I just want to clarify that the way we've been using the term "altered nuclear transfer" from the very beginning is that the alteration can be either in the cytoplasm or the nucleus or both.  So the procedure of knocking down the siRNA and the cytoplasm of the metaphase II oocyte would be a form of altered nuclear transfer.  What you've described would be altered nuclear transfer, not just what we would be interested in the nucleus.

DR. PELLEGRINO:  Thank you.

Dr. Foster.

DR. FOSTER:  I didn't have any comment.  I was just glad to get here. 

(Laughter.)

DR. FOSTER:  I was just waving, having waited seven hours to get a flight and then get canceled twice.

DR. PELLEGRINO:  Dr. Kass.

DR. KASS: Well, I want to say hi to Dan Foster and wave, too, and thank Dr. Schöler for really a remarkably exciting and illuminating presentation.

I have really basically two factual questions and then a more theoretical question.  First, these stem cells that you got from the Cdx2 altered cells, have they been tested for pluripotency by the gold standard tests and shown to be pluripotent?  The first question.

And second, neither you nor our colleague Dick Roblin in preparing the materials for this discussion referred to the publicized but not published work of Dr. Verlinsky.  These were fusion experiments done in humans with human embryonic stem cells, and I haven't seen any publications, but I wondered if one knows anything more about this.

Among the things that were striking about that report was that according to his experiments, it was the cytoplasts rather than the nucleoplasts that seem to contain the materials that could successfully produce the reprogramming of the somatic cell with which the cytoplasts were fused.

And then the more searching question has to do with your comment about the superiority of going all the way back to pluripotency, to totipotency, to —

DR. SCHÖLER:  To the oocyte.

DR. KASS: — to the very beginning oocyte and then coming forward.  Since it's clear that for therapeutic reasons one wants to have partial differentiation, as you pointed out at the beginning, to more specialized stem cells so that you don't have the tumorigenic concerns, why if you had a rather controlled process of de-differentiation to some place that was reproducible?  Wouldn't you be — and by these sort of cytoplasmic factors you knew what you were doing.  You knew to what stage you got it .- why does the fact that you haven't gone all the way back to the oocyte present a real liability?

That's a more searching question.  The other two were just questions of fact.

DR. SCHÖLER:  So first of all, I've just briefly scanned over that one slide because I was realizing that I'm talking very long.  So there was one slide that was showing that we have done our homework, that the ES cells derived from the Cdx2 treated pre-nucleated zygotes, fulfill all of the criteria of pluripotent cells.

The one I've put my head a little bit out of the window was saying that the most important thing is that you show that (unintelligible) navigation.  That's what we're currently so doing.

But all the others, chimerism and so on, that has already been done.  The nice thing about Cdx2 is that it also plays a role for intestine stem cells.  If that would have not been a transient effect, what we have done, the injection into the nucleide zygote and deriving ES cells, if that would have been a more stable effect, there should have been a problem with these intestine cells, and I've only shown you a picture with an intestine where you saw some blue cells.  This was from a ten months old mouse where we waited that long to make section, to show that this cell compartment is produced by derivatives of these injected cells.  So this is transient, which is the best indication that it's not a long term effect.

And if you think about what's going on is you're reducing Cdx2 in a compartment that might not even give rise to the embryonic stem cells.  It's an open question.  If the outer cells are confused cells because they still carry on material from the oocyte or if they, indeed, can be used to derive embryonic stem cells, we don't know that.

But by all means, we can get germ cells from these after injection and so on.  We think that they don't have a problem.

The second question, Verlinsky's and Strelchenko's work that they have published in the meantime in RB Online — in the meantime means, I think, half a year or so ago — I think if you read the publication, first of all, we only knew about that work from, I think, the scientists that were seeing a pattern, and we have read the pattern.  We didn't have an idea what was specific about how they did it, and so I didn't get it from the paper very well.  So I asked them how they exactly did it.

And the way I understand it is that they are doing this fusion on glass plates where the nuclei have been removed by centrifugation.

DR. KASS: Yes.  They plate, I think, the embryonic stem cells —

DR. SCHÖLER:  Yes.

DR. KASS: — on little glass cover slips.  They centrifuge them upside down.  The nuclei come out.  They're left with the cytoplasts and they fuse the somatic cells if I'm not mistaken.

DR. SCHÖLER:  Yeah, but what I couldn't get from the paper is that when they do this and do the fusion, if they can exclude, that this on the cover slip is forming something like a syncytium where you have cells fusing, and since the removal of nuclei is not complete, that you could have nuclear factors coming from the left over nuclei and doing the reprogramming.

And since the efficiency is extremely low, that's something which is clear from the paper.  You don't know what actually did the job.

So I think that what we had published, and I just briefly mention that publication, that where we have been using nuclei, we were hoping that would be the cytoplast.  That would have been easier.  You send vesicles to the clinics and they put in the nucleus and you get your embryonic stem cells.  That would have been a dream.  Maybe with other processes we can still get there.  We don't know.

But that's what we tried.  So we took embryonic stem cells apart and adult stem cells apart and recombined everything, and we could make a big sac out of the cytoplasts in a clean way.  Never did we get any reprogramming.  We needed the nucleus to do this.

And the work from Shinya Yamanaka is kind of confirming this because he needs four nuclear factors.  So I don't think that this will turn out to be a key paper, this cytoplast piece of work.

The other question concerning going back and forth again, I think that's my understanding.  This might turn out to be wrong at the end because somebody is doing a clever experiment to prove me wrong, but my understanding is that if you make tabula rasa, you clean up everything and then start building things up again.  It's easier than taking things out, things that at this stage we have no idea what you have to take up, the levels of repression that you need to get expression along one or the other lineage.

If you basically clean the table and then allow the things to develop by itself into the right direction, I think the different layers of gene regulation are then set by the oocyte.  They are doing the job.

If you push it to that point, since you might have more leftovers of things that are not perfectly reprogrammed, I think at the end that would be more difficult, and so far all the evidence is kind of suggesting that I'm right because whatever people have obtained by the procedures is not as good as nuclear transfer where the oocyte is being involved.

Is that okay?

DR. PELLEGRINO:  Dr. Rowley.

DR. ROWLEY:  I, too, want to thank you for a comprehensive overview of the area and the directions you and others are trying to pursue.

I have a couple of questions, one of which is a follow-on of Mike Gazzaniga's with the comments of Dr. Hurlbut and Robby George, and it does seem to me if you have to start with an oocyte and then you knock down a Cdx2, that the moral problems of using oocyte remains regardless of what you've done, and whether you've added siRNA or new nucleus to that oocyte to my mind isn't different.

And so to form that in the way of a question, is it different?  But I'm not sure.

I have two other questions.  One, is it really correct to call the primitive germ cell unipotent?  Because, in fact, if that cell is fertilized, it goes on to give rise to an embryo which gives rise to all three layers, ectoderm, mesoderm and endoderm.

So in my own view, I don't think of it as unipotent.  So that's a question.  Why do you call it or is it correct to call it unipotent?

And then more not philosophical but scientific question.  In your view why is it so difficult to begin to develop somatic cell nuclear transfer in humans or primates as compared with mice or other mammals?

We have the example of the South Koreans where at least at the present time it said that they use more than 2,000 oocytes and then get a single cell line, and there has to be some difference.  Do you have any insights as to what those differences are?

DR. SCHÖLER:  So first to the statement about that not changing the moral, ethical problems by still using oocytes, is this because of the problems of egg donation or is this from your point of view because of that we still have something which was supposed to give rise to an embryo?

DR. ROWLEY:  Well, I should really let somebody like Robby George answer that because I don't personally have a problem, but it would seem to me that for individuals who do have a problem with using oocytes that, in fact, it should still cause problems.

PROF.GEORGE:  Perhaps I could respond on that then.  I think that people on my side of this debate, and I've been a critic of the destruction of embryos for purposes of this research, are concerned not to destroy living human embryos.  So they're not concerned on that issue with oocytes as such, but rather with embryonic human life.

But then on the question of the use of oocytes, the concern is with how they're obtained and whether they would be obtained in a way that's potentially harmful for women and especially if it might result in the exploitation of women to obtain the larger number of eggs that would be required if therapeutic uses were found for these technologies.

So they're really two distinct questions that I think perhaps have been run together.  So that if there would be a way of obtaining oocytes without exploiting women or subjecting them to danger or harm, and if those oocytes could be used to produce embryonic or embryonic type stem cells without the destruction of embryos, then we would be out of the ethical problem as far as I'm concerned.

DR. ROWLEY:  Well, but then what Robby is sort of suggesting is if you go the way of John Gearhart, which is to use oocytes from fetuses and they are therapeutically aborted fetuses, but which raises another issue, but getting oocytes from them, then he has no problem

I think it's not an easy issue.  Certainly the question of egg donation by healthy women who do have to undergo hormonal treatment in order to release a lot of oocytes, that for me is a separate issue.  I agree from the ethical issue of using oocytes.

DR. SCHÖLER:  So with respect to egg donation, then let's say a combination between the ANT procedure and using what is now currently discussed a lot in Germany because of what the group in Newcastle has asked to do, nuclear transfer into bovine oocytes, with respect to egg donation at least, that should be fine; is that right?

PROF.GEORGE:  That's correct from my point of view, yes.  If I understand what you're saying correctly, where you would not use oocytes taken from female humans —

DR. SCHÖLER:  Yes.

PROF.GEORGE:  — you would rather be using non-human, animal oocytes for the procedure, do I have that correct?

DR. SCHÖLER:  Yes.

PROF.GEORGE:  Yeah, that does not strike me as — as long as we're not then creating an embryo, it doesn't strike me as having an ethical problem.

DR. SCHÖLER:  Okay.  So the first question concerning the primordial germ cells, they are considered to be unipotent because on their own they just give rise to germ cells.  At the end you have an oocyte or an egg, which are terminally differentiated cells.  These are the most exciting cells for me, but they are terminally differentiated.

The exciting thing is that if you bring them together, the clock is set back and now you're getting from two unipotent cells to combine a totipotent one, but as long as they're on their own, they are considered by scientists to be unipotent.

They're still on what I call the totipotent cycle because they give rise to an organism, and I call it now germline cycle because people have been confused by having cells which are unipotent on the totipotent germline cycle.

So that's what I would like to answer with respect to the potency of these cells.

And why is cloning so inefficient?  I think this has a lot to do with what you're depleting when you remove the chromosomes, and the spindle and so on might be something which is different between the different species.

So that might be a reason for problems.  This has been  nicely described by Jerry Schatten, why he thinks cloning is so inefficient, and I think from what he has been saying at that time, I think he has been correct.

It was just that, one, when he came out with this astonishing result, I thought, oh, maybe I was wrong, but I think he was right.

DR. PELLEGRINO:  Dr. Dresser.

PROF. DRESSER:  Thank you.

I had another question about, SCNT in humans, and I understand that one of the main basic science reasons people want to do it is to create stem cells from cells from patients with genetic disease so that they can learn more about the development of the disease and test drugs and so forth.

So, number one, it looks as though that will be difficult to do in humans.  So I wonder about alternatives to that.  I wonder if any of the procedures you describe offer a way to study that sort of problem.

And the other question I had was whether the difficultieswith SCNT would lead researchers to want to try to create embryos with genetic problems through IVF and whether that will be an emerging issue to address.

I suppose you have to have it confirmed on people who have genetic disease to try to make an embryo.  That would be a disease model that would be more efficient than SCNT or perhaps some of these other models.

DR. SCHÖLER:  Yes.  So actually to maybe start with the second question. I think if we come back to the laboratory of Verlinsky, he has derived an extensive (set of embryonics stem cell lines) from patients with genetic disease.  These from what I understand have been obtained by fertilization.  I think he has done preimplantation diagnosis and then correlated this with the disease.

I think this is, of course, far more efficient than doing nuclear transfer.  The big, big advantage if nuclear transfer would work is that you have the history of a patient is documented.  You know the outcome of what you're expecting to understand.  If you're doing it with one of these cell lines, you don't know how much genetics is playing a role in the outcome of what you're investigating.

Of course, there are other problems that we might not see at this stage, but if you know the disease, how the disease develops and you're trying to understand this process in the dish, I think that's the best way of going ahead.  It's like the reverse way of what you do in mice.  You destroy the mutated gene and then you're trying to see what happens. 

In this you know what's happening.  This is a disease that this patient is suffering, and if you're taking nuclei of, let's say, a handful of patients and trying to derive stem cell lines of this, you get to some degree of variation in what you might investigate in the dish.

And I think this will be a focused way of looking at disease.  Of course, you can't get to an understanding of what happens in the whole organism, but by correlating this understanding with the understanding that you have obtained from the patient, I think we will learn a lot.  That's what I think will be very important for the future.

Then the —

PROF. DRESSER:  But I guess my question is:  what if that proves to be too difficult?  Say it takes thousands of eggs or .-

DR. SCHÖLER:  Yes.

PROF. DRESSER:  — it's just not feasible.

DR. SCHÖLER:  That was basically your first question.  So for understanding disease, I couldn't do this approach in Germany anyway, from a practical point of view, but also in terms of the way you can screen for then successful results, the embryonic stem cells are — that's my understanding.  Again, I might be wrong, but that's my understanding — is the most potent system because you can plate cells.  You can look for selectable markers.  You can see if you can get a rare event.

And since embryonic stem cells have the power of proliferating in a way that you can get from one to millions and millions of cells, you can screen for rare events, and that you can't do with oocyte.  That's one problem which will remain in the future, that you can't really screen for something where you have a limited number of cells to start off with.

So I, indeed, will be trying to see if we can use the different reprogramming procedures that have been described with embryonic stem cells, if we can get disease this way into the dish, which might not be good enough to use these cells for the patients, but may be good enough to understand at least certain aspects of disease.

And then a final point to this is that in science it's very important that different people test different approaches.  This is our approach.  It might not work; it might work.  Others might be more successful with nuclear transfer, and we have to see at the end who is successful.  The patient will win if one of the processes is successful.

DR. PELLEGRINO:  I have Dr. Hurlbut and Dr. McHugh.

DR. HURLBUT: Just a comment on what you were saying.  That fertilization would work for certain dominant alleles, but it wouldn't necessary work as well for a lot of genetic diseases because it's the whole context of the existing genome that counts.

So obviously, the ideal would be to create an identical gene aligned from an identical genome, and this is sort of a question and a comment, just because there's something else I want to ask you.

Even with IVF embryos we don't know if we take the cells at the four-to-five cell stage, we don't know if those genomes can actually produce a full organism yet.  So is it something to consider, that even IVF might not produce perfect cell lines?

Why don't you answer that first?

DR. SCHÖLER:  So if we look how efficient preimplementation development is, at the end we are disappointed how inefficient it is.  I'm not sure exactly about the numbers, but it's in the range of, I think, 50 percent or so that are failing prior to implantation.

So if you take two germ cells and combine them and force these to do something which in vivo they might not have a chance to do, you're not really sure about the outcome of what you at the end have in hand.  So that is an important point.

But if you bring that up, you also have that problem with nuclear transfer because the problem with nuclear transfer is that you're skipping all of the selection that you have along gametogenesis because from my understanding, that if you have a problem with germ cells, germ cells basically don't try to repair what's going wrong.  They get rid of the germ cells.  There's a lot of apoptosis.

If you have a gene which is expressed in germ cell, during germ cell development, it's often that you see they are driven into apoptosis, active cell death, because my understanding is that when it wants to get rid of something, then to maintain it to start the next generation, if you're doing nuclear transfer, you're jumping over this.

So you might have at the end something which as a germ cell would have had no chance to get to that point.  That's also — we have have to talk about both sides.  And in vitro fertilization, actually the problem would be only a short problem, whereas nuclear transfer would be all the way through.

DR. HURLBUT: Yes, but the fact that you can get successful production of full organisms, in this case mice, using tetraploid complementation does confirm that at least some of the time you're getting a fully functional genome.

DR. SCHÖLER:  Yes, some.

DR. HURLBUT: And, by the way, I'm correct, aren't I, that when he did his experiments with altered nuclear transfer, Rudy Jaenisch did do tetraploid complementation studies?

DR. SCHÖLER:  Yes.

DR. HURLBUT: So those cells really were confirmed, and if I understood you earlier, none of the other methods, reprogramming or any other methods have established that you can get tetraploid complementation to work.

DR. SCHÖLER:  yes.

DR. HURLBUT: Is that right?

DR. SCHÖLER:  That's correct, but also, if you get to embryonic stem cells, you also have a selection for those embryos that give rise to embryonic stem cells.  It's also something you're — if you would try to force embryonic stem cells at earlier stages which the structures don't have a chance to get to the blastocyst stage, you maybe have more problems with these tetraploid complementation experiments.

I'm just saying that you always have to think about the selection you're doing or not doing in that process, and maybe cloning is inefficient because the genetic material, to some extent, because the genetic material is not as good as it is from germ cells.

So there's so many things we still don't know.

DR. HURLBUT: And finally, could you just say a little more about your statistics of increased efficiency of harvesting ES cell lines using the Cdx2 knock-down, was very interesting.  You got practically a doubling of the efficiency and at the eight cell stage, which is earlier than we had previously thought you could get ES cells.

Could you just expand on that a little bit?

DR. SCHÖLER:  Yes.  I realize at the end I have been too fast on that point.  The one astonishing experiment from Bob Lanza (phonetic) was that actually he can derive ES cells from (unintelligible) blastomere from an eight cell embryo...

So it is to me.

DR. HURLBUT: But his paper indicated his co-cultured the blastomeres.

DR. SCHÖLER:  Yes.

DR. HURLBUT: That's important because they were signaling.

DR. SCHÖLER:  I agree, but that that at all works.  So I wouldn't take it as too much of a surprise that if you take an eight-cell embryo and try to derive stem cells from there using also a feeder here in our case, that experiment, which we did also with the control treated one, but we wouldn't have had to do that because they grow on the outgrowing trophoblast cells, and so that would have been the feeder, but we don't have that in the knockdown experiment.  So to compare both, we had to use feeders in both cases.

So if you take these cells, these structures from the Cdx2 knock-down experiment, you had green cells throughout the structure, and we think that the increase of potential candidates to give rise to embryonic stem cells at the end started an increase of about 50 percent of an efficiency.

So it means if you take the control treated embryos, compare this with the structures, the Cdx2 treatment didn't worsen the derivation, but improved.  So our hope is that if you do the Cdx2 siRNA treatment at the metaphase II stage for nuclear transfer, that this would also result in an increase even.

So maybe this way we make nuclear transfer more efficient with respect to amniotic stem cell derivation.  That we have to see.  We don't know.

DR. PELLEGRINO:  Dr. McHugh.

DR. McHUGH:  I, too, want to thank you very much for your presentation, and I'm still thinking about it, and so I'm the slow member of the class trying to ask some simpler questions really simply, in part because of the important issues that you evoke for me, anyway, and for many other people about our existing science, our science base, and the directions we're going.

