Common heterozygous hemochromatosis gene mutations are risk factors for inflammation and fibrosis in chronic hepatitis C.

BACKGROUND: Chronic hepatitis C is frequently associated with increased hepatic iron stores. It remains controversial whether heterozygous mutations of hemochromatosis genes affect fibrosis progression. Therefore our aim was to assess associations between HFE mutations and hepatic inflammation and stage of fibrosis in German hepatitis C patients. METHODS: Liver biopsies from 166 patients were scored for inflammatory activity (A0-4) and hepatic fibrosis (F0-4). Gene mutations were determined by LightCycler, restriction fragment length polymorphism analysis, or direct sequencing. RESULTS: The frequencies of common HFE mutations C282Y and H63D are 4.2% and 21.3%, whereas the recently described S65C substitution and the Y250X mutation in the transferrin receptor 2 gene are very rare. In regression analysis, heterozygous carriers of C282Y or H63D mutations display significantly (P < 0.05) higher inflammatory activities and more advanced fibrosis than patients without mutations. For C282Y heterozygous patients, the odds ratios for marked inflammatory activity (A2-4) and advanced liver fibrosis or cirrhosis (F2-4) are 4.9 and 4.6, respectively, compared with patients carrying homozygous wild-type alleles. C282Y mutations are associated with significantly (P < 0.05) increased serum iron and aminotransferase levels, whereas H63D heterozygotes display higher transferrin saturation, serum iron, and ferritin concentrations compared to wild-type (P < 0.01). CONCLUSIONS: Common heterozygous hemochromatosis mutations are associated with higher grades of inflammation and more severe hepatic fibrosis. Our findings support a role of HFE mutations as primary risk factors for fibrogenesis and disease progression in chronic hepatitis C.     

Mutations of the HFE gene in patients with hepatocellular carcinoma

OBJECTIVE: Hepatocellular carcinoma (HCC) is a late consequence of severe liver disease. Patients with genetic hemochromatosis may be at risk for HCC, but limited information is available on the relationship of HCC and heterozygosity for the HFE gene mutations. METHODS: HFE mutations (C282Y and H63D) were assessed in 162 consecutive patients (131 men/31 women) with HCC. A total of 159 patients had cirrhosis. The most common etiologies of cirrhosis were chronic viral hepatitis (hepatitis C 39%, hepatitis B 9%) and alcoholic liver disease (36%). RESULTS: Five patients were C282Y homozygotes, four C282Y/H63D compound heterozygotes, and three H63D homozygotes. The C282Y and H63D allele frequencies in HCC were 8.3 (95% confidence limit = 5.3-11.3) and 11.1 (7.8-14.6), respectively, and not different from previously published data in healthy subjects or patients with chronic hepatitis C in Austria. Furthermore, there was no difference in the age at diagnosis in patients with or without HFE gene mutations. C282Y homozygotes had a 19-fold increased risk to develop HCC. In contrast, all other HFE allele constellations were not associated with such a risk. CONCLUSIONS: Except for C282Y homozygotes, HFE gene mutations do not increase the risk to develop HCC in patients with cirrhosis.     

HFE haemochromatosis gene mutations in liver transplant patients

BACKGROUND: The majority of patients with inherited haemochromatosis carry two mutant alleles of the recently discovered HFE gene. Individuals heterozygous for the HFE mutation could be predisposed to end-stage liver disease due to other causes. METHODS: The frequencies of the HFE gene mutations C282Y and H63D were determined in DNA samples obtained from 189 liver transplant patients and 225 healthy Finnish blood donors. RESULTS: 5% of the 189 liver transplant recipients were heterozygotes and 0.5% homozygotes for the C282Y mutation, while 16% were heterozygotes and 0.5% homozygotes for the H63D mutation. These figures were not increased in comparison to controls, of whom 11% were C282Y heterozygotes, 16% H63D heterozygotes and 0.9% H63D homozygotes. Among recipients with acute non-A-E hepatitis (n = 31), the frequency of the H63D allele was higher than in controls (21% versus 9.1%, P < 0.01). Perls' stain for iron in explanted liver specimens was positive in 28% of recipients with alcoholic cirrhosis, 26% of patients with acute non-A-E hepatitis and 14% in the rest of the recipients. The HFE genotypes did not correlate with the iron status. CONCLUSION: Individuals heterozygous for either the C282Y or H63D mutation of the HFE gene are not at increased risk of developing chronic end-stage liver disease. However, subjects heterozygous for the H63D mutation may have an increased risk to develop fulminant non-A-E hepatitis.     

