Prevalence of the C282Y mutation for haemochromatosis on the Island of Majorca

The C282Y mutation of the HFE gene has been reported to be present in most of the patients with hereditary haemochromatosis (HH) of Northern European ancestry. HH affects approximately 1/300 individuals, but it is not evenly distributed in the different European countries. In the present study, polymerase chain reaction (PCR) and restriction-enzyme digestion were used to analyse the frequency of the most important mutation in haemochromatosis (C282Y) in subjects from Majorca (Balearic Islands, Spain) and patients with haemochromatosis. The results were compared with other studies from Spain and Europe. A total of 420 Majorcan chromosomes were analysed and the C282Y mutation was observed at a frequency of 2.62%+/-0.8 (11 heterozygotes: eight men and three women). In the group of hereditary haemochromatosis probands, 13 out of 14 were homozygous for the C282Y mutation. In the distribution of the C282Y mutation, a north-west to south-east cline was detected, supporting the Celtic origin of this mutation.     

Vibrio vulnificus septicemia in a patient with the hemochromatosis HFE C282Y mutation

Vibrio vulnificus is an extremely invasive gram-negative bacillus found in marine waters that causes overwhelming bacteremia and shock that is associated with high mortality. Impaired iron metabolism has been implicated in the susceptibility to V vulnificus bacterial infections. We report a case of fatal V vulnificus sepsis in a 56-year-old man who died within 1 to 3 days after consuming raw seafood. At autopsy, he was found to have micronodular cirrhosis and iron overload. Postmortem genetic analysis revealed the presence of the hemochromatosis gene (HFE) C282Y mutation. To our knowledge, this is this first documented fatal case of V vulnificus infection in a patient proven to carry the HFE C282Y mutation. Because this patient was heterozygous for the major hereditary hemochromatosis mutation and was not previously diagnosed with clinical iron overload, the spectrum of clinical susceptibilities to V vulnificus infection may include carriers of the C282Y mutation.     

High incidence of the Cys 282 Tyr mutation in the HFE gene in the Irish population -implications for haemochromatosis

Abstract: A simple PCR-SSOP approach based on a single PCR product has been developed to screen the HFE gene for the haemochromatosis-associated mutations Cys 282 Tyr and His 63 Asp. Using this approach the prevalence of these mutations in a cohort (30) of haemochromatosis patients and normal controls (404) was determined. Ninety percent of the haemochromatosis patients were homozygous for the Cys 282 Tyr mutation. In the normal population we found an increased incidence of the Cys 282 Tyr mutation (17.3%; 95% confidence limits 0.136–0.209) which was also reflected in the higher frequency of Cys 282 Tyr homozygotes (1.24%; 95% confidence limits 0.0015–0.0232). Linkage disequilbrium analysis confirmed the association between A*03 and Cys 282 Tyr. However, strong linkage disequilibrium occurred with the HLA-A*03-associated allele HLA-B*14 but not the HLA-A*03-associated allele HLA-B*07. The His 63 Asp was found to be in linkage disquilibrium with HLA-A*29.     

C282Y mutation and hepatic iron status in hepatitis C and cryptogenic cirrhosis

BACKGROUND: Increased iron deposition in liver is seen in both primary and secondary hemochromatosis. However, it is not uncommon to see significant iron deposition in a liver biopsy, explant, or autopsy specimen without any significant clinical risk factor. Because of the discovery of the candidate gene (HFE) for hereditary hemochromatosis, we may now be able to screen high-risk patient populations for the abnormal mutation (C282Y). MATERIALS AND METHODS: In this study we analyzed the livers of 50 transplant patients with a diagnosis of either hepatitis C cirrhosis or cryptogenic cirrhosis for the prevalence of the more common C282Y mutation of the HFE gene and correlated the findings to hepatic iron concentration. RESULTS: Of the 26 cases of hepatitis C cirrhosis, 3 were found to be heterozygous for the C282Y mutation. Of the 22 cases of cryptogenic cirrhosis, 1 was found to be heterozygous for the C282Y mutation. Stainable iron was increased in hepatitis C cirrhosis (76.9%) as compared to cryptogenic cirrhosis (50%) (P =. 05). Of the 3 heterozygotes with hepatitis C cirrhosis, 2 showed hepatic iron concentrations of 3+ and 4+, and 1 showed 1+. CONCLUSIONS: We conclude that patients with hepatitis C have an increased tendency to accumulate iron in the liver, and mutations in the HFE gene play a minor role in hepatic accumulation of iron in these patients.     

Non-C282Y familial iron overload: evidence for locus heterogeneity in haemochromatosis.

