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Genetic mapping of the hemochromatosis locus on chromosome six
Genetic Mapping of the Hemochromatosis Locus on Chromosome Six C. Q. Edwards, G. E. Cartwright, M. H. Skolnick, and D. B. Amos
ABSTRACT: The purpose of this paper is to report a pedigree with hereditary hemochromatosif in which a recombination between the HLA-A and B loci occurred. Both the maternal and paternal HLA-A3, B7 haplotypes were carrying an allele for hemochromatosis. The A3,BI2 h~plotype of the proband *,,as a recombinant of the maternal A3,B7 and A28,BI2 haplotMm. The hemochromatosis locus segregated with the HLA-A locus and not with the HLA-B locus. Thus, the hemochromatosis locus maps in closeproximity to the HLA-A locus.
INTRODUCTION The discovery of an association of hemochromatosis with specific HLA antigens by Simon et al. [1] is now amply confirmed. A significant increase in the frequency of HLA-A3 (72oA. vs 21%), of HLA-B14 (28% vs 6o~), and of HLA-B7 (45% vs 18%) antigens has been observed in 152 unrelated patients with hemochromatosis from five different countries [2]. Close genetic linkage between the HLA region and the hemochromatosis locus has been demonstrated by Simon et al. [3], Lipinski et al. [4], and by our own group [5,6]. We obtained a lod score of ÷9.8 for linkage between the HLA haplotype and the hemochromatosis locus for a recombination fraction (0) of 0.0 and a gene frequency ,of 0.056, the gene frequency determined in the Mormon population in Utah. These studies indicate that hemochromatosis is inherited in an autosomal recessive fashion with the hemochro~atosis allele tightly linked to the HLA region. Although the recombination fraction between the hemochromatosis locus an,] the HLA region is very low, the hemochromatosis allele is not any of the known HLA alleles since the HLA markers may be different in different kindreds, A3 and B7 being the most frequent. Thus, it is now possible to predict with a high degree of probability heterozygosity and homozygosity for the hemochromatosis allele in siblings of an affected Froband on the basis of HLA genotyping. Furthermore, in our studies [6], abnormal
Prom the Departments of Medici~e and Me:tical Biophysics. Uni.versity of Utah College of Mediciu,. Salt Lake City. Utah, attd the Department of Mfi'robiolog~i and Immunology. Duke Unirersit), Medical Center, Durham. North Carolina. Address requestsfor reprints to: Dr. G. E. Cartwright. University Hospital. 50 N. Medical Drire. Salt Lake City, UT 84132. Received •979.
Human immunoloff~ 1,19-22 (1980) ~) El~vler North Holland, Inc., 1980 52 Vanderbilt A'~e., New York, NY 10017
19 0198-8859/80/01019-04 $2.25
20
C.Q. Edwards, G. E. Cat.right, M. H. Skolnick and D. B. Amos homozygotes could be clearly distinguished from heterozygotes on the basis of the degree of iron loading. The purpose of this paper is to report on a pedigree in which a recombination between the HLA-A and B loci occurred. These studies demonstrate that the hemochromatosis locus can be separated from the HLA-B locus and illustrate how HLA genotyping can be of value in genetic mapping even when critical members of a pedigree may not be available for study.
METHODS The standard N I H technique was used for the determination of HLA-A, B, and C specificities, modified by the addition of a wash step 1"7~ The serum panel used was defined by the 1975 workshop except for Aw36 but including the newer specificities Bw44, 45, 51, 52, and 53. The methods used for the measurement of serum iron, total iron-binding capacity, transferrin saturation, serum ferritin, urinary iron excretion after deferoxamine, hepatic parenchymal cell stainable iron, and hepatic iron concentration were described previously [8], along with normal values [6,8~ All blood specimens were drawn fasting at 8 AI~. Because of the lability of the serum iron and transferrin saturation, these determinations were performed on at least three blood samples drawn at least 1 wk apart. The means of the three o." more determinations were taken as a reasonable estimate of the serum iron and transferrin saturation. A major iron load was ~iefined as a transferrin saturation greater than 79% and a hepatic iron concentratio:, greater than 400 p.g/100 mg wet liver in males [6]. A minor iron load was defined as a transferrin saturation of 51 ~o 79% and/or a hepatic iron concentration between 30 and 400 p.g/100 mg wet liver in males.
