Mutation analysis of the HFE gene associated with hereditary hemochromatosis in a Venezuelan sample

The frequency of the C282Y, H63D and S65C alleles of the HFE gene was determined in a sample of the Venezuelan population. Two new sets of primers were tested for amplifying the regions mapping these mutations, and genotyping was performed by restriction fragment length polymorphism (RFLP). DNA sequencing was used to validate the RFLP analysis. Serum ferritin levels were also determined. Two hundred and fourteen individuals were tested, extracting DNA from whole blood cells (n=177) or from serum (n=37). The frequency of heterozygous subjects was 3.7, 18.2 and 1.7% for the C282Y, H63D and S65C mutations, respectively, and the allele frequencies were 0.019±0.01 for C282Y, 0.119±0.016 for H63D and 0.009±0.005 for S65C. The results suggest that the admixture of native populations with subjects of South European origin might have had an important role in the diffusion of HFE alleles in Venezuela. C282Y homozygous subjects were not found in this study. No HFE genotype studied here was associated with a significant elevation of serum ferritin concentrations, except for C282Y/H63D compound heterozygote found in one asymptomatic male. This finding supports the theory that the H63D mutation could be involved in alterations of iron parameters when inherited together with C282Y. Our results indicate that C282Y homozygotes will be rarely detected. Performance of HFE mutation analysis in individuals with high iron determinations would be recommended.     

Modifying factors of the HFE hemochromatosis phenotype

C282Y homozygosity is the only common HFE genotype able to produce a complete hemochromatosis phenotype. However, its biochemical penetrance is incomplete (75% in men and 50% in women) and its clinical penetrance is low, especially in women (1 vs 25% in men). Environmental (e.g., diet, alcohol, drugs and metabolic syndrome) and genetic (digenism, common polymorphisms in the bone morphogenetic protein pathway involved in the regulation of hepcidin synthesis) explain a part of the variability of the C282Y homozygous phenotype. All other common HFE genotypes – including C282Y–H63D compound heterozygosity – are not associated with significant biochemical and clinical expression in the absence of comorbid factors (e.g., alcohol, diabetes or steatohepatitis). Better identification of acquired and genetic modifiers of iron burden and iron-related organ damage is needed to improve the preventive, diagnostic and therapeutic management of HFE hemochromatosis.     

Mutations of the HFE gene among Turkish hereditary hemochromatosis patients

Since the discovery of the HFE gene, C282Y and H63D mutations have been reported as significantly correlated with clinically manifested hereditary hemochromatosis (HH). As the other genes involved in iron metabolism have been described, non-HFE cases of HH have been identified. Since in the general Turkish population, the C282Y mutation is not found and the H63D mutation is of high frequency, we aimed to determine mutations in the HFE genes in our patients with HH. The HFE gene of the five patients with HH were sequenced. C282Y mutation was absent, and all HH patients were heterozygote for H63D mutation. No other mutation was found in HFE gene by sequencing. Although the higher allele frequency of the H63D mutation in Turkish HH patients than in the general population implies a role of the H63D mutation in iron overload, there is a strong possibility that Turkish HH patients have non-HFE hemochromatosis.     

Mutation analysis of the HFE gene associated with hereditary hemochromatosis in African Americans

Homozygosity for the mutation Cys282Tyr in the HFE gene has recently been identified as a cause of hereditary hemochromatosis, a disorder resulting in the inappropriate absorption of iron. Approximately 10% of Caucasians are heterozygous for this mutation; however, the gene frequency in African Americans is unknown. A study of a control population of African Americans was performed to determine the frequency of the Cys282Tyr and His63Asp alleles in this ethnic group. The carrier frequency for each mutant allele in our African American population was 3.0%. DNA studies of four African-American hemochromatosis patients did not identify any individuals with the Cys282Tyr allele. These findings suggest that if the Cys282Tyr mutation confers susceptibility to hemochromatosis in Caucasians (as suggested by recent studies) there is an alternative mechanism for hemochromatosis in the American black population. Am. J. Hematol. 58:213–217, 1998. © 1998 Wiley-Liss, Inc.     

