Prevalence of the C282Y, H63D, and S65C mutations of the HFE gene in 1,146 newborns from a region of Northern Spain

In Spain, 85% of patients with genetic hemochromatosis (GH) are homozygous for the C282Y mutation of the HFE gene. H63D and S65C mutations of HFE may also play some role in the disease. The aim of this study was to establish the prevalence of C282Y, H63D, and S65C mutations of the HFE gene in newborns in Catalonia, Spain. One thousand one hundred forty-six newborn screening cards were selected randomly. DNA from these cards was extracted and HFE mutations were analyzed with the LightCycler equipment (Roche Diagnostics Gmbh, Mannheim, Germany). Sufficient DNA sample was obtained to screen for the three mutations in 1,043 cases (91%). The allelic frequencies of C282Y, H63D, and S65C mutations were 0.03 (IC 95% 0.022-0.037), 0.2 (IC 95% 0.19-0.22), and 0.01 (95% confidence interval [CI] 0.006-0.015), respectively. The frequency of C282Y homozygous newborns was 0.001 (95% CI 0.0005-0.0014). The frequencies of newborns doubly heterozygous for C282Y/H63D and C282Y/S65C were 0.01 (95% CI 0.005-0.02) and 0.002 (95% CI 0.0002-0.01), respectively. The allelic frequency of C282Y mutation is similar to that observed in Southern France, in the Czech Republic and in some areas of Italy. The allelic frequency of H63D mutation in Catalonia is the highest reported to date. Nevertheless, S65C is infrequent. These data should be kept in mind when designing hemochromatosis genotypic screening programs in Catalonia.     

Frequency of hemochromatosis gene (HFE) mutations in Russian healthy women and patients with estrogen-dependent cancers

Possible association between the C282Y and H63D mutations in the HFE gene and estrogen-dependent cancer risk was assessed. Genotyping was performed using PCR amplification followed by digestion of products with specific restrictases. In a population of 260 healthy women (permanent residents of the southwest European Russia), mutant allele frequencies at the C282Y and H63D sites were evaluated as 3.3 and 16.3%, respectively. In patients with breast, ovarian, and endometrial cancer, C282Y frequencies were also low (1.0, 1.3, and 3.8%, respectively), and no cancer risk associated with the C282Y mutation was found. Odds ratios for breast cancer risk associated with the H63D mutation increased significantly with age: 0.5 in women below 48 years old, 1.0 in a range of 48-57 years, and 4.4 in older women (P(trend)=0.002). The latter value was statistically significant (95% CI, 1.4-14.1), indicating that women bearing the H63D mutation may be at an increased breast cancer risk at an age above 57 years. Preliminary results obtained in patients with two other estrogen-dependent malignancies revealed the same tendency to OR increase with age in ovarian cancer patients (P(trend)=0.008), but no age-related OR differences in endometrial cancer patients.     

HFE genotype frequencies in consecutive reference laboratory specimens: comparisons among referral sources and association with initial diagnosis

We quantified HFE genotype frequencies in specimens submitted by physicians grouped by specialty and determined associations of genotypes with initial diagnosis based on phenotyping in patients evaluated at an iron disorders center. Of 526 specimens (519 from Alabama), these "typical" hemochromatosis-associated genotypes were detected: 85 C282Y/C282Y, 50 C282Y/H63D, and 27 H63D/H63D. Respective frequencies of C282Y/C282Y in specimens from an iron disorders center (n = 156), gastroenterologists (n = 147), hematologists/medical oncologists (n = 85), liver transplant surgeons (n = 11), endocrinologists and rheumatologists (n = 9), and "other sources" (n = 7) were greater (p < 0.05) than in population controls. In 44 patients from an iron disorders center initially diagnosed as "presumed hemochromatosis," 27 (61.4%) had C282Y/C282Y, 10 (22.7%) had C282Y/H63D, and 3 (6.8%) had H63D/H63D. C282Y/C282Y was not detected in 48 patients with "abnormality probably not an iron overload disorder." A total of 20.5% of 44 family members of patients had "typical" hemochromatosis-associated HFE genotypes (7.0% controls; p = 0.02). We conclude that most physicians who submitted specimens identify patients by phenotyping who have greater frequencies of "typical" hemochromatosis-associated HFE genotypes than controls, and that HFE mutation testing is useful in detecting hemochromatosis in family members of persons with hemochromatosis or iron overload.     