RESULTS The iron status and HLA haplotypes were determined on 29 individuals in three generations of pedigree N (Fig. 1). ll-1 and 1I-3 were the only members of the pedigree with clinically manifest hemochromatosis and/or a maior iron load. I-11 and II-8 were the only members of the pedigree with a minor iron load. The iron values on the remaining pedigree members were within normal limits. ~I-1, the 51-yr-old proband, had abnormal liver function studies and cirrhosis of the liver by biopsy. His alcoholic intake consisted only of an occasional drink on weekends. A maior iron load was present. His iron values were as follows: serum iron, 255 pg/100 ml; transferrin saturation, 100%; serum ferritin, 3600 ng/ml; 24-hr arinary iron excre6on after the intramuscular iniection of 15 mg of deferoxamine per kg of body weight, 9.7 rag; hepatic parenchymai cell stainable iron, grade 4; and hepatic iron concentration, 955 p.g/100 mg of wet liver. H-3, the 49-yr-old brother of the proband, had fibrosis but not cirrhosis of the liver by biopsy. H e did not consume alcohol. A major iron load was present. His iron values were as follows: serum iron, 243 ~t~/100 ml; transferrin saturation, 97%; serum ferritin, 855 ng/ml; 24-hr urinary iron excretion after deferoxamine, 4.4 rag; hepatic parenchymal cell stainable iron, grade 4; and hepatic iron concentration, 896 p~/100 mg of wet liver. II-8, the 39-yr-old brother of the proband (Fig. 1), was entirely well and the physical examination was unremarkable. He did not consume alcohol. Liver histology was normal. Only a minor iron load was present. His iron values were
Hemochromatosis Locus
21
PEDIGREEN
3,7 2.37
2,15 2.40
2,15 2,1.___~5 2,1.__~5 2,15 2 , 4 0 2 , 4 0 2,40 2.40
37
L
:
37
32.40
28.12
2,7
3,7
2,7
4
3.7_~2~,40
3,12 3,12 3,12 3,7 2,7 2,7 2,7 2,7
32~0
3,7 3,7 3,7 3,7 3;/' 3.7 3,7 3,7 2,40 ~4,7 24,7 2,40 24,7 24,7 2,40 ~
~6
3.7 28.12 24.18 24.18
FIGURE 1 Pedigree N. The arrow refers to the proband. Squares refer to males. circles to females. The solid squares refer to the two siblings with clinically manifest hemochromatosis. The slashed line indicates the individual is not living. The HIL.A haplotypes are given below the squares and circles. The first number refers to the HLA-A locus and the second number to the HLA-B locus. Parentheses around the HLA hapiotypes indicate that they were deduced and not determined.