Prevalence and clinical implications of HFE gene mutations (C282Y and H63D) in patients with chronic hepatitis B and C in Taiwan.

BACKGROUND/AIMS: The implication of hemochromatosis (HFE) gene mutations in chronic viral hepatitis remains controversial. We therefore studied the prevalence of HFE mutations and their impact on the progression of chronic viral hepatitis in Taiwan. PATIENTS & METHODS: H63D and C282Y mutations were screened by using polymerase chain reaction followed by restriction fragment length polymorphism in 152 chronic hepatitis B patients with various stages of liver disease, 87 chronic hepatitis C patients with various stages of liver disease, and 49 healthy controls. The distribution of each allele frequency was then compared among different groups of patients and in various stages of liver disease. RESULTS: All three groups of patients were C282Y wild type and the majority of H63D mutations were heterozygotes. Although statistically not significant, allele frequencies of H63D mutation in hepatitis B-related liver cirrhosis (6%) and hepatitis C-related liver cirrhosis (9.1%) were higher than those in healthy control (2%). After adjustment for age and sex, hepatitis B patients with H63D heterozygosity had a higher likelihood of cirrhosis than those with H63D wild type (odds ratios (OR): 3.2, confidence interval (CI): 0.49-20.5, P = 0.22). Similarly, hepatitis C patients with H63D homozygosity had a higher likelihood of cirrhosis compared with those with H63D wild type (OR: 2.35, CI : 0.19-28.5, P = 0.52). CONCLUSIONS: Almost all Taiwanese are C282Y wild type. H63D heterozygote and homozygote, occurring in less than 5% of the subjects, tended to be associated with the development of liver cirrhosis, irrespective of viral etiology. Screening for H63D mutation might be considered in patients with chronic viral hepatitis in Taiwan.     

HFE Haemochromatosis Gene Mutations in Liver Transplant Patients

Background: The majority of patients with inherited haemochromatosis carry two mutant alleles of the recently discovered HFE gene. Individuals heterozygous for the HFE mutation could be predisposed to end-stage liver disease due to other causes. Methods: The frequencies of the HFE gene mutations C282Y and H63D were determined in DNA samples obtained from 189 liver transplant patients and 225 healthy Finnish blood donors. Results: 5% of the 189 liver transplant recipients were heterozygotes and 0.5% homozygotes for the C282Y mutation, while 16% were heterozygotes and 0.5% homozygotes for the H63D mutation. These figures were not increased in comparison to controls, of whom 11% were C282Y heterozygotes, 16% H63D heterozygotes and 0.9% H63D homozygotes. Among recipients with acute non-A-E hepatitis (n = 31), the frequency of the H63D allele was higher than in controls (21% versus 9.1%, P &lt; 0.01). Perls' stain for iron in explanted liver specimens was positive in 28% of recipients with alcoholic cirrhosis, 26% of patients with acute non-A-E hepatitis and 14% in the rest of the recipients. The HFE genotypes did not correlate with the iron status. Conclusion: Individuals heterozygous for either the C282Y or H63D mutation of the HFE gene are not at increased risk of developing chronic endstage liver disease. However, subjects heterozygous for the H63D mutation may have an increased risk to develop fulminant non-A-E hepatitis.     

