Materials and Methods. Results

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1 GASTROENTEROLOGY 2002;122: A Population-Based Study of the Biochemical and Clinical Expression of the H63D Hemochromatosis Mutation PETER A. GOCHEE,* LAWRIE W. POWELL,* DIGBY J. CULLEN,, DESIRÉE DU SART, ENRICO ROSSI, and JOHN K. OLYNYK,# *Queensland Institute of Medical Research and The University of Queensland, Brisbane; Department of Gastroenterology, Fremantle Hospital, Fremantle, Western Australia; Busselton Population Medical Research Foundation, Perth; Murdoch Children s Research Institute, Melbourne; Pathcentre, Queen Elizabeth II Medical Centre, Nedlands; # Department of Medicine, The University of Western Australia, Western Australia, Australia Background & Aims: Two major mutations are defined within the hemochromatosis gene, HFE. Although the effects of the C282Y mutation have been well characterized, the effects of the H63D mutation remain unclear. We accessed a well-defined population in Busselton, Australia, and determined the frequency of the H63D mutation and its influence on total body iron stores. Methods: Serum transferrin saturation and ferritin levels were correlated with the H63D mutation in 2531 unrelated white subjects who did not possess the C282Y mutation. Results: Sixty-two subjects (2.1%) were homozygous for the H63D mutation, 711 (23.6%) were heterozygous, and 1758 (58.4%) were wild-type for the H63D mutation. Serum transferrin saturation was significantly increased in male and female H63D homozygotes and heterozygotes compared with wild-types. Serum ferritin levels within each gender were not influenced by H63D genotypes. Elevated transferrin saturation >45% was observed in a greater proportion of male H63D carriers than male wild-types. Male H63D homozygotes (9%) and heterozygotes (3%) were more likely to have both elevated transferrin saturation and elevated ferritin >300 ng/ml than male wild-types (0.7%). Homozygosity for H63D was not associated with the development of clinically significant iron overload. Conclusions: Presence of the H63D mutation results in a significant increase in serum transferrin saturation but does not result in significant iron overload. In the absence of the C282Y mutation, the H63D mutation is not clinically significant. Hereditary hemochromatosis is the most common inborn error of metabolism affecting individuals of northern European descent. 1 3 The progressive accumulation and deposition of iron into parenchymal tissues can lead to such diseases as hepatic cirrhosis, cardiomyopathy, and diabetes mellitus. Although the consequences of undetected hemochromatosis can be devastating, if identified early in its progression treatment by phlebotomy is simple and effective, and treated individuals can achieve a normal life expectancy. 4,5 Unfortunately, because of the insidious onset, wide variability in expression, and high frequency of nonspecific findings, diagnosis of hereditary hemochromatosis requires a high index of clinical suspicion and careful clinicopathological correlation. Recently, a major histocompatibility complex class I-like gene, termed HFE, has been described and 2 mutations have been associated with 60% 90% of all cases of hereditary hemochromatosis. 6 The first and major mutation, observed in up to 10% of alleles in the general population, results in a cysteine to tyrosine substitution at amino acid 282 and is termed C282Y. The second and more minor mutation, observed in up to 30% of alleles in the general population, results in a histidine to aspartic acid substitution at amino acid 63 and is termed H63D. 7,8 The high correlation of HFE to the development of hemochromatosis has persuaded many to consider HFE as a model for population-based genetic screening, as knowledge of an individual s HFE genotype may allow the rapid identification of individuals predisposed to the development of many preventable complications of hemochromatosis. However, recent expert panels have concluded that more in-depth studies are needed regarding the prevalence, penetrance, and natural history of both HFE mutations before population-based genetic screening can be advocated. 9,10 The decision on whether to screen for hemochromatosis by biochemical or genetic methods remains an active area of interest and debate We have previously shown the effects the C282Y mutation on total body iron stores in a population-based study. 14 Our results have shown that in individuals homozygous for the C282Y mutation, 50% have clinical Abbreviations used in this paper: PCR, polymerase chain reaction by the American Gastroenterological Association /02/$35.00 doi: /gast