as follows: serum iron, 120 /.~g/100 ml; transfi:rrin saturation, 36%; serum ferritin, 220 ng/ml; 24-hr urinary iron excretion after deferoxamine, 1.6 mg; h~;patic parenchymal cell stainable iron, grade 1; a~ad hepatic iron concentration, 169/zg/100 mg of wet liver. I-1 died at 53 yr o f age from biopsy-proven metastatic adenocarcinoma o f the prostate. There were no symptoms or signs suggestive of hemochromatosis in the hospital record. An autopsy was not performed. I-2 died at 66 yr o f age of a cerebral vascular .accident. H e r serum iron was recorded as 67/zg/100 ml with a transferrin saturation o f 36%. There were no symptoms or signs suggestive of hemochromatosis in the extensive hospital records. An autopsy was not performed. The proband (II-1) with clinically manifest hemochromatosis typed A3,B7,B12. A3,B7 and A 3 , B I 2 haplotypes appeared in his children, indicating that he was homozygous for A3. The brother (II-3) of the proband, also with clinically manifest hemochromatosis, typed A3,B7. All eight o f his children carried an A3,B7 haplotype, indicating with a high probability that ll-3 was homozygous for A3,B7. Furthermore, both a pater*,al and a maternal A3,B7 haplotype was identified in generation I. The third brother (II-8) with a.minor iron load and no clinical manifestations o f hemochromatosis carried A3,B7 and A28,B 12 haplotypes. Since II-3 was homozygous for A3,B7, each parent must have carried an A3,B7 haplotype. The maternal A28,B12 haplotype appeared in II-8. Therefore, I-2 was presumed to carry the A3,B7 and A28,B12 haplotypes. The second haplotype o f the father (I-1) was undetermined but this could not have been A3,B12 since four haplotypes were identified in his siblings and none o f these were A3,B12. Therefore, the A3,B12 haplotype of I1-1 must be a
22
C.Q. Edwards, G. E. Cartwright, M. H. Skolnick and D. B. Amos recombinant between the A3,B7 and A28,B12 haplotypes o f the mother (1-2). The only HLA-C specificity identified in the siblingship o f I-2 was Cw3.
DISCUSSION Both the paternal and the maternal HLA-A3,B7 haplotypes in this pedigree carried an allele for hemochromatosis. II-3 with clinically manifest hemochromatosis carried both A3,B7 haplotypes, l - l l carried the maternal A3,B7 haplotype and 1I-8 carried the paternal A3,B7 haplotype and both had a minor i,on load such as we have observed in some male heterozygotes [61 ll-1, the t~roband with cliaically manifest disease, carried the paternal HLA-A3,B7 t• aplotype and an HLA-A3,B12 recombinant haplotype from the mother (I-2). Thus, the hemochromatosis locus segregated with the HLA-A locus and not with the HLA-B locus. These data, along with the high degree of association between hemochromatosis and HLA-A3 reported in the literature [2], and the tight linkage which we have observed [6] between the hemochromatosis locus and the HLA region, indicate that the hemochromatosis locus is located in close proximity to the HLA-A locus. Whether it is located distal to the HLA-A locus or between the HLA-A and B loci cannot be determined from our studies. ACKNOWLEDGEMENTS These investigations were supported by the National Institutes of Health under Grants AM-20630, CA-16573, GM-10367, GM-07464, RR-0528, and FR-00064. The authors wish to thank Mrs. Alice Tustison, Miss Helen Ashenbrucker, and Miss Lisbeth Buskjaer for technical assistance. Dr. Rosina Dixon of Ciba-Geigy kindly provided the' deferoxamine (Desferal) for these studies. REFERENCES 1. Simon M, Bourel M, Fanchet R et al.: Association of HLA-A3 and HLA-VI4 antigens with idiopathic hemochromatosis. Gut 17:332- 334, 1976. 2. Batchelor JR, Morris PJ, eds: Seventh International Workshop on HLA and Disease. Idiopathic hemochromatosis, pp. 221-223, 1977. 3. Simon M, Bourel M, Genetet B e t al.: Idiopathic hemochromatosis. Demonstration of recessive transmission and early detection by family HLA typing. N Engl J Med 297:1017-1021, 1977. 4. Lipinski M, Hors J, Saleun JP et al.: Idiopathic hemochromatosis: Linkage to HLA. Tissue Antigens 11:471-474, 1978. 5. Kravitz K, Skolnick M, Cannings C et al.: Genetic linkage between hereditary hemochromatosis and HLA. AmJ Human Genetics 31:601-619, 1979. 6. Cartwright GE, Edwards CO., Kravkz K et al.: Hereditary bemochromatosis. Phenotypic expression of the disease. N Engl J Med 301:175-179, 1979. 7. Amos DB, Pool P: HLA typing. In: MR Rose, H Friedman, Eds. Manual of Clinical Immunology. Washington, DC, American Society for Microbiology, 1976, pp. 797804. 8. Edwards CQ, Carroll M, Bray P e t al.: Hereditarv hemochromatosis: Diagnosis in siblings and children. N Engl J Med 297:7-13, 1977.
