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INBORN ERRORS OF METABOLISM}
     
William A. Gahl, M.D., Ph.D., Principal Investigator
Yair Anikster, M.D., Research Fellow
Robert Keita, Ph.D., M.D., Clinical Fellow
Chanika Phornputkul, M.D., Clinical Fellow
Marjan Hulzing, Ph.D., Postdoctoral Fellow
Erin Strovel, Ph.D., Postdoctoral Fellow
Isa Bernardini, Senior Research Assistant
Katie Fitzpatrick, Predoctoral Fellow
Lynn Duffy, M.D., Guest Researcher
Wendy Introne, M.D., Guest Researcher
Donna Krasnewich, M.D., Ph.D., Guest Researcher
William Gahl
 
The Section on Human Biochemical Genetics, led by William A. Gahl, investigates rare and informative inborn errors of metabolism, including Hermansky-Pudlak syndrome, cystinosis, sialuria, and alkaptonuria.

Hermansky-Pudlak Syndrome
Bernardini, Anikster, Hulzing, Strovel, Fitzpatrick, Duffy, Gahl
Members of the section described the third patient in the world with Hermansky-Pudlak syndrome 2 (HPS-2) and mutations in ADTB3A, a gene encoding the b3A subunit of the adaptor complex AP3. This coat protein mediates the formation of vesicles that include the melanosome and platelet dense body, which HPS cells lack. Along with his albinism and platelet storage pool deficiency, the six-year-old boy with nonsense mutations in ADTB3A had frequent infections and neutropenia as an infant. In the process of finding the boy’s mutation, we also determined the genomic organization of the ADTB3A gene.

The section also studied HPS-2 melanocytes in collaboration with Dr. Ray Boissy (Dept. Dermatology, University of Cincinnati College of Medicine). In AP3-deficient cells, TYRP-1 displayed a normal melanosomal pattern of distribution. In contrast, tyrosinase exhibited a melanosomal (i.e., perinuclear and dendritic) pattern in normal cells but only a perinuclear pattern in HPS-2 melanocytes. In addition, tyrosinase exhibited a normal pattern of expression in HPS-2 melanocytes that had been transfected with cDNA encoding the b3A subunit of AP3. This suggested a role for AP3 in normal trafficking of tyrosinase to premelanosomes, consistent with the presence of a suitable dileucine recognition signal in the carboxy terminal portion of the tyrosinase molecule.
We also searched for a new gene responsible for a subtype of HPS. Using homozygosity mapping on pooled DNA of six families from central Puerto Rico, we localized an HPS susceptibility gene to a 1.6 cM interval on chromosome 3q24. The gene, HPS3, has 17 exons, a 3015-bp coding region, and a putative 113.7 kDa product with a clathrin binding motif and a possible endoplasmic reticulum retention signal. The homozygous, disease-causing mutation is a 3904-bp deletion involving exon 1, part of intron 1, and 2874-bp of up-stream sequence. The deletion apparently formed by homologous recombination within two Alu repeats; an ancestral haplotype analysis estimated the recombination event to have occurred approximately between 1880 and 1890 in the region of Aibonito, Puerto Rico. We also developed an allele-specific assay for diagnosing individuals heterozygous or homozygous for the deletion.

Subsequent mutation analysis performed on non–Puerto Rican HPS patients revealed eight individuals with mutations in HPS3. Five were Ashkenazi Jews. Three of the five were homozygous for a 1303+1G–›A splice site mutation causing a skip of exon 5, the deletion of an RsaI restriction site, and the production of decreased amounts of mRNA, as judged by northern blotting. Of 235 anonymous DNA samples from Ashkenazi Jews examined by a restriction enzyme-based assay, one was heterozygous for the 1303+1G–›A mutation. These findings have expanded the molecular diagnosis of HPS and suggest that other patients with mild hypopigmentation and decreased vision should be examined for HPS. Finally, we determined the sequence of the mouse HPS3 gene, which corresponds to cocoa.

We also studied therapy of the pulmonary fibrosis of HPS by using the investigational antifibrotic agent pirfenidone (5-methyl-1-phenyl-2-(1H)-pyridone). This drug inhibits TGFb-mediated fibroblast proliferation and collagen synthesis in cell culture systems. We initiated a double-blind, placebo-controlled trial of pirfenidone in 21 patients. The patients underwent an examination every four months at the NIH Clinical Center; change in forced vital capacity served as the outcome parameter. After 44 months, the evidence of a treatment effect was overwhelming and the trial was stopped. The pirfenidone-treated patients did better than the placebo patients by 3 percent per year, providing evidence for the first successful treatment of any type of pulmonary fibrosis.