HFE gene mutations, hepatic iron content, and histological severity in hepatitis C virus-induced chronic hepatitis]

OBJECTIVE: To study whether any relationship exists between the C282Y and H63D mutations of the HFE gene, iron liver content, and the severity of histological damage in patients with hepatitis C virus (HCV)-induced chronic hepatitis. MATERIAL AND METHODS: In 72 patients diagnosed with HCV-chronic infection, na?ve for antiviral therapy, and undergoing liver biopsy, the Knodell index was established, a morphometric evaluation of hepatic hemosiderin deposits was performed by using a semiautomatic method of image analysis, and mutations of the HFE gene were identified through a polymerase chain reaction on leukocyte genomic DNA by using specific restriction enzymes. The control group for the distribution of HFE genetic variants was composed of 181 healthy individuals with the same ethnic and geographical (white Spaniards) origin. RESULTS: (Cases/controls): 1. Genotype distribution: a) mutation C282Y: no homozygotes, 6/23 heterozygotes, 66/158 without the mutation (not significant, n.s.); b) mutation H63D: 2/5 homozygotes, 26/52 heterozygotes, 44/124 without the mutation (n.s.). compound heterozygotes 2/6. 2. Allele frequencies: a) mutation C282Y: 0.042/0.064 (n.s.); b) mutation H63D: 0.208/0.171 (n.s.). Four C282Y heterozygous patients had stainable liver iron (p=0.015 vs patients without mutations). Sixty-six patients were not carriers of the C282Y mutation; among them, 26.9% of 26 carriers and 15% of 40 non-carriers of the H63D mutation had liver stainable iron (n.s.). Knodell index score, gender, age at diagnosis, mode of transmission, and serum and liver iron values were not related to the HFE genotype. CONCLUSIONS: our results suggest that the C282Y mutation, but not the H63D mutation, of the HFE gene is frequently associated with stainable iron in the liver in HCV-related chronic hepatitis. The HFE genotype is not related to the histological severity of the disease.     

Could iron deposits in hepatocytes serve as a prognostic marker of HFE gene mutations?

BACKGROUND/AIMS: The diagnosis of hereditary hemochromatosis (HH) is based on qualitative measurement of tissue iron concentration and genetic tests. The aim of this study was to evaluate the correlation between the presence of iron deposits in the liver and the HFE gene mutations in patients with chronic liver diseases (CLD). METHODOLOGY: The 182 patients, age range 18-71 years, were hospitalized in Gdansk because of CLD. The C282Y, H63D and S65C HFE mutations were screened by PCR-RFLP analysis. Liver function tests, serological examinations for viral hepatitis, serum iron and ferritin concentration and semiquantitative assessment of liver iron were done in all subjects. Patients were divided into Group A without iron deposits in the liver, and Group B with deposits. The most frequent etiology of CLD was chronic hepatitis C. RESULTS: Biochemical parameters indicating iron storage and ALT activity were significantly higher in Group B. Either typical for diagnosis HH homozygotes C282Y/C282Y and combined heterozygotes C282Y/H63D or carriers of other HFE gene mutations were found significantly more frequently in Group B. CONCLUSIONS: The finding of iron deposits in routinely obtained liver specimen correlates with occurrence of the different HFE gene mutations.     

Hemochromatosis (HFE) gene mutations and hepatitis C virus infection as risk factors for porphyria cutanea tarda in Hungarian patients.

AIM: It is not clear whether the mutations in hemochromatosis (HFE) gene and hepatitis C virus (HCV) infection act independently in the pathogenesis of porphyria cutanea tarda (PCT). The prevalence of both risk factors varies greatly in different parts of the world. PCT patients from Hungary were evaluated to assess both factors. METHODS: The prevalence of C282Y and H63D mutations in the HFE gene was determined in 50 PCT patients and compared with the reported control frequencies. Furthermore, the presence of HCV infection was determined and related to the patients' HFE gene status. RESULTS: The C282Y mutation was found in 8/50 cases (three homozygotes and five heterozygotes), with an 11% allele frequency (vs. 3.8% control) (P<0.05). Seventeen patients were heterozygous, one was homozygous for the H63D mutation, allele frequency 19%, which did not differ significantly from the reported control prevalence of 12.3%. Twenty-two patients (44%) were HCV-RNA positive; six out of them were heterozygous for H63D mutation, one only for the C282Y mutation and one was compound heterozygous for both mutations. CONCLUSION: HCV infection and HFE C282Y mutation may probably be independent predisposing factors for development of PCT in Hungarian patients.     