Haemochromatosis (HC) is an autosomal recessive disease with progressive iron overload leading to midlife onset of clinical complications. The causal gene (HFE) maps to 6p, in close linkage with the HLA class I genes. An HFE candidate gene recently identified has two missense mutations (C282Y and H63D) associated with the disease. Here we document the phenotypic and genetic analysis of a nuclear family comprising two sibs with symptomatic and massive iron overload before the age of 25. The disease seemed to be recessively transmitted and fitted the agreed criteria for haemochromatosis, but was neither associated with the C282Y and H63D mutations nor linked with HLA markers. Our data strongly support locus heterogeneity in haemochromatosis by showing evidence that the gene responsible for juvenile haemochromatosis (JH) does not map to 6p. In the absence of clear cut phenotypic distinction from typical haemochromatosis, patients below 30 years of age and C282Y negative should be considered as putative juvenile cases. This has practical implications in genetic counselling and family management.     

Prevalence of C282Y mutation in patients with rheumatoid arthritis and spondylarthritis

Rheumatoid arthritis is an inflammatory joint and systemic disease believed to be of autoimmune origin. Predisposing factors also include genetic factors, such as the presence of alleles HLA-DRB1 *04, (HLA-DRB1 *0401, *0404, *0405 and *0408) and, in other ethnic groups, of subtypes DRB1 *0101, *0102 and DRB1 *1001. These genetic factors are believed to raise the risk of developing the disease. In rheumatoid arthritis, as in other chronic inflammatory diseases, iron metabolism dysfunction has been observed and attributed to inflammation. In hereditary hemochromatosis, tissue sideropexia is associated with a peculiar form of arthropathy. C282Y is a point mutation involving the replacement of a cysteine with a tyrosine at position 282 of the HFE protein. When found in homozygosis, there is a close association with hereditary hemochromatosis, accounting for one of the causes of iron metabolism dysfunction observed in this disease. The aim of this study was to compare the frequency of C282Y in patients with rheumatoid arthritis with that in patients with different forms of spondylarthritis and to correlate these findings with iron metabolism parameters. In the group of patients with rheumatoid arthritis, 2/24 (8.34%) were found to be positive for the C282Y mutation in the case of heterozygosis compared with 3/24 (12.5%) of patients with spondylarthritis. In patients with the C282Y mutation, ferritin levels were significantly higher than those in controls; conversely, serum iron levels were higher in patients with spondylarthritis. Serum transferrin levels, although slightly higher in rheumatoid arthritis patients, showed no statistically significant differences.     

Liver pathology in patients with primary hemochromatosis--homozygous carriers of C282Y mutation

Liver pathology was studied in 3 patients with primary chemochromatosis. In two cases so-called iron free foci with signs of hepatocytes with feature of dysplasia were found. Many siderosomes were found ultrastructurally in the cytoplasma of hepatocytes. Histological markers of virus infection were absent in a patient with positive serum HbsAg and HCV-Ab. Alcohol did not produce typical histological changes. In this case grave liver reticuloendothelial hemosiderosis typical for secondary hemochromatosis and overloading with iron of spleen pulp according to MR imaging were observed.     

Homozygosity for the predominant Cys282Tyr mutation and absence of disease expression in hereditary haemochromatosis.

A candidate gene for hereditary haemochromatosis, HLA-H, has recently been presented. Two missense mutations in the HLA-H gene sequence are predicted to account for nearly 90% of all cases of the disease. The aim of this study was to correlate the presence of these missense mutations with the expressivity of the disease, as assessed by standard biochemical evaluation of serum iron parameters. Detection of the known mutations in haemochromatosis, Cys282Tyr and His63Asp, was undertaken in a large pedigree showing variable expression of the disease in successive generations. In three sibs with overt disease (one male, two female, aged 50 to 53 years), homozygosity for the predominant G to A transition (Cys282Tyr) in HLA-H was detected. However, homozygosity for this mutation was also detected in an asymptomatic male sib, aged 50, harbouring an identical genotype. The finding of an asymptomatic homozygous Cys282Tyr subject, haplo-identical to affected sibs, indicates that clinical expression of symptomatic disease is variable, even in middle aged Cys282Tyr homozygotes. This has profound implications for the future use of genetic screening for haemochromatosis.     