2 March 2002 EXPRESSION OF THE HEMOCHROMATOSIS H63D MUTATION 647 evidence of hemochromatosis and an additional 25% show biochemical evidence of iron overload. To date, a population-based study to determine the effects of the H63D mutation, in the absence of the C282Y mutation, on total body iron stores remains to be assessed. In this study, we accessed a well-defined, non blood bank population of northern European descent and, in the absence of the C282Y mutation, determined the frequency of the H63D mutation and its influence on total body iron stores to ascertain its importance in population-based genetic screening for iron overload. Materials and Methods Patients Busselton is a town in the southwest of Western Australia with a population predominantly of Anglo-Celtic descent that has been prospectively studied since 1966 and is in many respects similar to the Framingham population. The most recent follow-up study of this population was in During the 1994 population-wide assessment, all subjects were clinically evaluated and whole blood and serum samples were obtained from approximately 5000 white subjects. All blood tests were performed in the fasting state. We randomly selected 3011 nonrelated subjects aged 20 to 79 years, regardless of age strata, and from this group 2571 individuals were identified who were negative for the C282Y mutation. 14 Clinical, biochemical, and genotypic information was collected on all subjects. Iron studies were defined as significantly elevated when either the serum transferrin saturation was 45% and ferritin level 300 g/l or the ferritin-alone was 1000 g/l. These subjects were reviewed by one of the investigators and the serum iron studies repeated while fasting. Permission was granted for this study by the Busselton Population Medical Research Foundation and The Committee for Human Rights at the University of Western Australia. Measurement of Serum Indices Serum iron levels were measured using a standard colorimetric method, and transferrin concentration was determined by rate immunoturbidimetry on a 917 analyzer (Hitachi, Tokyo, Japan). Serum transferrin saturation was calculated from these results as follows: transferrin saturation (Serum iron/2 transferrin) 100. Serum ferritin levels were measured by chemiluminescence immunoassay on an ACS-180 analyzer (Chiron, Norwood, MA). Determination of the H63D Mutation in Hemochromatosis Gene Genomic DNA was extracted from 2571 Guthrie cards by standard methods. Duplex PCR using allele-specific primers for the normal (5 - CAG CTG TTC GTG TTC TAT TAT C-3 ) and mutant alleles (5 - GCG GGC AGG GCG GCG GGG GCG GGG CAG CTG TTC GTG TTC TAT TAT G-3 ) for the H63D mutation and a common reverse primer (5 -CAG TGA ACA TGT GAT CCC ACC CTT TCA G-3 ) was performed on a BioRad I-Cycler Real-Time PCR machine. Sybr Green I fluorescence was incorporated into the PCR product during PCR to allow detection. The primers were designed to produce different melting temperatures for each of the normal and mutant alleles. A GC-rich tag was added to the mutant primer, which increased the melt temperature for the mutant PCR product by 5 C. Melting-curve analysis was performed at the end of the PCR to allow genotyping. As a quality-control measure, 10% of samples were selected randomly and verified by a standard restriction digest and gel electrophoresis assay. 15 Data Presentation All data are presented as the mean SEM unless otherwise specified. Comparisons between groups were made using the Mann Whitney U test. All P values are 2-tailed. Results Patient Demographics Demographic information on the age and sex distribution for study subjects is shown in Table 1. Of 2571 individuals initially identified as wild-type for the C282Y mutation, H63D genotype was determined for 2531 and unable to be determined for the remaining 40. There were 1255 females and 1276 males uniformly distributed between the ages of 20 to 79 years. Distribution of H63D Genotypes H63D genotype frequencies are detailed in Tables 2, 3, and 4. There were 62 (2.1%) H63D homozygotes, 711 (23.6%) H63D heterozygotes, and 1758 (58.4%) individuals who were HFE wild-type, which, when combined with our previous data, 14 yield H63D and HFE wild-type frequencies of 15.1% and 77.2%, respectively. The H63D allelic frequencies were not significantly different between age groups, within each gender. Table 1. Age and Sex of the 2531 Study Subjects Age Male (n 1276) Female (n 1255) Median (yr) Age group yr yr yr yr yr yr NOTE. All subjects were wild-type for the C282Y mutation in the HFE gene.