ABSTRACT: The purpose of this paper is to report a pedigree with hereditary hemochromatosif in which a recombination between the HLA-A and B loci occurred. Both the maternal and paternal HLA-A3, B7 haplotypes were carrying an allele for hemochromatosis. The A3,BI2 h~plotype of the proband *,,as a recombinant of the maternal A3,B7 and A28,BI2 haplotMm. The hemochromatosis locus segregated with the HLA-A locus and not with the HLA-B locus. Thus, the hemochromatosis locus maps in closeproximity to the HLA-A locus.
INTRODUCTION The discovery of an association of hemochromatosis with specific HLA antigens by Simon et al. [1] is now amply confirmed. A significant increase in the frequency of HLA-A3 (72oA. vs 21%), of HLA-B14 (28% vs 6o~), and of HLA-B7 (45% vs 18%) antigens has been observed in 152 unrelated patients with hemochromatosis from five different countries [2]. Close genetic linkage between the HLA region and the hemochromatosis locus has been demonstrated by Simon et al. [3], Lipinski et al. [4], and by our own group [5,6]. We obtained a lod score of ÷9.8 for linkage between the HLA haplotype and the hemochromatosis locus for a recombination fraction (0) of 0.0 and a gene frequency ,of 0.056, the gene frequency determined in the Mormon population in Utah. These studies indicate that hemochromatosis is inherited in an autosomal recessive fashion with the hemochro~atosis allele tightly linked to the HLA region. Although the recombination fraction between the hemochromatosis locus an,] the HLA region is very low, the hemochromatosis allele is not any of the known HLA alleles since the HLA markers may be different in different kindreds, A3 and B7 being the most frequent. Thus, it is now possible to predict with a high degree of probability heterozygosity and homozygosity for the hemochromatosis allele in siblings of an affected Froband on the basis of HLA genotyping. Furthermore, in our studies [6], abnormal
Prom the Departments of Medici~e and Me:tical Biophysics. Uni.versity of Utah College of Mediciu,. Salt Lake City. Utah, attd the Department of Mfi'robiolog~i and Immunology. Duke Unirersit), Medical Center, Durham. North Carolina. Address requestsfor reprints to: Dr. G. E. Cartwright. University Hospital. 50 N. Medical Drire. Salt Lake City, UT 84132. Received •979.
Human immunoloff~ 1,19-22 (1980) ~) El~vler North Holland, Inc., 1980 52 Vanderbilt A'~e., New York, NY 10017
19 0198-8859/80/01019-04 $2.25
20
C.Q. Edwards, G. E. Cat.right, M. H. Skolnick and D. B. Amos homozygotes could be clearly distinguished from heterozygotes on the basis of the degree of iron loading. The purpose of this paper is to report on a pedigree in which a recombination between the HLA-A and B loci occurred. These studies demonstrate that the hemochromatosis locus can be separated from the HLA-B locus and illustrate how HLA genotyping can be of value in genetic mapping even when critical members of a pedigree may not be available for study.
METHODS The standard N I H technique was used for the determination of HLA-A, B, and C specificities, modified by the addition of a wash step 1"7~ The serum panel used was defined by the 1975 workshop except for Aw36 but including the newer specificities Bw44, 45, 51, 52, and 53. The methods used for the measurement of serum iron, total iron-binding capacity, transferrin saturation, serum ferritin, urinary iron excretion after deferoxamine, hepatic parenchymal cell stainable iron, and hepatic iron concentration were described previously [8], along with normal values [6,8~ All blood specimens were drawn fasting at 8 AI~. Because of the lability of the serum iron and transferrin saturation, these determinations were performed on at least three blood samples drawn at least 1 wk apart. The means of the three o." more determinations were taken as a reasonable estimate of the serum iron and transferrin saturation. A major iron load was ~iefined as a transferrin saturation greater than 79% and a hepatic iron concentratio:, greater than 400 p.g/100 mg wet liver in males [6]. A minor iron load was defined as a transferrin saturation of 51 ~o 79% and/or a hepatic iron concentration between 30 and 400 p.g/100 mg wet liver in males.