The recently identified type 2A juvenile haemochromatosis gene (HJV), a second candidate modifier of the C282Y homozygous phenotype

The most common form of hereditary haemochromatosis is an adult-onset condition usually associated with the HFE C282Y/C282Y genotype. The phenotypic expression of this genotype is heterogeneous and depends on a complex interplay of genetic and non-genetic factors. The aim of the present study was to determine if mutations in the recently identified HJV gene were associated with more severe iron overload phenotypes in C282Y homozygous patients. From a cohort of 310 C282Y homozygous patients, we found nine (six males and three females) with an additional HJV missense mutation in the heterozygous state (S105L, E302K, N372D, R335Q or the previously described L101P and G320V). The iron indices of eight patients appeared to be more severe than those observed in C282Y homozygous patients of identical sex and similar age ranges. The mean serum ferritin concentration of the six males with an HJV mutation was significantly higher than that of C282Y homozygous males without an additional mutation [2350.3 (sigma=1429.9) versus 1227.2 (sigma=1130.1) microg/l; P=0.0233, Student's t-test]. We have recently reported that mutations in the gene that encodes hepcidin (HAMP) could explain one part of the C282Y/C282Y-related phenotypic heterogeneity by accentuating the iron burden. Our new data reveal that mutations in the HJV gene could be associated with a similar effect. Taken together, these results emphasize that a search for modifier genes could enable us to more precisely distinguish those C282Y homozygous patients with a higher risk to develop a severe iron overload and, consequently, clinical complications.     

A PCR-SSP method for detecting the Cys282Tyr mutation in the HFE gene associated with hereditary haemochromatosis.

Hereditary haemochromatosis is a common genetic disorder that causes hyperabsorption of dietary iron, leading to increased deposition and various organic diseases. Early diagnosis is important if effective treatment is to be applied and the iron overload corrected before the onset of clinical symptoms. Recently, a candidate gene has been identified in which a single point mutation shows a very close association with hereditary haemochromatosis. A polymerase chain reaction method using sequence specific primers (PCR-SSP) is described that, in conjunction with a simple DNA extraction method, would provide a specific diagnostic test or rapid screening procedure for this putative haemochromatosis associated mutation.     

The 16189 variant of mitochondrial DNA occurs more frequently in C282Y homozygotes with haemochromatosis than those without iron loading

Background:Patients with hereditary haemochromatosis (HH) are usually homozygous for the C282Y mutation in the HFE gene. They have variable expression of iron overload and present with a variety of complications, including liver disease, diabetes, arthropathy, fatigue, and cardiomyopathy. The mitochondrial 16189 variant is associated with diabetes, dilated cardiomyopathy, and low body fat at birth, and might contribute to genetic predisposition in further multifactorial disorders. The objective of this study was to determine the frequency of the 16189 variant in a range of patients with haemochromatosis, who had mutations in the HFE gene.

Methods:Blood DNA was analysed for the presence of the 16189 variant in British, French, and Australian C282Y homozygotes and controls, with known iron status, and in birth cohorts.

Results:The frequency of the mitochondrial 16189 variant was found to be elevated in individuals with haemochromatosis who were homozygous for the C282Y allele, compared with population controls and with C282Y homozygotes who were asymptomatic (42/292 (14.4%); 102/1186 (8.6%) (p = 0.003); and 2/64 (3.1%) (p = 0.023), respectively).

Conclusions:Iron loading in C282Y homozygotes with HH was exacerbated by the presence of the mitochondrial 16189 variant.

     

C282Y and H63D mutation of the hemochromatosis gene in German porphyria cutanea tarda patients

BACKGROUND AND AIMS: Patients with porphyria cutanea tarda (PCT) have a susceptibility to reversible inactivation of hepatocyte uroporphyrinogen decarboxylase, which can be triggered by alcohol, hepatitis C virus, and other agents. Inherited factors that may predispose to PCT include the C282Y mutation in the hemochromatosis (HFE) gene. METHODS: We analyzed the hemochromatosis mutations C282Y and H63D in liver biopsies and serum samples of 190 German patients (mean age 48+/-12.5 years) with sporadic PCT. The hepatic iron concentration was determined within the liver tissue. Age-matched healthy blood donors (115 donors) served as controls. RESULTS: The C282Y and H63D mutations were found in 75 (39%) and 85 (45%) of 190 patients with PCT, respectively. Twenty-two patients (12%) were homozygous for the C282Y mutation, and eighteen patients (9%) were compound heterozygotes, displaying both the C282Y and the H63D mutation. Within the control group, 3 of 115 patients were heterozygous for C282Y (3%) and 12 for H63D (10%). Serum and hepatic iron, ferritin, transferrin saturation, or liver enzymes did not differ significantly between patients with or without HFE mutations. CONCLUSIONS: The high frequency of homo- and heterozygosity for the C282Y and H63D alleles strongly suggests that these mutations are important predisposing factors for PCT in German patients.     