3 648 GOCHEE ET AL. GASTROENTEROLOGY Vol. 122, No. 3 Table 2. Transferrin Saturation (TRS) and Serum Ferritin Levels (Ferritin) in Male and Female Subjects Who Were Wild-Type for the C282Y Mutation Male Female TRS Ferritin TRS Ferritin H63D/H63D a a Range, (n 33) Range, (n 33) Range, (n 29) Range, (n 29) H63D/H63D a 94 4 a Range, 8 74 (n 356) Range, (n 356) Range, 5 60 (n 355) Range, (n 355) Wild-type/wild-type b a,b 96 4 a Range, (n 887) Range, (n 887) Range, 3 61 (n 871) Range, (n 871) NOTE. Subjects have been analyzed according to the presence of H63D mutations in the HFE gene. a P 0.01 vs. male group of same H63D genotype. b P 0.01 vs. H63D/H63D and H63D/wild-type of same column. Correlation of Serum Transferrin Saturation and Ferritin Levels to H63D Genotype The mean transferrin saturation and serum ferritin levels for both male and female subjects are detailed in Table 2. The serum transferrin saturation was significantly increased in male and female subjects who were homozygous (35.1% 1.9% and 29.6% 1.6%, respectively) or heterozygous (31.5% 0.5% and 26.7% 0.5%, respectively) for the H63D mutation over male and female subjects wild-type for both the H63D and the C282Y mutation (28.4% 0.3% and 25.0% 0.3%, respectively) (P 0.01). Male H63D homozygotes, heterozygotes, and wild-types had significantly increased serum transferrin saturation over females with the same genotype (P 0.01). The serum ferritin levels were increased in male vs. female H63D homozygotes ( and g/l, respectively), heterozygotes ( and /L, respectively), and H63D wild-types (213 7 and 96 4 /L, respectively) (P 0.01). No significant difference in serum ferritin levels Table 3. Prevalence of Elevated Transferrin Saturation (TRS 45%) and Serum Ferritin Level ( 300 ng/ ml) in Male Subjects Who Were Wild-type for the C282Y Mutation According to the Presence of H63D Mutations in the HFE Gene Elevated TRS Elevated ferritin Both elevated H63D/H63D (n 33) 5 (15%) 12 (36%) 3 (9%) H63D/wild-type (n 356) 42 (12%) 82 (23%) 12 (3%) Wild-type/wild-type (n 887) 43 (5%) a 177 (20%) 6 (0.7%) a Total (n 1276) 90 (7%) 271 (21%) 21 (2%) a P 0.01 vs. H63D/H63D and H63D/wild-type of same column ( 2 test). was observed between H63D genotypes within each sex in the absence of the C282Y mutation. A greater proportion of male H63D homozygotes and heterozygotes (15% and 12%, respectively) had elevated serum transferrin saturation levels 45% than male HFE wild-types (5%) (P 0.01, Table 3). A significant difference was not observed in the proportion of male H63D homozygotes (36%) and heterozygotes (23%) with elevated serum ferritin 300 g/l vs. male HFE wild-types (20%), though a trend toward significance was observed (P 0.09). A greater proportion of male H63D homozygotes and heterozygotes (9% and 3%, respectively) had both elevated serum transferrin saturation and elevated serum ferritin than male wild-type HFE (0.7%) (P 0.01). No significant difference was observed between the proportion of female H63D homozygotes, heterozygotes, and HFE wild-types, and elevated serum transferrin saturation or elevated serum ferritin (Table 4). No confounding variable was significantly different between H63D homozygotes or heterozygotes and wild-type HFE (Table 5). Table 4. Prevalence of Elevated Transferrin Saturation (TRS 45%) and Serum Ferritin Level ( 300 ng/ ml) in Female Subjects Who Were Wild-type for the C282Y Mutation According to the Presence of H63D Mutations in the HFE Gene Elevated TRS Elevated ferritin Both elevated H63D/H63D (n 29) 2 (7%) 4 (13%) 0 (0%) H63D/wild-type (n 355) 11 (3%) 7 (2%) 0 (0%) Wild-type/wild-type (n 871) 19 (2%) 39 (4%) 2 (0.2%) Total (n 1255) 32 (3%) 50 (4%) 2 (0.1%)

4 March 2002 EXPRESSION OF THE HEMOCHROMATOSIS H63D MUTATION 649 Table 5. Prevalence of Confounding Variables Observed in H63D Homozygotes and Heterozygotes and HFE Wild-Type Subjects Variable Wild-type/ wild-type H63D/ wild-type H63D/ H63D Age (yr) Gender Male (%) Female (%) Body mass index (kg/m 2 ) Diabetes treatment (%) Cholesterol (mmol/l) HDL cholesterol (mmol/l) Smoking Non (%) Ex (%) Light (%) Heavy (%) Drinking Never (%) Ex (%) Light (%) Medium (%) Heavy (%) Hemoglobin (g/100 ml) NOTE. Cigarette smoking was classified as light/moderate ( 15 cigarettes per day) and heavy ( 15 cigarettes per day). Alcohol consumption was classified as light ( 140 g alcohol per week), moderate ( g alcohol per week), or heavy intake