The overlapping of local iron overload and HFE mutation in venous leg ulcer pathogenesis

Chronic venous stasis determines red blood cell extravasation and either dermal hemosiderin deposits or iron-laden phagocytes. Several authors have suspected that iron could play a role in the pathogenesis of venous leg ulcers. They hypothesized that local iron overload could generate free radicals or activate a proteolytic hyperactivity on the part of metalloproteinases (MMPs) or else down-regulate tissue inhibitors of MMPs. However, they were unable to explain why iron deposits, visible in the legs of patients with chronic venous disease (CVD), cause lesions in only some individuals, whereas in others they do not. We hypothesized that such individual differences could be genetically determined and investigated the role of the C282Y and H63D mutations of the HFE gene. C282Y mutation significantly increases the risk of ulcer in primary CVD more than six times (OR = 6.69; 1.45-30.8; p = 0.01). Patients carrying the H63D variant have an earlier age of ulcer onset, by almost 10 years (p > 0.004). The increased risk of skin lesion and the early age of onset of the disease in HFE carriers confirm in a clinical setting that intracellular iron deposits of mutated macrophages have less stability than those of the wild type. We hypothesize that the physiologic iron protective mechanisms are affected by the HFE mutations and should be investigated in all diseases characterized by the combination of iron overload and inflammation.     

Frequency of HFE gene mutations C282Y and H63D in Bosnia and Herzegovina

Genetic epidemiology studies of hereditary hemochromatosis (HHC) have shown a high prevalence of the C282Y mutation in individuals of the North Western European origin, whereas lower prevalence of HFE gene mutations was detected in the populations from southern European countries. However, no HFE mutation prevalence data have been provided for the population of Bosnia-Herzegovina so far. Therefore, the aim of this study was to determine the frequency of the C282Y and H63D HFE gene mutations in the population of Bosnia-Herzegovina. Among 200 analysed subjects 8 (4%) were C282Y heterozygotes; no C282Y homozygotes were found. The frequency of the H63D allele was 11.5%. There were 33 (16.5%) heterozygotes and 6 (3%) homozygotes for the H63D mutation. One (0.5%) compound heterozygote C282Y/H63D was identified. The observed C282Y and H63D allele frequency was 2.25% (95% confidence interval: 1.2-4.2) and 11.5% (95% confidence interval: 8.7-14.9), respectively. The prevalence of the C282Y and H63D mutations was estimated in Bosnia-Herzegovina, which fit well in the European northwest-to-southeast gradient of the C282Y mutation distribution. In addition, these results have an important implication for clinical evaluation of HHC in Bosnia-Herzegovina.     

The risk of new‐onset cancer associated with HFE C282Y and H63D mutations: evidence from 87,028 participants

To investigate the association between mutation of HFE (the principal pathogenic gene in hereditary haemochromatosis) and risk of cancer, we conducted a meta‐analysis of all available case–control or cohort studies relating to two missense mutations, C282Y and H63D mutations. Eligible studies were identified by searching databases including PubMed, Embase and the ISI Web of Knowledge. Overall and subgroup analyses were performed and odds ratios (ORs) combined with 95% confidence intervals (CIs) were applied to evaluate the association between C282Y mutation, H63D mutation and cancer risk. Sensitivity and cumulative analyses were used to evaluate the stability of the results. A total of 36 eligible studies were included, comprising 13,680 cases and 73,348 controls. C282Y was significantly associated with elevated cancer risk in a recessive genetic model (OR: 1.991, 95% CI: 1.448–2.737). On subgroup analysis stratified by cancer type, statistically significantly increased cancer risks were found for breast cancer, colorectal cancer and hepatocellular carcinoma in a recessive model. When stratified by territory, a significantly increased risk of cancer was found in Oceanic populations in a recessive model and in Asian populations in an allele model and dominant model. H63D mutation did not significantly increase overall cancer risk in any genetic model. However, when, stratified by territory, an increased cancer risk was found in the Asian population in an allele and dominant. C282Y but not H63D mutation was related to elevated cancer risk. Further large‐scale studies considering gene–environment interactions and functional research should be conducted to further investigate this association.     