Elevated MCP-1 serum levels are associated with the H63D mutation and not the C282Y mutation in hereditary hemochromatosis

Monocyte chemoattractant protein-1 (MCP-1) is a major lymphocyte and inflammatory chemokine associated with persistent inflammatory states. Several abnormalities in the immune status of patients with hereditary hemochromatosis (HH) have been reported, suggesting an imbalance in their immune function. This may include persistent production of, or exposure to, inflammatory cytokines contributing to the pathogenesis of this disorder. The aim of this study was to assess MCP-1 levels in patients with HH and correlate these results with HFE status and iron indexes. One hundred and thirty-nine subjects diagnosed with HH (C282Y homozygotes = 87, C282Y/H63D = 26 heterozygotes, H63D homozygotes = 26), 27 healthy control subjects with no HFE mutation (N/N), and 18 normal subjects heterozygous for the H63D mutation served as age- and sex-matched controls. Ferritin and transferrin saturation and the presence of HFE mutation status were correlated with MCP-1 levels. Full white blood cell count analysis was also performed. We found a strongly significant decrease in MCP-1 protein levels in the C282Y homozygotes compared with the H63D homozygotes (P = 0.0009) and C282Y/H63D heterozygotes (P = 0.002). Similarly, MCP-1 protein levels in the C282Y homozygotes were decreased compared with the healthy controls (P = 0.00076). Furthermore, MCP-1 serum levels were elevated in H63D patients compared with the healthy controls (P = 0.0008). This study suggests for the first time that a differential expression of MCP-1 protein in patients with HH is associated with the specific HFE genetic component for iron overload. Therefore, these findings offer a possible explanation in the variable clinical spectrum of pathogenesis in patients with HH through abnormalities of an imbalance in the immune states of patients with HH.     

Prevalence of the C282Y and H63D polymorphisms in a multi-ethnic control population.

Recently a candidate gene for hereditary hemo-chromatosis, HFE, was identified. The finding raises the possibility for genetic testing to provide earlier detection and more complete genotypic evaluation of hemochromatosis affected individuals. We determined the frequency of the HFE polymorphisms, C282Y and H63D, in a randomly selected multi-ethnic control population for establishment of a hemochromatosis genetic testing program. Prevalence was determined by PCR amplification and restriction enzyme digestion of HFE in 100 Caucasians, 100 Hispanics, and 56 African Americans. Heterozygosity for C282Y was detected in 8% of Caucasians, 3% of Hispanics, and 2% of African Americans. Homozygosity for C282Y was detected in 1% of Caucasians. Heterozygosity for H63D was detected in 24% of Caucasians, 15% Hispanics, and 3.5% of African Americans. Homozygosity for H63D was present in 4% of Caucasians and 1% of Hispanics. One Hispanic case was double heterozygous for C282Y and H63D. These results indicate the highest prevalence of C282Y and H63D in the Caucasian population. Additionally, we demonstrate C282Y and H63D polymorphisms in our Hispanic and African American populations, groups in which prevalence rates remain less defined. Our results support the need for thorough interpretation of genetic results for hereditary hemochromatosis in various ethnic populations.     

Simultaneous detection of HFE C282Y, H63D and S65C mutations associated with type 1 haemochromatosis using a multiplex luminex bead assay

Type 1 hereditary haemochromatosis (HH) is a common genetic disorder in Caucasoids resulting from mutations in the HFE gene. Routine diagnostic testing for type 1 HH involves genotyping for two of these described HFE mutations, C282Y and H63D. In some cases typing of a third mutation, S65C is also performed. Several techniques have been reported for HFE genotyping and these include polymerase chain reaction (PCR)-sequence-specific primers (SSP), PCR-restriction fragment length polymorphism (RFLP), PCR-sequence-specific oligonucleotide probe (SSOP), real-time PCR followed by melting curve analysis and TaqMan assay. The aim of this study was to develop an alternative method to both conventional PCR and real-time PCR/TaqMan assay to detect all three HFE mutations in a single assay using Luminex technology. DNA controls of known genotypes (n = 109) were used to evaluate this approach. These controls were selected to represent the three possible genotypes (wild type, mutant, heterozygous) for each mutation. Subsequently, blind DNA samples (n = 100) were used to validate this method. This new assay was then compared with current techniques (in-house PCR-SSP and TaqMan assay). Comparison of genotypes obtained with the Luminex method with those previously reported by both in-house PCR-SSP and TaqMan assay showed 100% concordance for both DNA controls and blind DNA samples and no discrepancies were observed. Allelic frequency for C282Y, H63D and S65C mutations were 22%, 16% and 2%, respectively. We report here a high-throughput, accurate and robust multiplex luminex bead assay for routine clinical testing of C282Y, H63D and S65C mutations in the HFE gene.     

