Expression of HLA-Linked Hemochromatosis in Subjects Homozygous or Heterozygous for the C282Y Mutation

Transcription

1 GASTROENTEROLOGY 1998;114: Expression of HLA-Linked Hemochromatosis in Subjects Homozygous or Heterozygous for the C282Y Mutation DARRELL H. G. CRAWFORD, ELIZABETH C. JAZWINSKA, LARA M. CULLEN, and LAWRIE W. POWELL Clinical Sciences Unit, Queensland Institute of Medical Research and University of Queensland, Brisbane, Australia Background & Aims: In the absence of a genetic test, diagnostic criteria for hereditary hemochromatosis have been imprecise. The identification of the HFE gene and the C282Y mutation allow definition of expression of this disease and reassessment of diagnostic criteria. The aim of this study was to analyze the concordance between the genetic diagnosis and the previous clinical diagnosis in families with hemochromatosis. Methods: Three hundred subjects were tested for the C282Y mutation and were grouped as homozygous, heterozygous, or homozygous normal. Results: All adults previously diagnosed as homozygous or heterozygous for HLA-linked hereditary hemochromatosis carried at least one C282Y mutation. Two adolescents, previously thought to be homozygous, had no C282Y mutation. Of 127 subjects homozygous for the mutation, 105 met criteria for diagnosis. Iron overload was not expressed in 6.7% of homozygous men and 32.7% of homozygous women. The iron indices in 8 of 171 subjects heterozygous for the C282Y mutation were within the range previously regarded as indicative of homozygosity. Seven of these 8 carried the H63D mutation. Conclusions: In Australia, 17.3% of subjects homozygous for the C282Y mutation do not express iron overload to meet current diagnostic criteria of hemochromatosis. In subjects heterozygous for the mutation, 4.8% have iron overload in the range previously diagnosed as homozygous. Nonexpression is common, particularly in women. HLA-linked hereditary hemochromatosis (HHC) is one of the most common inherited diseases in white populations, having a prevalence of approximately 1:300 and a carrier rate of 1:12. 1,2 The disease is characterized by an inappropriately high level of iron absorption, and excess iron is deposited in the liver, pancreas, heart, and other organs of affected individuals. 3 The phenotypic manifestations of HHC are variable and subject to genetic and environmental influences. Thus, the diagnosis of HHC may be imprecise in a proportion of subjects. Recently, Feder et al. described a strong candidate gene for HHC. 4 Eighty-three percent of their American patients showed a G-to-A base mutation resulting in a substitution of tyrosine for cysteine at position 282 (C282Y) of a novel gene (HFE) located on chromosome 6p and in linkage disequilibrium with HLA-A. Nine patients (5%) were heterozygous for this mutant allele, and 21 (12%) carried only the normal allele. Feder et al. explained the lack of a causative mutation in 12% of their patients by proposing genetic heterogeneity with a second locus unrelated to chromosome 6p. However, because the ultimate test for a candidate gene is the clear segregation of a mutation with the disorder in all patients, particularly in families, we recently studied the concordance of the C282Y mutation with disease status in 26 well-characterized families that included at least 2 affected individuals. 5 The C282Y mutation was identified in 100% of homozygous patients, thus indicating that HFE is the primary locus for HHC in Australia. Virtually identical findings were reported from France. 6 A second H63D mutation was described by Feder et al. 4 The exact significance of this mutation is uncertain because some investigators consider it likely to be a simple polymorphism, whereas others have suggested that it may represent a marker of another mutation. 4 7 These recent findings provide an opportunity to reassess the extent of genotypic and phenotypic expression of HHC in a large series of patients referred to our center for evaluation of iron overload. In addition, these new data provide an opportunity to evaluate current diagnostic criteria for this common disease. Materials and Methods Inclusion Criteria We studied a total of 300 subjects in 109 families referred for diagnosis and management of iron overload. One hundred fifteen subjects met clinical diagnostic criteria for HHC 3 and were previously classified as homozygous affected for the disease. One hundred eighty-five subjects, all siblings or parents of affected individuals, were previously classified as heterozygous on the basis of iron indices and HLA typing, i.e., Abbreviations used in this paper: HHC, hereditary hemochromatosis; HIC, hepatic iron concentration; HII, hepatic iron index by the American Gastroenterological Association /98/$3.00