RESULTS The iron status and HLA haplotypes were determined on 29 individuals in three generations of pedigree N (Fig. 1). ll-1 and 1I-3 were the only members of the pedigree with clinically manifest hemochromatosis and/or a maior iron load. I-11 and II-8 were the only members of the pedigree with a minor iron load. The iron values on the remaining pedigree members were within normal limits. ~I-1, the 51-yr-old proband, had abnormal liver function studies and cirrhosis of the liver by biopsy. His alcoholic intake consisted only of an occasional drink on weekends. A maior iron load was present. His iron values were as follows: serum iron, 255 pg/100 ml; transferrin saturation, 100%; serum ferritin, 3600 ng/ml; 24-hr arinary iron excre6on after the intramuscular iniection of 15 mg of deferoxamine per kg of body weight, 9.7 rag; hepatic parenchymai cell stainable iron, grade 4; and hepatic iron concentration, 955 p.g/100 mg of wet liver. H-3, the 49-yr-old brother of the proband, had fibrosis but not cirrhosis of the liver by biopsy. H e did not consume alcohol. A major iron load was present. His iron values were as follows: serum iron, 243 ~t~/100 ml; transferrin saturation, 97%; serum ferritin, 855 ng/ml; 24-hr urinary iron excretion after deferoxamine, 4.4 rag; hepatic parenchymal cell stainable iron, grade 4; and hepatic iron concentration, 896 p~/100 mg of wet liver. II-8, the 39-yr-old brother of the proband (Fig. 1), was entirely well and the physical examination was unremarkable. He did not consume alcohol. Liver histology was normal. Only a minor iron load was present. His iron values were
Hemochromatosis Locus
21
PEDIGREEN
3,7 2.37
2,15 2.40
2,15 2,1.___~5 2,1.__~5 2,15 2 , 4 0 2 , 4 0 2,40 2.40
37
L
:
37
32.40
28.12
2,7
3,7
2,7
4
3.7_~2~,40
3,12 3,12 3,12 3,7 2,7 2,7 2,7 2,7
32~0
3,7 3,7 3,7 3,7 3;/' 3.7 3,7 3,7 2,40 ~4,7 24,7 2,40 24,7 24,7 2,40 ~
~6
3.7 28.12 24.18 24.18
FIGURE 1 Pedigree N. The arrow refers to the proband. Squares refer to males. circles to females. The solid squares refer to the two siblings with clinically manifest hemochromatosis. The slashed line indicates the individual is not living. The HIL.A haplotypes are given below the squares and circles. The first number refers to the HLA-A locus and the second number to the HLA-B locus. Parentheses around the HLA hapiotypes indicate that they were deduced and not determined.
as follows: serum iron, 120 /.~g/100 ml; transfi:rrin saturation, 36%; serum ferritin, 220 ng/ml; 24-hr urinary iron excretion after deferoxamine, 1.6 mg; h~;patic parenchymal cell stainable iron, grade 1; a~ad hepatic iron concentration, 169/zg/100 mg of wet liver. I-1 died at 53 yr o f age from biopsy-proven metastatic adenocarcinoma o f the prostate. There were no symptoms or signs suggestive of hemochromatosis in the hospital record. An autopsy was not performed. I-2 died at 66 yr o f age of a cerebral vascular .accident. H e r serum iron was recorded as 67/zg/100 ml with a transferrin saturation o f 36%. There were no symptoms or signs suggestive of hemochromatosis in the extensive hospital records. An autopsy was not performed. The proband (II-1) with clinically manifest hemochromatosis typed A3,B7,B12. A3,B7 and A 3 , B I 2 haplotypes appeared in his children, indicating that he was homozygous for A3. The brother (II-3) of the proband, also with clinically manifest hemochromatosis, typed A3,B7. All eight o f his children carried an A3,B7 haplotype, indicating with a high probability that ll-3 was homozygous for A3,B7. Furthermore, both a pater*,al and a maternal A3,B7 haplotype was identified in generation I. The third brother (II-8) with a.minor iron load and no clinical manifestations o f hemochromatosis carried A3,B7 and A28,B 12 haplotypes. Since II-3 was homozygous for A3,B7, each parent must have carried an A3,B7 haplotype. The maternal A28,B12 haplotype appeared in II-8. Therefore, I-2 was presumed to carry the A3,B7 and A28,B12 haplotypes. The second haplotype o f the father (I-1) was undetermined but this could not have been A3,B12 since four haplotypes were identified in his siblings and none o f these were A3,B12. Therefore, the A3,B12 haplotype of I1-1 must be a
22
C.Q. Edwards, G. E. Cartwright, M. H. Skolnick and D. B. Amos recombinant between the A3,B7 and A28,B12 haplotypes o f the mother (1-2). The only HLA-C specificity identified in the siblingship o f I-2 was Cw3.
