Tissue Antigens (1978), 11,471-474 Published by Munksgaard, Copenhagen, Denmark No part may be reproduced by any process without written permission from the author(s)

Idiopathic Hemochromatosis: Linkage with HLA Marc Lipinski’, Jacques HOB’ , Jean-Pierre Saleun’, Raymond Saddi,3 Philippe Passa4, Sophie Lafaurie’, Nicole Feingold’ and Jean Dausset’ Unitd INSERM, U.93, HBpital Saint-Louis, Paris, Centre Dipartemental de Transfusion Sanguine, Brest, Institut de Pathologie Moldculaire (CNRS- INSERM) CHU Cochin Pans, Service d’Endocrinologie Mdtabolique, Hopital Saint-Louis and Unit6 INSERM U.155, ChLteau de Longchamp, Paris, France

Forty-eight unrelated patients with idiopathic hemochromatosis were found to have a significantly higher frequency of three HLA antigens (A3, B7 and B14) than 591 healthy controls. A significant association between HLA haplotypes and disease segregations was demonstrated in 14 family studies. A recessive inheritance of a strongly A3-linked disease gene responsible for abnormal iron stores in the heterozygote state is postulated. The lod score value (4.415for 0 = 0.025) is compatible with this hypothesis. However, the excess of HJA-identical pairs of affected sibs does not exclude the possibility of a pseudo-recessiveness due to two codominant genes both HLA-linked. For the first time, a means of screening for high risk subjects is available and therefore offers the possibility of a preventive approach. Received for publication 18 October, revised, accepted 5 December 1977

Since Sheldon’s report (1935), idiopathic hemochromatosis (I.H.) has been known to be a genetic disease. The association between I.H. and HLA antigen A3 was first described by Simon et al. (1975). Since then, a strong association between I.H. and A3 has been confirmed (Walters et al. 1975, Shewan et al. 1976) whereas the disease was found to show a weaker association with the B locus: B7 (Walters et al. 1975, Shewan et al. 1976) and B14 (Simon et al. 1975). However, the genetical mode of transmission is not yet clearly defined. Different models have been proposed: dominant gene (Balcerzack

1966) recessive gene (Saddi & Feingold 1974, Simon et al. 1976, 1977) two genes (Bomford et al. 1977) and multiple factor heredity (Grace & Powell 1974). We now report findings from a series of unrelated patients and multiple-case families with well defined I.H. in whom we examined the HLA-A and -B loci. The results were evaluated in relation to clinical features and different genetical analyses. Material and M e t h o d s Patients Fourteen multiple-case families affected

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LIPINSKI ET AL.

with I.H. and 34 unrelated diseased individuals were studied. All patients were Caucasians. Clinical, biological and histological criteria for diagnosis are described elsewhere (Lipinski et al. 1978). In the families, subjects were divided into three classes: (1)patients with a major form: overt

idiopathic hemochromatosis (O.I.H.) proved by liver biopsy (in 1 7 out of 24) showing strainable iron and unequivocal clinical and biological arguments. (2) healthy persons (serum iron < 140 pg/100 ml, iron binding capacity saturation < 40%, no clinical abnormality). (3) and a third category of subjects considered as intermediate idiopathic hemochromatosis (I.I.H.) showing a slight elevation of serum iron (from 140 t o 190pg/100 ml) and an increased saturation of the iron binding capacity (from 40 to 80%) without any clinical trouble.