And so I just wanted to ask simply three or four questions that came out from what you said.  The first thing you said was that, you know, using existing embryonic stem cells we're really quite able to do wonderful things, and you said we have kilograms of these cells now available.

Now, does that mean that, in fact, the cells that were made available really at the birth of this Council by President Bush when he said that he would give federal funding, anyway, to research that based itself on those cells, that in point of fact those cells are adequate for the work that is being done?

Are they adequate in number and in character?  That's the first question.

The second question is, of course, like everyone else I'm very amazed and think it is a wonder, this Cdx2 business.  I want to be really sure that we've answered the question that we're not dealing as we might have been in other situations with essentially a wounded embryo.

I think that's really what we come down to, and from your slide that you began with, you showed that, in fact, in the mouse like in every other creature, the cycle of life begins with a fertilized ovum.  I want to be sure if I understand correctly that in this situation we are not beginning with a fertilized ovum.  We've done the treatment beforehand.

The third thing is that you mentioned the possibility that neurotrophic factors and the like would be helpful in reprogramming things, and of course, that's a very exciting prospect for all of us because if trophic factors are there, they ultimately can be synthesized because they're chemicals, and how close are we to getting to synthesize them?  And then we don't even have to have anything to do with cells.  After all, we won't use the penicillin mold anymore.

And then the last little one, it's a tiny point, but you mentioned that the laws in Germany were going to change in some respects in relationship to this research.  Are they changing in such a fashion that IVF embryos now can accumulate in Germany the way they have accumulated in other countries and be a source of problems for us?

Thank you.

DR. SCHÖLER:  So the adequacy of the cell lines.  So as long as people like Rudolf Jaenisch, Doug Melton, and so on are able to publish in Cell, Nature, Science with these cell lines for basic research, I think you can do quite a lot, and that's why I was trying to distinguish between what you can do to quite some extent in basic research in comparison to applied science.

And if you would, just one scenario, if you would try to use the existing cell lines, which have been cultured for a very long time, have been cultured in the presence of animal cells, sera, and so on, and you would use a primate model; let's say you want to try to cure or at least alter the phenotype of monkey model for Parkinson's, and you transplant these cells and you have the tumor or the cells are rejected.  At the end you will not be able to know is this because my procedure has not been developed in an appropriate way or is it because the cells were not good enough for the procedure to start off with?

If you have a tumor, is it possible that cells that you differentiated from the embryonic stem cells, let's say you have progenitors for neuro; let's say you have neuro stem cells.  In embryonic stem cells you might not see that the genes have been mutated over the many passages, but once you get to an intermediate stage or even to the different shaded stain, maybe it's then when they exert their problems.

So if you think about testing this in an animal model, you rather want to start this with genetic material which is perfect.

DR. McHUGH:  But at the level of the basic science, which is often the complaint about the Bush proposal, that it would block basic science, as far as you say at the basic science study of stem cell they're adequate you're saying.

DR. SCHÖLER:  So for the type of research I am doing —

DR. McHUGH:  Yes.

DR. SCHÖLER:  — basic research I'm doing, I don't have a problem.  The problem I have with the cells now is that in Germany — I'm talking about the problems I have as a scientist — I have a problem with interacting with scientists in other European countries because if we're getting cells, let's say, from Y cell, we are signing contracts.  We have our own cell lines.  So why don't we use our cells and we have less of a problem maybe there.

DR. McHUGH:  Right.

DR. SCHÖLER:  So then it happened with the Sixth Framework that because they wanted to include certain German scientists, they had to use the old cell lines and not the newer ones and said, "But we have our own."

So in the end they say, "Why don't we leave Germans out of, the Italians out of the scheme because we can happily work together?" and so on.  So that's another issue, but that's a German one.

DR. PELLEGRINO:  Thank you very much, Dr. Schöler.  We've been working you very, very hard. 

I'd like to just for a moment ask the Council if they have any comments they want to make on the summarization of alternate procedures that was prepared for us by the staff, Adam Schulman and Dick Roblin, and also if you have any comments on the relationship of these alternatives and also the comments by Professor Schöler to our publication on alternate sources.

That's a little complicated question, but I think any aspect of that we'd appreciate your guidance on.

DR. GAZZANIGA:  Dr. Pellegrino, just before maybe going there, I think this wounded embryo question is central to a lot of the conversation this morning, and maybe he could answer that before we move on to the general.

DR. PELLEGRINO:  All right.

DR. SCHÖLER:  So if you would consider the pronuclear state zygote, the pronuclear zygote as an embryo, then you would say we would be doing something to an embryo, and then it would be in your view a wounded embryo.

So that's why I was saying to address that question one would have to do the treatment earlier, before fertilization, but that's —

DR. GAZZANIGA:  Which is possible to do.

DR. SCHÖLER:  And there is no reason to believe why it's not possible to do, but before you have seen the results, you don't know.

But as I said, we want to go as early as the metaphase II.  That's the point where you would put in the nucleus.  That would be the earliest that we have seen that there's obviously a Cdx2 there.  So I don't see why it should not work.

DR. ROWLEY:  Well, but I think it's very critical to emphasize that he hasn't done it.

DR. SCHÖLER:  Yes.

DR. ROWLEY:  An it may be that Cdx2 at that point activity or gene expression is critical for the process of fertilization.  Because you have to ask why is it so high.

DR. McHUGH:  That's right.  That's very interesting.

DR. ROWLEY:  And it's not high for no reason, I would suspect, and it's just that we're jumping to the conclusion that you can do it at this stage, and until it's actually done and shown to be successful and as efficient as a later stage, this is an unanswered question.

DR. McHUGH:  Yes, Janet.  I agree with that.  The whole reason for putting forth the concept of wounded embryo is to get us into this discussion of where the science is and what we can depend on and what we can't depend upon.  And that's most helpful; your comment is most helpful in that respect.

DR. SCHÖLER:  If I may add something to this, this experiment I've shown you to give you an idea about the outcome.  So this has not been described, I think, to that extent in the development paper by Janet Rossant and also not in the Nature paper by Rudolf Jaenisch.  That's the outcome.

But Rudolf Jaenisch has shown a way to address that question not having a wounded embryo, like doing this at a stage, at the metastage II phase to transfer a nucleus in there.

I assume that we have a very similar outcome, and I think from what is published that we have a more severe problem.  So if you can accept what they have done, I think ours is going to be the effect of Cdx2 knock-down is going to be worse even at the pronucleoid stage.

So if you do it earlier, we have to wait for the outcome, but I don't see why that should be better.  And if it's not working there, then you say that's unacceptable.  That's the outcome, but we have to see.

DR. HURLBUT: Not worse; better though for our purposes is what you mean, right?

DR. SCHÖLER:  Yes.

DR. HURLBUT: Better in preventing an embryo from coming into being.

DR. SCHÖLER:  Yes.  Thanks for clarifying that.

DR. HURLBUT: Yes.

DR. SCHÖLER:  The procedure works better.

DR. PELLEGRINO:  Other questions or comments?

DR. SCHÖLER:  So I have not all of those four questions.  Should I?

DR. KASS: Mr. Chairman, I think our guest has one more thing he wanted to add in response to Paul.

DR. PELLEGRINO:  Did I?

DR. KASS: No, I think Dr. Schöler.

DR. SCHÖLER:  That was the change in Germany if this resides now more in IVF embryos.  I don't think that this — first of all, we're not allowed to derive our own embryonic stem cells.  So there's no reason to believe that by any of these procedures that we have an increase of in vitro fertilized embryos.

So first of all, that and I think the numbers that are officially in German fridges are extremely low, but Dr. Tinori might also add something.  I think in Germany they're extremely low.

DR. McHUGH:  That's what we had heard previously, and we just wanted to know or I just wanted to know whether this was going to change and that the number of IVF embryos would now accumulate in Germany like they have here.

DR. SCHÖLER:  No.

DR. McHUGH:  With the implications that they bring to our discourse.

DR. PELLEGRINO:  Leon.

DR. KASS: Well, I think I would like to sort of put on the record my own delight and to some extent astonishment that we are November '06.  The Council's white paper was published in May of '05.  A quick subtraction, 18 months, right?  In 18 months we've had peer reviewed publications showing proof of principle, depending on what you think about the reprogramming studies, how much of proof of principle that we've seen.

But there's been active research in all of these areas, and I don't think from the conversations we've had around here that the doubts and the skepticisms have been completely set aside.  There are large scientific questions that remain, and there are, of course, questions that the people who are keen on working with embryonic stem cells have been too polite to press in the discussion, but from their point of view it's not clear why the research need wait for the development of these alternative sources.  That research goes on.

And I think I appreciate very much your suggestion that the full understanding of what's going to work in this area will depend upon the work that's done with adult stem cells and with pluripotent stem cells derived whether from embryos or from some of these alternative sources.

So I think this research would have gone on I'm sure without the Council's prodding.  I think we owe Bill Hurlbut, I think, a considerable debt of gratitude for having insisted that we somehow lift this possibility that science might find a technical way around an ethical dilemma so that the research, if this is successful, could go forward at a very high level in ways that no one will feel morally compromised.  I think this is very exciting.

Whether there's enough here to warrant our saying anything more other than having this very fine update from you on this occasion, I have my doubts.  I mean, I think it would be very nice if the larger community could have been here to hear this really very elegant presentation and also the summaries that Dick Roblin has prepared on the literature I think are helpful reminders to all of us as to what's going on really in the last two to three years in this area, and maybe there is a way to use our Website to call attention to this presentation and to these publications, but since I guess there was partly a discussion last time as to whether we, the Council, need another publication  at this particular juncture on this subject, I myself don't see it.

DR. PELLEGRINO:  Thank you.

Any further comment on Leon's comment or any other related to the alternative sources?  Yes.

DR. GAZZANIGA:  Ever since the alternative sources have been proposed, there has been no, I don't think, doubt that various kinds of biologic experiments could be applied to those questions.  I don't think that's an issue.

I do think that the concern of some of us is that we're dealing with what the economists think is called lost opportunity cost, that there is a way laid out in the scientific community to go forward, and we are allocating resources, limited as they are, to approaches that for some of us don't seem to really answer the moral question, which were why these ideas came up.

So we just have learned that the Cdx2 experiments, in fact, do have the moral problems that were raised by many on the Council.  Robby George is the most articulate with that view, I think.

And I think the de-differentiation suggestion, if you really think about it has similar problems for those who are concerned about a viable entity that could be a human being.

And one could go through on the other two cases and make other moral arguments.  So I guess I, for one, would be more relaxed about these efforts if I actually thought the people who have these concerns truly are at rest with the moral dimensions of it as they had proposed.

Nonetheless, it was a great review, and we thank you.

DR. SCHÖLER:  Thank you.

DR. PELLEGRINO:  Dr. George.

PROF.GEORGE:  Yes.  I think —

DR. PELLEGRINO:  And then Dr. Meilaender and then Dr. Gómez-Lobo.

PROF.GEORGE:  When you call on them I thought they were going to be —

DR. PELLEGRINO:  No, no.  In the order in which I call them out.

PROF.GEORGE:  Thank you, Dr. Pellegrino.

I think I should just say something in response to Mike Gazzaniga's last comment, and perhaps I misunderstood Mike, but just to be clear about my own position, I found myself reassured by what Dr. Schöler was saying, that in the relevant alternative methods that he was discussing, we are not talking about the creation and destruction of embryos. 

So far from being persuaded that the methods he's discussing have the same moral problems, it seems to me that he was arguing that they don't; that, in fact, we not only don't have an embryo in the research he was discussing, but we don't even have an organism.

So we have, you know, some sort of a tissue culture perhaps, a collection of cells that lack the organization and self-direction of an embryo, but if that's right, then we don't have that moral problem.

Now, we still might have the moral problem with the use of oocyte, but Dr. Schöler has also proposed without going into nearly as much detail that there may be alternative sources of oocytes that will not involve exposing women to super ovulation and the possibility of exploitation.

So have I misunderstood you, Mike?

DR. GAZZANIGA:  Well, no.  There's a little bit of a unclarity with this.  Paul McHugh asked the question driving home the point of whether these were wounded embryos.  I happened to notice you were out of the room, and Dr. Schöler answered, well, if you consider a union of an egg and a sperm the beginnings of life and an entity that's on the trajectory towards whatever organism you're talking about, then, in fact, by introducing the RNAs, we do have a wounded embryo.

So this was the answer to the question which finds me stating the thing that I stated.

PROF.GEORGE:  Well, let me see then if I understand Dr. Schöler 's position, and he can simply clarify this.  Take, for example, the kind of work done by Jaenisch.  In your opinion has Jaenisch created wounded embryos or has Jaenisch created non-embryonic sources of pluripotent cells?

DR. GAZZANIGA:  Just to be clear, we're talking about his experiments, and we're talking about his Cdx2 experiments.

PROF.GEORGE:  Well, maybe you could tell us your opinion in both cases.

DR. SCHÖLER:  So what I tried to show here is the outcome of the Cdx2 experiment under these conditions, and to avoid that one has a wounded embryo according to what you said.  One would have to do that at an earlier stage.  I don't see any reason by the outcome should be any different.  If at all, it would be more severe, even more severe.

PROF.GEORGE:  But would that mean you don't have an embryo at all?

DR. SCHÖLER:  No.

PROF.GEORGE:  No?

DR. SCHÖLER:  If you do it at an earlier stage, you definitely don't have a —

PROF.GEORGE:  Definitely don't have an embryo.

DR. SCHÖLER:  — an embryo, and it's a matter of what — now we caught in different cultures or systems.  And according to the German law, law, not church, law, the polukiat (phonetic) zygote is not considered to be an embryo, whereas Hans Schöler, a developmental biologist, would say as soon as you fertilize the oocyte you're starting the process.  Ergo, I would consider this to be an embryo.

Ergo, you would be right by saying that's a wounded embryo, but if I, Hans Schöler, would say is that something that should be protected or not, I would say no.

But from what you've been saying, this reprogramming, bringing back, you're not even getting close to something, what is an embryo.

DR. GAZZANIGA:  Well, just to make my point clear, if we go back to somatic nuclear cell transfer and you have a moving a film forward, you go through stages where you do have cells that could become, if implanted, an animal or a human being, depending on the organism in question. 

So that was a moral concern to many people on this Council, that possibility.   So you have to understand the context of the arguments that have been here.

DR. SCHÖLER:  Absolutely.

DR. GAZZANIGA:  So by dedifferentiating a human cell back to some point, some totipotent place, there you will go again.  You'll have those cells that if implanted could be a human being.

So the de-differentiation technology doesn't answer this deep problem that some people have, and that's my simple point.

DR. SCHÖLER:  These are two different areas.  If you talk about de-differentiation, we are talking about cells.

DR. GAZZANIGA:  Yes, correct.

DR. SCHÖLER:  If you look at transfers, that's not de-differentiation.  That's reprogramming because what you're doing is you're putting a nucleus into an oocyte.  The very second if you say de-differentiation, I'm thinking about cocktail of factors and pushing it through the pluripotent state, and I've been answering —

DR. GAZZANIGA:  But you have got those same totipotent cells.

DR. SCHÖLER:  No, you get to a stage which is pluripotent, and what I was saying to the kind of scientific question, that where would I bet my money on is that with respect to therapy, I was saying if you go all the way back and then forward, that would be in my opinion the better way of doing this for of the reasons that I've been mentioning.

And if you can do this the way Dr. Hurlbut has proposed it, then you wouldn't get to something which is an organism embryo.  That's the difference.

DR. McHUGH:  Since I coined the term, can I come back at it then?  I'm concerned ethically with using wounded embryos, but I am not concerned with changing gametes.  Okay?  So that from those gametes we could produce cells.  As I understand it, you're saying, and Janet was talking back and forth with me about how this has to be proven that the Cdx2 could work on the gametes, but it has not been demonstrated yet.  If it was with the gametes, then Robby, me and everyone doesn't think we've got a wounded embryo that we're working with here.

So I gathered from what you said that at least it looks like you can use the Cdx on gametes, and gametes and oocytes are unfertilized, and then go from there.  If that's the case, you don't have an organism and I don't have a problem.

DR. SCHÖLER:  Yes.  So you can be assured that actually when I'm back to my computer I will push that we're going to test even earlier, but that was not the scientific question.

DR. McHUGH:  No, I understand that.  I just want my terms to be clear, why I used the terms I did.

DR. ROWLEY:  Well, wait.  So you're saying it's okay to use a wounded zygote, a wounded oocyte that will then be fertilized by a normal sperm to get a zygote that has the genetic defect.  So you're using a wounded oocyte.

I mean, you know, I don't think this is going anywhere, but I just want to point out that —

DR. McHUGH:  I think it's going somewhere.

DR. ROWLEY:  — the manic morass we've gotten ourselves into.

DR. McHUGH:  Yes, I think we're going somewhere.  I think that at the level of cell reproduction and cell things, if they cannot — if you have a wounded gamete, it cannot be fertilized and turned into an organism it seems to me.

DR. ROWLEY:  Yeah, but if it can't be fertilized, then he can't get an eight cell stage structure to use, and he's betting that, in fact, a wounded oocyte plus a normal sperm will give you this Cdx2 deficient eight cell stage organism or structure that will be useful for cell lines.

DR. SCHÖLER:  I personally have a real, real problem to the terminology "wounded" with respect to a cell.

(Laughter.)

DR. SCHÖLER:  I've seen wounded people, but I think this is creating pictures in our heads, which I don't consider to be absolutely valid.

DR. PELLEGRINO:  The Chair just wants a word for a moment.  We want to give everybody a chance to talk.  There are a lot of hands waving desperately.  I must say I've lost the order a little bit.  The order we had was Meilaender, Gómez-Lobo and Bloom and others who want to get into it put your hand up.  Leon, you had your hand up.  I think, Dr. Foster, you had your hand up, did you?

DR. FOSTER:  I was just going to make one comment without making — the only thing is, you know, I was very encouraged to hear this morning from Robby, for example, for the first time.  Nobody is more concerned about wounded embryos and so forth, if you want to use that, than he is, and he's very encouraged by this.

There are going to be some people who think that if you mutate DNA, you've stopped an embryo.  I thought it was tremendously encouraging for the first time we've been in this Council to say we may be able to go forward along these things.

I think we ought to drop this thing about seeing what cells are wounded.  I mean, it's sort of silly, but there are going to be people in this world that no matter what you do, you'll never do a stem cell research, and you just have to accept that they are going to do it.

So I thought the comments were tremendous from this side of the room and very different from the comments early on because of different circumstances.

Thank you for letting me interrupt.

DR. PELLEGRINO:  Yes, you did.  Thank you.  Thank you for speaking while you were interrupting.

We have Meilaender, Gómez-Lobo, Bloom and anybody else that wants to get on the last time, ask Professor Schöler, who has been very, very kind to agree to wait and not respond until you've all made your comments and then he'll put his response together because I suspect there may be some overlap.

Dr. Meilaender.