Expression of the HFE hemochromatosis gene in a community-based population of elderly women

BACKGROUND AND AIM: Recent studies suggest that the clinical penetrance of associated hereditary hemochromatosis, defined as either the C282Y homozygote or compound heterozygote HFE genotype status, is much lower than previously thought. METHODS: We investigated the clinical penetrance and phenotypic expression of HFE-associated hereditary hemochromatosis in a community-based population of 1352 elderly female subjects with a mean age of 75 years. Serum transferrin saturation and ferritin levels were determined on all subjects bearing a C282Y mutation and a subset of wild-type C282Y subjects. RESULTS: The prevalences of the C282Y homozygous and compound heterozygous HFE genotypes were 0.15% (2/1352) and 2.0% (27/1352), respectively. The observed prevalence of 0.15% for C282Y homozygotes borders on significance (P = 0.054) for deviation from the Hardy-Weinberg population equilibrium calculations, which predict a prevalence of 0.49%, whereas the observed and predicted compound heterozygote prevalences were not significantly different. Clinical symptoms of hemochromatosis were absent in both the C282Y homozygote subjects. Of the compound heterozygous subjects, 2/27 (7%) had elevated serum transferrin saturation and ferritin values; however, clinical symptoms of hemochromatosis were absent in both. Considered as a whole, the compound heterozygous subjects had markedly elevated means for serum iron (19.4 vs 16.0 micromol/L, P = 0.0008), transferrin saturation (34.8% vs 25.2%, P < 0.0001) and ferritin (157 vs 92 microg/L, P = 0.002) compared with the wild-type subjects. CONCLUSION: The C282Y homozygous HFE hereditary hemochromatosis genotype was under-represented in this elderly cohort, whereas the compound heterozygous genotype was not. None of the homozygous or compound heterozygous subjects expressed the phenotype of iron overload disease.     

Frequency of the HFE gene mutations in five Italian populations

Genetic hemochromatosis is an autosomal recessive disorder characterized by iron overload and a variety of clinical manifestations such as liver cirrhosis and arthropathy. It is the most common genetic disease of northern European populations. The principal gene responsible for hereditary hemochromatosis, designated HFE, is located on chromosome 6 in the HLA region. The single point mutation 845A, changing cysteine at position 282 to tyrosine (C282Y), in this gene has been identified as the main genetic basis of hereditary hemochromatosis. Two other mutations, 187G, a histidine to aspartate at amino acid 63 (H63D), and 193T, a serine to cysteine at amino acid 65 (S65C), appear to be associated with milder forms of hereditary hemochromatosis. There is a high prevalence of the C282Y mutation in northern European populations, whereas in those of the Mediterranean basin the prevalence seems low and almost absent in Far East countries. This mutation seems usually to occur on the ancestral haplotype 7.1. Accordingly, a Celtic origin of this mutation has been suggested. The aim of this study was to determine the frequency of HFE gene mutations in five geographic regions in Italy. Samples were tested for C282Y, H63D, and S65C mutations of the HFE gene according to methods of each laboratory and the results were standardized with the exchange of typed samples between the different laboratories. In addition, C282Y-positive DNA samples were typed for D6S105 allele 8 and HLA-A3 by ARMS-PCR. We have found that the allele frequency of the C282Y mutation decreases from northeast Italy (Friuli, 6%) to northwest Italy (Piedmont, 4.8%) and to central Italy (Emilia-Romagna, 1.7%). However, this mutation is lacking in the two regions of the Mediterranean basin's center (Sicily and Sardinia). Accordingly, a significant difference in the frequency of the mutation was observed between these Italian regions (P = 0.07 x 10(-3)). In contrast, no difference was observed in allele frequency of H63D in the five Italian regions. Finally, as regards the S65C mutation a very low frequency was observed in Friuli, Emilia-Romagna, and Sardinia, whereas in Sicily and Piedmont we have not found this mutation. In conclusion, these data are consistent with the hypothesis that the C282Y mutation occurred in Caucasian populations of Celtic origin, whereas the H63D mutation is more ancient as demonstrated by the ubiquitous distribution.     