H L A Typing HLA typing was done by a modified microcytotoxicity technique (Mittal et al. 1968) using antisera for 11 alleles of the A locus and for 17 of the B locus. Genetical Analysis The phenotypic frequencies of the different antigens in the 48 patients (one from each family plus 34 unrelated) with O.I.H. were compared with the controls by means of the chi square test; P values were corrected by multiplying by the number of comparisons tested (28 antigens). A gene was considered disease susceptible in both recessive and dominant models according to a previously reported method (Thomson & Bodmer 1977). The HLA genotype distribution of affected sibs

Table 1 H L A antigens in idiopathic hemochromatosis

HLA antigens A3

B7 B14

Patients N=48

Controls N = 591

P

75% 38% 29%

26%

< < 0.05 < 0.05

19% 09%

P is corrected by multiplying by the numbers of

antigens tested

within the families was compared with that expected if there were no link between HLA and I.H. Lod score values were calculated (Morton 1956) in the recessive model for eight out of the 1 4 families. Five families were eliminated because one of the parents of the affected sibship had suffered from I.H., and a sixth because the haplotype determination was not done unequivocally. Results The family data are published elsewhere in full detail (Lipinski et al. 1978). The most significant association was found between HLA-A3 and O.I.H. (Table 1) (75% versus 26% in the control group: P < The frequencies of B7 and B14 were also increased but less significantly ( P < 0.05) (Table 1). All the patients with B7 and/or B14 were A3-positive. The phenotypic associations A3, B7 and A3, B14 were consequently increased (respectively 21% and 5.3% versus 12% and 0.6%) with an important linkage disequilibrium: A = A = 0.06 (A3-Bl4); 0.105 (A3-B7); A2, B12 and Bw40 were slightly but not significantly increased, whereas the other antigens had a lowered incidence. Fourteen families with a total of 24 overt forms (O.I.H.), (i.e. the proband of each family and 10 more related affected individuals), 39 intermediate forms (I.I.H.) and 35 healthy subjects were studied.

IDIOPATHIC HEMOCHROMATOSIS A N D HLA

____

O.I.H. I.I.H.

473

Table 2 Relationship between H L A genotypes and idiopathic hemochromatosis

_____

.-

____.-_.

HLA

HLA Semi-

HLA

identical

identical

different

Total

8 (5) 1

2 (2)

0

10 (7) 36 (14) 20 (1)

31

Healthy subjects

1 (0)

(13) 16 (1)

Total

10 (5)

49 (16)

(0)

(0) 4

(1) 3 (0)

7

66

(1)

(22)

O.I.H. Overt idiopathic hemochromatosis. I.I.H. Intermediate idiopathic hemochromatosis. The 66 related subjects are distributed according to their HLA ideqtity with the proband and according

to the existence of a hemochromatosis. Numbers in brackets correspond to males over 17.

From nine affected sib pairs, seven shared both HLA haplotypes and two shared only one, a significantly non-random result ( P < 0.001). Of the 10 related O.I.H. (in one family in which three affected sibs were HLA-identical, only one pair was considered), eight were HLA-identical and two were semi-identical t o the proband (Table 2). Most with the intermediate form (31/36) had one HLA haplotype in common with the proband (Table 2). Lod score values for 0 of 0.020, 0.025 and 0.030 were 4.403, 4.415 and 4.410 respectively . Discussion Our data show that the association of I.H. with the antigen HLA-A3 is strikingly stronger than the association with HLA-B7 or B14. The excess of affected sib pairs sharing two HLA haplotypes is significantly non-random. These facts indicate that I.H. is genetically controlled by at least one gene located close to the HLA-A locus and in linkage disequilibrium with HLA-A3. These results are

closer to a recessive gene model than t o a dominant one (Thomson & Bodmer 1977). Furthermore, the segregation of the HLA haplotypes in the families fits with the transmission of the disease in 64% of the cases (Table 2). Since females are physiologically protected and children under 17 may be too young to contract the disease, the relationship reaches 82% when only males over 17 are considered. Out of the eight families tested, seven had a positive lod score while one had a negative one. Thus an 0 value of 0.025 is not incompatible with a single recessive I.H. gene closely linked to HLA-A locus, and responsible for abnormal iron stores (I.I.H.) in the heterozygote state and for an bvert form (O.I.H.) in the homozygote state. According to this hypothesis, some people are less affected than expected (Table2) but most of them (18119) are under 1 7 andlor females. Six patients are more affected than expected. Several hypotheses can be considered: a) Marriages between homozygote and heterozygote parents should not be rare in those overaffected selected families. In one family