PROF. MEILAENDER:  Three comments, each very short.  The first, I suspect that actually not a one of us lacks clarity on what we've been talking about here.  I mean, I don't know anything about cell biology, but we all understand that what Dr. Schöler reported on with respect to his own research is something that might perhaps be called an embryo that had then been disabled, but that he thinks — he's actually fairly confident — that it would be possible to do similar research in a way that didn't do it.

I think we're all clear on that, and I don't see any need to run around that pole indefinitely.

The second point, I want to say a word, Mike Gazzaniga, about your lost opportunity cost point.  I thought the mantra all along had been good science proceeds on as many fronts simultaneously as possible.  Therefore, you don't know; you can't quantify opportunity cost lost or gained until you're at the end of the process.

So it seems to me or at least I've always been told that good science proceeds on different fronts at the same time, and therefore, this is not a case of lost opportunity cost but good science going in various directions.

And then third, just sort of to second what Leon said earlier, I don't know that I see that there's something new to say particularly.  I mean, undoubtedly one could do little riffs on different parts of the white paper, but it seems to me that we did a pretty good job actually on that, and it has stood the test of time quite well.

DR. PELLEGRINO:  I have Gómez-Lobo, Bloom, and, Leon, I have you down here.  I wasn't trying to cut you off.

Anybody else who wants to get on the list, and Bill Hurlbut, but let's go again according to procedure, and we'll ask Professor Schöler to hold off until we've heard from all of you.

Gómez-Lobo.

DR. GÓMEZ-LOBO:  Thank you.

First of all, a question of terminology.  I would drop the word "wounded" altogether.  I think it is more or less standard to talk about mutilated or disabled embryo.

Now, if that's the case, I think we're talking here about the question is whether we have a mutilated embryo or non-embryonic structure at all.  Now, if this is obtained by modifying the gametes, from a moral point of view I don't see any problem.  In fact, I'm a bit disheartened by Mike Gazzaniga's remark because precisely here there has been a convergence.  I mean, there's many of us who are concerned about the intentional destruction of human embryos that are seeing a light here, are seeing a possibility of doing this research in a morally acceptable way.

If there's no embryo and if the de-differentiation does not lead to an embryo, there are no more problems whatsoever.  In fact, I was encouraged even by Bob Lanza's experiments because in principle he was accepting the idea that it made sense to try to derive embryonic stem cells without killing embryos.

Now, he didn't do it, but that was the point.

DR. PELLEGRINO:  Thank you.

Dr. Bloom.

DR. BLOOM:  This is not necessarily for Dr. Schöler 's presentation nor comments, but I wanted to expand the possibility of the list that Dick has been tracking for us to mention the work of Diana Bianchi, which deals with pregnancy associated progenitor cells, fetal cells of origin that persist in the maternal circulation for years and seem to be stem-like cells that can repair injured organs in the mother.

Since they are fetal cells, they will have both maternal and paternal genomes represented, and it seems to me that this is a very underexplored area.  It has been largely in the clinical literature, and it seems to me there is some very exciting cell biology that could be done with this because these are stem cells that don't require the destruction of any kind of embryo at all.

DR. PELLEGRINO:  Thank you very much.

Dr. Hurlbut.

DR. HURLBUT: Hans, I understood you to be saying — I think you used this very term that what is created is not an embryo but a single lineage cell culture if you start with the oocyte; is that correct?  That's the kind of terminology you would accept?

DR. SCHÖLER:  Yes.

DR. HURLBUT: Also, do I understand you correctly saying that even though there are barriers in the procurement of oocytes making SCNT or any kind of ANT work in non-human primates and then later with humans, and — well, suffice it those two, the oocytes and the SCNT.  Those are all barriers right now, but I understood you to say that you thought those were technical problems that by reasonable scientific estimation they could be overcome.  Without knowing for sure, it's reasonable to say that these will —

DR. SCHÖLER:  Could you repeat the last part?

DR. HURLBUT: Well, we will find ways eventually to derive oocytes without having to superovulate women.  You've made that point several times, and also that you thought it was reasonable that we could, not certain, but reasonable that we could make nuclear transfer work in primates.

DR. SCHÖLER:  Yes.

DR. HURLBUT: So given those positions, I just want to underscore what Gil was saying.  There's lost opportunity cost if we don't pursue these methods because whether one agrees or not, there's a political impasse, and pursuing these methods could open up what isn't open now.  We could get stem cell lines that could qualify for federal funding and that would be wonderful because they could be used with good ethical oversight.  They could be used on a national and international level for collaboration, and if the techniques work, we could get unlimited source of genotypes to work with.

So that strikes me as an opportunity that's worth pursuing, but not to mention the fact that how much better would it be for our civilization if we could go forward with this research with social consensus?  Besides the fact that this is a bitter controversy, the fact is that if this ever does come to cell therapies, it would be so much better if every patient who entered the hospital and would partake of these cell therapies felt comfortable morally with the way the therapies were developed.

So on both the social and a personal level, it seems like a very positive thing to see an alternative source of these cells, and from what you've said, it sounds like scientifically there's strong reason to believe that's possible.  Is that all a fair statement?

DR. PELLEGRINO:  Can you hold?

Dr. Kass and then we'll have Dr. Schöler.

DR. KASS: Pass.

DR. PELLEGRINO:  Dr. Kass passes.

Dr. Schöler.

DR. SCHÖLER:  So first of all, for me its something very important to have social consensus.  That's something that I came back to Germany from the States about two years ago I had a lot of discussions along these lines, and it's from me something that one person whom I really, really respect has been a major player  in the Bundestag, Fraufen Renessa  (phonetic) with whom I discussed this point, that society should be not torn apart by what we scientists are doing, and I think this is very important.

I also think it's for the sake of scientists themselves very important.  I've been in Germany as a student then the Sony Gevirin (phonetic), that is, the angry viruses — that's how the people were called who burned down labs in Germany.  I remember what Oliver Bristler had had to suffer because his name was put in the newspaper in a way that he had to be under police protection.

And so I think there will be certain groups in society who try to take this as an excuse to do harm to others, and for all these reasons, for more reasons, for these reasons and so on, I think as scientists we had a responsibility to take care that society is not torn apart.  I think that's very important.  That is for me a very important issue.

Regardless if I have certain points which differ with respect to other points, I think as a scientist I have to agree to the view which is providing the most consensus on these very critical issues.

And that's why I think going along these lines where people have less, I'm sure that you're never going to satisfy everyone, but by getting the problems solved in a way that more and more people have less of a problem with that type of research I think is extremely important, and that's why if I'm being faced by other scientists to say you're wasting your time because you're not going to please anyone, I think that's wrong.

Some people will try to hold up the stick higher and higher and say, "Now jump over this one," if you see how high it has to be until you stumble.

But I think having said this to the other part of your question, I think a lot of these things are really technical problems.  If you find a drug that disassembles the things that you right now remove together with the chromosomes, that's something that you have to do research on that, and you're not even going to find the question.

I think what science has shown, that normally there are solutions to problems.  It's just like it takes smart scientists together with people who know more about certain issues.  That's a teamwork effort, and we're going to get to solutions.

I'm very optimistic that we're going to find solutions.  If we are patient enough right now to wait until we are through this bottleneck, if we say — that's something that has been discussed a couple of times — that it's important that we move on as many fronts as  possible to see which one is the most successful, and what we are as a society willing to accept or not.  Maybe this is more promising, but we don't want to have it.  We at the end have to say if we will see if we were right or wrong what we're doing.

But where I see myself in the dilemma, I think a lot of things are possible here that are not possible even in Germany, and in Germany at the end things will be imported; therapies will be imported from countries where things were developed and Germans did not participate, and we're using the fruits of what has been done.  That's where I personally, Hans Schöler, will see a problem

Did that answer yours?

DR. PELLEGRINO:  Thank you very much, Dr. Schöler, for a very, very comprehensive and stimulating and obviously provocative presentation, which I think has been very useful to all of us.  We're much indebted to you.

Thank you.

DR. SCHÖLER:  Thank you.

(Applause.)

DR. PELLEGRINO:  We recess until two.

(Whereupon, at 12:21 p.m., the meeting was recessed for lunch, to reconvene at 2:00 p.m., the same day.)

SESSION 3: OVERVIEW: GENETIC RESEARCH AND CLINICAL APPLICATIONS

DR. PELLEGRINO:  As you all know, we've been having discussions over the last six or eight, nine months on various aspects of screening of newborns.  We're going to continue that discussion this afternoon, looking at some of the broader aspects than we've engaged before and the clinical aspects and some of the social aspects.

Our first speaker is Dr. Robert Nussbaum of the University of California at San Francisco, and again, Dr. Nussbaum, our custom has been not to provide a long curriculum vitae,  so you won't have the schizophrenic experience of saying, "Who is it they're talking about?"

(Laughter.)

DR. PELLEGRINO:  But we do have your curriculum vitae in the book.  It's in the book and you can all refer to it.

Dr. Nussbaum is going to address us on the clinical aspects of gene medicine, genomics.

DR. NUSSBAUM:  Thank you all for inviting me.  It's really a great pleasure.

So what I'll be talking about briefly and trying to leave plenty of time for discussion of, is a broad overview of just one aspect, one particular application of the Human Genome Project, and that is a public health application in an area that is often referred to as personalized medicine.

Before I begin, I did a search on this, and it's interesting.  The first time that I could find anyone use the term "personalized medicine" — I'm sorry it's cut off at the bottom as we have Macintosh PC problems.  It's a different kind of PC — in that it was back in 1990, and it was a paper called "Rewarding Medicine, Good Doctors and Good Behavior."

And what they said was we need to choose persons for medical careers who will find patient-centered care rewarding, and we need to provide those persons the training and socialization and underscore the value of personalized medicine.  That was 1990, but that was also the time that the Human Genome Project was just getting off the ground.

Fifteen years later, when the Genome Project was pretty much complete, personalized medicine took on a very different tone.  This is a quote from my former boss, Francis Collins, NHGRI.  "At its most basic, personalized medicine refers to using information about a person's genetic makeup to tailor strategies for the detection, treatment or prevention of disease," and it is in this context, in this definition of personalized medicine that I'll be referring to for the rest of this talk.

So I also want to say what my sort of bedrock supposition is here and one that I think we should all agree upon, and that is that gene variants are not completely determinative of disease or phenotypes of various kinds; that we're dealing with an interaction between genes and environment.

In some diseases, environment is a minor contributor.  For example, in cystic fibrosis your genotype is the predominant determinant of whether you're going to get this disease or not, although, of course, there's variation in your genotype, which particular mutant alleles can have an effect on the severity of disease, and there are also clearly environmental factors.

On the other end of the spectrum, we have a disorder like AIDS where the environmental impact of the virus infection is the overwhelming factor, but there's still a strong genetic contribution, for example, whether or not one has a mutant allele for a cell surface receptor that blocks the ability of the AIDS virus to get into the cell.

And in most disorders, for example, Type 2 diabetes or  Type 1 diabetes, we have an interaction between environment and genes in some very complex way that we are just now starting to try to dissect out, but it is a very difficult and challenging job to dissect out the environmental and genetic contributions.

But I'd like to point out that these are not mutually exclusive; that if we can understand, for example, the genetic contributions to disease, that will make us much smarter in being able to ferret out what are the environmental factors because we will then have some context.

As hard as it is to find these genetic contributions, we do have only about 25,000 genes, but the environmental impact, the environmental factors are broader.  They occur over time, and it's actually in some ways a much more challenging problem.

Okay.  So variation between two chromosomes.  We have these variants.  The most common are polymorphisms that are called SNPs or single nucleotype polymorphisms, and so here's an example of three polymorphic SNPs on a stretch of DNA.

On average, we have about one SNP for every 1,250 nucleotides.  We're talking about close to three million between any two chromosomes that one inherits from a father and a mother.

But if you look through all of humankind, we're talking about a lot more SNPs, already identified, ten million SNPs that have been found.  These are single-based changes in different populations across the world.

So there's a significant amount of variation, and of course, one of the challenges is how many of these SNPs actually have functional significance.  Some are going to be in neutral areas, and some are going to affect gene expression in extremely subtle ways.  Some of them may be actually in the coding regions of genes and change the amino acid that is encoded by that gene.  Others will change the way the gene is spliced.  Others will change the way the promoter works, and others affect the function of the gene in ways that we do not understand at this point.

And of course, the non-coding parts of the genome are the vast majority of all the DNA.  Only  a very small percentage of all of your DNA actually has the triplet code that codes for amino acids.

So the other important point I'd like to make about these SNPs is that as a result of the genome project and the catalogue of SNPs, we are starting to be able to trace the history of DNA variants: where they come from, and in particular, that there are groups of variants that stay together and have stayed together for tens of thousands of years.

And so that if you carry one variant in that location, you are very likely to carry other neighboring variants, and what location are we talking about?  Well, that's all over the genome, and it includes regions that are perhaps anywhere from 2,000 to 3,000 base pairs up to 10,000 or even 50,000 base pairs, depending on what part of the genome you're talking about and what population you're talking about.  The population history plays an enormous role in this degree of what we call linkage disequilibrium, that is, having multiple variants that are nearly always found together on a single chromosome.

So the idea here is that a variant may arise on a certain founder chromosome, and this would be in a small group of people.  Then as the population expands, and of course, we as human beings are actually quite recently developed as a huge population.  We started from a much, much smaller group of people in Africa.

So you have population expansion, and what happens of course is that every time you make eggs or sperm, chromosomes cross over with each other.  So you end up with this shuffling of parental chromosomes in the offspring.

But what happens is that when these regions are close together, the chance of shuffling gets lower and lower and lower to a point where these yellow bars represent regions of genome that have stayed together over hundreds of generations, and so all of the variants present here will be found together on a given chromosome in what's called linkage disequilibrium, the importance of which I'll bring up in a minute.

So let's talk about, first of all, the basic science.  You start with DNA sequencing, and you identify variation.  The next question you want to ask is to what extent do those variants contribute to susceptibility to disease.  That's really the basic science questions.

The basic tool for trying to understand that is association analysis.  It's an epidemiological tool, and it's very simple in some ways, and in other ways it is very challenging.

You take a group of people and you ask simply how many of them have the disease and how many of them don't or how many will develop the disease and how many don't develop disease.  If you follow people over time and you ask among those what fraction have the genetic variant that you're interested in and how many of them don't have it, and this variant can be two copies of a variant allele on both chromosomes or one copy on one chromosome.

This is essentially what epidemiologists call the exposure.  You're either exposed to the variant or you're not, and you divide up the population in this way, and the fundamental question when asked is: what is the relative risk?  What is the fraction of people with a variant who get the disease versus the fraction of people without the variant who get the disease?

And what is that?  Well, here's our two-by-two table again for association study, and this is the basic issue, the relative risk.  This would be A over A plus B.  It's the fraction of people with the disease who have the variant.  That's the people who have the variant, divided by the fraction of people with the disease who don't have the variant.  Okay?  That's the relative risk ratio.

When it's greater than one, and it's statistically significantly greater than one, there's an association between the variant and the disease.

Now, you can have a variant that's highly associated with a disease in a population for a number of different reasons, some of them biological and some of them artifactual.

What are the biological reasons?  Well, the first is the variant you're testing for association is actually responsible for the susceptibility.  It's because of that variant affecting the way the gene is being regulated or expressed or the way the protein looks that you're actually affecting the function and, therefore, the susceptibility.

It could also be as I described, that the variant you're asking about is in linkage disequilibrium with the variant that's actually responsible for the susceptibility.  The association will still be there, but not the functional connection.

And it's also possible that this association that you see is actually an artifact, an artifact of the way you put the study together.  You can have what's called stratification artifacts, and I don't want to go into any of the details, but this is one of the reasons why with association studies people have repeatedly said with good reason that an association needs to be replicated.  You need to see it happen in more than one population and make sure that it's not either a statistical quirk or actual artifact of the way you put your study together.

So here is, I think, an example of a recent association study that has been quite interesting and replicated and, I think, important, and that is this gene called TCF7L2.  It's a transcription factor.  It's expressed in the beta cells of the pancreas, the cells that make insulin.

About the common variants in introns associated with increased risk: now this is not in the coding part of the gene.  This is in the spacers between the coding part.  Common variants in introns are associated with an increased risk of Type 2 diabetes, and this was found through a study by DeCode Genetics, which is the company that's working in Iceland comparing genetic information and clinical information obtained through medical records in the country of Iceland.

What's interesting is the effect appears to be pan-ethnic.  So once this was found in Iceland, obviously a number of other people jumped to look.

Oh, excuse me.  This is the extent of the relative risk.  So if you have no copy, that's defined as being one.  If you have one copy of the variant, your relative risk is one and a half times, and if you have two copies of the variant, it's about 2.3 or 2.4 times.

So that's the degree of relative risk, and I want to stress that this is a very significant finding, and yet its effect on relative risk is moderate.  We're not talking about if you have this variant you're 100 times more likely to develop Type 2 diabetes than if you didn't have the variant.  We're talking about one and a half to two and a half times.

Okay.  So this has been repeated now in Indian-Asians and Afro-Caribbeans, and these numbers are not relative risks.  They're actually odds ratios, which are very similar to relative risks, and I'm happy to explain what the difference is, but it has to do with the way the study is designed.

But the important point is that all of these numbers are greater than one.  They're all significantly greater than one.  They all increase when you go from one copy to two copies.  So I think that this is a real finding, that variants in this gene in the entrons are associated with Type 2 diabetes in more than one ethnic group.

So what are the basic science questions?  Are the entronic variants the actual functional variants responsible for susceptibility or are they an LD, linkage disequilibrium, with the responsible variants?  Those questions are being answered.

Whatever the variant is that's responsible for the positive association, what effect do the responsible variants have on gene function?  Why do these variants increase your risk for Type 2 diabetes?

And finally, how does this effect on gene function increase susceptibility?

Okay.  So future progress.  You certainly want to know what are the susceptibility variants for a variety of common disorders.  Type 2 diabetes is one.  There are many others.  We'd like to know what those variants are.

In addition, there's a whole other area which I'm very happy to talk about, and that is identifying the variants that don't increase your susceptibility for diseases, but increase your risk of an adverse drug reaction or increase your risk for not having effective drug therapy.

And so this whole area of pharmacogenetics now is becoming extremely interesting and important, and the search for such variants is ongoing.

Okay.  So we talked a little bit about the basic science.  We want to find the susceptibility variants, but how do you use them?  And that's really where I'd like to spend the rest of the time.

Clearly, if you can identify susceptibility variants, what's the translational science?  Well, you certainly would like to be able to do individual risk assessment, in other words, test patients that come to your office for these susceptibility variants who may not have any disease at all, but would allow you to identify people who are at increased risk for developing disorders so that you could prevent it, intervene in some way either medically, behavioral changes, life style changes, whatever.

Also, if you can identify susceptibility variants, it may help you understand how to treat people better who actually have the disease.  If we understood why that transcription factor alteration affects Type 2 diabetes, we might be able to then treat people with Type 2 diabetes more effectively and more rationally.

And this, of course, is something that people talk about a lot, have talked a lot about, and I'm not going to in this context.