Recent concepts in iron metabolism and HFE mutations

Abstract   Our understanding of the mechanisms by which the body regulates iron absorption and iron homeostasis has improved greatly in recent years with the identification of key molecules such as HFE, ferroportin, transferrin receptor 2, hepcidin and hemojuvelin. Hereditary hemochromatosis, a primary inherited disorder of iron metabolism leading to progressive iron loading of parenchymal cells of the liver and other organs, can result from mutations in any of these genes. However, HFE-associated hereditary hemochromatosis accounts for over 90% of cases in Caucasian populations. Penetrance is incomplete with variable clinical expression. The majority of cases demonstrate biochemical expression but a lower proportion develops advanced disease. Clinical disease, especially hepatic fibrosis, is related to the level of body iron stores which is reflected primarily in the liver. The available evidence indicates that adequate screening and diagnostic strategies can ensure that early case detection and treatment occur prior to the development of irreversible end-organ damage. The most cost-effective methods of early case detection are family (cascade) screening and evaluation of potential cases by primary care physicians with a high index of clinical suspicion.     

Frequency of the C282Y and H63D mutations of the hemochromatosis gene (HFE) in a cohort of 1,000 neonates in Madrid (Spain)

For centuries in Europe, population movements have contributed to ethnic groups, cultures, and consequently, inheritance mixing. There are certain genetic diseases such as hereditary hemochromatosis whose distribution is directly related to the population movements. The objective of the present investigation was to determine the C282Y and H63D mutation frequency of the HFE gene in a cohort study of 1,000 neonates in the Community of Madrid (Spain), thus contributing to the HFE gene mutations distribution research in Europe and establishing the origin of the mutations in Spain. The allelic frequency of C282Y mutation was 1.7% (CI 95% 1.1–2.3) and the H63D allele was present in 16.4% of chromosomes (CI 95% 14.8–18). In Spain, the presence of C282Y mutation and its distribution could be due more to Celtic than to Viking legacy, whereas it is assumed that the one in relation to the H63D variant occurred in the Basque Country during the Paleolithic Period.     

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.     

Hereditary spherocytosis associated with mutations in HFE gene

We report on a Spanish family in which three members of different generations were diagnosed with hereditary spherocytosis (HS). Additionally, one of them II-I (44-years-old), presented iron overload with hepatic deposit and needed treatment with periodic phlebotomies. The rest of the family members presented normal analytical values in iron metabolism. To investigate the presence of H63D and C282Y mutations in the HFE gene, patient II-I was found to be compound heterozygous and was the only family member presenting HS and this genetic condition in HFE. We propose a synergistic effect of HS and mutations in HFE as the cause of the iron deposits.     

Analysis of HFE gene mutations (C282Y, H63D, and S65C) in the Ecuadorian population

Type 1 hemochromatosis is a disorder of iron metabolism mostly related to the HFE gene mutations. In the present study, we performed a mutation analysis to determine the frequencies of the HFE gene mutations (C282Y, H63D, and S65C) in DNA samples of 100 healthy Ecuadorian individuals. We used the polymerase chain reaction (PCR) to amplify exons 2 and 4 of the HFE gene and then the restriction fragment length polymorphism (RFLP) method to detect the mutations. The results revealed that the mutations in the normal Ecuadorian population have frequencies of 0.0, 0.035, and 0.04 for C282Y, H63D, and S65C, respectively. We also searched for these mutations in 12 hemochromatosis patients, and the frequencies that we found were 0.0 for C282Y, 0.167 for H63D, and 0.042 for S65C. We found differences [using the chi-square (²) test] in the frequency of the H63D mutation between the control group and the group of hemochromatosis patients (p<0.01). This suggests that in Ecuador, type 1 hemochromatosis is more influenced by the H63D mutation than the other two mutations that we analyzed. Given that in a Caucasian population hereditary hemochromatosis is mostly related to the C282Y mutation, it is possible that the findings for the Ecuadorian population are due to geographical differences between the populations.     