Initial screening transferrin saturation values, serum ferritin concentrations, and HFE genotypes in whites and blacks in the Hemochromatosis and Iron Overload Screening Study

We compared initial screening data of 44,082 white and 27,124 black Hemochromatosis and Iron Overload Screening (HEIRS) Study participants. Each underwent serum transferrin saturation (TfSat) and ferritin (SF) measurements without regard to fasting, and HFE C282Y and H63D genotyping. Elevated measurements were defined as: TfSat more than 50% (men), more than 45% (women); and SF more than 300 ng/ml (men), more than 200 ng/ml (women). Mean TfSat and percentages of participants with elevated TfSat were significantly greater in whites than in blacks. Mean SF and percentages of participants with elevated SF were significantly greater in blacks than in whites. TfSat and SF varied by gender and age in whites and blacks. Prevalences of genotypes that included either C282Y or H63D were significantly greater in whites than in blacks. The prevalence of elevated TfSat and SF plus genotypes C282Y/C282Y, C282Y/H63D, or H63D/H63D was 0.006 in whites and 0.0003 in blacks. Among whites with HFE C282Y homozygosity, 76.8% of men and 46.9% of women had elevated TfSat and SF values. Three black participants had HFE C282Y homozygosity; one had elevated TfSat and SF values. Possible explanations for differences in TfSat and SF in whites and blacks and pertinence to the detection of hemochromatosis, iron overload, and other disorders with similar phenotypes are discussed.     

Prevalence of C282Y and H63D mutations in the haemochromatosis (HFE) gene in Tunisian population

The studies of the HFE mutations: H63D and C282Y in North African populations have revealed the extreme rarity or even the absence of the C282Y mutation. We have examined 1140 chromosomes (570 Tunisian people) for the presence of the two HFE mutations by PCR-RFLP analysis. We have found that the allele frequencies are, respectively, 15.17% (+/-2.1%) for the H63D and 0.09% (+/-0.17%) for the C282Y. These results are consistent with the worldwide spread of the H63D mutation and the north European restriction of the C282Y. This study will be completed by determining whether homozygote trait for H63D and associated risk factors (beta thalassémia) can lead to iron overload in Tunisia.     

Genetic hemochromatosis, a Celtic disease: is it now time for population screening?

In populations of northern European ancestry, hereditary hemochromatosis (HH) is tightly linked to mutations within the hemochromatosis gene (HFE gene). Over 93% of Irish HH patients are homozygous for the HFE gene C282Y mutation, providing a reliable diagnostic marker of the disease in this population. However, the prevalence of the C282Y mutation and that of the second HFE gene mutation, H63D, have yet to be determined within the Irish population. The objective of this study was to identify the true prevalence of the genetic form of HH in the Irish population. DNA was extracted from 1002 randomly selected newborn screening cards and analyzed for the C282Y and H63D mutations within the HFE gene. Complete results were obtained from 800 cards. Mutations were identified in 364 (46%) neonates. Eight (1%) neonates were homozygous for C282Y and 8 (1%) were homozygous for H63D. One hundred and fifty-five (19%) neonates were C282Y heterozygous and 226 (28%) were H63D heterozygous. Of these, 33 (4%) carried one copy of both C282Y and H63D mutations, i.e., compound heterozygous. Allele frequencies for C282Y and H63D were 11% and 15%, respectively. The high C282Y allele frequency in the Irish population together with its close linkage to HH indicate that C282Y genotyping is the preferred screening strategy for this disease in Ireland.     