Okay.  So what are the translational science questions for the TCF7L2 variant Type 2 diabetes?  How can we use knowledge of susceptibility variance and devise new drugs or behavioral therapies?  And what does having a positive test for a variant mean to an individual person whether they have the disease or not?

Let's talk first of all about the ones who don't, and this leads us to this basic area, which I call the three "-itys":  analytical validity, clinical validity, and clinical utility of any genetic test.  You want to try to analyze these.  What are they?

Analytical validity I'm not going to spend any time talking about.  It's essentially the technical aspects of "Do you get the test right?"  Do you know how to do the test?  Can you find the variant that you think is there through the laboratory test?

Clinical validity is, all right, suppose you've got the right genotype.  You know what variant the person is carrying.  How well does that predict the phenotype, the disease?

And then finally, if you successfully predicted phenotype, what's the usefulness of it?  What's the clinical utility?  Does it result in an improved outcome to that person?

Clinical validity.  How predictive of disease is a positive test for any one patient?

Well, for that you really need these basic pieces of information, what's called the positive predictive value.  That's the fraction of people with a test who have or will develop the disease, and the negative predictive value, the fraction of people who are negative on the test who will not have the disease.

In other words, you can rule out or reduce their risk by doing the test and find they have a negative test.

Well, this is a busy slide, but it is, I think, the best way for me to demonstrate this.  You've got three factors you need to take into account.  One is how frequent is the variant in the population.  Is it a rare or common variant?

Number two, how common is the disease in the population?  So that's disease prevalence.  So here I've got one in 1,000 people have the disease, one in 100 people have the disease, one in ten people have the disease. That would be obviously very common.

Here's the genotype frequency, one in 1,000, one in 100, one in ten.

And then finally, what is the relative risk conferred by having that variant?  And I've generated these relative risks, everything from 1.5 or two, which is where we were for the Type 2 diabetes variant, up to 100, which would be a very substantial relative risk.

And what I've plotted here on the vertical axis is the positive predictive value, and I think that what you can see here is until you are at very high relative risks and common disorders, having a positive test has very little positive predictive value. 

I mean, down here, for example, if you have a relative risk of, let's say, one and a half or two with a disease that affects one in 100 people and with a variant that is present in one in ten or one in 100 people in the population, we're talking about positive predictive values well below ten percent. 

In other words, out of every 20 people or so walking into your office and you test them and they test positive, 19 out of 20 will not develop this disease.  Only one in 20 will.

So for most multigenic disorders that we're dealing with, these are common disorders where the disease prevalence is high, relative risk ratios are modest, one and a half, twofold.  Positive predictive values are very low, and the non-genetic factors are going to be very important.

So these are clinical validity issues.  So how about for the Type 2 diabetes?  These are the calculations that I did for preparing for this.  Disease prevalence, about six percent I think is a reasonable assessment for Type 2 diabetes.  The allele frequency for this variance has been found.  It's about.28.  So 28 percent of the population will carry either one or two copies.

The positive predictive value of carrying one copy is 7.5 percent.  So 92 and a half percent of people who carry one of those variants won't develop the disease. And for two copies, 11 percent, or 89 percent won't develop the disease.  Eleven percent will.

So you can see from a clinical usefulness point or I should say from a clinical validity point of view, this test is not very good at predicting your chance of developing disease.

Okay.  But now that leads us to the other issue, which is suppose even so, you do the test and you find people have this genotype.  How useful is it?  So this is an interesting quote from Kari Stefansson.  He is a CDO of DeCode.  "It's terribly important to know if you have this gene variant.  It gives you an added incentive to exercise and eat right."

And so the question really is: given that people might carry this variant, how is that going to change what you actually tell the patient sitting in your office about what that person should do?

Now, there are a number of common variants and common diseases that have been identified, and I put these down because they really in some ways cover the gamut.  One is ApoE4 for Alzheimer's disease, the disorder for which we can do susceptibility testing, but we can't intervene in any way.  We don't have any way of trying to suppose you identify someone with increased susceptibility.

On the other hand, we have Factor V Leiden, which is an alteration in one of the coagulation factors; it increases your susceptibility for deep vein thrombosis and possible pulmonary embolists, clots in the lung.  That's something you can intervene on.  You can intervene with anticoagulation.

And down the list, these all vary to a greater or lesser extent.  Hemochromatosis, you can intervene by removing blood and taking iron off of people, et cetera.

Okay.  So this leads us of the clinical utility.  Assuming the result is interpreted properly, is having an individual's test results useful or harmful?  That's really the essence of clinical utility.  What good is knowing the information?

And is that utility evidence-based?  Do you actually have retrospective data at a minimum, prospective data preferably, i.e., data that affects health outcome and economic and also has a beneficial effect on economic factors?

Of all the areas where this sort of genetic testing seems to be closest to really coming to use is in pharmacogenetics, and probably the number one area that people are looking at this very carefully now is in the use of the cumadin or warfarin, the blood thinning drug.

This is a drug that has a very high rate of adverse events.  We're talking about significant bleeding occurring per year in a few percent of people on this drug: three to five percent of people on this drug, some estimates as high as ten percent will have a significant bleed per year that they're on it.

A lot of people are on it: people with atrial fibrillation, people with deep vein thrombosis,  a variety of other people that are at risk for clots going to their lungs are on this drug.

The drug is metabolized through a variety of enzymes that have variants in the population, that are common, common variants, and depending on what your variants are, the proper dose for this drug can vary by as much as fivefold.

So that it is possible for someone to sit in your office, two people.   You see them back to back in your office.  You give them the same dose of warfarin, and one is going to bleed and the other is going to clot because one dose isn't enough.

Now, what do you do?  Well, what physicians who use warfarin do is they start the drug and then they monitor people closely.  They look at their anti-coagulation by doing what's called an INR.  It's the degree of blood thinning.

And people have gotten very, very good at following the INRs and adjusting the dose.  Despite that, we still have a significant level of harmful outcomes from coumadin use or warfarin use, and so the question is:  do we have any actual evidence that if we genotype the people for the variants that affect warfarin metabolism, would it improve care?

And it's sort of amazing that the FDA is right now in the process of thinking about changing the labeling for warfarin, and yet we actually don't have any clear prospective evidence that it actually affects outcome and very little retrospective evidence.

There is excellent evidence that if you genotype people you can get their INRs within range more quickly and more stably.  So if your outcome is the blood test, the degree of blood thinning, pharmacogenetic analysis is helpful.  If your outcome is serious bleeding, we don't know. 

The same is true for other drugs.  For example, there's a chemotherapeutic agent, arinatikan (phonetic), which is metabolized by an enzyme that has some significant variation in the population.  You can give people this same drug and one person will drop their white counts and get bone marrow suppression.  Another person won't.  That is now on the FDA label.

Another drug, mercaptopurine used in leukemia chemotherapy, also a tenfold difference in the proper dose of that drug depending on what your genetic makeup is.

So I think from the science, clinical utility point of view, pharmacogenetics is right at the top, and it's what we are going to see coming into clinic now.

Once you step back from that and you start asking, all right, well, what difference does it make to an overweight patient who is at risk for Type 2 diabetes whether they have the variant or not in that transcription factor if the positive predictive value is ten percent or eight percent?  First of all, what harm would you do that person by genotyping them at that locus?  One question.

What harm would you do by labeling people as being a "susceptibility carrier" if they actually are never going to end up getting diabetes?  Would you then draw back and say, "Well, it's not so important for you to lose weight and change your diet and your life style because you don't carry the variant."  What other sorts of problems might you be then allowing this person to suffer?

How good are we at using genetic information to motivate patients?  Would it actually motivate patients?  There's very little information about this.

There's one interesting study that was done by my former colleague, Colleen McBride who looked at variants in an enzyme that metabolizes some of the constituents of cigarette smoke, and she did this study among an African American population in North Carolina where they genotyped them for these variants and then tried to use that information to try to intervene and convince people that they should stop smoking because they're at greater risk for bad outcomes.

At six months after instituting this prospective study, the people who had gotten genotypic information had a better rate of quitting smoking.  By 12 months it was gone.

And so our ability to intervene with behavior modification is questionable as to whether this genotype information is going to help or not.  I am not one of these people who says it's useless because I just don't think we know.  It has never really been put to the test.

So in summary: We're in the era of personalized medicine.  Common genetic variants increase susceptibility rather than cause disease.  Genetics empowers the basic science investigations and drug discovery in a very important way, and I do not want to downplay the significance of this for basic science.

The direct application to patient care requires evidence of validity and utility, and this has to be done on a case-by-case basis, disease by disease and locus by locus.

And with that I'll stop and be happy to answer questions and discussion.

(Applause.)

DR. PELLEGRINO:  I'll ask Dr. Janet Rowley to open the discussion.  Will you do this for us, Janet?

DR. ROWLEY:  Thank you, Doctor Pellegrino.

Well, I'm sure I speak for all of my colleagues, Bob, when I thank you for a thoughtful, logical, but very sobering primer related to genetics.  I wasn't here for the last meeting.  So I've likely missed some of the pertinent discussion then which has, I believe, led to some of this afternoon's presentations, but it seems to me that much of what Bob Nussbaum discusses is tangential to some of our earlier discussions on prenatal genetic testing.

Therefore, at present as far as I know, although Kathy may disagree, PGD is done for single gene disorders with a clear association for a particular disease, usually one with a sufficiently serious or fatal outcome so the parents with to avoid having a child with that disorder, realizing, of course, as Bob pointed out that the accuracy of the test and the degree of penetrance certainly makes clear-cut associations difficult.

When we come to the era of personalized medicine, the decision to have any kind of genetic testing is complex, and it depends on the individual, on social factors, particularly the family, and the information regarding the disorder and the genetic complexity of the disease.

But I think that it's important to separate out single gene disorders, such as Tay-Sachs, with high penetrance from some of the others that we've been talking about, and I'm sure that there are good data available, though I don't know.

What's the proportion of individuals at risk of Tay-Sachs, say, amongst the Ashkenazi Jewish population? How many of those patients were screened, and what kind of impact did it have?

You've indicated that related to smoking the long term impact was little.  My impression is in Tay-Sachs with a very educated and committed and concerned population, maybe the answers are somewhat different.

You also raised the question of the interaction of environmental factors and also the interaction of other genes with a particular gene in question, and these are critical factors that we don't know.

So I think that — and this is something that we've discussed in the past — that genetic testing is going to be very unlikely for determining a person's height, athletic prowess or pulchritude, but the screening is going to be limited, in general, to serious genetic diseases.

And I think that one of the other issues is whether therapy is available, and we can test for Huntington's disease, but many very intelligent individuals who are at risk for Huntington's disease don't want to know whether they've got the disease or not or are at risk of having the disease because there isn't any treatment for it in any case.

So this goes back to your question as to what is the utility of this, and we had some readings under Tab 60 that you provided, Bob, that have different points of view.  Holtzman and Marteau are relatively negative about the impact of genomics on medicine, whereas Guttmacher and Collins are quite understandably more positive.

I think it's worth noting that the former was written in 2000 and the latter in 2005, and given the rapidity with which the field is moving, I think the difference is critical.

And you raised linkage equilibrium.  Certainly the development of the HapMap, which really defines how these blocks of DNA — not defines, but gives us information about these blocks of DNA and their inheritance in different populations — is going to be extremely important because rather than asking about a single genetic variant, one can say for a particular disease or group of patients who have, say, heart disease, are certain blocks inherited more frequently in the affected population than in those that don't have the disease?

And whereas it doesn't give you the gene because as Bob indicated, some of these blocks can be rather large, they certainly narrow down regions that we should be paying attention to.

And I think we also have to remember that some genes are associated with decreased susceptibility rather than increased susceptibility so that we're really a mixture and a balance, if you will, of those that decrease or susceptibility with other genes that increase susceptibility, and this just goes to confirming the complexity of complex diseases.

If every factor accounts for one or two percent in an individual, you know, you have to have 50 or 60 factors, probably not that many, but a large number of factors that are going to be involved.

So I think that Bob's points about genetic variance may increase the susceptibility rather than cause the disease is a very critical one, and then going to the question of whether the variants are functionally involved in the disease and if so, how their altered function is associated with the disease is very critical.

Now, my own view is that personalized medicine already has an impact on cancer treatment, and it isn't one  you mentioned, Bob, but in part I think it's because we're further along in our understanding of the genes or genetic changes that are associated with cancer.

So you know, we know not all of the individual genes that increase the risk, but also regions that are gained or lost that are associated with malignancy.  So I disagree with Francis' statement in his paper at Tab 8 that in the future we're going to sequence tumors.  I think instead what we're going to do is for large classes of tumors, breast cancer, prostate, et cetera, we'll have a series of known genes or chromosomal segments of interest, and we're going to monitor them in the tumor from the individual patient and then tailor treatment depending on what the answer is.

I do think in the future that we're going to do the same for common diseases, and then partly the question isat what age should monitoring begin.  The simplest thing is as you're drawing blood for Guthrie test and others, you draw blood for at least a HapMap.

Francis' dream is that we're going to have the $1,000 genome sequence, but then whether it's worthwhile spending $1,000 on every newborn to get the sequence, I'm not sure that we're there, but I can see reasons for doing a HapMap on children, and it certainly is going to depend on the disease.  And only time is going to tell whether personalized medicine really can fulfill some of its promises.

but I think in some areas it has already shown that it's important.

DR. PELLEGRINO:  Thank you very much.

Dr. Nussbaum.

DR. NUSSBAUM:  Well, I thank Janet very much for underscoring what I think is really a very important distinction, and I chose not to talk about single gene disorders of extremely high penetrants, such as Tay-Sachs disease.

There the relevant risks are infinite.  I mean, you essentially get the disease if you have two copies of defective gene.  The effect on a population screening, heterozygote carrier in Tay-Sachs has been among, for example, a targeted population, a self-targeted population, Ashkenazi Jews, has been very high.  The rate of Tay-Sachs in the last ten or 15 years has dropped  to, I believe, somewhere around five percent of what it was before based on carrier detection and people either choosing prenatal diagnosis or in some cases the arranged marriage is disarranged.

So this has had a very serious effect, a very significant effect.  I was really trying to focus much more on the multi-factorial complex disorders and the issue of really the validity and utility of that sort of testing for common disorders, and it's really a different area.

DR. PELLEGRINO:  Thank you.

DR. NUSSBAUM:  I was going to say the other thing is that I think the effect of genetic analysis in cancer has been profound, but once again, I'd like to make a distinction.  I think most of what's been done has been on the cancer cells, and so it has allowed us a lot of information and is going to provide even much more information about how to treat a cancer.

And in some ways that's a lot like what we have already been doing since the discovery of sulfa and penicillin, and that is studying the microbe to see what they're sensitive to and susceptible to so that we pick the right treatment.  That is where the cancer treatment is going, and I think it already is having a significant effect.

What hasn't happened in cancer yet, I think, is a screening where we are finding people who are constitutionally susceptible to cancer and then intervening in some way.

You know, there are people that are carriers for atxitillangetasia mutations, Bloom's Syndrome mutations that have a significant increased risk  for various cancers, but in the modest range, similar to what I showed you before, quite different, for example, through BRCA-1 and 2 where we have a highly penetrant gene depending on what study between 50 and 80 percent of people who carry this gene will develop breast cancer and/or varying cancer, and there personalizing the medicine for that family, identifying the mutations and counseling people on an individual basis I think is already having a very substantial effect.

So there's a difference between the single gene and the complex is one that's worth keeping in mind.

DR. PELLEGRINO:  Thank you very much.

Janet, do you want to respond?  Your light is on.

DR. ROWLEY:  I agree with him.

DR. PELLEGRINO:  Open for general discussion now.

PROF. DRESSER:  Thank you very much.  That was elegant and very clear to a non-scientist.

You mentioned that finding out more about genetic susceptibility would help discover environmental factors, but isn't it always going to be very difficult?

I mean, cellular Type 2 diabetes susceptibility, so that you've narrowed it down some, but you still have  a huge percentage from environment.

DR. NUSSBAUM:  I mean, for example, something near and dear to my heart and that's Parkinson's disease and trying to understand Parkinson's disease.  We have already identified single gene defects which cause a small percentage of Parkinson's disease.  By understanding the pathway that's affected, we can now look and see, all right, well, what does this pathway interact with, and is it involved with how pesticides are handled?  Is it involved with the way reactive oxygen species are dealt with?

So I think that the genes will shed light on pathways that they will help be smarter about asking about environment because as a non-epidemiologist, as a geneticist, I find environment very, very daunting.  It is not like the Genome Project where 25,000 genes and ten million variants.  I mean, that's a lot, but you can get your hands around it.

DR. PELLEGRINO:  Other questions?  Gil.

PROF. MEILAENDER:  Well, this is sort of a quirky question.  So make of it what you will, but as I was thinking about what you said, you know, the example about the smoking case that after I think it was 12 months the information seemed to have ceased to affect behavior.

But it also strikes me that even if for many of us information like that would cease to affect our behavior, if without it costing us too much because, say, our insurance paid for it or something, the information were available, lots of people would want to know, and those are strange things to try to put together, kind of.  You know, a lot of us would want to know this information, and that it wouldn't make a lot of difference in the long run in our behavior.

Now, I don't have any evidence really, but does that strike you as true?  And what, if anything, should one conclude from that?

I realize this goes beyond the kinds of sort of technical questions you were raising, but I'd just be interested in hearing you talk about that.

DR. NUSSBAUM:  Yeah, I'd be happy to.  I think it's really a very interesting point.

Some people, and I think there are differences between different people in terms of their personality, are very big into control.  They like to feel like they're in control and to be told, for example, that they can't be tested for a susceptibility to cancer from smoking.  Whether it affects their behavior or not, what they will say is, "I want the information, and then it is up to me to decide whether I want to act on it or not and how I want to act on it," and it's a matter of personal control.

I remember very clearly when the BRCA-1 gene was first cloned, first identified, and some of the first mutations were found.  We really didn't have a good handle on what the penetrance was.  So if you carried one of these variants, how likely was it you were going to develop breast cancer?

And so that the push came from NIH and from other areas that we need a study to find that out before people got tested, and I remember very clearly there was a letter to the editor from a breast cancer survivor saying, "Don't patronize me.  I don't want that sort of paternalism.  I want to know and I should be able to go get the testing now."

And I think that there is some validity to that approach that people have.

On the other hand, you do have to be careful because information can be dangerous.  It can hurt people.  It can hurt people's self-image.  It can hurt them in terms of employment, insurance, and a whole variety of other ways.  So if they're being tested for genetic variance and increased susceptibility, they have very little positive predictive value.  What are you doing to  — what good are you doing for them?

So you have to balance their feeling of "I want."  You know, it's about me.  It's my body, my DNA.  I want this information.

On the other hand, what is having that information going to do from a negative and positive point of view?  My view of it is that it's not clear cut at all and that it's going to vary from person to person, just in the same way as Janet brought up.  Even with a disorder like Huntington's disease, there are people that say, "I want to know."