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Lipinski, M., Hors, J., Saleun, J. P., Saddi, R., Passa, Ph., Feingold, J., Lafaurie, s. Dausset, J. (1978) Hemochromatose idiopathique, liaison avec le s y s t h e HLA. Diubkte Mktab. (in press). Mittal, K. K., Mickey, M. R., Singal, D. P. & Terasaki, P. I. (1968) Serotyping for homotransplantation. XVIII. Refinement of microdroplet lymphocyte cytotoxicity test. Transplantation 6, 913-927. Morton, N. E. (1956) Sequential tests for the detection of linkage. Amer. J. hum. Genet. 7,277-318. Saddi, R. & Feingold, J. (1974) Idiopathic haemochromatosis: an autosomal recessive disease. Clin. Genet. 5, 234-241. Sheldon, J . H. (1935) Haemochromatosis. London, Oxford University Press. Shewan, W. G., Mouat, S. A. & Allan, T. M. (1976) HLA antigens in hemochromatosis. Brit. med. J . 1,281-282 Simon, M., Bourel, M., Alexandre, J . L., Brissot, P., Hita De Nercy, Y.,Scordia, C., Fauchet, R., Genetet, N. & Genetet, B. (1976) HCrCditC de I’hCmochromatose. Hdmochromatose: sa liaison avec le systhme HLA. Nouv. PresseMkd. 5 , 1976. Simon, M., Bourel, M., Genetet B. & Fauchet R. (1977) Idiopathic Hemochromatosis. Demonstration of recessive transmission and early detection by family HLA typing. N. Engl. J . Med. 237,1017-1021. Simon, M., Pawlotsky, Y.,Bourel, M., Fauchet, R. & Genetet, B. (1975) Hemochromatose idiopathique. Maladie associde A l’antig4ne tissulake HLA-A3? Nouv. Presse Mkd. 4, 1432. Thomson, G. & Bodmer, W. (1977) The genetic analysis of HLA and disease associations. HLA and Disease pp. 84-93, Munksgaard, Balcerzack, S. P., Westeman, M. P., Lee, R. E. & Copenhagen. Doyle, A. P. (1966) Idiopathic hemochroma- Walters, J. M.,Watt, D. W., Stevens, F. M. & tosis, a study of three families. Amer. J. Med. McCarthy, C. F. (1975) HLA antigens and 40,857-913 hemochromatosis. Brit. med. J. 4, 520. Bomford, A., Kennedy, L. A., Eddleston, A. L. W. F., Batchelor, J. R. & Williams, R. (1977) Address: Histocompatibility antigens as markers of Marc Lipinski abnormal iron metabolism in patients with idiopathic hemochromatosis and their Laboratoire d’Immuno-h6matologie. Institut Gustave Roussy relatives. Lancet i, 327-329. Grace, N. D. & Powell, L. W. (1974) Iron storage 16bis. rue Paul Vaillant Couturier disorders of the liver. Gastro-enterology 67, 94800 Villejuif France 1257-1 283.

especially, in which two affected sibs were sharing only one haplotype, the father died of hemochromatosis and was therefore carrying the postulated I.H. recessive gene on both haplotypes. b) the occurrence of a crossing-over between I.H. gene and HLA region; c) Besides concealed external factors (alcohol), a genetical heterogenity of I.H. is possible. In a few families (the family with a negative lod-score ?), the disease could result from another gene without linkage with the HLA complex; d) Finally, a model with two dominant complementary genes both located on the sixth chromosome, the one closely linked to HLA-A, the other further from the HLA region, has t o be considered: in most cases in trans-position they would support a recessive model; a cis-position would explain the few apparently conflicting results. Such a pseudo-recessive model would explain the excess of the HLA identities in the affected sibs. For the first time in I.H., our data suggest that HLA typing can provide an opportunity for a preventive detection of high risk subjects. By paying the closest attention to suspected individuals, imposing an alcohol-free diet, early phlebotomies and genetical counselling, a decrease in the disease incidence should be possible. References