Hereditary hemochromatosis gene (HFE) mutations C282Y, H63D and S65C in patients with idiopathic dilated cardiomyopathy

BACKGROUND: Hereditary hemochromatosis (HH), a common autosomal recessive disease, leads to excessive iron accumulation in some organs, including the heart. It is therefore not surprising that cardiomyopathy is one of the most severe complications of HH. The HFE gene defects have been thought to contribute to idiopathic dilated cardiomyopathy (IDCM) in some patients, even though the results of genotype analyses have so far been contradictory. Hence we set out here to evaluate the prevalence and potential role of HFE mutations in patients with IDCM. METHODS: A total of 91 IDCM patients and 102 controls were subjected to HFE mutation analyses, in which C282Y, H63D and S65C mutations were determined for each patient. We also analyzed the impact of the C282Y and H63D mutations on the left ventricular end-diastolic diameter (LVEDD), left ventricular ejection fraction (LVEF) and New York Heart Association (NYHA) functional classes. RESULTS: The prevalences of heterozygosity for the C282Y, H63D and S65C mutations in the IDCM patients were 13.2%, 22.0% and 2.2%, respectively. LVEDD was significantly higher (P=0.037) in those with the C282Y mutation at the end of the follow-up period than in those with no mutation. CONCLUSIONS: Our data showed no significant deviations in C282Y, H63D and S65C mutation frequencies between the IDCM patients and controls, suggesting that these mutations do not increase the risk of IDCM. Heterozygosity for the C282Y mutation may nevertheless be a modifying factor contributing to LV dilatation and remodeling.     

Non-classical hereditary hemochromatosis in Portugal: novel mutations identified in iron metabolism-related genes

The most frequent genotype associated with Hereditary hemochromatosis is the homozygosity for C282Y, a common HFE mutation. However, other mutations in HFE, transferrin receptor 2 (TFR2), hemojuvelin (HJV) and hepcidin (HAMP) genes, have also been reported in association with this pathology. A mutational analysis of these genes was carried out in 215 Portuguese iron-overloaded individuals previously characterized as non-C282Y or non-H63D homozygous and non-compound heterozygous. The aim was to determine the influence of these genes in the development of iron overload phenotypes in our population. Regarding HFE, some known mutations were found, as S65C and E277K. In addition, three novel missense mutations (L46W, D129N and Y230F) and one nonsense mutation (Y138X) were identified. In TFR2, besides the I238M polymorphism and the rare IVS5 −9T→A mutation, a novel missense mutation was detected (F280L). Concerning HAMP, the deleterious mutation 5’UTR −25G→A was found once, associated with Juvenile Hemochromatosis. In HJV, the A310G polymorphism, the novel E275E silent alteration and the novel putative splicing mutation (IVS2 +395C→G) were identified. In conclusion, only a few number of mutations which can be linked to iron overload was found, revealing their modest contribution for the development of this phenotype in our population, and suggesting that their screening in routine diagnosis is not cost-effective.     

Association of HFE mutations (C282Y and H63D) with iron overload in blood donors from Mexico City

Background and objective: Iron overload has been associated with HFE mutations (C282Y and H63D). We investigated the association between these mutations and high serum ferritin in a sample of healthy adult men.Design and methods: We enrolled unrelated blood donors from three hospitals in Mexico City in a cross-sectional study. Serum ferritin (SF) was determined to define iron overload, and HFE gene mutations were identified by PCR–RFLP.Results: We evaluated 2524 male blood donors and included 246 individuals for each group. We identified 108 individuals with HFE gene mutation, 20.5 % were heterozygote (wt/H63D or wt/C282Y) and the remaining homozygote (H63D/ H63D). The genotype wt/C282Y was observed in two cases, none cases with C282Y/C282Y. The allelic frequency of H63D and C282Y was 0.115 and 0.002, respectively. We observed different association for H63D allele with iron overload (OR 1.54, CI 95 %1.16-2.03) and none in allele C282Y. Although values averages were different, the extreme dispersion of serum ferritin not showed statistically significant differences between H63D and C282Y alleles and ferritin concentrations.Conclusions: The male unrelated blood donors from Mexico City with iron overload prevalence of 13.8% hold similarities with other populations from Europe o America continent, respecting the allele frequency H63D. Nevertheless, allele frequency C282Y is lower than that observed in descendents from northern Europe. We have not observed statistic difference of SF or iron overload frequency by effect of both alleles.     