I mean, I counseled a man two months ago who had through a research study gotten the information.  He wasn't supposed to get the information back, but he insisted, and in fact, the researchers couldn't deny it to him, that he was a homozygote ApoE4 carrier for apoepiprotein E, and therefore had an increased risk for development Alzheimer's somewhere between 15 to 25 years earlier than the general population, and he demanded to know that information.  He wanted the information because he was making life decisions.  He knew that there was nothing he could do to intervene, but you know, should I sell my house and buy a condo?  You know, there are certain things he wanted to know and had to do if — having control over his life by knowing this information.

So that's my view of it.

DR. PELLEGRINO:  On this point, Gil?

PROF. MEILAENDER:  Just to follow it up,  I mean, that was a nice and helpful response.

If we had some kind of national health insurance program and we put you on the committee to decide how we should rank, what sort of a lexical ranking we should come up with even though we can't fund everything in the world, how high would — sort of how important would be paying to test for some of these multi-factorial diseases that you talked about.  I mean, obviously they're of interest to you.  You've studied them, but now we've put you on this committee that's got to make this other kind of decision.  Where would it come?

DR. NUSSBAUM:  I hope I don't get put on that committee, but if I were and since you've just put me on it, I would try to test for those variants that reasonable clinical validity, and that they have utility.  Can you intervene so that you can improve the outcome of this person?  Can you benefit economics?  Is it going to in the long run save us, save society money by knowing?

And so, for example, very high on the list, I think my personal feeling would be everybody who comes in for the routine physical at some point is going to have a complete pharmacogenetic survey done.  That information only has to be done once.  It goes into that person's record, and then it will inform all drug therapy after that.

So that in the long run adverse drug reactions are an enormous source of morbidity and mortality and cost in this country.  Billions of dollars a year are spent because of adverse drug reactions.  If we could understand what the genetic basis for those are and prevent them prospectively by knowing the genetic information, I think we could have a major impact on well-being and economics.  So that would be high on my list.

So I think things with decent validity and demonstrated utility.

DR. PELLEGRINO:  I have Drs. Lawler, Kass, and Carson, in that order.

DR. LAWLER:  So, for example, diabetes, it would seem to pass the two tests but in a relatively trivial way, right?  For example, as a physician, as someone comes into your office, how would you judge, for instance, the diabetes?  Would it be the genetic test or the fat gut?

I think the fat gut test would be much more telling, especially if it's fat in a certain way, as you know.  So this diabetes information, I think, by itself just wouldn't be striking enough to me, the genetic information, to cause me to exercise more, and my doctor could tell me to lose weight without that genetic information.

So the genetics goes two for two on the test.  Nonetheless, if you were on this committee, would you bother?

DR. McHUGH:  I'd like to actually make two comments.  One is that one should not forget what is probably the single biggest personalized medicine intervention that we've had for years, and that is the family history, and so a family history of diabetes, I think, would play a role, and there is information  that people's behavior can be motivated to some extent by a family experience.

See, family history has two effects.  One is it demonstrates that there is a low susceptibility variance in that family that your patient is at risk for inheriting, but the other is the social aspect of it, which is that this person will have known somebody who has had this disorder and may have seen Uncle Joe end up with an amputated limb.

And so family history is a major effect.  I'm not sure at this point that the variant for Type 2 diabetes make it at the clinical utility level.  I mean, we really don't know that.

On the other hand, I'd love to see some well funded, decent prospective studies that really go at it.  I mean, that study with the genetic variants on the glutationous transferase that was done by Colleen McBride is one of the fe studies I can find in the literature where people have actually tried to do it and actually put it to a test, in essence, a randomized trial of genetic information to affect behavior.  We need more of those sorts of trials because for one thing, it may teach us how to do it better.

DR. ROWLEY:  Can I just intervene here?  So if you had ten or 15  factors for diabetes, and actually there are a few additional genes that I guess are more of Type 1 than Type 2, what would your answer be to Peter?

DR. NUSSBAUM:  Yeah, so the answer would be twofold.  One is if you had a constellation of variance that raised the relative risk very substantially, then I think the positive predictive value and the clinical validity would go up.

However, what has to be factored in is that the more variance you have, the rarer you are in the population, and so that the impact from a public health point of view is probably reduced.  so that's the tradeoff.

DR. PELLEGRINO:  Leon.  Dr. Kass.

DR. KASS: Thank you.

And thank you for that wonderful presentation.

I want to, I guess, continue on this theme of clinical utility, which I think you presented quite admirably, and it is sort of of two parts.  The study that you cited, and I don't know how many such studies there have been, it seems to me it would be interesting to replicate these things to see what the comparison is between the fear that might be generated by a genetic risks factor from other sorts of things that could be held up as a way of providing changes to the incentives to change behavior.

It's not enough, I think, to sort of simply look at does this genetic knowledge somehow lead to a greater incentive to quit smoking and displaying, you know, photographs of cancers and taking someone to visit, you know, the hospital.?

Since in so many of these things which are not single gene disorders with high penetrants, where the environment plays a large role, it seems to me that it would be very desirable to have some kind of well thought out, prospective disorders to see what is the efficacy of genomic knowledge compared to other sorts of things.

And I wondered if you could comment on that, and then I guess second — well, a footnote to that.  The change of behaviors that would be required will differ a lot.  I mean, it's one thing for someone who for a variety of reasons likes to eat and likes to eat to excess.  The loss, the calculations of present pleasures versus future risks, very different.  Much harder to motivate certain kinds of people to exercise than others.

And so it would seem to me that to really do this study right, you would a great deal of multi-variables in terms of the environmental things, and not all diseases are going to look the same.

The other thing is I wondered if genomic knowledge and genetic knowledge — maybe this will change — still has a kind of mystique about it, not necessarily to the scientists who work on it, but to lots of people in the public, and you can tell them till you're blue in the face this is not a determinant.  This is part of a susceptibility.

They hear this as there's a certain element of fatedness about this, and I wondered to what extent that is beneficial or misleading in the source of doing your own sort of clinical counseling, especially when you're dealing with things with the penetrants as low and the meaning of this genomic knowledge to you will differ from its meaning to the people to whom you give it.

There wasn't a clear question in there.  I'm sorry, but it sort of circles around the questions of how do the people receive this kind of knowledge in contrast to other sorts of knowledge, and if you're interested in clinical utility, how will a profession that might come, notwithstanding all of your caveats, to regard the genomic element as very high?  How are we going to know that that really is the best way to try to begin to influence the behaviors that would make for really clinical usefulness?

It wasn't as clear as I would have liked, but you nod.  So maybe you can do something with that.

DR. NUSSBAUM:  No, I think those are all very useful and important points that you're making.  In terms of the first part, I'm not a behavioral scientist, and I just think we need to do a lot more work.  I can put a small plug in here for my former colleagues, Colleen McBride and Larry Brody at the National Human Genome Research Institute in the intramural program, that are undertaking now, I think, a very interesting prospective trial where they are typing people for variants that are thought to affect things like bone density and so for a risk of osteoporosis and a variety of other such complex disorders.

From the point of view of trying to really study how do you communicate that information and what do people remember about it and how do they use it and do they used it, I just think we need a lot more of that.

The other point you're making is actually one of the areas where I was told in my charge that I was supposed to particularly identify areas that are of ethical issues, ethical dilemmas, and I think you put your finger on a major one, this issue of genetic determinism and what negative effects that will have or could have on society.

So in terms of thinking about genetic variants that increase susceptibility to disease, there have been a lot of studies done looking at do people take a fatalistic point of view or do they take a sort of empowerment point of view.

And the answer is yes.  Different people, different perspectives, different responses, and it's really quite fascinating.  The genetic counseling literature has a lot of such studies.

Unfortunately the vast majority of them are sort of hypothetical.  You would go to someone and say, "Suppose we had genetic variants that increase susceptibility for alcoholism.  What do you think about that?  Would you want to be tested?" et cetera, et cetera, et cetera.

Once we start actually finding variants, then I think those studies are going to take a very different kind of tone.

The other area, I think, is in genetic variants that change our susceptibility not so much for type 2 diabetes, but things like alcoholism, drug addiction and other sorts of traits that have a disease component, but also have more social effects.

And there I'm very concerned about over stressing of genetic determinism for traits that have, you know, major social impacts.  And so I think it's really incumbent upon everyone who does genetics and people that are interested in genetics to continue to repeat the message that a complex trait is a complex trait with environmental effects that can be intervened in through environmental ways, and that if we were at the end of the day to have a situation where people thought they could be tested and then this would make a prediction as to whether they would have violent behavior or whether they would or would not become alcoholics with high positive predictive value, that I think would be a serious disservice.

DR. PELLEGRINO:  Dr. Carson.

DR.CARSON:  I'd like to add my thanks for that clear and interesting presentation.  My initial question was really along the same lines as Leon's and you sort of answered it to a degree.

But one question or I assume that you're quite pro, you know, genetic testing.  It certainly seems like a worthwhile thing to do, and yet it's really in my opinion not that different from many things we've been doing for decades, you know, some of the enzymatic testing, for instance, that we do on newborns.  You know, every man when he has his annual physical gets a PSA, which is not necessarily 100 percent predictive, but certainly can provide some guidance in terms of clinical utility.

Doesn't it seem to you like we could use very much the same type of argument for genetic testing?  It's just maybe perhaps a little more sophisticated than what we've been doing in the long run, but in principle it's no different.

DR. NUSSBAUM:  So I think you're making an excellent point, and to use that old, hackneyed phrase, the devil is in the details.  So in newborn screening, for example, if we  successfully identify a child at risk for PKU, that child will develop PKU, and it's not a matter of having a  low positive predictive value.  It's a screening test that obviously needs to be followed up, but I'm talking about the whole system, not just the one Guthrie test, but the whole system or the one tandem aspect, the whole system results in a test which is very predictive and which we can intervene on, an enormous clinical utility, enormous clinical validity.

And so that, I think, is in a different pot.  I shudder to talk about PSAs with someone with the kind of experiences that you've had as a surgeon, although I guess prostate surgery is probably not your area — well, yeah, the other end.

(Laughter.)

DR.CARSON:  But my understanding is PSA testing also is an issue, and to what extent should it be done and at what age, and what really is the clinical utility and validity of testing people over age 60 or 65 with PSA?

And so in that sense I think they're very similar and the same kinds of questions should be applied.

The argument, and I think Dr. Kass brought this up, too, which is this genetic exclusivity or the specialness of genetic testing.  I think what it comes down to, to some extent, is that a lot of the testing that we do is to test for the early signs of a developing phenotype like abnormal glucose tolerance or an elevated blood pressure, well before there is any disease from it, but at least there's a measurable change in the phenotype of that patient.

With genetic testing, there is no phenotype yet, and there may never be, and so I think that's the area where we really have to apply real critical thinking and decide.

In some situations I'm absolutely convinced that genetic testing is going to be life saving.  It's going to reduce economic cost.  It's going to reduce hospitalization.  As I said, I think the top of the list is pharmacogenetic testing from my point of view, although even that requires more demonstration.

In other areas, until the science changes, and of course, what would I be saying sitting here five years from now if within the next five years we develop an effective treatment that prevents the onset of Alzheimer's?  then I think whether one would test for ApoE4 or not becomes a very different question.

DR. PELLEGRINO:  Dr. Hurlbut.

DR. HURLBUT: I appreciate your emphasis on the utility of the single cause model and the causal web and the polygenic nature of many traits.  What I want to ask you is sort of a slightly different angle on the clinical utility question.

Am I right in thinking that when it comes to recombination events that there are hot spots, that it isn't just simple random recombination?

DR. NUSSBAUM:  It depends on the scale.  So if you're looking at the whole chromosome level or even down to a few megabase level, it's quasi random.  There are some differences.  The tips of chromosomes have a higher recombination area than the area around centromeres, but in general it looks pretty uniform.

But just like with a digital picture, once you get up really close and start seeing the pixels, then you start seeing very different recombination frequencies, and there is some evidence that the linkage disequilibrium blocks that have been detected as statistical associations have biological reality in that the boundaries between those blocks have been at least in some cases demonstrated to be areas of high recombination.

DR. HURLBUT: The reason I ask that is because it strikes me that it would be very clever of nature to have several genes contributing to a single trait, segregating together consistently.  That way you could select for the trait, and that's the reason I wanted to ask you the question.

Because in an earlier report we worked on preimplantation genetic diagnosis, and we pointed out rightly in that report that  selecting for traits for most things we care about wasn't very likely; that it's much easier to select for a gene that's a broken link in a chain and, therefore, cause of a disease, but for positive traits or desired variations, that's a lot harder.

But now what you're saying implies that there might be haplotypes that could be selected for, in which case the idea of doing genetic testing for  reasons other than disease analysis might have some attraction here.

PARTICIPANT:  It becomes more realistic.

DR. HURLBUT: Yeah.  Do you think there's anything to that?

DR. NUSSBAUM:  I guess I think that the element that's missing from that picture is that it's very likely that it's going to be multiple haplotypes distributed throughout the genome rather than one single one.

DR. HURLBUT: But 50,000 base pairs could still subsume quite a few genes.

DR. NUSSBAUM:  Yes, in the area where we already know that there's linkage disequilibrium and significant effects over long ranges in the MAC, the HLA region where there are alleles that move together, and for reasons that are unclear that may not have to do with whether recombination is random or not, but have to do with selection for certain alleles being kept together.

And so I don't think we really know.  I think you're raising an important scientific question, is that certain alleles in regions in multiple genes may have been kept together for reasons other than failure of recombination to occur.

DR. PELLEGRINO:  Dr. George.

PROF.GEORGE:  Dr. Nussbaum, I wanted to follow up — oh, was somebody ahead of me? — on some of the comments and questions of Professor Meilaender and Kass, and really the question I have is one that people in your business probably ask very frequently.  It's the forbidden knowledge question.

And here what I have in mind is not whether an individual is better off not knowing something about himself or a family is better off not knowing something about a family member, but rather the more general question:  are there some questions about genetics that we, in general, are better off not asking?  Is there genetic knowledge that we are better off as a society not knowing about?

And we divide those two questions along another axis.  If there's anything to the idea of forbidden knowledge, are there some things that, for example, it's better off not knowing absolutely, that there are no circumstances in which a decent society would really want to know because of the bad things likely to happen if we do know.

And the other would be a category of things that we're better off not knowing now because knowing it is dangerous, potentially harmful before we know other things.  Now, maybe after we learn other things, the possession of the first body of knowledge would not be dangerous.

Now, I ask this not as a rhetorical question.  I myself have a strong aversion to the very idea of forbidden knowledge, but you're working right smack in the area, and I bet you've asked yourself the question, and I'll bet it is kicked around.  So what are your own thoughts and what do people say about this?

DR. KASS: Are you going to give an example?

PROF.GEORGE:  Yeah.  Do you have one, Leon?  Maybe if you have one.

DR. NUSSBAUM:  I'd love one.

PROF.GEORGE:  Things having to do with links of genetics with crime.  There's a term, but is it called criminogenic, a criminogenic basis for behavior, that kind of knowledge?

DR. NUSSBAUM:  So I can approach this two ways.

PROF.GEORGE:  Or even just your own — sorry to interrupt again —

DR. NUSSBAUM:  Sure.

PROF.GEORGE:  — your own example that you were talking about with alcoholism.  I think you were getting near raising a question about whether we really are better off as a society knowing about it, knowing about a genetic link with alcoholism or a predisposition to alcoholism because of the nature of alcoholism is not simply a disease, although there is a disease component to it, I guess, by the prevailing account.

DR. NUSSBAUM:  Major account.

PROF.GEORGE:  But there's so many other things connected with it, social factors connected with it.

DR. NUSSBAUM:  Right.

PROF.GEORGE:  I wish I could think of a better example.  If somebody has a better one, toss it in, but this is the general thrust of my question.

DR. NUSSBAUM:  Right.  It's a very difficult question. It's actually one that bothers me at two o'clock in the morning when I wake up and I'm having trouble sleeping.

So is there such a thing as forbidden knowledge in genetics?  I approach it sort of in two ways. One is I can kind of assuage my concern by just reminding myself that we're not talking about genetic determinism.  We're talking about susceptibility variants, which are going to be, as Dr. Rowley pointed out, probably balancing acts of various kinds.  Certain variants that might increase one's relative risk for certain things and others that would decrease it, and so that knowing those at an individual locus-by-locus or gene-by-gene way might not necessarily have a major impact on the individual.  There's going to be a mixture of these things.

So there's that.  However, I still personally have concern about what I think is the major challenge facing complex genetics now, and that is what are we learning about human origins and, in particular, human geographic origins, and what is the overlap between the science of human genetic origins and the social construct that we call race.

That, I think, is a major, significant, serious societal issue, and one could imagine — and this has already happened to some extent.  Janet brought up the question about Tay-Sachs disease among Ashkenazi Jews.  Ashkenazi Jews were very fast to promote heterozygote screening.  Ten, 15, 20 years later, a very different attitude among quite a few Ashkenazi Jews about the breast cancer gene.  It was actually quite different, even though they have a significant allele frequency for certain variants that predispose to breast cancer.  The feeling that this was a variant that was stigmatizing them as a social group.

DR. PELLEGRINO:  Dr. Foster.

DR. NUSSBAUM:  Is that even close to addressing?

PROF.GEORGE:  Yes.  Indeed, it is, and the only follow-up I would have is what about the potential uses.  Do you ever worry about potential uses of genetic knowledge, for example, in a military context that could make matters worth — what weaponizations that are made possible by virtue of advances in genetic knowledge?  Is there anything there to worry about?

DR. NUSSBAUM:  That has not occurred to me, and I can't imagine it, but I mean, maybe someone could convince me, but I don't know of it.

PROF.GEORGE:  It sounds like Janet knows something about it.

DR. ROWLEY:  I don't, but you didn't really ask the question of forbidden knowledge or answer the question, and I'm curious as to what your thoughts are on that matter.  Are there some things — say, take the example of alcoholism — are we better off not knowing about susceptibility to alcoholism or drug abuse as a society rather than understanding that?

That's just one example that was tossed out.

DR. NUSSBAUM:  So my answer to that is that I think that those are not examples where we should — those are not examples of where we should not go. I'm sorry to do the double negative, but I think those are examples of where we should go, but the individual autonomy of being able to choose whether one wants to be personally tested or not, needs to be absolutely protected.

But I think that that knowledge of susceptibility to alcoholism, drug abuse, other sorts of traits that have societal impact is valuable.  I'm actually not that worried about that.

I guess the example I come back to often is, I mean, I'm an Ashkenazi Jew, and there are, quote, Jewish diseases.  There are rare carriers of rare disorders for a whole host of disorders, Gauche disease, Canovan disease, Tay-Sachs disease.  You can go down the list. 

My own personal feeling is I'm grateful to have that knowledge so that we can do something about it.  I don't feel stigmatized because I know that every group around the world has their own sect, and it's just to some extent we've discovered them.  In many situations we haven't discovered them.  So we're all in the same boat together.  It's just that we're sitting in different places in the boat, but we're all in the boat.