Effects of HFE C282Y and H63D polymorphisms and polygenic background on iron stores in a large community sample of twins

The aim of this study was to assess and to compare the role of HFE polymorphisms and other genetic factors in variation in iron stores. Blood samples were obtained from 3,375 adult male and female twins (age range 29-82 years) recruited from the Australian Twin Registry. There were 1,233 complete pairs (562 monozygotic and 571 dizygotic twins). Serum iron, transferrin, transferrin saturation with iron, and ferritin were measured, and the HFE C282Y and H63D genotypes were determined. The frequency of the C282Y allele was.072, and that of the H63D allele was.141. Significant sources of variation in the indices of iron status included age, sex, age-sex interaction, body-mass index, and both the C282Y and H63D genotypes. The iron, transferrin, and saturation values of CC and CY subjects differed significantly, but the ferritin values did not. After correction for age and body-mass index, 23% and 31% of the variance in iron, 66% and 49% of the variance in transferrin, 33% and 47% of the variance in transferrin saturation, and 47% and 47% of the variance in ferritin could be explained by additive genetic factors, for men and women, respectively. HFE C282Y and H63D variation accounted for <5% of the corrected phenotypic variance, except for saturation (12% in women and 5% in men). We conclude that HFE CY and HD heterozygotes differ in iron status from the CC and HH homozygotes and that serum transferrin saturation is more affected than is serum ferritin. There are highly significant effects of other as-yet-unidentified genes on iron stores, in addition to HFE genotype.     

HFE alleles in an Irish cystic fibrosis population

The variable clinical manifestations of cystic fibrosis (CF) suggest the influence of modifier genes. Genetic and environmental factors that determine whether an individual will develop associated complications are still being determined. It has been proposed that the gene for hemochromatosis, HFE, may be a modifier locus for CF disease phenotype. Recent research has suggested a relationship between mutations to the HFE gene and the development of meconium ileus (MI) and liver disease in CF. This study aims to expand our knowledge of the HFE mutations C282Y and H63D carrier rate in an Irish population of CF allele carriers. PCR restriction enzyme analysis was performed on blood samples from CF patients to identify the C282Y and H63D mutations. HFE status of CF allele carriers and CF patients (Delta F508) homozygotes with and without meconium ileus was determined. The carrier frequency for C282Y was 30.8% for the Delta F508 homozygote MI positive group, as compared to 12.5% for the non-Delta F508 MI positive group but did not reach statistical significance (p = 0.27). Interestingly, no Delta F508 homozygote patients were homozygous for the C282Y mutation.     

Lisinopril attenuates renal oxidative injury in l-NAME-induced hypertensive rats

Genetic factors related to cancer have been extensively studied and several polymorphisms have been associated to breast cancer. The FGFR4, MTHFR, and HFE genes have been associated with neoplastic diseases development. The current report outlines the analysis of the polymorphisms G388A (FGFR4), C677T (MTHFR), C282Y, and H63D (HFE) in Brazilian breast cancer patients. We studied 68 patients with invasive ductal and operable breast carcinoma and 85 women as a control group. The polymorphism frequencies in the breast cancer and control groups were analyzed, but no significant difference was observed by comparing the two groups. The presence of each polymorphism was analyzed according to the clinical features and markers already established as prognostic in the breast cancer group. The C677T, H63D, and G388A polymorphisms were not associated to histological grade, age of diagnosis, expression of HER2 receptor, or estrogen and progesterone receptor. The H63D polymorphism showed a significant association (P = 0.02) with the presence of p53 mutations, and C667T showed association to lymph node involvement (P = 0.05). Lymph node involvement, G388A polymorphism, and histological grade were independently associated to metastasis/death. Our data suggests that the polymorphisms G388A, C677T, and H63D are not useful in breast cancer diagnosis, but they may be significant additional prognostic markers related to breast cancer survival.     

Hereditary hemochromatosis in Spain

The C282Y mutation of the HFE gene has been reported as the main cause of hereditary hemochromatosis (HH). Another missense mutation (H63D) has also been detected at an increased frequency in a compound heterozygote state with the C282Y mutation in HH patients. However, these two mutations are not present in all of the HH patients, indicating that other mutations in the HFE gene, or in other loci, should exist. The present study reports the frequencies of the C282Y and H63D mutations in 74 Spanish HH patients and the results of the sequencing analysis of the HFE exons, intron-exon boundaries, and 588 bp of the 5' region in 5 patients negative for the C282Y mutation. We have detected a high frequency of the C282Y mutation (85.1%) in Spanish HH patients, indicating that this mutation is the most common defect associated with the disease in Spain. The screening of the HFE regions in our patients without the C282Y mutation has revealed the presence of five polymorphisms. However, no other pathological mutations have been found. Therefore, further efforts to characterize the unscreened part of the HFE gene or other loci should be taken to identify the potential genetic factors causing HH in the C282Y-negative patients.     