HFE基因多态性检测判断慢性丙型肝炎患者疾病活动的价值*

目的 观察遗传性血色素沉着症候选基因HFE多态性检测判断CHC疾病活动的价值。方法 2016年11月～2018年11月我院收治的257例丙型肝炎病毒感染者,其中病毒感染者131例和慢性丙型肝炎(CHC)患者126例。采用酶结合免疫吸附法测定血清铁蛋白(SF)水平,使用ABIPrismsTM-7900实时荧光定量PCR仪和TaqMan-MGB荧光探针,以实时定量PCR法检测HFE基因rs2071303和rs9366637位点基因型。结果 病毒感染者SF水平为(97.5±4.1)μg/L,显著低于CHC患者【(202.1±24.5)μg/L,P＜0.05】,血清ALT水平为(34.0±4.5)U/L,显著低于CHC患者【(88.4±5.6)U/L,P＜0.05】,AST为(37.5±4.2)U/L,显著低于CHC患者【(70.0±5.4)U/L,P＜0.05】;病毒感染者HCV基因非Ⅰb型频率为29.8%,显著高于CHC患者13.5%(P＜0.05),病毒感染者HCV基因型中混合型频率为9.1%,显著低于CHC患者的21.4%(P＜0.05);病毒感染者rs2071303位点GG基因型患者SF水平为(97.6±4.2)μg/L,显著低于CHC患者【(199.5±45.4)μg/L,P＜0.05】,GA基因型SF水平为(97.6±4.1)μg/L,显著低于CHC患者【(207.5±34.7)μg/L,P＜0.05】,AA基因型SF水平为(96.7±3.7)μg/L,显著低于CHC患者【(198.0±44.8)μg/L,P＜0.05】;病毒感染者rs9366637位点TT基因型患者SF水平为(97.4±4.0)μg/L,显著低于CHC患者【(206.4±35.6)μg/L,P＜0.05】,TC基因型SF水平为(97.2±4.0)μg/L,显著低于CHC患者【(208.5±34.0)μg/L,P＜0.05】,CC基因型SF水平为(99.1±4.5)μg/L,显著低于CHC患者【(178.5±58.6)μg/L,P＜0.05】;病毒感染者rs2071303位点AA基因型频率为13.7%,显著低于CHC患者的21.4%(P＜0.05),病毒感染者AC单倍型频率为3.1%,显著低于CHC患者的8.3%(P＜0.05)。结论 HFE基因多态性与CHC疾病活动密切相关,临床应引起足够的重视。     

Human leukocyte antigen haplotypes and HFE mutations in Spanish hereditary hemochromatosis and sporadic porphyria cutanea tarda

BACKGROUND AND AIMS: It has been postulated that the HFE C282Y mutation (linked to human leukocyte antigen [HLA]-A3-B7 haplotype) is not only responsible for hereditary hemochromatosis; HLA class I alleles would also contribute to the disease pathogenesis. In addition, H63D mutation linked to HLA-A29-B44 would also be pathogenetic, particularly in the Mediterranean Basin and throughout the world. However, sporadic porphyria cutanea tarda (s-PCT) has also been linked to these HFE mutations. In the present work, we have studied HFE mutations and HLA genes to test these hypotheses. METHODS: C282Y and H63D mutations together with HLA genetic typing have been performed in Spanish hereditary hemochromatosis (n = 98) and PCT (n = 63) patients. The etiologic fraction (delta) has been used to determine the absolute strongest gene linkage to both diseases. RESULTS: The Spanish frequent HLA-A29-B44 haplotype is not significantly associated to the H63D mutations in hereditary hemochromatosis patients (although it is found more frequently in patients than in controls). Sporadic porphyria cutanea tarda patients do not show a significant association to H63D mutations, although it is also more frequent than in controls; however, compound H63D/C282Y subjects seem to bear a significant risk to s-PCT. Allelic C282Y (and not H63D) frequencies show a significant association with s-PCT. CONCLUSIONS: The postulated additional risk of hereditary hemochromatosis given by class I HLA antigens may be secondary to the HFE gene linkage disequilibrium with certain class I alleles or to the existence of other neighboring genetic pathogenetic factors in our Spanish sample.     