Mutation Analysis of the HFE Gene in German Hemochromatosis Patients and Controls using Automated SSCP-based Capillary Electrophoresis and a New PCR-ELISA Technique

Background: The diagnosis of hereditary hemochromatosis (HH) before the onset of iron-overload has been difficult in the past. However, a convincing candidate gene for HH: HFE has been described recently. The aims of this study were: 1) To determine the prevalence of the hemochromatosis associated mutations C282Y and H63D of the HFE gene in patients from Southern Germany with hemochromatosis phenotype; and 2) to test two new, time- and cost-saving methods: automated SSCP-based capillary electrophoresis (SSCP-CE) and a PCR-ELISA technique for the analysis of HFE mutations. Methods: HFE genotype was studied in 36 unrelated HH patients and 126 controls from Southern Germany. In addition, family screening was performed in 76 relatives. The C282Y and H63D mutations were detected using SSCP-CE and restriction length polymorphism (RFLP). The C282Y mutation was additionally analysed by a PCR-ELISA. Results: Twenty-six (72%) HH patients were homozygous for mutation C282Y, and three compound heterozygous for C282Y and H63D. One patient was homozygous for H63D. By performing family screening, six additional patients with the +/+ C282Y mutation were identified. The results of the SSCP-CE and the PCR-ELISA analysis agreed completely with data obtained by RFLP. Conclusions: SSCP-CE and PCR-ELISA analysis proved to be reliable methods for HFE genotyping and therefore represent cost- and time-effective alternative methods to the widely used restriction analysis allowing large populations to be screened for HH associated with HFE mutations. Surprisingly, only 72% of our HH patients had the C282Y +/+ genotype. This indicates that hemochromatosis in Southern Germany is genetically more heterogeneous than in other regions. A challenge for the future will be to define the genetic or environmental factors responsible for ironoverload in HH patients who do not show typical alterations of the HFE gene.     

Mutation analysis of the HFE gene in German hemochromatosis patients and controls using automated SSCP-based capillary electrophoresis and a new PCR-ELISA technique.

BACKGROUND: The diagnosis of hereditary hemochromatosis (HH) before the onset of iron-overload has been difficult in the past. However, a convincing candidate gene for HH: HFE has been described recently. The aims of this study were: 1) To determine the prevalence of the hemochromatosis associated mutations C282Y and H63D of the HFE gene in patients from Southern Germany with hemochromatosis phenotype; and 2) to test two new, time- and cost-saving methods: automated SSCP-based capillary electrophoresis (SSCP-CE) and a PCR-ELISA technique for the analysis of HFE mutations. METHODS: HFE genotype was studied in 36 unrelated HH patients and 126 controls from Southern Germany. In addition, family screening was performed in 76 relatives. The C282Y and H63D mutations were detected using SSCP-CE and restriction length polymorphism (RFLP). The C282Y mutation was additionally analysed by a PCR-ELISA. RESULTS: Twenty-six (72%) HH patients were homozygous for mutation C282Y, and three compound heterozygous for C282Y and H63D. One patient was homozygous for H63D. By performing family screening, six additional patients with the +/+ C282Y mutation were identified. The results of the SSCP-CE and the PCR-ELISA analysis agreed completely with data obtained by RFLP. CONCLUSIONS: SSCP-CE and PCR-ELISA analysis proved to be reliable methods for HFE genotyping and therefore represent cost- and time-effective alternative methods to the widely used restriction analysis allowing large populations to be screened for HH associated with HFE mutations. Surprisingly, only 72% of our HH patients had the C282Y +/+ genotype. This indicates that hemochromatosis in Southern Germany is genetically more heterogeneous than in other regions. A challenge for the future will be to define the genetic or environmental factors responsible for iron-overload in HH patients who do not show typical alterations of the HFE gene.     

Mutations in the HFE gene and their interaction with exogenous risk factors in hepatocellular carcinoma