DR. PELLEGRINO:  Dr. McHugh.

DR. McHUGH:  On the forbidden knowledge thing, you have to remember that sometimes the very study of the possibility of a connection to behavior and genes has provoked many people to be beset by anger about this.

The XYY study, you remember, we were looking to see whether young children with XYY were criminal, and I think quite rightly we said that was perhaps stigmatizing them right at the start and putting before them the possibility that might not be there if we weren't, in fact, studying it.

So it became a forbidden form of study.  Fortunately, it turned out that they weren't.

DR. NUSSBAUM:  Well, I should say that my very close friend and actually my mother-in-law —

(Laughter.)

DR. McHUGH:  That's pretty close.

DR. NUSSBAUM:  Yeah.  — was involved in the study in Denver run by Arthur Robinson, which was a prospective study of newborns looking for SEC (phonetic) chromosome abnormalities, and as opposed to what happened with the Walter study, that study was allowed to continue, and they ended up following 40 or 50,000 newborns, and it was done as a therapeutic interaction between social workers, geneticists and the families.

And what it demonstrated was that knowledge could be used for intervention, particularly in the area of education for the children who actually have learning disabilities which are not the 47 XYYs, but the 47 XXYs nd the 47 XXX females.

DR. PELLEGRINO:  Dan, you've been waiting very patiently.  Thank you.

DR. FOSTER:  Well, that's right.  I want to just make a brief comment that's not meant to be amusing or anything about it, but there is another form of genetic terminism that is rampant in clinical medicine, and you know, it really is in the old phrase that, you  know, my genes made me do it, and probably the most important generalized disease in the world right now has to do with obesity and Type 2 diabetes.  When you get the metabolic syndrome you might — I can't remember whether I said this last meeting or not — but the leading cause of liver disease in the world is non-alcoholic fatty livers.  It gives you more cirrhosis than you get with alcohol and so forth.

If you take care of patients with diabetes, as I do, and Type 2 is the common thing, and all are overweight, and it's an absolutely curable disease right now.  You don't need a kidney transplant.  You don't need anything.  You prevent the eye disease.  It's an environmentally cured disease, but they always say they see this new gene for Type 2 diabetes and said, "I always knew that the reason that I couldn't lose weight was because of this gene."

And as a consequence, a defense against the cure of major diseases, like lung cancer and smoking, coronary artery disease, almost all of these things have a huge environmental component.

Now, we learn other things about doing the genes.  I've been involved in the discovery of an effect on the insulin fairly recently in Asian Indians, which gives you the metabolic syndrome without obesity.  It's a very interesting thing.  There's a little review coming out fairly soon about that.

So you learn these things, but a very great amount of the disease that we deal with right now can be controlled without the genes, and this is not to say that, you know, endocanibinioid receptors and hedonic pleasure networks are not important, you know.  I mean, we now think that the eating signals are hooking up with the same place marijuana goes and so forth so that there are things that drive you.

But these are curable diseases, and so we have to — I think we have to be careful to not give an excuse to do the things that we can do is the minor point that I want to make.

Mike Brown, who works at our place, always tells the medical students that disease is a consequence of both genes and environment, and he said, for example, a person that was just hit by the car out in front of the medical school and broke his hip had a genetic disease.

And the students all say, "Well, wait a minute, Dr. Brown.  How can you say that?  I mean, that's a pure environmental disease.  The car hit you and he broke the thing."

And he says, "Well, if you had better hearing or better seeing, you know, you would have gotten out of the way and you wouldn't have had it."

But I do want to say we have to be a little bit careful about a defense against things that we can do right now on a genetic basis because of the assumption that these polymorphisms and whatever they are are going on are true, and we need to do all of those things, I think.  I mean, you're going to learn how to maybe do specific treatments in that.

DR. NUSSBAUM:  I think it's a fascinating question in behavior science to ask the question does knowing that information make one more motivated to act on it or retreat into a fatalism and say, "Because of my genes there's nothing I can do about it."

I think it's an open question.  It's probably different for different people, but what I think is really a fascinating question is can we  now build on that to do behavioral science research and figure out how better to get people to intervene in the environmental factors and cure these diseases that they're susceptible to.

DR. PELLEGRINO:  Other questions?

PROF. SCHAUB:  Do you have views on direct to consumer genetic testing, given your concerns about misapplications or misunderstandings of the information?

DR. NUSSBAUM:  Yeah, I do have strong issues about it, and actually I was at a meeting that Dr. Hudson organized quite recently precisely on that topic. 

So I think there are some potential benefits of certain kinds of testing in terms of empowering people, is what I was speaking to Dr. Meilaender about, which is people want to know information and perhaps use it.

On the other hand, there's a lot of direct to consumer testing that, first of all, is false in terms of clinical validity.  It is without any clinical utility, and on top of it all, in some situations is bound together with completely unproven neutroceutical interventions that I think are essentially something that the Federal Trade Commission should look hard at in terms of false advertising.

So, yeah, I do have strong feelings about it.

DR. PELLEGRINO:  Leon.

DR. KASS: This is an indulgence, but I don't see other hands.  So if you'll indulge me, this is back to the subject of dangerous knowledge.  Just an anecdote.

I was a young staff person on an NRC committee just like this one in 1970 to 1972, and I was stunned when the report review committee of the academy decided to censor the report that our little committee had produced on the grounds that if people read that document they would cut off all funds for biomedical research.  That was dangerous knowledge, number one.

(Laughter.)

DR. KASS: But the more interesting one was it was coincident with another example of self-censorship, one which I applauded, when William Shockley proposed that the academy come out in favor of a study of race and IQ, then I don't think was anything like the genomics studies, which now could in principle at least be undertaken, but Dubjanski was appointed the head of a serious committee, and it was a model, just a model of prudence saying that there was absolutely no good that could come from this kind of a study, and we ought not to give it our blessings.

And it seems to me — and you've touched on this already — we may yet face certain kinds of difficulties.  The African American community did not take to the proposals for routine screening for sickle cell disease the way the Jewish community did over Tay-Sachs partly because of all kinds of other concerns about what this meant in terms of stigmatization, absence of care, and I think not without cause.

DR. PELLEGRINO:  Dan.

DR. FOSTER:  All racial things are not problems at all.  I hope you didn't mean to do that because it would be just like the sickle cell thing.  Helen Hobbs has discovered a new gene that means that if you're carrying it if you're an African American, you don't get coronary artery disease for the same level of cholesterol and so forth.

I mean, this is a terrific thing that came out of the Dallas heart study.  I mean, you know, and the Dallas heart study was deliberately aimed at why African Americans have more heart disease than other things.

You know, we measure blood pressures in barber shops.  I mean in other words, what they — I don't have anything to do with this, except it's at my school — and the whole community, not only the religious community, but it turns out if you want to get your blood pressures checked regularly, go to the barbers for men.  It's the men that are a problem, you know, to do that.

And so we have these little things all over town, and the enthusiasm of the community because things are coming out that may save their lives and so forth.  So it's one thing to talk about — I mean I agree with  you wholeheartedly about minor changes in intellect that might say that somebody who lived in Texas was going to be more stupid than somebody who lived in Massachusetts or something, you know, but these other things, I don't want to leave the impression that racial studies in and of themselves are dangerous or should not be done.

DR. KASS: I agree with you completely.  I didn't mean to imply otherwise.

DR. FOSTER:  Well, I was sure you didn't mean to imply that.

DR. KASS: I'm glad to have it on the record.

DR. PELLEGRINO:  I think it's time for a break until 3:45.  See you all then.

(Whereupon, the foregoing matter went off the record at 3:30 p.m. and went back on the record at 3:51 p.m.)

SESSION 4: OVERVIEW: GENETIC ETHICS AND PUBLIC POLICY

DR. PELLEGRINO:  Next, this is an overview of genetic ethics and public policy.  We have the privilege of hearing Dr. Kathy Hudson, the Director of Genetics and Public Health Policy at Johns Hopkins University.  She knows we don't give long introductions and she's pleased with that.

So I'll ask you to jump right into the matter at present.

DR. HUDSON:  Thank you very much for the invitation to be with you today. 

I have a narrow subject about genetics ethics and public policy, and what I thought I'd do is divide my remarks into three sections and talk about ethics and policy issues in genetics research, in clinical practice, and in non-medical contexts.

So first, in talking about genetics research issues, there are a number of policy issues and ethics issues which are really garden variety issues and are common to all biomedical research and really don't pose immediate problems in genetic research, and some examples are given there.

Then there are issues that are special but manageable issues in genetics research, including impacts on family members, including non-paternity ownership of specimens and data, and intellectual property issues which, while present in all biomedical research, are particularly acute, I think, in genetics research.

And then there's the really tough issues, and I think some of these really tough issues are emerging as a consequence of the rapid proliferation of very large cohort studies with large biobanks and databases.

So Bob had talked a little bit about the intersection between genetic factors and environmental exposures and life style and behavior.  And in order to dissect out the weak genetic contributors, probably numerous genetic contributors to any specific health outcome, and the numerous environmental exposures, lifestyle and behavior inputs, it has been proposed that in order to unpack that problem, that we do a large scale, population-based study where we collect information about all of these inputs.

So the proposal has been made, but not funded and probably won't be funded for some time, to study a very large cohort of people in America, and I should mention that this has already been under way in a number of countries around the world, including Iceland, which is where the diabetes allele that Bob mentioned was found.

So in the U.S. it has been proposed that half a million people be followed, that DNA and biological specimens be collected, that clinical data be collected, lifestyle and behavioral information be collected, environmental exposures, and that folks be followed over a decade, and this is all to provide a very large research resource in which people can use that resource in order to be able to identify weak genetic, environmental, and behavioral contributors to health outcomes.

I should mention that in terms of the technologies for being able to do this, the genetic technologies are really ripe to be able to do the genetic component of this.  The technologies for accurately assessing lifestyle behavior and environmental exposures, I think, are really sort of akin to where we were in the '80s with genetics, where we really don't have very precise measures of some of these things.  They are coming along.  So, for example, sensors that can be worn that measure air quality, for example.

So what issues are raised by such a study?  There are issues in terms of whether or not the primary data is returned to the individual research participants, and if information is revealed, that places that participant at high risk, imminent risk.  What is the obligation of the researchers to provide immediate care?

What kind of research or what kind of consent is provided for this secondary research, with this very large database?

As Bob in the discussion mentioned, the personal and social reactions to potential group findings, findings that are relevant to different social groups, and then, of course, how do we protect the participants in terms of privacy discrimination.

And certainly within the study information will be collected about people's participation in illegal or stigmatizing behaviors.

So in many large DNA studies oftentimes the samples and the information is de-identified and whereby it becomes no longer subject to some of the rules and regulations that guide human subjects research.  And just to remind you that human subjects research guidelines defined a human subject as somebody who's alive, somebody from whom data is collected through an intervention or interaction, and it contains private identifiable information.

The office at HHS responsible for implementing and enforcing these rules has said that it doesn't consider coded private information to involve human subjects if the information was not initially collected expressly for the purpose of the second study or third study or 105th study, and that the investigator cannot readily assess the identity.

So it's not that anybody can't ascertain the identity.  The investigators can't readily identify the individuals.  And I think that raises some issues for us collectively, is whether severing the link between researcher and participant is a good thing or a bad thing.  Is DNA ever really not identifiable?

Amy Maguire and Richard Gibbs have published a paper recently in Science in which they argue that we might need to reconsider the rules governing the use of de-identified samples in the absence of consent.

Specifically in a proposed large cohort study severing the link between the participant and the researcher may in the end sever the ability of individuals who participate to receive information about that study that may be relevant to their own health.

So I'm going to move now to clinical genetics issues, and I'm going to just give three little tidbits of information, I think, that are relevant to the clinical genetic situation in terms of policy and ethics.

And the first, and just to remind everyone, the number of genetic tests is increasing steeply.  Most of the genetic tests prior to the present day were for rare Mendelian genes and mutations.

More recently, they are for more common variants that contribute to complex diseases and for pharmacogenetic tests, and you can see that the slope is getting steeper on this line, and I think that's likely to continue.

And there has been projections that we will have handy-dandy devices that can read out our entire genomes within the next few years.  This is an article by George Church that was in a recent Scientific American.

So this committee has considered the issue of preimplantation genetic diagnosis in the past.  To remind you embryos produced through in vitro fertilization have a single blastomere removed.  Genetic analysis is performed, and based on that analysis embryos are selected for transfer back into a woman's uterus.

This committee, when it issued its report, "Reproduction and Responsibility," said, and I quote or maybe I'll paraphrase, that there really wasn't enough information about PGD to help the committee or other policy makers formulate policies to govern this area of clinical practice and research, and the committee recommended that studies be undertaken to really get a good handle on what was going on in terms of preimplantation genetic diagnosis.

In the wake of that recommendation and our own work, we conducted a survey that I would like to share just a couple of top line results from, where we surveyed 415 assisted reproductive technology clinics in the United States, had a 45 percent response rate, and what we learned was that three-quarters of the IVF clinics are performing pre-implantation genetic diagnosis.

We ask them to estimate the number of cycles of PGD — this is not babies.  This is cycles of PGD — in 2005 and had among our group 3,000 cycles reported, and we estimate that that's about four to six percent of all the IVF cycles in the United States.

This committee has talked a lot about for what purposes preimplantation genetic diagnosis is performed, and so we asked clinics whether or not they offered PGD for these different purposes, aneuploidy testing to look at abnormalities in chromosome number, autosomal disorders, chromosomal rearrangements, X-linked diseases, non-medical sex selection, adult onset disease, HLA typing in combination with a single gene test, and HLA typing in the absence, and finally to select a disability.

And you can see here that overwhelmingly aneuploidy testing is the most.  Most clinics that are performing PGD are offering PGD for aneuploidy.

Of interest, of note is that 42 percent of the clinics indicated that they are offering preimplantation genetic diagnosis for non-medical sex selection.

We also asked them about how many cycles they perform for each of these purposes, and you can see that there's a big drop notably in everything except for aneuploidy.  You can see that only nine percent — well, 42 percent of the clinics are offering it.  Only nine percent of the clinics actually performed such a cycle in 2005.

We asked clinic directors.  There have been a number of really heartbreaking stories about misdiagnosis in preimplantation genetic diagnosis.  We asked clinic directors about their awareness of inconsistencies between PGD results and subsequent genetic testing.  And nearly a quarter of the clinic directors said they were aware of such a circumstance.

That doesn't mean that 21 percent of the cases are missed diagnosis.  It means 21 percent of the directors had been aware of such a case at some point.  It may have been their own.  It may have been another laboratory's.

So data I think is important, and this sort of reiterates your own recommendations, is needed for informed patient decisions, for quality improvement in PGD, and for evidence-based policy.

And as  a result, we are in the process of putting together a voluntary registry for preimplantation genetic diagnosis, and we are working collaboratively with the American Society of Reproductive Medicine, the Society for Assisted Reproductive Technology, and the PGD International Society.

We have the data fields all collected.  We know what we want to collect.  We have the collaboration and cooperation of the leadership of these organizations, and are now seeking funding for this registry.

So moving to my second issue, is one that's near and dear to my heart, which is the quality of genetic testing.  We talked about the clinical utility of tests and focused on that, and Bob's remarks on the clinical validity of tests.  I'm going to focus somewhat on the analytic validity, that "-idity" of tests.

So as background, genetic testing laboratories are governed by the Clinical Laboratory Improvement Amendments, which were put in place in the wake of bad Pap smear test results going back to women in the '80s.

The responsibility for implementing CLIA is given to the Centers for Medicaid and Medicare Services, and CLIA was really intended to assure analytic validity.  When a laboratory does a test and says there's a mutation there, you want to be quite confident that they're right.  Analytic validity, whether or not that mutation has an association with the health outcome and if there's something useful that you can do with it are the two other "-ities."  I'm talking about the first "-ity."

So the law directs the government to issue standards to assure consistent quality performance and including a whole bunch of measures that you would expect would be in laboratory quality, including proficiency testing.

Of note, there is a special category for high complexity tests and all genetic tests are high complexity tests, and specific requirements can be developed for specific types of tests through the creation of a specialty area.  For example, there are specialty areas for microbiology, toxicology, immunology, chemistry, et cetera.

There has been no specialty created for genetic testing despite the fact that I believe it is the fastest growing area of the diagnostics market, and creating a specialty area really is a prerequisite for mandating proficiency testing programs which Congress believed was the best way to directly measure whether or not a laboratory can get the right answer consistently.

So we're not the first people to notice that this is a problem.  Advisory committees over time have pointed out that there needed to be enhancements in laboratory quality for genetic testing.  The NIH-DOE task force nearly a decade ago, the Secretary's Advisory Committee on Genetic Testing in 2000, specifically recommended the creation of a genetic testing specialty, and in 2000 HHS said, "Yes, we're going to create such a thing," and create tailored standards for these complex set of tasks.

After six years went by and no regulation came out, we looked at the comments that were submitted in response to that notice of intent and found, in fact, that most people were supportive, and we were pleased when we communicated with the department that they said that they were planning to publish a proposed rule for genetic testing as soon as possible, and that was in January.

A couple of months later they put it on their regulatory agenda, which is their signaling we're going to do this in a formal way, but then there was an abrupt change within CMS and more recently they have first privately and more recently publicly they have indicated that they have no intention to create special standards for genetic testing.

And according to a CMS official earlier this week at another advisory committee meeting, they said that genetics is moving very fast, and that's true. 

They said that CLIA does not address clinical validity, and that's true.

They said that CLIA does not address all of the complicated ethical, legal, and social issues, and that, too, is true. 

They said that there are not many samples available or formal programs for proficiency testing, and that is also true.

They said that there is not an evidence of a problem, which I do not believe is true, and that genetic testing laboratories participate in other specialty areas, the relevance of which is unclear to me.  If you can do a blood chemistry test, it doesn't tell me that you can do a genetic test.

So we did a survey of genetic testing laboratories and found that there are deficiencies in genetic testing laboratories and that the more a laboratory participates  in proficiency testing, the fewer analytic errors they observe.

So proficiency testing is doing exactly what it was intended to do.  It's reducing analytic errors.  CLIA was intended to reduce analytic errors so that when you get a test result and you make a profound decision based on that test, you know the answer is right.

We document this sort of sad history in a report that I think was included in your briefing book, and we also have formally requested that the agency move ahead with rulemaking, along with Public Citizen and the Genetic Alliance, and we're awaiting a response to our petition.

So that's the laboratory end of things.  What's FDA's responsibility here?  Genetic tests can be done as home brews.  That's a laboratory developed test where the lab makes all of the ingredients itself.  They don't really buy anything except for general purpose reagents.

Then there's home brews using analyte  specific reagents which are purchased, and then there are genetic tests using kits that are premanufactured.  And FDA regulates both analyte specific reagents and they regulate kits.

So of the 1,000 or so genetic tests that are are available out there, only five have been reviewed and approved by the Food and Drug Administration.  Actually a couple of these  Bob talked about.  Cyp450 is a pharmacogenetic test.  UGT1A1 is the test that will tell you whether or not you are at risk for an adverse reaction to irinotecan for colon cancer.