2 1004 CRAWFORD ET AL. GASTROENTEROLOGY Vol. 114, No. 5 they shared one HLA haplotype in common with an affected proband. No patient had evidence of any other cause for increased iron stores, in particular hemolysis or an iron-loading anemia. DNA was extracted from buffy coats, and the C282Y mutation was analyzed in all subjects using polymerase chain reaction followed by SnaB1 restriction enzyme cleavage and agarose gel electrophoresis as described previously. 5 H63D mutation was analyzed as reported previously. 5 Iron indices were then reviewed in all of the subjects who were shown to be homozygous for the C282Y mutation to determine if they met previously accepted clinical diagnostic criteria for overt (expressed) hemochromatosis, i.e., the presence of at least two of the following: (1) hepatic iron concentration (HIC) of 80 µmol/g dry weight, (2) hepatic iron index (HII) of 2, (3) stainable iron in the liver (grade 3 4), or (4) 4 g of iron removed by quantitative phlebotomy The clinical expression was also assessed in those subjects heterozygous for the C282Y mutation. All subjects were treated in a single institution under the direct supervision of clinicians who were experts in the field of iron metabolism. In contrast to our previous analysis, 5 in the present study not all homozygous subjects were members of a multiplex family with at least 1 other affected member. Assessment of Iron Status and Liver Histology Transferrin saturation and serum ferritin concentration were measured as described previously. 10 Transferrin saturation was performed on morning samples after an overnight fast. HIC was measured by atomic absorption spectrophotometry after acid digestion. 10 The HII was calculated by dividing the HIC (in µmol/g dry wt) by the patient s age in years. 10,11 Morphological evaluation of liver biopsy specimens was performed after H&E staining. Semiquantitative assessment of iron stores was performed using Perls Prussian blue stain and a grading of 0 4 according to the method of Scheuer et al. 12 Statistical Analyses Measurements of transferrin saturation, HIC, and HII were expressed as means SD, and comparisons between groups of subjects were made with Student s t test. Serum ferritin values were expressed as median and range, and comparisons between groups were made after logarithmic transformation to normalize the distribution of data values. Informed Consent The study was approved by the Institute Ethics Committee, and all subjects gave written informed consent, including those who agreed to undergo a liver biopsy. Results Of the 300 subjects studied, 127 were homozygous for the C282Y mutation, 171 were heterozygous for the mutation, and 2 previously thought to be homozygous because of increased iron stores had no mutation (aged 12 and 15 years, respectively). Details of biochemical indices of iron metabolism in homozygotes and heterozygotes are shown in Table 1. Homozygous men had significantly greater serum ferritin concentration than homozygous women. There were strong trends in favor of higher HIC and HII in homozygous men. In the heterozygous population, men also had higher serum ferritin and transferrin saturation than heterozygous women. Homozygous C282Y Subjects Who Did Not Meet Current Clinical Diagnostic Criteria for Homozygous Hemochromatosis We identified 22 subjects (5 men and 17 women) who were homozygous for the C282Y mutation but who did not meet current diagnostic criteria for overt HHC (Table 2). Of the 5 men, 2 had an HII of 2.0 (reflecting partial expression) but did not meet additional diagnostic criteria used for expressing homozygous HHC. In 1 man (case 18), the body iron stores (as suggested by serum ferritin concentration) were too low to justify performing liver biopsy at this stage. One man had an HIC and HII of 23.3 and 0.5 µmol/g, respectively. These latter 2 subjects were clearly nonexpressing in terms of iron accumulation. However, all homozygous men had a transferrin saturation level of 40% (the lowest was 41%). Table 1. Iron Indices in Subjects Homozygous or Heterozygous for the C282Y Mutation n Age ( yr) TS (%) SF ( g/l) HIC ( mol/g ) HII Homozygotes ( ) Men ( ) a b Women ( ) Heterozygotes (6 1605) d d Men c ( ) Women (6 1605) TS, transferrin saturation; SF, serum ferritin. a P , men vs. women. b P 0.10, men vs. women. c P 0.003, men vs. women. d Only 12 heterozygous subjects underwent liver biopsies.