Two novel mutations in the SLC40A1 and HFE genes implicated in iron overload in a Spanish man

The most common form of hemochromatosis is caused by mutations in the HFE gene. Rare forms of the disease are caused by mutations in other genes. We present a patient with hyperferritinemia and iron overload, and facial flushing. Magnetic resonance imaging was performed to measure hepatic iron overload, and a molecular study of the genes involved in iron metabolism was undertaken. The iron overload was similar to that observed in HFE hemochromatosis, and the patient was double heterozygous for two novel mutations, c.-20G>A and c.718A>G (p.K240E), in the HFE and ferroportin (FPN1 or SLC40A1) genes, respectively. Hyperferritinemia and facial flushing improved after phlebotomy. Two of the patient's children were also studied, and the daughter was heterozygous for the mutation in the SLC40A1 gene, although she did not have hyperferritinemia. The patient presented a mild iron overload phenotype probably because of the two novel mutations in the HFE and SLC40A1 genes.     

Human platelets express hemochromatosis protein (HFE) and transferrin receptor 2

OBJECTIVES: While body iron status may influence platelets, little information is available about platelet expression of proteins regulating iron homeostasis. HFE, the protein defective in hereditary hemochromatosis, and transferrin receptor 2 (TfR2) are two novel protein candidates that could be involved in mechanisms of iron transport across the platelet plasma membrane. METHODS: The expression and localization of HFE, TfR1 and TfR2 proteins in human platelets were examined using Western blotting and immunocytochemistry. RESULTS: Human platelets expressed HFE and TfR2, whereas no signal for TfR1 was found. The positive reactions for HFE and TfR2 were mainly confined to the platelet plasma membrane. CONCLUSIONS: Expression of HFE and TfR2 proteins in human platelets may indicate that the mutations in the corresponding genes could influence platelet count, size and/or activation. The presence of TfR2 and absence of TfR1 suggests that HFE may serve a different function in platelets compared with the other HFE-positive cell types, e.g. enterocytes, macrophages and syncytiotrophoblasts.     

Identification of heterozygous p.Y150C and p.V274M mutations in the HJV gene in a Japanese patient with a mild phenotype of juvenile hemochromatosis: A case report

Juvenile hemochromatosis (JH) is known as a progressive iron‐storage disease, and causes severe organ impairments, including cardiomyopathy and liver cirrhosis. However, JH is a rare genetic disorder, and information for genetic mutations and phenotypes is limited. Here, we report a case of JH with heterozygous p.Y150C and p.V274M mutations in the HJV gene. A 39‐year‐old Japanese man was referred to Kurume University Hospital, Kurume, Japan, for fatigue and liver injury, which first appeared at the age of 25 years. There was no history of alcohol abuse and medication, and viral hepatitis, autoimmune liver diseases, and Wilson's disease were absent. However, transferrin saturation, serum ferritin, and fasting serum hepcidin levels were 98.4%, 6421 ng/mL, and 7.4 ng/mL, respectively. Furthermore, a marked reduction in signal intensity of the liver in T1/T2‐weighted magnetic resonance images was seen and the R2* maps showed hepatic iron overload. Family history of hemochromatosis and severe organ impairment, such as cardiac dysfunction and diabetes mellitus, were negative. In addition, the HFE and HAMP genes did not show any mutation. However, we identified novel heterozygous p.Y150C and p.V274M mutations in the HJV gene in the patient. The p.Y150C and p.V274M mutations were seen in his mother and father, respectively. After phlebotomy, fatigue disappeared and serum transaminase levels were normalized. Furthermore, R2* maps showed a reduction of hepatic iron concentration. We first demonstrated heterozygous p.Y150C and p.V274M mutations in the HJV gene of patients with a mild JH phenotype. Thus, genetic testing should be considered even in patients with a mild phenotype of hemochromatosis.