Cystinosis
Bernardini, Anikster, Keita, Gahl
Members of the section demonstrated that the variant forms of cystinosis are allelic with the nephropathic form, i.e., exhibit mutations in CTNS. Four ocular or non-nephropathic cystinosis patients each had one severe mutation and one mild mutation, the latter consisting of either a 928G–› (G197R) mutation or an IVS10-3 C–›G splicing mutation resulting in the insertion of 182 bp of IVS10 into the CTNS mRNA. In collaboration with Dr. Jess Thoene of Tulane School of Medicine, we determined that six patients with intermediate cystinosis also displayed one severe mutation and one milder mutation in CTNS. The CTNS mutations in four African American cystinosis patients were also elucidated. Furthermore, members of the section defined the promoter sequence for CTNS and identified three cystinosis patients with promoter defects causing their disease.
The section has maintained its expertise in cystinosis by following approximately 60 pre- or post-transplant patients. We perform mutation analysis on patients new to the protocol, accumulate longitudinal data on treatment regimens, and detect complications of cystinosis in both the pre- and post-transplant groups. We also work in close collaboration with Dr. Kaiser-Kupfer of the NEI and with Sigma-Tau Pharmaceuticals to bring cysteamine eye drops to New Drug Approval. The NDA application should be submitted to the FDA some time in 2002.

Sialuria
Bernardini, Keita, Hulzing, Krasnewich, Gahl
The section serves as the center for studies of sialuria, a disorder of unregulated sialic acid synthesis. In collaboration with Dr. Jules LeRoy of Ghent, we reported the world’s sixth known sialuria patient, a four-year-old Belgian boy, and the seventh patient, his 34-year-old mother. The boy’s heterozygous R266Q mutation was detected in the patient’s mother, who has similarly increased urinary free NeuAc, confirming for the first time the dominant mode of inheritance of this inborn error. These findings call for expansion of the phenotype to include adults and for more extensive assaying of free NeuAc in the urine of children with mild developmental delay.

Alkaptonuria
Bernardini, Introne, Anikster, Gahl
We have extensively examined 57 patients with alkaptonuria during one-week admissions to the NIH Clinical Center. The clinical data collected so far are currently undergoing analysis. Using SSCP and direct sequencing, we have already identified the homogentisic acid oxidase gene mutation in approximately 60 percent of our patients. A collaboration with the Rehabilitation Medicine Department of the NIH Clinical Center has revealed that alkaptonuria patients exhibit muscle involvement on ultrasound examination, a condition not previously reported.

Other Disorders
Bernardini, Anikster, Keita, Hulzing, Strovel, Gahl
In collaboration with Dr. Tzipora Falik-Zaccai of Haifa, Israel, members of the section have described the clinical characteristics of two siblings in a genetic isolate of Moslem Bedouins with Gray Platelet syndrome, or GPS. The disorder is characterized by thrombocytopenia and large platelets that lack a-granules and their contents. The detailed description of these cases provides a basis for future differentiation of the various types of GPS and for attempts to isolate the gene causing GPS in this genetic isolate.

 
 