3 May 1998 CLINICAL EXPRESSION OF HEMOCHROMATOSIS 1005 Table 2. Subjects Homozygous for the C282Y Mutation With Incomplete Clinical Expression No. Sex Age ( yr) TS(%) SF( g/l) HIC ( mol/g ) HII Grade of iron Iron removed Comments 1 F Crohn s disease 2 F F F Pregnancies 5 F F F M F Pregnancies 10 M F Blood donor; 4 children 12 F F F M F F Children 18 M F F M F TS, transferrin saturation; SF, serum ferritin;, not performed. Twelve of the 17 women were premenopausal, and 2 of the postmenopausal women had clear reasons for not accumulating iron: 1 with diagnosed inflammatory bowel disease and another had had multiple pregnancies. In 1 woman (case 20), the HIC was 2.0, but no other criteria were met. In 13 women, the serum ferritin concentration was too low to justify performing liver biopsy, and these women were regarded as nonexpressing. Subjects Heterozygous for the C282Y Mutation Who Met Clinical Diagnostic Criteria for Homozygosity Eight patients (all unrelated men) were found to be heterozygous for the C282Y mutation, despite meeting clinical diagnostic criteria for HHC (Table 3). All subjects had a transferrin saturation of 40%, and the range of the serum ferritin concentration was µg/l. The HIC was measured in 7 of these subjects and ranged from 63 to 126 µmol/g. The range of the HII was and was 2.0 in the 5 subjects. Seven of the 8 subjects carried the H63D mutation. However, testing of members of these families identified an additional 7 compound heterozygotes, none of whom met diagnostic criteria for expressing disease. Assessment of Previously Accepted Diagnostic Criteria In no men and in only 4 women homozygous for the C282Y mutation was the serum transferrin saturation 40% (Table 2). Twenty percent of heterozygote subjects had a transferrin saturation of 40%, confirming previous reports of transferrin saturation in such subjects. Because of the widespread use of the HII as a diagnostic test for homozygous HHC, we analyzed the distribution of patients who were homozygous for the C282Y mutation according to this index. All patients with an HII of 4.0 were homozygous for the mutation. In contrast, 7 Table 3. Subjects Heterozygous for C282Y With Clinical Diagnostic Criteria for Homozygous Hemochromatosis No. Sex Age ( yr ) TS(%) SF HIC ( mol/g ) HII Grade of iron Iron removed Alcohol a ( 80 g/day ) H63D 1 M No 2 M NA NA Yes 3 M No 4 M No 5 M No 6 M No 7 M No 8 M NA No TS, transferrin saturation; SF, serum ferritin; NA, not available. a Alcohol intake per day.