of alcohol ( 420 g alcohol per week). Plus minus values are means SD. Causes of Significantly Elevated Iron Indices in C282Y Noncarriers Elevation of both transferrin saturation levels 45% and serum ferritin 300 g/l on initial biochemical screening was observed in 23 subjects. Of these, 6 subjects had persistent and significantly elevated serum iron studies with repeat biochemical screening (2 H63D heterozygotes and 4 wild-type). Clinical evaluation and assessment was conducted on these 6 subjects, and where appropriate, liver biopsy was recommended. Following clinical assessment, these subjects comprised: one 66- year-old female with lymphoma; 2 middle-aged male subjects with alcohol-related liver disease; 1 subject with mild elevation of serum -glutamyl transpeptidase and liver biopsy showing grade 2 hepatic iron deposition, hepatic iron concentration 49 g/g dry weight and hepatic iron index 0.9; 1 male subject aged 77 years who refused biopsy and was offered therapeutic phlebotomy; and 1 subject with no obvious cause of elevated iron indices elucidated at clinical assessment and who is currently being followed-up with annual iron studies. Sensitivity and Specificity of H63D Screening for Iron Overload Initial biochemical screening identified 15 of 773 H63D allele carriers and 8 of 1758 H63D allele noncarriers with elevated serum transferrin saturation and serum ferritin levels (Tables 3 and 4). These data provide a sensitivity of , a specificity of , a positive predictive value of and a negative predictive value of for determining iron overload by screening for the presence of at least 1 H63D allele in the absence of the C282Y allele. Discussion This study shows in a true population-based sample the effects of the H63D mutation on serum transferrin saturation and ferritin levels, in the absence of the C282Y mutation. We have shown that individuals who possess the H63D mutation have increased serum transferrin saturation compared with individuals of the same gender who are wild-type for both mutations in HFE. Male H63D carriers are more likely to have transferrin saturation 45% vs. male HFE wild-types. Serum ferritin levels were not significantly different between H63D genotypes within each gender, though ferritin levels were higher in males compared with females of the same H63D genotype. Male H63D homozygotes and heterozygotes were more likely to have both elevated transferrin saturation 45% combined with elevated serum ferritin 300 g/l than male HFE wild-types and a trend toward significance was observed in the proportion of male H63D homozygotes and heterozygotes with elevated serum ferritin 300 /L. Moreover, though our results show a high specificity in genetic screening for iron overload by the presence of the H63D allele in C282Y allele noncarriers, this must be tempered with an extremely low sensitivity and more than 98% of identified H63D carriers will not have biochemical indication of increased iron stores, defined as both elevated serum transferrin saturation and elevated serum ferritin. The results of this study extend previous work by analyzing the effect of H63D, in the absence of C282Y, on transferrin saturation and serum ferritin in a non blood bank cohort selected randomly from a well-characterized population. The results are in agreement with previous reports of the frequency of H63D mutations. In a study of 1653 individuals in the workplace, McDonnell et al. 16 report 3.5% of subjects as H63D homozygous and 23.9% as heterozygous. Whitefield et al. 17 reported an H63D allelic frequency of 15.7% and report a significant increase in serum transferrin saturation in H63D carriers, both male and female, and no differences in the

5 650 GOCHEE ET AL. GASTROENTEROLOGY Vol. 122, No. 3 mean serum ferritin between H63D genotypes, irrespective of gender. Beutler et al. 18 report increased serum transferrin saturation and serum ferritin in H63D carriers in an assessment of 10,198 individuals from a health appraisal clinic, although the increase did not appear to be clinically relevant. Additionally, Steinberg et al. 8 reported H63D homozygous and heterozygous weighted prevalence estimates of 2.15% and 23.55%, respectively. However, H63D genotypes were not correlated with phenotypic expression in this study. Initially considered an HFE polymorphism, the H63D mutation has subsequently been reported to increase the relative risk for development of iron overload and predispose to hemochromatosis. 