Synergy between the C2 allele of transferrin and the C282Y allele of the haemochromatosis gene (HFE) as risk factors for developing Alzheimer's disease

Background: There is evidence that iron may play a role in the pathology of Alzheimer's disease (AD). There may be genetic factors that contribute to iron deposition resulting in tissue damage thus exacerbating AD.

Methods: We have genotyped 269 healthy elderly controls, 191 cases with definite or probable AD, and 69 with mild cognitive impairment (MCI) from the OPTIMA cohort.

Results: We have examined the interaction between the C2 variant of the transferrin (TF) gene and the C282Y allele of the haemochromatosis (HFE) gene as risk factors for developing AD. Our results showed that each of the two variants was associated with an increased risk of AD only in the presence of the other. Neither allele alone had any effect. Carriers of both variants were at 5 times greater risk of AD compared with all others. The interaction was significant by logistic regression (p = 0.014) and by synergy factor analysis (p = 0.015, synergy factor = 5.1). Further, carriers of these two alleles plus apolipoprotein E ε4 (APOE4) were at still higher risk of AD: of the 14 tri-carriers of the three variants, identified in this study, 12 had AD and two MCI.

Conclusion: We suggest that the combination of TF C2 and HFE C282Y may lead to an excess of redox-active iron and the induction of oxidative stress in neurones, which is exacerbated in carriers of APOE4. Since 4% of Northern Europeans carry the two iron-related variants and since iron overload is a treatable condition, these results merit replication.

     

A hemochromatosis-causing mutation C282Y is a risk factor for proliferative diabetic retinopathy in Caucasians with type 2 diabetes

Iron metabolism might be involved in the pathogenesis of type 2 diabetes and in the pathogenesis of diabetic retinopathy. C282Y and H63D mutations in the hemochromatosis (HFE) gene are associated with increased serum iron levels and consequently with hereditary hemochromatosis. In the present study, we searched for a relationship between C282Y and H63D gene mutations and the development of proliferative diabetic retinopathy in Caucasians with type 2 diabetes. For this purpose, 90 subjects with type 2 diabetes with proliferative diabetic retinopathy (PDR) were compared to 133 diabetic subjects without PDR. There was a significantly higher frequency of the C282Y heterozygotes in patients with PDR compared to subjects without it (OR=3.0, 95% CI=1.2–8.0; p=0.02), whereas no association was demonstrated between PDR and H63D genotypes (OR=1.1, 95% CI=0.6–2.2; p=0.7). Logistic regression analysis revealed that the C282Y mutation was a significant independent risk factor for the development of PDR (OR=6.1, 95% CI=1.2–30.5; p=0.027). These data suggest that heterozygosity for C282Y might be a novel risk factor for PDR in Caucasians with type 2 diabetes.     

The C282Y mutation may have been positively selected as it mitigates the infertility of celiac disease

Celiac disease and C282Y homozygous hemochromatosis have a similar increasing incidence across Europe. Both have gradients of frequency which increase from Turkey to North West Europe and culminate with a high frequency in Ireland. These two gradients follow the path of the Neolithic settlers who reached the edge of Europe at Ireland. Celiac disease and C282Y hereditary hemochromatosis have opposite effects on iron absorption and probably on the absorption of some other divalent metals including copper. The C282Y mutation is estimated to be some 2000 years old. Celiac disease is likely a much older disorder. The C282Y mutation may have been positively selected for as it increases absorption of divalent metal ions in celiac disease and thus has a mitigating effect on the infertility which may be associated with celiac disease.     

HFE gene mutation, C282Y causing hereditary hemochromatosis in Caucasian is extremely rare in Korean population

Hereditary hemochromatosis (HFE), which affects 1 in 400 and has an estimated carrier frequency of 1 in 10 individuals in Western population, results in multiple organ damage caused by iron deposition, and is treatable if detected early. C282Y mutation in HFE gene has been known to be responsible for the most hereditary hemochromatosis cases and 5-10% of white subjects are heterozygous for this mutation. However, the prevalence of hemochromatosis in the Asian population was reported to be very low and ethnic heterogeneity has been suspected. The aim of our study was to determine the prevalence of heterozygosity and homozygosity for the C282Y HFE gene mutations in 502 unrelated Koreans. Results revealed that none of them had the mutant gene, suggesting a significant ethnic difference when compared with Caucasians. Our study excluded underlying possibility of hereditary hemochromatosis in Korean which could mimic the findings of alcoholic liver disease with iron overload or liver cirrhosis with chronic hepatitis C.