4 1006 CRAWFORD ET AL. GASTROENTEROLOGY Vol. 114, No. 5 subjects heterozygous for the C282Y mutation had an index of 2.0 (upper limit, 3.7). Discussion This study analyzes the relation between genotype and phenotypic expression in subjects homozygous or heterozygous for HHC. Of the 127 subjects homozygous for the C282Y mutation in the HFE gene, 82.7% met previously accepted clinical criteria for the disease. In addition, 9 subjects (5.3%) who were heterozygous for the C282Y mutation met clinical diagnostic criteria for expressed disease. Two subjects (aged 12 and 15 years, respectively) previously thought to be homozygous for the disease had no mutation. These 2 children (both without other affected family members) are examples of previously reported juvenile hemochromatosis whose disease would thus appear to be unrelated to the C282Y mutation. 13 Whether other mutations in HFE or mutations in other loci account for this condition is not known. We identified 22 subjects (17 women and 5 men) homozygous for the C282Y mutation who did not meet current diagnostic criteria for HHC and who did not express the disease to any significant extent. The majority of such patients had evidence of pathological blood loss or were women of childbearing age. This group represented 17.3% of the homozygous subjects studied and indicated that 6.6% of the men and 32% of the women homozygous for the C282Y mutation did not express the disease to such an extent as to meet current clinical criteria for overt disease. These findings in a large series of patients referred for diagnosis and management of abnormal iron indices extend results of our previous study, which was confined to multiplex families in whom there were 2 or more affected individuals. 5 That study showed that in the 26 well-characterized families analyzed, all individuals with HHC carried two copies of the C282Y mutation, confirming that HLA-H is the primary locus for this disease. Of particular interest in the present study is the group of patients previously diagnosed as homozygous for the disease who carried only one copy of the C282Y mutation. In each case, the degree of iron overload was modest and reassessment of HLA and family studies, and in some cases retesting a further sample for the mutation, supported their status as heterozygous for HHC. Recently, we showed evidence that heterozygotes differ from the healthy population because the mean transferrin saturation of a large heterozygous population was greater than in a healthy population. 14 Similar results were obtained in a larger study by Bulaj et al. 15 The data in the present report suggest that a small group of heterozygotes can show phenotypic expression to accumulate a modest increase of iron in the liver. Of interest is our finding that 7 of these 8 subjects who met diagnostic criteria also carried the H63D mutation. Although this could support the hypothesis that the H63D mutation is more than a simple mutation polymorphism and can affect disease expression, 4,6,7 our finding of an additional 7 compound heterozygotes in these families who did not meet diagnostic criteria for the disease indicates that H63D is of low penetrance or is linked to HFE modifiers. Although the levels of hepatic iron in this group could be associated with symptoms of lethargy and fatigue, it is unlikely in the absence of other hepatotoxins, such as chronic viral hepatitis or alcohol, that such patients will develop fibrosis and cirrhosis because the rate of iron accumulation is comparatively slow. 10 This is in agreement with the recent study of Bulaj et al. 15 who found that histological abnormalities on the liver biopsy specimen in heterozygous subjects were generally associated with hepatitis, porphyria cutanea tarda, or alcohol abuse. Of the subjects heterozygous for the C282Y mutation with phenotypic expression of iron overload, only 1 drank alcohol to excess. This is consistent with the view that heavy alcohol ingestion does not accentuate iron overload in heterozygous subjects. 16 However, this important question is still unresolved and further more extensive studies are required before this can be clarified. We and others have shown previously that patients homozygous for the ancestral haplotype have more severe disease expression than patients with none or one copy of the ancestral haplotype. 