News and Publications

Division of Hematology

Ernest Beutler, M.D., Division Head

Human Genetic Diseases

E. Beutler, D. Balicki, L. Forman, T. Gelbart, C. Halloran, E. Boas, P. Lee, C. West

The main thrust of our laboratory continues to be the understanding of human genetic diseases at the molecular and biochemical level. Whenever possible, we want to use the information that can be gleaned from human mutations ("experiments of nature") to better understand not only the disease associated with a specific mutation but also normal physiology. In the past year, we investigated (1) the HFE mutation that causes the iron storage disease, hereditary hemochromatosis; (2) mutations of glucose-6-phosphate dehydrogenase (G6PD), pyruvate kinase, glucosephosphate isomerase, and triosephosphate isomerase, all causes of hemolytic anemia; (3) mutations of glucocerebrosidase that cause Gaucher disease; and (4) mutations of the gene for UDP-glucuronyltransferase 1 that causes jaundice.

HEREDITARY HEMOCHROMATOSIS

About 82% of U.S. patients with hereditary hemochromatosis are homozygous for the 845A mutation of the gene HFE. HFE, the product of this gene, is a class 1 MHC protein, and its role in regulating iron absorption is unknown. We are attempting to answer a number of important but difficult questions about HFE. What is its role in iron metabolism? What is the range of phenotypic expression of patients who inherit the 845A mutation and of those who inherit the more common 187G mutation? To try to answer these questions and better understand the entire mechanism of intestinal iron absorption, we studied the association of HFE with other proteins and sequenced HFE and associated genes, such as those for ß₂-microglobulin and calreticulin, in patients with hemochromatosis. So far, no additional mutations have been found that either cause hemochromatosis in the patients who do not carry the 845A mutation or modify the disease in those who do.

We also determined the structure of Nramp2, a gene involved with the transport of iron and other metals. We found that this gene exists in splice forms that do and do not contain a 3´ iron-responsive element. It is tempting to speculate that the form that contains the element is important in iron transport and that the form that lacks the element may play a role in the transport of other metals such as zinc and cadmium. We found polymorphic markers in the gene. Along with these markers and previously defined intron-exon boundaries, direct sequencing may lead us to mutations that cause or modify human disease.

Study of the epidemiology and natural history of patients with HFE mutations can only be achieved by investigating large numbers of subjects. In a collaboration with Kaiser-Permanente in San Diego, we are establishing a screening program in which we will do mutation analyses in 60,000 patients as part of a health screening. We should be able to obtain important data on the expression of iron storage disease in subjects who are homozygous for the 845A mutation and in subjects who are heterozygous for 845A/187G.

DEFICIENCIES OF RED CELL ENZYMES

Mutations of the genes for the enzymes G6PD, pyruvate kinase, glucosephosphate isomerase, and triosephosphate isomerase are causes of hemolytic anemia. We have identified many new mutations in these genes in patients with these enzyme deficiencies. The effects of these mutations on the structure of the protein may provide some insights on structure-function relationships.

Mutation analysis is the best, and in the case of pyruvate kinase deficiency, the only means for accurate prenatal diagnosis of enzyme deficiencies. Because severe pyruvate kinase deficiency is a devastating disorder, prenatal diagnosis is often important to parents who already have a child with this disorder. Our laboratory is the only one that offers prenatal diagnosis of this deficiency.

THE UDP-GLUCURONYLTRANSFERASE-1 GENE

Gilbert syndrome is a mild, symptomless form of jaundice that occurs in 5--10% of the population. In most cases, the syndrome is due to a mutation in the promoter region of the gene for UDP-glucuronyltransferase 1 in which 1 additional thymine-adenine is inserted in a region that contains 6 thymine-adenine repeats. The most serious clinical consequence of G6PD deficiency is neonatal jaundice. In some, but by no means all, infants with G6PD deficiency, severe jaundice develops that occasionally leads to kernicterus, a disorder characterized by serious, permanent neurologic damage. Why do severe jaundice and even kernicterus develop in some infants, whereas the majority of infants have no ill effects?

Working in collaboration with M. Kaplan and his associates at the Shaare Zedek Hospital in Jerusalem, we found that severe neonatal jaundice develops only in G6PD-deficient infants that have a second mutation, the 7 repeats of the promoter for UDP-glucuronyltransferase 1. This finding is the first clear example of human disease due to the interaction of 2 different mutations. Either mutation alone has benign, clinically insignificant consequences. Together, the 2 mutations produce a potentially devastating, even fatal, disorder.

We extended our study of the mutation in the promoter for UDP-glucuronyltransferase 1 and discovered that persons of African origin not only have genes with 6 and 7 thymine-adenine repeats but also have genes with 5 and 8 thymine-adenine repeats. We measured the activity of these promoters in cell lines derived from human liver and showed that the strength of the promoter decreases progressively as the length of the thymine-adenine repeat increases. The form with 5 repeats has the highest promoter activity; the form with 8 repeats has the weakest activity (Fig. 1).