PUBLICATIONS

  1. Anikster Y, Huizing M, White J, Bale S, Gahl WA, Toro J. Mutation of a new gene causes a unique form of Hermansky-Pudlak syndrome in a genetic isolate of central Puerto Rico. Nat Genet 2001;28:376-380.
  2. Anikster Y, Kleta R, Shaag A, Gahl WA, Elpeleg O. Optic atrophy plus (3-methylglutaconic aciduria type 3): identification of the OPA3 gene and its founder mutation in Iraqi Jews. Am J Hum Genet, in press.
  3. Anikster Y, Lacbawan F, Brantly M, Gochuico BL, Avila NA, Travis W, Gahl WA. Pulmonary dysfunction in adults with nephropathic cystinosis. Chest 2001;119:394-401.
  4. Aula P, Gahl WA. Sialic acid storage diseases. In: Scriver CR et al., eds. The metabolic and molecular bases of inherited disease, 8th edition. New York: McGraw-Hill, Chapter 200, 2001;5109-5120.
  5. Enns GM, Seppala R, Musci TJ, Weisiger K, Ferrell LD, Wenger DA, Gahl WA, Packman S. Clinical course and biochemistry of sialuria. J Inher Metab Dis 2001;24:328-336.
  6. Falik-Zaccai TC, Anikster Y, Rivera CE, Horne MK, Schliamser L, Phornphutkul C, Attias D, Gahl WA. A new genetic isolate of Gray Platelet Syndrome (GPS): Clinical, cellular and hematologic characteristics. Mol Genet Metab, in press.
  7. Gahl WA. Cystinosis. GeneClinicsTM Website. Available at http://www.geneclinics.org, 2001–2003.
  8. Gahl WA. Genotypes and phenotypes [editorial]. Genetics Med, in press.
  9. Gahl WA. New therapies for Fabry Disease [editorial]. New Engl J Med 2001;345:55-57.
  10. Gahl WA, Thoene J, Schneider JA. Cystinosis: A disorder of lysosomal membrane transport. In: Scriver CR et al., eds. The metabolic and molecular bases of inherited disease, 8th edition. New York: McGraw-Hill, Chapter 199, 2001;5085-5108.
  11. Hahn SH, Yoo OJ, Gahl WA. Effect of metal ions on the stability of metallothionein in the degradation by cellular fractions in vitro. Exper Mol Med 2001;33:32-36.
  12. Huizing M, Anikster Y, Fitzpatrick DL, Jeong AB, D’Souza M, Rausche M, Kaiser-Kupfer MI, White JG, Gahl WA. Hermansky-Pudlak syndrome type 3 in Ashkenazi Jews and other non-Puerto Rican patients with hypopigmentation and platelet storage pool deficiency. Am J Hum Genet 2001;69:1022-1032.
  13. Huizing M, Anikster Y, Gahl WA. Hermansky-Pudlak syndrome and Chediak-Higashi syndrome: disorders of vesicle formation and trafficking. Throm Haemostasis 2001;26:233-245.
  14. Huizing M, Anikster Y, White JG, Gahl WA. Characterization of the murine gene corresponding to human Hermansky-Pudlak syndrome type 3: exclusion of the subtle gray (sut) locus. Mol Genet Metab, in press.
  15. Huizing M, Didier A, Walenta J, Anikster Y, Gahl WA, Kramer H. Molecular cloning and characterization of human VPS18, VPS11, VPS16, and VPS33. Gene 2001;264:241-247.
  16. Huizing M, Gahl WA. Disorders of vesicles of lysosomal lineage: the Hermansky-Pudlak syndromes. Curr Mol Med, in press.
  17. Huizing M, Sarangarajan R, Strovel E, Zhao Y, Gahl WA, Boissy RE. AP-3-dependent vesicles carry tyrosinase, but not TRP-1, in cultured human melanocytes. Mol Biol Cell 2001;12:2075-2085.
  18. Huizing M, Scher CD, Strovel E, Fitzpatrick DL, Hartnell L, Anikster Y, Gahl WA. Nonsense mutations in ADTB3A cause complete deficiency of the a3A subunit of adaptor complex-3 and severe Hermansky-Pudlak syndrome type 2. Pediatr Res 2002, in press.
  19. Kaiser-Kupfer MI, Gahl WA. Cystinosis. In: Gold DH, Weingeist TA, eds. Color atlas of the eye in systemic disease. Gaithersburg MD: Lippincott Williams & Wilkins, Chapter 73, 2001;295-297.
  20. Kleta R, Anikster Y, Lucero C, Shotelersuk V, Huizing M, Bernardini I, Park M, Thoene J, Schneider J, Gahl WA. CTNS mutations in African American cystinosis patients. Mol Genet Metab, in press.
  21. Lee J, Jiao X, Hejtmancik JF, Kaiser-Kupfer M, Gahl WA, Markello TC, Guo J, Chader GJ. The metabolism of fatty acids in human Bietti’s crystalline dystrophy. Invest Ophthalmol Vis Sci, in press.
  22. Leroy JG, Seppala R, Huizing M, Dacremont G, De Simpel H, Van Coster RN, Orvisky E, Krasnewich DM, Gahl WA. Dominant inheritance of sialuria, an inborn error of feedback inhibition. Am J Hum Genet 2001;68:1419-1427.
  23. Lukong KE, Landry K, Trudel S, Seyrantepel V, Ahmand A, Gahl WA, Lefrancois S, Morales CR, Pshezhetsky AV. Intracellular distribution of lysosomal sialidase is controlled by the internalization signal in its cytoplasmic tail. J Biol Chem, in press.
  24. Maurer-Spurej E, Dyker K, Gahl WA, Devine DV. A novel immunocytochemical assay for the detection of serotonin in platelets. Br J Haematol, in press.
  25. Phornphutkul C, Anikster Y, Huizing M, Braun P, Brodie C, Chou JY, Gahl WA. The promoter of a lysosomal membrane transporter gene, CTNS, binds Sp-1, shares sequences with the promoter of an adjacent gene (CARKL), and causes cystinosis if mutated in a critical region. Am J Hum Genet 2001;69:712-721.
  26. Sarangarajan R, Budev A, Zhao Y, Gahl WA, Boissy RE. Abnormal translocation of tyrosinase and tyrosinase related protein-1 in cutaneous melanocytes of Hermansky-Pudlak syndrome and in melanoma cells transfected with anti-sense HPS1 cDNA. J Inves Derm, in press.
  27. Tsilou ET, Rubin BI, Reed GF, Iwata F, Gahl WA, Kaiser-Kupfer MI. Age-related prevalence of anterior segment complications in patients with infantile nephropathic cystinosis. Cornea 2002, in press.