14,17 We hypothesized that the presence of the ancestral haplotype was associated with a unique mutation in the hemochromatosis gene and that patients with this mutation had a more severe phenotype. Several questions are raised and are presently unresolved by the finding of a single mutation in some subjects expressing iron overload. First, why does extensive phenotypic variation exist? Second, why is there concordance of disease expression between siblings independent of exposure to environmental factors that may influence body iron stores? 18 Third, why is there an association between disease severity and the presence of the ancestral haplotype? Whether HFE-like genes exist in the immediate vicinity of HFE and account for the association between the ancestral haplotype and more severe disease expression remains to be determined. Similarly, our data suggest that full clinical expression may be attenuated by nonenvironmental factors. No explanation for incomplete expression could be determined in 8 of the 22 subjects homozygous for C282Y. It is possible that genetic factors are responsible for such reduced clinical expression. This

5 May 1998 CLINICAL EXPRESSION OF HEMOCHROMATOSIS 1007 hypothesis requires extensive analysis of larger numbers of pedigrees of nonexpressing C282Y homozygotes. A further practical issue resulting from the isolation of this novel gene is the clinical application of a DNA-based diagnostic test. HHC is a condition par excellence for genetic testing. The disease has an insidious onset and, if unrecognized, results in hepatic cirrhosis, hepatocellular carcinoma, or life-threatening impairment of other organs, including the heart or pancreas. Early diagnosis and therapeutic intervention by phlebotomy improve symptoms and restore life expectancy to normal. 3,19 The place of a genetic test, in contrast to the phenotypic marker transferrin saturation, in the diagnostic work-up of patients suspected of having HHC has not been studied previously. Assuming that the cost of a genetic test will be comparatively high, analysis of our large number of affected families supports a strategy in men of using a transferrin saturation of 40% as an initial screening test. We found that all men who were homozygous for mutant HFE had a transferrin saturation of 40%, whereas only 20% of patients heterozygous for HFE had a transferrin saturation greater than this value. These results confirm previous large population analyses. 2,20 Therefore, if a male subject is being investigated for hemochromatosis, we suggest that the transferrin saturation should be the initial test. If the transferrin saturation is 40%, the C282Y mutation could then be analyzed. If the patient is homozygous for this mutation, an assessment of iron burden (by serum ferritin) should be undertaken. As discussed above, some heterozygotes for the C282Y mutation and some compound (C282Y/ H63D) heterozygotes may show some degree of iron accumulation, but the available evidence would indicate that these subjects do not develop fibrosis or cirrhosis of the liver. If the clinician is at all suspicious of underlying cirrhosis on the basis of age, clinical features, degree of elevation of serum ferritin, abnormal liver function test results, or the presence of cofactors, a liver biopsy should be performed. Our data 21 suggest that cirrhosis is unlikely if the patient is younger than 30 years of age and if the serum ferritin concentration is 500 µg/l with normal liver function test results. In all other circumstances, liver biopsy should be performed because liver biopsy is the only reliable means of diagnosing hepatic cirrhosis, and the presence of cirrhosis substantially increases the risk of complications, including hepatocellular carcinoma, thus altering the clinical management of the patient. 3,19 A level of 40% for transferrin saturation warranting further investigation is less stringent than previously proposed, but in view of our findings in subjects defined genetically as homozygous or heterozygous, the cost benefits of this approach are likely to be significant. 