The possible role of iron in facilitating the development of liver cancer is still debated. The aims of this study were to define the prevalence of the mutations 845G --> A and 187C --> G (C282Y and H63D) in the HFE gene associated with hereditary hemochromatosis in Italian patients with hepatocellular carcinoma occurring in cirrhosis and to analyze the interaction between these mutations and other established risk factors for hepatocellular carcinoma. The HFE gene mutations, performed by polymerase chain reaction, were analyzed in 81 patients (63 males, 18 females) with hepatocellular carcinoma. None of the patients had a phenotype compatible with homozygous hereditary hemochromatosis. Interaction between HFE mutations and exogenous risk factors was analyzed by collecting information on alcohol consumption, hepatitis B and C virus infections, and iron status at the time of diagnosis of chronic liver disease. This analysis was performed only in males to rule out gender influence on patients' iron status by using the case-only approach specifically designed to estimate departure from multiplicative risk ratios under the assumption of independence between genotype and environmental exposure. The prevalence of the C282Y mutation was significantly higher in patients with hepatocellular carcinoma than in normal controls (8.6% vs 1.6%, P < 0.03). At univariate analysis, iron overload was significantly associated with both HFE mutations (P < 0.0001), whereas ongoing hepatitis B virus infection was associated with the C282Y mutation (P < 0.05). By multivariate analysis, a trend for an increased risk of being positive for hepatitis virus markers (OR 2.9, CI 95% 0.9-9.5) and of having been alcohol abusers (OR 3, CI 95% 0.7-14) was observed in patients heterozygous for the HFE mutations. These data indicate that the prevalence of the main mutation associated with hereditary hemochromatosis is significantly higher in cirrhotic Italian patients with hepatocellular carcinoma compared to a normal population and suggest that heterozygotes for HFE mutations exposed to hepatitis virus infections or who had been alcohol abusers could have an increased risk of developing cirrhosis and later liver cancer than people without the mutations exposed to the same risk factors.     

HFE genotype in patients with hemochromatosis and other liver diseases.

BACKGROUND: Hereditary hemochromatosis is a common inherited disorder of iron metabolism. The gene HFE, which contains two missense mutations (C282Y and H63D), was recently identified. OBJECTIVE: To determine how HFE genotyping for the C282Y and H63D mutations contributes to the diagnosis of hemochromatosis and to determine the prevalence of HFE mutations in a group of patients with liver disease. DESIGN: Cross-sectional study. SETTING: Academic medical center. PATIENTS: 66 patients with hereditary hemochromatosis and 132 referred patients with other liver diseases. MEASUREMENTS: At initial diagnosis, fasting transferrin saturation, ferritin level, routine chemistry panel, and complete blood count were determined. Percutaneous liver biopsy was done on all patients for histologic analysis and measurement of hepatic iron concentration and hepatic iron index. HFE genotyping for the C282Y and H63D mutations was done on all patients by using genomic DNA samples. RESULTS: Of the 66 patients with hemochromatosis diagnosed on the basis of serum iron studies and liver biopsy findings, 60 (91%) were C282Y homozygotes, 2 (3%) were compound heterozygotes, 1 (1.5%) was a C282Y heterozygote, 2 (3%) were H63D heterozygotes, and 1 (1.5%) was negative for both mutations. Of the 132 patients with liver disease, 6 (5%) were C282Y homozygotes, 8 (6%) were compound heterozygotes, 6 (5%) were C282Y heterozygotes, 5 (4%) were H63D homozygotes, 20 (15%) were H63D heterozygotes, and 87 (66%) were negative for both mutations. All 66 C282Y homozygotes had an elevated hepatic iron concentration, and 65 of the 66 patients (98%) had a transferrin saturation of at least 45%. Ten of the 66 patients (15% [95% CI, 7.5% to 26%]) had a hepatic iron index less than 1.9 mmol/kg per year; hemochromatosis was not suspected in 6 of the 10 patients before genotyping. Cirrhosis or substantial hepatic fibrosis was not seen in any (0% [CI, 0% to 18%]) of the 19 patients younger than 40 years of age who were homozygous for the C282Y mutation. CONCLUSIONS: All 66 patients homozygous for the C282Y mutation of HFE had an elevated hepatic iron concentration, but approximately 15% of these patients did not meet a previous diagnostic criterion for hemochromatosis (hepatic iron index > 1.9 mmol/kg per year). Determination of HFE genotype is clinically useful in patients with liver disease and suspected iron overload and may lead to identification of otherwise unsuspected C282Y homozygotes.     