HISTONE H2A-FACILITATED GENE TRANSDUCTION

Gene transfer is one of the unfulfilled promises of the 20th century. In many diseases, permanent transduction of target cells is required, and vectors that integrate the transferred gene are needed. In some diseases, such as Gaucher disease, transiently expressed normal genes might accomplish the desired clinical goal: expression of the normal glucocerebrosidase gene for just a few weeks might be sufficient to remove accumulated lipid from macrophages. We discovered that complexing DNA with histone H2A increases the efficiency of transduction by several orders of magnitude. Other histone classes are ineffective. We hope to learn what the unique properties of histone H2A are and whether it functions by improving entry of genes into cells, by facilitating transport of genes into the nucleus, or by improving gene expression. Histone H2A or a domain within this histone may be a useful tool in gene therapy.

PUBLICATIONS

Balicki, D., Beutler, E. Histone H2A significantly enhances in vitro DNA transfection. Mol. Med. 3:782, 1997.

Beutler, E. The biochemical, molecular, and population genetics of Gaucher disease. In: Advances in Jewish Genetic Diseases. Desnick, R.J. (Ed.). Oxford University Press, New York, 1998.

Beutler, E. Commentary: Targeted disruption of the HFE gene. Proc. Natl. Acad. Sci. U.S.A. 95:2033, 1998.

Beutler, E. Enzyme replacement therapy for Gaucher disease. Baillieres Clin. Haematol. 10:711, 1997.

Beutler, E. Gaucher disease phenotypes outflanked. Genome Res. 7:950, 1997.

Beutler, E. Stem cell transplantation in the twenty-first century. In: Hematopoietic Cell Transplantation, 2nd ed. Thomes, E.D., Blume, K.G., Forman, S.J. (Eds.). Blackwell, Boston, 1998, Chap. 104.

Beutler, E. Susumu Ohno: The father of x-inactivation. Cytogenet. Cell Genet. 80:16, 1998.

Beutler, E., Laubscher, K. Glucose-6-phosphate dehydrogenase deficiency. In: Clinical Studies in Medical Biochemistry. Glew, R.H., Ninomiya, Y. (Eds.). Oxford University Press, New York, 1997, p. 91.

Beutler, E., West, C. New diallelic markers in the HLA region of chromosome 6. Blood Cells Mol. Dis. 23:219, 1997.

Beutler, E., West, C., Britton, H.A., Harris, J., Forman, L. Glucosephosphate isomerase (GPI) deficiency mutations associated with hereditary nonspherocytic hemolytic anemia (HNSHA). Blood Cells Mol. Dis. 23:402, 1997.

Boas, E., Forman, L., Beutler, E. Phosphatidyl serine exposure and red cell viability in red cell ageing and in hemolytic anemia. Proc. Natl. Acad. Sci. U.S.A. 85:3077, 1998.

Burke, W., Thomson, E., Khoury, M., McDonnell, S.M., Press, N., Adams, P.C., Barton, J.C., Beutler, E., Brittenham, G., Buchanan, A., Clayton, E.W., Cogswell, M.E., Meslin, E.M., Motulsky, A.G., Powell, L.W., Sigal, E., Wilfond, B.S., Collins, F.S. Hemochromatosis: Gene discovery and its implications for population-based screening. JAMA 280:172, 1998.

Demina, A., Beutler, E. Six new Gaucher disease mutations. Acta Haematol. 99:80, 1998.

Demina, A., Boas, E., Beutler, E. Structure and linkage relationships of the region containing the human L-type pyruvate kinase (PKLR) and glucocerebrosidase (GBA) genes. Hematopathol. Mol. Hematol. 11:63, 1998.

Demina, A., Varughese, K.I., Barbot, J., Forman, L., Beutler, E. Six previously undescribed pyruvate kinase mutations causing enzyme deficiency. Blood 92:1, 1998.

Kaplan, M., Renbaum, P., Levy-Lahad, E., Hammerman, C., Lahad, A., Beutler, E. Gilbert syndrome and glucose-6-phosphate dehydrogenase deficiency: A dose-dependent genetic interaction crucial to neonatal hyperbilirubinemia. Proc. Natl. Acad. Sci. U.S.A. 94:12128, 1997.

Vlachos, A., Westwood, B., Lipton, J.M., Beutler, E. G6PD Mt. Sinai: A new severe hemolytic variant characterized by dual mutations at nucleotides 376 and 1159. Hum. Mutat. 6(Suppl. 1):S154, 1998.

Weimer, T.A., Salzano, F.M., Westwood, B., Beutler, E. G6PD variants in three South American ethnic groups: Population distribution and description of two new mutations. Hum. Hered. 48:92, 1998.