19,20 Individuals who compound heterozygous for H63D and C282Y are at an increased risk of iron overload, with an estimated clinical penetrance of 1% 2%. 1 Homozygosity for H63D has been reported to increase the relative risk for phenotypic expression of hemochromatosis by as high as 9-fold over HFE wild-type subjects. 16 However, there is a high degree of variability in expression among H63D homozygotes and compound heterozygotes, and it has been postulated that additional factors may influence phenotypic expression. Such factors may include alcoholism, viral hepatitis, and other genetic factors, although many of these remain to be elucidated. 17,21,22 Indeed, a recent study by Aguilar-Martinez et al. 22 found no association between iron overload in H63D homozygosity and an influence of other HFE related mutations. Our results confirm and extend the concept that the H63D mutation in isolation is insufficient in itself to cause clinically significant iron overload, and that the presence of an additional modifying factor may be necessary for the expression of overt iron overload. Hereditary hemochromatosis meets nearly all of the World Health Organization s criteria for populationbased testing. 10,23 The disease poses an important health problem that has cost-effective treatment readily available for individuals recognized early in its progression. Both phenotypic and genetic tests exist for detection of hemochromatosis. Phenotypic screening is based on measurements of serum transferrin saturation and serum ferritin levels, although recent evidence suggests unsaturated iron binding capacity may be an additional lowcost alternative for determining iron status. 24,25 Screening via serum transferrin saturation generally requires 2 separate tests, an initial screening and a second fasting re-test for individuals with an elevated initial test. Unfortunately, up to 20% of individuals with elevated initial transferrin saturation do not return for a second test, thereby potentially missing the diagnosis of iron overload in a significant proportion of expressing individuals. 26 Genetic testing for hereditary hemochromatosis focuses on the detection of mutations in HFE. An obvious advantage of genetic testing is the detection of individuals presently expressing iron overload, as well as individuals who may present later in life. Although genetic tests will identify all C282Y homozygotes, as well as other HFE genotypes, several problems arise with its advocacy. Not all individuals identified as C282Y homozygotes, or other HFE genotypes associated with an increased risk of hereditary hemochromatosis, will develop overt iron overload. Indeed, recent evidence indicates that among C282Y homozygotes, 50% have clinical evidence of hemochromatosis, and an additional 25% express biochemical evidence of iron loading. 4,14 Additionally, in certain populations a significant proportion of hereditary hemochromatosis is associated with other factors, and testing exclusively for mutations in HFE would not identify these affected individuals. 27 However, cost-benefit analysis indicates that genetic screening for hereditary hemochromatosis based on HFE genotype is cost-effective, even if only 20% of identified individuals would have developed life-threatening symptoms, and that genetic screening can be more cost-effective than phenotypic screening, if the cost of the genetic test is less than $ Although questions regarding the prevalence and penetrance of HFE genotypes are beginning to be answered, additional issues remain. Psychosocial aspects of genetic screening need to be addressed, as diagnosis based on genetic testing raises many concerns, such as stigmatization, insurance and employment discrimination, and diminished self-worth. Indeed, anecdotal evidence of insurance and employment discrimination has been reported. 29 As physicians, scientists and policy makers, it is imperative that we consider all aspects of populationbased genetic screening and not focus solely on the biological ramifications of future screening decisions. The results of this study provide support that while the presence of H63D influences the mean serum transferrin saturation level, these effects are not clinically significant. In screening for iron overload by genetic methods, initial detection of C282Y is warranted before pursuing the presence of H63D. References 1. Bacon BR, Powell LW, Adams PC, Kresina TF, Hoofnagle JH. Molecular medicine and hemochromatosis: at the crossroads. Gastroenterology 1999;116: Powell LW, Subramaniam VN, Yapp TR. Haemochromatosis in the new millennium. J Hepatol 2000;32: Bacon BR. Hemochromatosis: diagnosis and management. Gastroenterology 2001;120:

6 March 2002 EXPRESSION OF THE HEMOCHROMATOSIS H63D MUTATION Olynyk JK. Hereditary haemochromatosis: diagnosis and management in the gene era. Liver 1999;19: Niederau C, Erhardt A, Haussinger D, Strohmeyer G. Haemochromatosis and the liver. J Hepatol 1999;30: Feder JN, Gnirke A, Thomas W, Tsuchihashi Z, Ruddy DA, Basava A, Dormishian F, Domingo R Jr, 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, Morikang E, Wolff RK. A novel MHC class I-like gene is mutated in patients with hereditary haemochromatosis. Nat Genet 1996; 13: Merryweather-Clarke AT, Pointon JJ, Shearman JD, Robson KJ. Global prevalence of putative haemochromatosis mutations. J Med Genet 1997;34: Steinberg KK, Cogswell ME, Chang JC, Caudill SP, McQuillan GM, Bowman BA, Grummer-Strawn LM, Sampson EJ, Khoury MJ, Gallagher ML. 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N Engl J Med 1999; 341: Burt MJ, Upton JD, Morison IM, Chapman BA, Faed JM, George PM. Molecular analysis of HLA-H gene mutations in New Zealand patients with haemochromatosis. N Z Med J 1997;110: McDonnell SM, Hover A, Gloe D, Ou CY, Cogswell ME, Grummer- Strawn L. Population-based screening for hemochromatosis using phenotypic and DNA testing among employees of health maintenance organizations in Springfield, Missouri. Am J Med 1999;107: Whitfield JB, Cullen LM, Jazwinska EC, Powell LW, Heath AC, Zhu G, Duffy DL, Martin NG. Effects of HFE C282Y and H63D polymorphisms and polygenic background on iron stores in a large community sample of twins. Am J Hum Genet 2000;66: Beutler E, Felitti V, Gelbart T, Ho N. The effect of HFE genotypes on measurements of iron overload in patients attending a health appraisal clinic. Ann Intern Med 2000;133: Beutler E. Genetic irony beyond haemochromatosis: clinical effects of HLA-H mutations. Lancet 1997;349: Risch N. Haemochromatosis, HFE and genetic complexity. Nat Genet 1997;17: Moirand R, Jouanolle AM, Brissot P, Le Gall JY, David V, Deugnier Y. Phenotypic expression of HFE mutations: a French study of 1110 unrelated iron-overloaded patients and relatives. Gastroenterology 1999;116: Aguilar-Martinez P, Bismuth M, Picot MC, Thelcide C, Pageaux GP, Blanc F, Blanc P, Schved JF, Larrey D. Variable phenotypic presentation of iron overload in H63D homozygotes: are genetic modifiers the cause? Gut 2001;48: Wilson JMG, Junger G. The principles and practice of screening for disease. Public Heath Paper 34. Geneva, Switzerland: World Health Organization, Adams PC, Kertesz AE, McLaren CE, Barr R, Bamford A, Chakrabarti S. Population screening for hemochromatosis: a comparison of unbound iron-binding capacity, transferrin saturation, and C282Y genotyping in 5,211 voluntary blood donors. Hepatology 2000;31: Hickman PE, Hourigan LF, Powell LW, Cordingley F, Dimeski G, Ormiston B, Shaw J, Ferguson W, Johnson M, Ascough J, Mc- Donell K, Pink A, Crawford DH. Automated measurement of unsaturated iron binding capacity is an effective screening strategy for C282Y homozygous haemochromatosis. Gut 2000;46: McDonnell SM, Phatak PD, Felitti V, Hover A, McLaren GD. Screening for hemochromatosis in primary care settings. Ann Intern Med 1998;129: Pietrangelo A, Montosi G, Totaro A, Garuti C, Conte D, Cassanelli S, Fraquelli M, Sardini C, Vasta F, Gasparini P. Hereditary hemochromatosis in adults without pathogenic mutations in the hemochromatosis gene. N Engl J Med 1999;341: Adams PC, Valberg LS. Screening blood donors for hereditary hemochromatosis: decision analysis model comparing genotyping to phenotyping. Am J Gastroenterol 1999;94: Alper JS, Geller LN, Barash CI, Billings PR, Laden V, Natowicz MR. Genetic discrimination and screening for hemochromatosis. J Public Health Policy 1994;15: Received August 21, Accepted November 21, Address requests for reprints to: John Olynyk, M.D., University Department of Medicine, P.O. Box 480, Fremantle 6959, Western Australia, Australia. jolynyk@cyllene.uwa.edu.au; fax: (61) Supported in part by a Healthways Health Promotion Grant, Western Australia; the Great Wine Estates of Western Australia; and Perpetual Trustees.