So laboratories are not required to use test kits if they're available, which creates an unequal system in the marketplace, and there are two paths to the market.  People for good reason take the path of least resistance.

FDA has recently jumped into this fray and has said that they will regulate one specific type of laboratory developed test, which they call in vitro diagnostic multivariate index assays, if you can say that five times real fast, and so they've sort of caused quite a lot of consternation, I would say, in the laboratories and in genetic testing companies and in the biotech industry and in the patient community because it's unclear where FDA is going here.

Why did they jump into IVDMIAs?  The guidance is really based on the technology used and not the risk necessarily posed by these kinds of tests, and it's very unclear what the big picture plan is and how can we ensure quality and also ensure access as we move forward.  Sort of what's the big strategy here for how we move forward?

So CMS has thrown in the towel and gone home.  FDA has put its toe in the pool.  It's not clear what the overall strategy here is, and so we all are getting conflicting signals or at last confusing signals.

So we need transparency.  We need quality.  We need a level playing field.  We need to reward innovation, and we need to ensure access, and we need a good plan for how to do that, which we don't yet have.

We talked — you talked — a little bit about direct to consumer testing, and I'm going to end the clinical chunk by talking a little bit about this and not the specifics of the oversight system that's in place for these, but rather to give you a couple of examples of what's on the market.

There is a test available for women that can tell you whether your child will be male or female at five weeks of pregnancy by looking supposedly at fetal DNA circulating in maternal blood.  There have been a lot of complaints about this.  Some report that they get the right answer about 50 percent of the time.

(Laughter.)

DR. HUDSON:  More disturbingly, the company has contacted women who have had the test and told them, "Your fetus has severe chromosomal abnormalities.  You need to see a doctor immediately."  People have gone through intensive testing and screening and then given birth to health babies with normal karyotypes.  So there's some troubling characteristics here.

DNA Direct offers a number of genetic tests, including for people who are desperately trying to have a child, fertility testing, where they look at chromosomes in Factor V testing.  Of course, the first thing you really should do is go to your doctor and make sure that you're producing the two key reagents, oocytes and sperm.

(Laughter.)

DR. HUDSON:  Factor V testing they say is a common genetic variance.  I think "common" in genetic parlance and "common" to the lay public has very different meaning, and they talk about women having recurrent miscarriages may carry this particular mutation.

In fact, I think most of the scientific literature points to this mutation only being associated strongly with third trimester pregnancy losses, and the overwhelming majority of pregnancy losses are first trimester.

There's a stress gene test, and I know I've got it.

There's the Alzheimer gene test that's available, despite the widespread agreement that this is not ready for prime time.

And then there's my favorite, CyGene Direct, which can give you a genetic test for your athletic performance.  Some of us don't need a genetic test to tell us that.

And then this test is no longer available, although the offer has popped up in a new company offering similar testing. 

"Are you concerned about your child's future?  Does your child have a genetic trait that leads to disruptive addictive personality?  DNA testing can help you understand and manage your child's behavior before it gets out of control.  Imagene will test a panel of dopaminergic related Reward Deficiency Syndrome genes."

And the physicians in the crowd, I'm sure, learned a lot about reward deficiency syndrome in medical school.

So we can talk about what we need to do or not need to do about direct to consumer testing in the conversation.  I'm going to move quickly to the non-medical uses of genetics, and probably the most common use of genetics outside of a medical context is in law enforcement, identification of suspects with DNA presented as evidence.

The Innocence Project having successfully exonerated a number of people who were wrongly accused or convicted. 

More troubling, I think, or somewhat troubling, I think, are the increasing use of DNA dragnets where DNA is asked to be voluntarily supplied by people in a particular area or meeting a particular eyewitness description.

And then DNA profiling, where people, in fact, a company will take the genotype and give you the probable phenotype of the suspect.

Although this hasn't happened much lately, there's some reasonable chance, I think, that genetic information will be used in the courtroom, especially in the sentencing phase in determining culpability.

So to talk about the other non-medical issues, I want to tell a little story of this family, and we're going to call this woman down here Beth.  And Beth's father has pre-senile dementia and is now being principally taken care of by her mother, and her two brothers who are older than her have early symptoms, very similar to what her father had in earlier years.

Her mom learns about a test that's available for presenile dementia.  This is one of those cases where there's nothing you can do about it, like ApoE4.  In this case my made up gene is presenilin-1, which is a real gene, which also leads to presenile dementia.

So the family gets tested except for Beth, and in fact, the affected family members are found to have a mutation in the presenilin-1 gene.  So Beth is thinking, "Should I get tested, too?"

So if there were an intervention, the whole equation would change, right?  If there were something she could do to prevent the onset of dementia, I think what she would be thinking about and their magnitude would be very different.

One thing she might be thinking about is whether or not this information might be used against her, specifically in the health insurance context, but luckily Congress, with some foresight in passing the Health Insurance Portability and Accountability Act, included genetic information as among the factors that group health plans cannot use to deny coverage or increase rates.  So if Beth is in a group health plan, she's protected.

What else might she be thinking about?  Well, she might be thinking about whether or not her employer can get this information. 

There has been a debate over the last probably decade about whether or not the Americans with Disabilities Act provides sufficient protection for predictive genetic information, and specifically, the Equal Employment Opportunities Commission has said that predictive genetic information would be covered under the so-called third prong of the ADA and that people with predictive genetic information, if they were discriminated against, would be regarded as having a disability.

There has been some cases that called that into question, and most courts are now very narrowly construing what meets the definition of having a disability under the ADA, and so in the wake of that lack of clarity in 2000, President Clinton signed an executive order which remains in place today that the federal government as an employer cannot deny jobs or employment benefits based on genetic information.

And when he signed that order, he said, "I'm trying to set an example for the private sector," and he called on the Congress to pass an equivalent law.

Unfortunately, his example was not followed, and one year and one day later there was a case of Burlington Northern-Santa Fe Railroad where they were surreptitiously testing employees for whether or not they had a genetic predisposition for Carpal Tunnel Syndrome.

There has been a bill pending for a long time in the House and the Senate.  Its most recent iteration would prevent genetic discrimination in employment and in the individual health insurance market.  It passed in the Senate by 98 to zero, not much opposition there.  It has been stalled in the House despite the fact that it has 244 sponsors.  It's very likely that in the next Congress this bill will be reintroduced in both the House and Senate and pass pretty quickly.

So that means that when Beth goes to make her decision, her doctor can tell her with absolutely clarity that this information cannot be used against her in health insurance and employment, something that right now is having a very negative impact on genetic research and clinical practice.

My last little example here is assuming that Beth is in the military, she joined up to serve in Iraq and she has this mutation or she may have this mutation.  Would she be protected?

The Department of Defense provides benefits to our Armed Service men and women, including providing medical and disability benefits for retired service men and women, but they have this funny little policy that any injury or disease discovered after a service member enters active duty is presumed to have been incurred in the line of duty, with the exception of congenital and hereditary conditions.

I met this young man, Jay Platt, a number of years ago.  He had served in the Marines two tours of duty in the Gulf War, had been diagnosed with a number of cancers, was diagnosed with von Hippel-Lindau disease, which is a cancer syndrome.

He requested a medical discharge.  It was denied, which meant he would not receive benefits, and only because of his perseverance and only because the NIH intervened on his behalf and argued a technicality, frankly — we argued that he lost function in the other allele, maybe, because of something he was exposed to in the war — and he got his benefits reinstated.

Most people aren't as clever as Jay is.  I think that this policy is not viable over the long term, and it's certainly not a just policy if you think that the people whose genetic — where their genetic contribution is known today don't get benefits, and if your genetic contribution is not yet known, you do get benefits.  It doesn't make sense to me.

So what do we need to do for Beth?  There's a lot of stuff we need to do for Beth, and the most important one is to develop an effective intervention.  That's thing one.

So we need to support a robust research pipeline.  We need to make sure that she and other members of the public are confident in the research enterprise and confident in the medical enterprise.

We need to demand that genetic testing is of exceptionally high quality by creating a genetic testing specialty, rationalizing the FDA system, tracking outcomes over time which can then feed back into the clinical utility question.  It would be much easier if we had electronic health records.

We need to provide health provider tools so that health care providers know who to test with what test and what to do based on that test, and we need to protect against privacy and misuse of genetic information, and perhaps reconsider the standards for de-identifying samples.

I'm going to close with a word of caution.  I'm not sure exactly what the discussions have been about this Council taking up issues in genetics more broadly outside of the reproductive context where you have done such great work in the past.  But I want to remind you that there are a number of other committees who take genetics issues quite seriously.

Most of these are within the Department of Health and Human Services, and they are listed here, some with quite unpronounceable acronyms.  If anyone can pronounce that, I'd be interested in hearing it.  These are all committees that are focused, this newly created actually this week; all four of these committees are focused expressly on genetics issues, and then the Advisory Committee on Human Research Protections focused more broadly on biomedical research issues.

And so with that, I'd like to thank you and look forward to the discussion.

(Applause.)

DR. PELLEGRINO:  Thank you very much, Dr. Hudson.

Dr. Schaub, would you be kind enough to open the discussion?

PROF. SCHAUB:  Thank you very much for that presentation.

My remarks and questions are based on the two advanced readings that we received from you, and I think I'll leave it to my colleagues to follow up on some of the new information and policy proposals in your talk.

The first report that you supplied to us calls for the creation of a genetic testing specialty under the CLIA, arguing that it's critical to the public's health.

The second report suggests, in addition, that the FDA expand its purview to insure that all genetic tests are analytically and clinically valid.

It would certainly be odd to say that one is opposed to folks being competent at their jobs.  So if a designated specialty with standard procedures and ways to test both the tests and the tester would improve the accuracy of genetic information being supplied to individuals, then that seems like a good thing.

However, accurate genetic information is only a good thing to the extent that genetic information itself is a good thing, and I guess I think that in addition to these policy options that you put before us, there are some prior inquiries that our council may want to take before joining in the quest for accuracy.

I would want to ask whether and in what cases and for whom the information is desirable in light of the fact that our ability to test for disease or increased risk for disease is so far in advance of our ability to actually treat or cure these diseases.  I'm not certain that better information about ones future fate is better for the human beings concerned.

Know thyself is a human desideratum, but I have some doubts as to whether individualized genetic information contributes to self-knowledge or happiness.

Indeed, I'm not even sure that it contributes always to health.  Both reports assert that reliable tests are critical to the public's health, and you give five, in one of the reports, you give five illustrative instances of the serious consequences that laboratory errors can lead to.

Two of those involved prenatal genetic testing and a third one involved parental testing with a view to  procreation.  In each case parents were wrongly informed that their child would not have a particular disorder.

The implication is that had they had the correct information, they would have aborted the fetus.  I'm not sure where precisely the threat is here to the public's health, unless we mean that allowing unhealthy individuals to be born is the threat.

In other words, genetic information pretty quickly lends itself to eugenic uses, fueled in these instances not by government mandate, but by the longing of parents for unblemished offspring.

You know, if your insurance company finds out what you're going to develop certain genetic diseases, it won't insure you.  If your parents find out, they may not welcome you into their arms.

I was very struck by what we learned at the last Council meeting about testing for Huntington's and the efforts that are made to protect the privacy of the young, at least once born, even against the parents by not permitting testing until age 18.  So I think there are some real questions to be raised about the ethics of testing not oneself, but another, although another who is, in the case of parents, admittedly also one's own.

Can you tell us what proportion of the genetic testing being done today is prenatal?

In the examples that were given, the errors all led to individuals being born who otherwise might not have been.  I suppose that the errors also occur in the other direction.  A fetus is diagnosed with a genetic disorder.  The fetus is aborted, and then perhaps found to be quite healthy.

Does that happen or do we not know since follow-up testing is not done?

In the second reading, you suggest that a focus on the quality of testing could actually help us to answer questions about who should have access to which tests, along with these questions about advertising and commercialization.

If we went that route right now,  and required greatly increased federal regulation and oversight, would the effect be a dramatic scaling back in the availability of genetic testing, at least a temporary dramatic scaling back, since at present only four of the 900-some genetic tests have FDA approved test kits, would laboratories have to close off access, especially this direct to consumer access until FDA approval is secured and appropriate guidelines are developed?

Finally, I want to just say something about the art work by Dennis Ashbaugh which accompanies the article in Issues in Science and Technology.  I thought the paintings were very beautiful and the colors were very beautiful, but they seemed to me to illustrate one of the perils of genetic testing.  To me at least the paintings displayed a form of misreading that goes beyond the misreading committed by insufficiently trained technician.

The misreading that I'm worried about lodges in the popular imagination.  Dennis Ashbaugh says that the point of his DNA paintings is to "reveal the inner code beneath appearances."  And on page 64, there's a reproduction of a painting entitled "Son of Sam."  Presumably it shows a section of the notorious mass murderer's DNA.

And while the scientists might assure us that Son of Sam was not coded for mass murder and while a scientist might tell us that the relation between the genotype and the phenotype is more complicated than the inner code beneath the appearances, I suspect that non-scientists will not really get the message.

Indeed, we've been told that even physicians often have a very sketchy grasp of the meaning of genetic test results that are, you know, returned to them.

Human beings have always sought knowledge of their individual fate.  The Greeks visited the Oracle at Delphi.  Other peoples looked to the stars and astrology for predictive power.  Yet others have turned to evidence supposedly offered by the body itself as in palm reading or phrenology.

I certainly don't mean to suggest that genetic testing is fraudulent in the way that these earlier fortune tellers were.  Not at all.  Part of the danger today may be that genetic testing will be embraced by the public not for its real, albeit limited, value, the sort of thing that was sketched out for us by Professor Nussbaum in talking about pharmacogenic results, but rather that it will be embraced as a scientifically valid version of palm reading.

In seeking more detailed information about our bodily fate, in doing that, will we become a nation of fatalists? 

Even when genetic information is sought in order to stave off or to avert one's fate, one is nonetheless obsessed with fate, and in that sense a fatalist.  Alexis de Tocqueville predicted that democratic peoples would be strongly inclined toward both fatalism and materialism.  And he argued that it would be important for democratic legislators not to contribute to this doctrine of fatality.

So in the Council's consideration of the ethical meaning of genetic testing and the public policies to be adopted, I would hope that we would remember Tocqueville's warning that it is a question of elevating souls and not completing their prostration.

DR. PELLEGRINO:  Thank you very much.

DR. HUDSON:  Thank you very much for those comments.

So this committee has previously dealt a lot with the reproductive uses of genetic testing, and I think that while I'm not aware of any concrete data of the proportion of all genetic tests that are performed in the reproductive context, I'm fairly confident that it represented the majority of testing certainly up until the present time.

And part of the reason for that is the inability, and there's sort of a range of genetic tests as information only about which you can do nothing, to genetic information where you can intervene, such as we hope for in Beth's case, but she doesn't yet have.

And so in the absence of being able to do anything for the individual, reproductive uses of this technology, whether to prepare for the birth of an affected child or to terminate a pregnancy, have been very commonly used.

One would hope, and maybe it is but a hope, that as we move forward and understand the molecular mechanisms underlying some of these diseases that we can develop interventions whereby we're treating the individual as a living person and there will be less focus on the reproductive context.

So certainly today and doing genetic testing for Coumadin dosing, for example, outside of the reproductive context.  The Cyp 450 that I listed as one of the FDA approved tests is testing for enzymes that are involved in the metabolism of a huge proportion of prescription drugs and presumably could decrease adverse drug effects and the cost of those substantially.

So it's my hope that we move outside of the reproductive context for most of our focus in genetics.  They're hard choices no matter how you feel on the pro life/pro choice question.

The issue of quality and whether or not our focus on quality would reduce access I think is something important to keep in mind.  We certainly would not want to suddenly have a reduction in the access of patients to get tests that are so vital to their futures.

There are some proposals that are being developed.  Senator Kennedy has a draft bill that has been circulated now where — and I haven't read the most recent draft carefully — but where he proposes allowing genetic tests to remain on the market and therefore accessible while everybody lines up and goes through a review.  And so once your number is up and you go to the deli counter, you can no longer be on the market if you don't pass FDA's seal of approval.

But until that time, nothing is taken off of the market.  I think there may be an exception in the bill for direct to consumer testing.  There are, as I showed, some very questionable tests that are being offered, and sort of this intersection of the Internet and genetic technology giving rise to this new business model.

Dr. Nussbaum suggested that the Federal Trade Commission has a role here.  I think at a minimum if we could guarantee that people had access to information about what those tests can do and what they can't do, then at a minimum people have appropriate information.

A lot of these tests that are being offered on the Internet and even by laboratories not over the Internet, it's very hard to get information about what is the gene, what is the variant, what is its prevalence, what's the positive predictive value, how many people were in the study that demonstrated that there is this correlation.  It's very hard to get at this information.

And so at a minimum if we could get at some transparency in the system, I think we could facilitate good decision making and good patient decision making.

And with regard to the art, we didn't pick it.  We didn't see it until it came out.

DR. PELLEGRINO:  Thank you.

Any comments?  Yes, Janet.

DR. ROWLEY:  Well, I'm sort of surprised that you think that most genetic testing is related to reproduction.  I guess I would have thought that most of it is Guthrie type testing or maybe you don't.

DR. HUDSON:  Newborn screens.

DR. PELLEGRINO:  Dr. George.

PROF.GEORGE:  Yes, just to be clear and to follow up on what Diana was saying, when you say in the reproductive context, does that mean predominantly for eugenic purposes?

DR. HUDSON:  Without commenting on what is and is not eugenic —

PROF.GEORGE:  Well, I mean with a view — well —

DR. HUDSON:  — so the most — so in 2001, for example, the American College of Obstetricians and Gynecologists adopted a guideline, health professional guideline, that indicated to obstetricians and gynecologists that they should offer cystic fibrosis carrier testing to all couples of a reproductive age.

As it turns out, in practice that test is most frequently offered after a couple already has a pregnancy underway, when, in fact, it makes much more sense and was the guideline to do that testimony prior to initiating a pregnancy.

So I don't think there's any concrete data on the absolute number of CF carrier tests that are being performed today, but it has got to be a vast, vast number now, not to the extent of newborn screening, but it's a big number.

PROF.GEORGE:  Do you happen to know why things went awry in that one example that you used?  Why did it end up being the case that most testing was done after conception rather than before?

DR. HUDSON:  I'm not a medical doctor.

PROF.GEORGE:  It wasn't anticipated?

DR. HUDSON:  Yeah.  I think part of it is that when a woman shows up for her first prenatal visit obstetricians are accustomed to offering sort of a series of tests, and that's the time when they do that test, when in fact women, many, many women go in for their annual Pap smear and that's the only doctor visit they see, and in theory it should be at those visits that the gynecologist says, "Hey, are you thinking about — let's talk about — let me give you some information about."

Unfortunately, that's not yet happening, and maybe testing will move earlier.  Certainly ACOG is making every effort to see that happen.