DISCUSSION Both the paternal and the maternal HLA-A3,B7 haplotypes in this pedigree carried an allele for hemochromatosis. II-3 with clinically manifest hemochromatosis carried both A3,B7 haplotypes, l - l l carried the maternal A3,B7 haplotype and 1I-8 carried the paternal A3,B7 haplotype and both had a minor i,on load such as we have observed in some male heterozygotes [61 ll-1, the t~roband with cliaically manifest disease, carried the paternal HLA-A3,B7 t• aplotype and an HLA-A3,B12 recombinant haplotype from the mother (I-2). Thus, the hemochromatosis locus segregated with the HLA-A locus and not with the HLA-B locus. These data, along with the high degree of association between hemochromatosis and HLA-A3 reported in the literature [2], and the tight linkage which we have observed [6] between the hemochromatosis locus and the HLA region, indicate that the hemochromatosis locus is located in close proximity to the HLA-A locus. Whether it is located distal to the HLA-A locus or between the HLA-A and B loci cannot be determined from our studies. ACKNOWLEDGEMENTS These investigations were supported by the National Institutes of Health under Grants AM-20630, CA-16573, GM-10367, GM-07464, RR-0528, and FR-00064. The authors wish to thank Mrs. Alice Tustison, Miss Helen Ashenbrucker, and Miss Lisbeth Buskjaer for technical assistance. Dr. Rosina Dixon of Ciba-Geigy kindly provided the' deferoxamine (Desferal) for these studies. REFERENCES 1. Simon M, Bourel M, Fanchet R et al.: Association of HLA-A3 and HLA-VI4 antigens with idiopathic hemochromatosis. Gut 17:332- 334, 1976. 2. Batchelor JR, Morris PJ, eds: Seventh International Workshop on HLA and Disease. Idiopathic hemochromatosis, pp. 221-223, 1977. 3. Simon M, Bourel M, Genetet B e t al.: Idiopathic hemochromatosis. Demonstration of recessive transmission and early detection by family HLA typing. N Engl J Med 297:1017-1021, 1977. 4. Lipinski M, Hors J, Saleun JP et al.: Idiopathic hemochromatosis: Linkage to HLA. Tissue Antigens 11:471-474, 1978. 5. Kravitz K, Skolnick M, Cannings C et al.: Genetic linkage between hereditary hemochromatosis and HLA. AmJ Human Genetics 31:601-619, 1979. 6. Cartwright GE, Edwards CO., Kravkz K et al.: Hereditary bemochromatosis. Phenotypic expression of the disease. N Engl J Med 301:175-179, 1979. 7. Amos DB, Pool P: HLA typing. In: MR Rose, H Friedman, Eds. Manual of Clinical Immunology. Washington, DC, American Society for Microbiology, 1976, pp. 797804. 8. Edwards CQ, Carroll M, Bray P e t al.: Hereditarv hemochromatosis: Diagnosis in siblings and children. N Engl J Med 297:7-13, 1977.