HFE mutations and chronic hepatitis C: H63D and C282Y heterozygosity are independent risk factors for liver fibrosis and cirrhosis

BACKGROUND/AIMS: The impact of heterozygous HFE mutations on the course of chronic hepatitis C and iron indices was studied. METHODS: Ferritin, transferrin saturation (TS), serum iron, C282Y and H63D mutations were determined in 401 patients with chronic hepatitis C virus (HCV) infection and 295 healthy controls. Liver histologies were available in 217 and HCV genotypes in 339 patients. RESULTS: Allele frequencies of the C282Y and H63D mutation did not differ between HCV patients and healthy controls (6.95 vs. 6.2%; 14.75 vs. 16.4%; n.s.). HFE heterozygous HCV patients had higher ferritin (349+/-37 vs. 193+/-15 microg/l; P<0.0005), TS (38+/-2 vs. 32+/-1%; P<0.0005), serum iron (144+/-6 vs. 121+/-3 microg/dl; P<0.0005), semiquantitative liver iron staining (0.26+/-0.07 vs. 0.09+/-0.03; P<0.006) and fibrosis scores (1.9+/-0.2 vs. 1.4+/-0.1; P<0.003) compared to HFE wildtypes. By multivariate regression analysis odds ratios for liver cirrhosis were 5.9 (confidence interval (CI) 1.6-22.6; P<0.009) for C282Y heterozygotes and 2.9 (CI 1.0-8.4; P<0.05) for H63D heterozygotes compared to HFE wildtypes. Considering all HFE heterozygous HCV patients, odds ratios of 3.6 (CI 1.4-9.3; P<0.009) for cirrhosis and 3.1 (CI 1.3-7.3; P<0.009) for fibrosis were calculated. CONCLUSIONS: C282Y or H63D heterozygosity is an independent risk factor for liver fibrosis and cirrhosis in HCV infected individuals. Screening for HFE mutations should be considered in HCV infection.     

Two novel nonsense mutations of HFE gene in five unrelated italian patients with hemochromatosis

BACKGROUND & AIMS: Most hemochromatosis patients of Northern European descent are homozygous for the C282Y mutation of HFE gene. In Italy, many patients with iron overload are not homozygous for C282Y, and the presence of other mutations or other genetic determinant has been suggested. METHODS: Five unrelated Italian patients heterozygous for C282Y with the classic hemochromatosis phenotype were studied. The entire coding sequence and the exon-intron boundaries of the HFE gene were analyzed. Chromosome 6p haplotypes were defined in each patient by analysis of D6S265, D6S105, and D6S1281 microsatellites. RESULTS: Two novel nonsense HFE mutations were identified in exon 3 in the C282Y negative chromosome. The first one, a G-to-T transition at codon 168, was detected in 3 probands; the second, a G-to-A transition at codon 169, was detected in the others. CONCLUSIONS: The 2 nonsense mutations in the compound heterozygous state with C282Y result in the classic hemochromatosis phenotype in several unrelated Italian patients. This confirms that hemochromatosis in Italy is not as homogeneous as in northern Europe and suggests that other mutations can exist in C282Y or H63D heterozygotes with iron overload. These findings have practical implications for diagnostic and screening strategies for hemochromatosis.     

Prevalence of hemochromatosis gene (HFE) mutations in Greece

The frequencies of the hereditary hemochromatosis gene (HFE) mutations C282Y and H63D vary between different populations. There are a limited number of reports regarding the frequency of these mutations in populations of southeastern Europe. Two hundred and sixty-four adult individuals of Greek origin were examined for the C282Y and H63D mutations to determine the allele and genotype frequencies. The HFE gene region of DNA samples extracted from peripheral leukocytes was amplified by the polymerase chain reaction. Restriction enzyme analysis was performed using RSAI for C282Y and MBOI for H63D. None of the 264 individuals carried the mutation C282Y. Forty-three individuals (16.2%) were heterozygous carriers of the H63D allele and 2 were homozygous for this mutation (0.75%). The overall H63D allele prevalence is thus estimated at 8.9%. HFE mutation frequencies were low in the population studied and this may explain, in part, the relative rarity of clinical cases of hereditary hemochromatosis in Greece.     