22,23 However, whether this strategy is appropriate in female subjects in whom the transferrin saturation is lower than in males remains to be determined. References 1. Edwards CQ, Griffen LM, Goldgar D, Drummond C, Skolnick MH, Kushner JP. Prevalence of hemochromatosis among 11,065 presumably healthy blood donors. N Engl J Med 1988;318: Leggett BA, Halliday JW, Brown NN, Bryant S, Powell LW. Prevalence of haemochromatosis amongst asymptomatic Australians. Br J Haematol 1990;74: Powell LW, Jazwinska E, Halliday JW. Primary iron overload. In: Brock JH, Halliday JW, Pippard MJ, Powell LW, eds. Iron metabolism in health and disease. Philadelphia: Saunders, 1994: Feder JN, Gnirke A, Thomas W, Tsuchihashi Z, Ruddy DA, Basava A, Dormishlan F, Domingo R, Ellis MC, Fullan A, Hinton LM, Jones NL, Kimmel BE, Kronmal GS, Lauer P, Lee VK, Loeb DB, Mapa FA, McClelland E, Meyer NC, Mintier GA, Moeller N, Moore T, Morlkang E, Prass CE, Quintana L, Starnes SM, Schatzman RC, Brunke KJ, Drayna DT, Risch NJ, Bacon Br, Wolff RK. A novel MHC class 1 like gene is mutated in patients with hereditary haemochromatosis. Nat Genet 1996;13: Jazwinska EC, Cullen LM, Busfield F, Pyper WR, Webb Si, Powell LW, Morris CP, Walsh TP. Haemochromatosis and HLA-H. Nat Genet 1996;14: Jouanolle AM, Gandon G, Jézéquel P, Blayau M, Campion ML, Yaouanq J, Mosser J, Fergelot P, Chauvel B, Bouric P, Carn G, Andrieux N, Gicquel I, Le Gall J-Y, David V. Haemochromatosis and HLA-H. Nat Genet 1996;14: Beutler E. Genetic irony beyond haemochromatosis: clinical effect of HLA-H mutations. Lancet 1997;349: Summers KM, Tam KS, Halliday JW, Powell LW. HLA determinants in an Australian population of hemochromatosis patients and their families. Am J Hum Genet 1989;45: Bassett ML, Halliday JW, Powell LW. HLA typing in idiopathic hemochromatosis: distinction between homozygotes and heterozygotes with biochemical expression. Hepatology 1981;1: Bassett ML, Halliday JW, Powell LW. Value of hepatic iron measurement in early hemochromatosis and determination of the critical iron level associated with fibrosis. Hepatology 1986;6: Summers KM, Halliday JW, Powell LW. Identification of homozygous hemochromatosis subjects by measurement of hepatic iron index. Hepatology 1990;12: Scheuer PJ, Williams R, Muir AR. Hepatic pathology in relatives of patients with haemochromatosis. J Pathol Bacteriol 1962;84: Perkins KW, McInnes IWS, Blackburn CRB, Beal RW. Idiopathic hemochromatosis in children. Report of a family. Am J Med 1965;39: Crawford DHG, Powell LW, Leggett BA, Francis JS, Fletcher LM, Webb SI, Halliday JW, Jazwinska EC. Evidence that the ancestral haplotype in Australian hemochromatosis patients may be associated with a common mutation in the gene. Am J Hum Genet 1995;57: Bulaj ZJ, Griffen LM, Jorde LB, Edwards CQ, Kushner JP. Clinical and biochemical abnormalities in people heterozygous for hemochromatosis. N Engl J Med 1996; Powell LW. The role of alcoholism in hepatic iron storage disease. Ann N Y Acad Sci 1975;252:

6 1008 CRAWFORD ET AL. GASTROENTEROLOGY Vol. 114, No Piperno A, Arosio C, Fargion S, Roetto A, Nicoli C, Girelli D, Sbaizi L, Gasparini B, Boari G, Sampietro M, Camaschella C. The ancestral hemochromatosis haplotype is associated with a severe phenotype expression in Italian patients. Hepatology 1996; 24: Crawford DHG, Halliday JW, Summers KM, Bourke MJ, Powell LW. Concordance of iron storage in siblings with genetic hemochromatosis: evidence for a predominantly genetic effect on iron storage. Hepatology 1993;17: Niederau C, Fischer R, Purschel A, Stremmel W, Haussinger D, Strohmeyer G. Long-term survival in patients with hereditary hemochromatosis. Gastroenterology 1996;110: McLaren CE, Gordeuk VR, Looker AC, Hasselblad V, Edwards CQ, Griffen LM, Kushner JP, Brittenham GM. Prevalence of heterozygotes for hemochromatosis in the white population of the United States. Blood 1995;86: George DK, Crawford DHG, Powell LW. Liver function tests and liver damage in haemochromatosis (abstr). Gut 1997;40(Suppl 1):A Baer DM, James MD, Simons JL, Staples RL, Rumore GJ, Morton CJ. Hemochromatosis screening in asymptomatic ambulatory men 30 years of age and older. Am J Med 1995;98: Bassett ML, Leggett BA, Halliday JW, Webb SI, Powell LW. Analysis of the cost of population screening for haemochromatosis using biochemical and genetic markers. J Hepatol 1997;27: Received July 8, Accepted January 13, Address requests for reprints to: Lawrie W. Powell, M.D., Ph.D., Queensland Institute of Medical Research, 300 Herston Road, Brisbane, Australia Fax: (61) Dr. Crawford s current address is: Department of Gastroenterology and Hepatology, Princess Alexandra Hospital, Brisbane, Australia.