PROF.GEORGE:  What's the normal way that that information is communicated so that changes in practice actually take place?

DR. HUDSON:  Well, professional guidelines, and actually the CF testing guideline is a rarity; so with 1,000 genetic tests out there increasingly for common diseases and conditions, there's only a tiny handful of professional guidelines that are available right now.

There are some efforts underway funded by CDC to develop the evidence base that would facilitate health professional guideline development, but it takes a lot of resources and intensity for those guidelines to be developed.  The CF guideline was supported by federal funding from the NIH, and I think that there is data about how long it takes from the time that a health professional guideline comes out to when a majority of practitioners are actually following it, and it's a fairly substantial lag time.  It's sort of just the normal diffusion time.

DR. PELLEGRINO:  Rebecca.

PROF. DRESSER:  Kathy, I was wondering about CF and these home brew tests.  would the Kennedy bill get any jurisdiction over that?

And do they say they don't have jurisdiction because there isn't interstate commerce or I don't understand.

DR. HUDSON:  Yeah, yeah.  So they actually years ago, they said in the preamble to some regulation, they said we believe that laboratory developed genetic tests are medical devices, and they are subject to the Food, Drug and Cosmetic Act devices amendments.

But we are using enforcement discretion and saying we're not going to pay attention to them.  So for years and years and years they said, "We're not paying attention to them, but we have jurisdiction."  There was sort of a silence for a period of time when the General Counsel at FDA was rumored to believe that they were not under FDA's jurisdiction.

At a hearing in July, on direct to consumer testing, an FDA official shocked us all when he said, "Not only do I believe we should have jurisdiction, but we do have jurisdiction," and shortly thereafter they put out this draft guidance which would cover one subset of laboratory developed tests.  So sort of shook up the world.

PROF. DRESSER:  Did they explain anything about why they chose that limited kind of a test?

DR. HUDSON:  Yes.  These are tests that are looking at multiple analytes at a time.  So think of microray either looking at DNA variants or expression patterns where it's not just a binary answer.  They're using some sort of computer algorithm to develop a risk profile, a recurrence profile.

One of the companies that's out there that would presumably be an IVDMIA MIA is Genomic Health, which looks at gene expression from a number of genes and calculates a recurrence rate for breast cancer.  So they view this algorithm as being sort of a black box where no well trained health professional would be able to understand really how they got the answer, and so that's sort of their hook.

Whether or not that's higher risk than getting the wrong result on a Huntington's test I'm not sure.       

DR. PELLEGRINO:  Dr. Rowley.

DR. ROWLEY:  Well, I just wanted to make a comment about this de-identified samples, and I don't think that the general public really understands what a serious medical problem this is. 

Well, let me give you two examples.  One is from a very well respected investigator at Harvard who could get DNA samples from women de-identified, and he found five of 100 women had BRCA-1 mutations.

Now, because the samples were de-identified, he didn't have any idea which of the five women were actually at risk, and in order to find that out, one would have to go back and do the tests all over again.

So I think that de-identified samples are a bad idea, and particularly in cancer as we're trying to associate genetic abnormalities in tumors with survival.  If you are given de-identified samples, you have no idea once you find the genetic abnormality what its consequences are.

So we've talked a lot about patient privacy, but I think there are a number of very important examples where patients are actually done badly by having de-identified samples.

DR. HUDSON:  I don't know that I have a formulated opinion yet about the costs and benefits of de-identification, but I agree with you that severing that link does deny anybody the ability to get back with important health information.

It seems to me that with information technology and the Internet, that some process of sort of an ongoing, rolling consent model might be preferable to just absolutely severing this link and the set of responsibilities that researchers and participants have towards one another.

DR. PELLEGRINO:  Dr. Kass.

DR. KASS: Thank you very much, Kathy for a very fine presentation.

I have a couple, maybe three questions.  One, you cited the Council's "Reproduction and Responsibility" report and the call for, among other things, longitudinal studies, the effects of PGD on the children born.  In your own study that you cited then, the word "outcomes" appears, and it's a collaborative study involving ASRM.

How are you going to get the ASRM people to pay attention to more than just "a live baby was produced here?"  I mean, the really interesting things I think one needs to have evidence for are pediatric studies and things going further on.  I'm wondering if that has been taken into account.  Okay?

The second question, I was very struck as you pointed it out, the percentage of the IVF clinics that are offering PGD for non-medical sex selection.  I think the number was 42 percent, although they haven't at all done it.

This ought to raise some further doubt in case one didn't have it already about the efficacy of the practice guidelines because the ASRM is on record on this subject.  They are also on record not enforcing these guidelines, and I wonder whether — I mean, one would like where possible to rely on professional self-regulation, but I wonder whether or not the experience there is a kind of warning to us if we're sort of thinking about the degree to which we can rely on practice guidelines unenforced, especially where the commercial interests become very, very large to do the job of protecting the public.

Finally, and this is just a factual question, you put up a slide of the things that the CMS said when they sort of drew back from where you thought they were going.  Four of the items you said were true, and the fifth most important one, they deny that there's a problem.  It seemed to be false.

Do you know — this is a political question — do you know what happened and is there powerful, organized economic lobbying, that if one wants to think about public policy in the area of testing that one should address, we certainly met these lobbies with respect to other things that we were engaged in?

And if you could help us think about that, I at least would be grateful.

DR. HUDSON:  In terms of the registry and how it might help track and assess children over time, we know and you certainly know that IVF clinics currently really have no reach into the family with the baby and so, for example, the data they collect on malformation rates among children of IVF are lower than in general population.

So the data is of very poor quality on the health of the babies born after IVF.  So what we are proposing to do here is that when data is entered into the registry, there will also be entered whether or not the family is providing their consent for recontact for subsequent studies and the registry would provide a research resource for subsequent investigators to actually construct, devise, and carry out studies of the sort that would be needed to really assess the children's outcomes.

Now, there's not that many PGD babies in the United States, and there have been studies that have been designed in the past where children have been assessed from various different technologies and where people have gotten in their vans and driven around the country and actually done direct health assessments of children.

So this would enable that.  It would not in itself do it, and the registry we would propose would have a set of research priorities so that entry into — being able to access the information and the patients would be based on the priorities that the registry governance body had created, and this is the number one priority.

Oh, and then in terms of the non-medical sex selection and whether or not professional guidelines are sufficient in the absence of a big stick, I'm going to quote Joe Leigh Simpson here, who I think has spoken to the Council in the past, former president of ASRM and a prominent geneticist, and he has recently published an article where he has talked about the PGD registry and proposed that it may be a means of identifying and eliminating, quote, outliers.

Whether or not that can actually come to fruition and how that would come to fruition, I'm not sure, but I just put before you what Joe Leigh Simpson has proposed.

And then lastly, what happened with CMS?  A mystery, somewhat of a mystery.  The personalized medicine coalition which is pharmaceutical companies, biotech, academic organizations, large organizations, has supported the creation of a specialty.   The American Society of Human Genetics has supported the specialty.  The majority of our survey respondents, the regulated community, has supported the creation of a specialty.

Probably the most compelling reason that I have heard, and apparently it got yanked at CMS.  So it never went — I was thinking, oh, it was the Office of Information and  Regulatory Affairs, it was OMB, it was somewhere else.  I have heard it was within CMS that the decision was made, and that it was based on their competing priorities.  So it was not viewed within the agency as not that important.

DR. PELLEGRINO:  Dr. Hurlbut.

DR. HURLBUT: Kathy, can you say a little more about the issue that's brought up in one of your reports of surreptitious testing and also the need for required counseling?

This strikes me as a very worrisome — very, very worrisome, and then I have a follow-up question.

DR. HUDSON:  Thank you very much for raising the question.  There's an interesting issue which has not been real enough until recently to really worry about, which is that you leave DNA everywhere, right?  And there are now companies that will test various clothing to tell you whether or not you might have had infidelity in your family.  Parents, perhaps disgruntled parents, can test their children without their permission or consent to find out whether or not that child is actually theirs.

So there is this non-permitted, non-consented taking and examination of DNA that is permitted right now, and it may be that we are approaching a time where we need to think very seriously about whether or not there should be some limits on whether or not it should be permitted lawful to do genetic testimony except under certain circumstances, for example, at a crime scene.

I can't read my handwriting.  So I can't remember just —

DR. HURLBUT: Required counseling.

DR. HUDSON:  Counseling.

DR. HURLBUT: I know of a case where an elderly woman was told that she carried apolipoprotein E4 allele, and she told me that she went through over a year of waking up in the night every night crying and worrying about arranging her whole life around the reality that she was going to get Alzheimer's disease, and then finally just mentioned something from the doctor about when is it going to come on.

I mean, it just strikes me as an amazingly tragic potential out there, and especially combined with what Dr. Nussbaum mentioned about the over interpretative determinism of these tests.

By the way, just to add a little element, you were talking about tests not to do.  I do think we ought to do tests in this, but it struck me that just think of the impact of not just tests like Huntington's disease, which by the way sometimes people who have gotten results that said they weren't going to get the disease have had decompensations that were quite severe.

But it strike me that there are quite a few grayer zones with polygenic traits like depression, for example, that — I mean, if you're already susceptible to depression, hearing a depressing result might not do you much good.

(Laughter.)

DR. HURLBUT: And counseling seems to me to be really crucial here.

DR. HUDSON:  Yeah, particularly for serious diseases for which there is no intervention.  I think the standard paradigm of pre and post test counseling really needs to be adhered to, but it's really about what's the content of that counseling and how much are counselors really able to get the individual to think about "what will you do with the test result if it's this way and that way."

And even with that, I think there is the reality that what you think you're going to do when you have a piece of information and what you actually do when you have that piece of information don't always line up, and that's just the reality.

Because genetics, we've been in this state, this sort of uneasy state for such a long time with being able to, you know, tell parents what their recurrence risk is for having a child with a specific genetic disease.  Now we're entering a different phase, albeit slowly, and so in some ways it's time to sort of question the paradigm of genetic counseling.  Do you really need pre and post test counseling to tell you that you're a fast metabolizer, for example?  Do you need to think about the implications for  your family of you being a fast metabolizer?

So we need to sort of realize that genetics isn't all on that one end of the spectrum anymore of serious diseases where you can't do anything about it but across the spectrum and sort of attenuate our expectations for what health care providers do.

The one other thing I'd say is people who provide genetic counseling, which are often not genetic counselors, don't get paid for what they do really.  The time, you know, you can't evaluate with somebody what are you going to do if you find you have the ApoE4 allele in 15 minutes.  And so how we are coding and reimbursing for genetic services and genetic tests is, I think, a significant issue.

DR. HURLBUT: Can I have one follow-up on that?

DR. PELLEGRINO:  Yes, yes.

DR. HURLBUT: Kathy, from what we heard earlier, it seems realistic that there might be the $1,000 genome in the future, and actually you could do much easier and quicker and cheaper analysis of 100,000 or 200,000 sailient alleles or locations, coding zones.

And it strikes me that all of this individualized testing may be outmoded in a couple of years, not a couple, but maybe ten or 12 years, and our policies might just be coming into place then.

It strikes me we need to anticipate that possibility, and by the way, what a nightmare scenario for counseling because now you're looking at 20,000 genes with various percentage probabilities.  Do you see what I'm saying?

DR. HUDSON:  Sort of to reinforce that, I have heard that there is a company that's going to be launching soon that will be looking at a large number of variants, in the thousands, and be providing that information back to people and then providing them sort of a  Web portal to do their own investigation about what each of those variants means.

So stay tuned and get ready.

DR. PELLEGRINO:  Dr. Carson.

DR.CARSON:  Thank you for that presentation.

You know, the thing that worries me a little bit is the whole concept of mission creep.  You know, as a pediatric neurosurgeon, I remember many years ago I would get referrals of babies who in utero were diagnosed by ultrasound with anencephaly.  Well, you know, that was pretty easy.

And then it was hydroanencephaly.  You know, they had a little bit of a cortical-matter, but not much function, and then it just became, you know, hydrocephalus, and then it became questionable ventriculomegaly.

And the question at each stage was, you know, what should be done with this baby, and you know, what recommendation would you have to keep the same kind of mission creep from happening as we develop more of this genetic information and people not wanting to risk, you  know, abnormalities?

DR. HUDSON:  I'm afraid I don't have a concrete answer.  I will reinforce the problem by sharing stories that my genetic counseling friends have shared with me, which is that during amniocentesis when you just look at the chromosome, you look at a karyotype; when you find a chromosomal rearrangement, a little tip of chromosome that's sitting on the tip of another chromosome, for example, a chromosomal rearrangement that you haven't seen before, and so the family, you know, — you tell the family that there's this chromosomal rearrangement, and they say, "What does it mean?"

And you say, "We don't know," right?  What do parents do in that circumstance?  And that's the nature of the analysis, the information that you get and the information that parents get and make decisions on.

Sort of related to this, there was a bill that, well, is still a bill, a bill introduced by Senator Brownback that suggested that parents when making the decision to have prenatal genetic testing be given better, more comprehensive information about the conditions that are being tested for, specifically Down's Syndrome.

And it's no doubt true that in genetics because it's easier to identify the extreme phenotype that that's how we define things, right?  We define things by the extreme phenotype and not so much by the gradations in phenotype, and so the emphasis there was how can we provide more complete information about what this really means, looking parents up to families that have children with that condition as a means of trying to help people make informed decisions and not sort of lump everything together.

And I didn't understand what any of those terms were that you said.

DR. PELLEGRINO:  Any other comments?

PROF. LAWLER:  At the end of the day, given all of the problems you talk about, given the need for more federal leadership, does this Council provide any of this federal leadership or should these problems we addressed somewhere else?

DR. HUDSON:  I think that there are a number of these issues that are being seriously undertaken by others, some of the issues that I talked about that were seriously undertaken by others, and yet there are some where especially I think sort of the more anticipatory issues, the ones that aren't here right now but that might come to become more prominent, like Bill mentioned, the sort of unauthorized taking and testing, I think, are potentially some issues here.

And it might be worth reviewing what's on the agenda for those committees who are currently focused on genetics issues to see whether or not there are issues that the Council is interested in that are not being considered or not on the prospective agenda for those groups.

DR. PELLEGRINO:  Paul.

DR. McHUGH:  I, too, thank you for what you've done, and I'm raising just really two issues to get your information on this.

The first one is what I tend to refer to as materialism in the woman, and that is being pregnant today is a much tougher task and a more frightening task for women than it ever was before, primarily because of the information we've given about the material.

And at this Council's meetings and at other meetings, I've protested about the psychological burden that women bear with the triple test that gets them to change their odds about Down's Syndrome, and the failure of genetic counselors and the like to help these women even when they've had an amniocentesis and they've got at least that test that shows that they don't have a Down's Syndrome child.

But the encouragement that they get to press on in these ways, and their sense of defect which seems to be a real frightening burden that women carry, and I'm really surprised that our government and our Public Health Services haven't been studying this matter more carefully and seeing the burden that's come for women in this matter.  So that's the first thing I wanted to ask you, if there's anything going on there.

The second thing that was interesting to me, you pointed out that we have made great advances in genetics but we may not be making any advances or we may be back in the 1980s on our studies of behavior and life styles, and you put it, and I think quite correctly, that part of the reasons for being in that is the difficulties in maintaining privacy and the like.

But I wondered whether you had looked into the work, particularly done in the NORC Center at the University of Chicago, where they have worked out ways with interviewers to interview people about the most intimate matters of their life in kind of dueling computers, have been able to take that information in and then disperse it into a body so that the people can be assured not only are they private, but they're even private in the interview itself, which is very hard, which is a very important thing to get the information.

I just wondered whether those things were coming to the fore.  So those are the two questions.

DR. HUDSON:  I think you're quite right that there is a real burden of information on women.  There was a beautiful article in the New York Times ten years ago by Natalie Angier where she talks about the burden of information on women as they're pregnant, and it was beautifully, beautifully written.

And at the time I was actually pregnant and I had chicken pox during my first trimester of pregnancy, and that ostensibly linked to various forms of birth defects, and you know, when you know too much you can know too much.  So I knew way too much and had the phone call from my doctor after a sonogram telling me to please call the office.  There were abnormal results.

That was 6:30 at night when I got the message.  You can imagine how much I slept, and he later indicated to me that there was an abnormal interocular distance in the fetus, and I said, "Well, what does that means?"

And he said, "We don't know, but we need to do more testing," which we politely declined, deciding that if our son looked like Lyle Lovett that was okay with us.

(Laughter.)

DR. McHUGH:  By the way, I'm surprised that you got just this information at 6:30 and by not responding till the next morning you didn't get ten more messages between 6:30 and 5:00 a.m. because the obstetrician is so fearful that if he doesn't let you know this, he'd be sued.

DR. HUDSON:  There's going to be a lawsuit, right.

And then in terms of the privacy technology, I think there are wonderful ways of getting accurate information from individuals, and particularly, you know, there is an effect when you actually see a human being.  You give them the response that you think that they want to hear, and so you get very different responses from people when you actually take the other person out of the room.

So Internet based or paper based surveys and information collection devices are much more effective than actually having a person sitting across from you because I want to give you the answer that I think you think is okay.

In terms of the privacy though, when you link that with DNA it's still kind of identifiable, and I'll give you an example of how it can be identifiable.

There was a case of a man whose father was a sperm donor, and he wanted to contact his father and find out who his biological father was, and so he himself put his DNA into one of these genealogical databases where you can trace your ancestry and who's related to whom, and he found out that there were a group of people who were genetically related to him in a certain part of the country.  He contacted those people, asked if there are any young gentleman family members who happened to be in the Boston region or whatever city it was in the year that he was born, and managed to locate his father.

DR. McHUGH:  Good for him.

(Laughter.)

DR. McHUGH:  By the way, as I was saying, the Nork thing, although it is face to face, the dueling computers made it possible.  There are advantages, of course, to having somebody speaking to somebody and at the same time having that somebody not have any clue as to what your answer is.

So this kind of development of technology and appreciating the data I'll follow with great interest, and I'll look up that article in the New York Times.

DR. PELLEGRINO:  Any other questions or comments on this subject?

(No response.)

DR. PELLEGRINO:  If not, let me thank you, Dr. Hudson, for again a very, very excellent presentation.

(Applause.)

DR. PELLEGRINO:  And let me ask the Council for a moment tomorrow morning we'll be going over a paper by Eric Cohen and Sam Crowe with suggested policies having to do with some aspects of organ transplantation.  I'd like to be very specific about that tomorrow and have us concentrate on it, and so I would suggest just for that if you could some time look at page 1 and 2 for the guidelines that are now being used in organ transplantation and then look at the recommendations that are being made, and I'd like to find your opinions and get your opinions specifically on those you think that are important, those that may not be of significance.

Speaking now of the specific recommendations made by Sam Crowe and  Eric Cohen rather than the guidelines that are current, except as to background against which you would want to think about the proposed policy changes.

Thank you very much.   Have a good evening.

(Whereupon, at 5:06 p.m., the meeting was adjourned, to reconvene Friday, November 17, 2006.)