HFE genotyping demonstrates a significant incidence of hemochromatosis in undifferentiated arthritis

OBJECTIVE: Hereditary hemochromatosis is a common autosomal recessive disorder of iron metabolism. Among Northern Europeans the carrier frequency is estimated to be 1 in 10, while up to 1 in 200 is affected by the disease. Arthropathy is one early clinical manifestation of this disease, but the articular features are often misdiagnosed. In this study the two frequent mutations of the HLA-linked hemochromatosis gene (HFE) were investigated in a rheumatology clinic population. METHODS: Two hundred and six consecutive patients (mean age 57.7 years; 38 male/168 female) attending a rheumatology clinic over a period of 14 months were screened for HFE mutations (C282Y and H63D). All standard diagnostic procedures were used to identify the aetiology of the arthropathy. Mutations were evaluated by separation on PAGE of digested PCR amplificates of DNA (by SnapI and Bcl-I, for C282Y and H63D, respectively) obtained from PBMCs. RESULTS: The C282Y and H63D allele frequencies were 4.5 and 12.8 in patients with rheumatic diseases. Five patients were homozygote for H63D (2.4%), and one for C282Y (0.5%). Five patients were compound heterozygous (2.4%). The observed C282Y allele frequency in rheumatic patients with undifferentiated arthritis was 12.9 and exceeded that of healthy subjects (p = 0.01). CONCLUSIONS: Determination of the HFE genotype is clinically useful in patients with arthritis of unknown origin, to allow early diagnosis of hemochromatosis.     

Idiopathic hemochromatosis with the mutation of Ala176Val heterozygous for HFE gene.

Most patients with hereditary hemochromatosis are homozygous for C282Y in the HFE gene in populations of Celtic origin, but the genetic cause of this disease is unknown in Japan because of its rarity. A 48-year-old Japanese patient was recently diagnosed with idiopathic hemochromatosis. Analysis of the entire coding region of the patient's HFE by RT-PCR showed a heterozygous nucleotide substitution at nucleotide 527 from C to T, which resulted in A176V amino acid substitution. Another mutation at nucleotide 942 from T to C was observed, but this was a nonsense mutation. C282Y and another mutation, H63D, were not found in the patient. The mutation may have a possible role on the cause of hemochromatosis in this Japanese case.     

Transferrin receptor-2 gene and non-C282Y homozygous patients with hemochromatosis

More than 80% of the patients affected by hereditary hemochromatosis, a common inherited iron disorder, are homozygotes for the 845G --> A (C282Y) mutation of the HFE gene. However, depending on the population, 10-20% of hereditary hemochromatosis can be linked either to other HFE genotypes, particularly the compound heterozygous state for C282Y and the 187 C --> G (H63D) mutation, or to mutations of new other genes. Recently, Camaschella et al. (Nat. Genet. 25, 14-15, 2000) identified a stop mutation (exon 6 nt 750 C --> T, Y250X) on the transferrin receptor-2 (TFR2) gene in two unrelated Sicilian families with hereditary hemochromatosis. The TFR2 gene is a transferrin receptor gene homologue that seems to be involved in iron metabolism. Moreover, one of the patients described by Camaschella et al. was a H63D homozygote. H63D homozygosity can be associated with various phenotypes from asymptomatic subjects to patients with a typical form of hereditary hemochromatosis. Thus, the Y250X mutation could be the molecular defect responsible for hereditary hemochromatosis in subjects with atypical HFE genotypes. We have searched for the Y250X mutation in 63 unrelated French subjects. Forty-three had a diagnosis of hereditary hemochromatosis based on classical criteria. This group included 12 H63D homozygotes, 3 C282Y heterozygotes, and 3 patients with none of the two most prevalent HFE mutants. These 18 patients had no other HFE sequence change and were subsequently subjected to DNA sequencing of the 15 last exons and flanking sequences of the TFR2 gene. The 25 remaining hereditary hemochromatosis patients who were tested for the Y250X mutant were compound heterozygotes for the C282Y and H63D mutations. Finally, we also tested for this TFR2 mutation 20 H63D homozygotes with milder manifestations of iron overload and no acquired cause of iron overload. None of the 63 tested subjects had the Y250X mutation. Concurrently, none of the 18 hereditary hemochromatosis patients who had their TFR2 gene sequenced had any deleterious mutation. Thus, TFR2 mutations are not responsible for hemochromatosis in non-C282Y homozygous patients of our area.