Hereditary hemochromatosis in a Brazilian university hospital in São Paulo State (1990-2000)

Hereditary hemochromatosis (HH) is the most common genetic disease among individuals of European descent. Two mutations (845G-->A, C282Y and 187C-->G, H63D) in the hemochromatosis gene (HFE gene) are associated with HH. About 85-90% of patients of northern European descent with HH are C282Y homozygous. The prevalence of HH in the Brazilian population, which has a very high level of racial admixture, is unknown. The aims of the present study were to identify individuals with diagnostic criteria for HH among patients with a body iron overload attended at the university hospital of the Faculty of Medicine of Ribeirao Preto from 1990 to 2000, and to evaluate the prevalence of HFE mutations. We screened first-degree relatives for HFE mutations. Four of 72 patients (three men and one woman, mean age 47 years) fulfilled the criteria for HH. HFE mutations were studied in three patients [two C282Y homozygotes (patients 1 and 2) and one H63D heterozygote]. Patient 1 had four children (all C282Y heterozygotes with no iron overload) and seven brothers and sisters: two sisters (66 and 76 years old) were C282Y homozygotes and both had an iron overload (a liver biopsy in one showed severe iron deposits), one sister (79 years old) was a compound heterozygote with no iron overload, one brother (78 years old) was a C282Y heterozygote with no iron overload, two individuals were H63D heterozygotes (one brother, 49 years old, obese, with a body iron overload and abnormal liver enzymes - a biopsy showed non-alcoholic steatohepatitis, and one 70-year-old sister with no iron overload). Patient 2 had two children (22 and 24 years old who were C282Y heterozygotes with no iron overload) but no brothers or sisters. These results showed that HH was uncommon among individuals attended at our hospital, although HFE mutations were found in all patients. Familial screening is valuable for the early diagnosis of individuals at risk since it allows treatment to be initiated before the onset of the clinical manifestations of organ damage associated with HH.     

Polymorphism in the Matrix Metalloproteinase-2 Gene Promoter is Associated with Cervical Neoplasm Risk in Mexican Women

Matrix metalloproteinase-2 (MMP-2) is an enzyme with proteolytic activity on matrix proteins, and previous studies have revealed a strong association between the MMP-2 −1306C→T polymorphism and the risk of several types of cancer. Our study looked at whether this polymorphism contributed to the development of cervical neoplasia by analyzing 54 patients with invasive squamous cell cervical cancer, 100 patients with cervical intraepithelial neoplasia, and 126 control subjects. The MMP-2 CC genotype was more frequent in the cancer patients when compared with the control group (OR 2.57; 95% CI 1.15–5.86). The association of cervical cancer with the CC genotype was more pronounced in women who had first coitus at an early age (OR 3.96; 95% CI 1.46–11.06). The CC genotype was associated with intraepithelial neoplasia only in women with first coitus at 19 years old or younger. The data suggest that the MMP-2 −1306C→T polymorphism contributes to the development of squamous cell cervical cancer in the population studied, especially in women who had first coitus at an early age.     

A primer for predicting risk of disease in HFE-linked hemochromatosis

Since the discovery of the hemochromatosis gene (HFE) in 1996, there has been increasing interest in diagnostic testing for the C282Y and H63D mutations. The high frequency of these two alleles and their incomplete penetrance in homozygotes and compound heterozygotes make genetic counseling for hemochromatosis different from some other autosomal recessive conditions in that parents and children may also be at risk for iron overload, while homozygotes may remain asymptomatic. We provide a guideline for genetic counseling in HFE-linked hemochromatosis based on the genetic probability of inheriting HFE mutations and known information about expression of iron overload in various HFE genotypes. Genetic probabilities were based on allele frequencies derived from large population studies and Hardy-Weinberg equilibrium estimates. Expression of iron overload in those of various genotypes was based on available estimates of serum ferritin from population screening studies. Estimates for the likelihood of clinical iron overload requiring follow-up screening or treatment are provided by gender and genotype. The probability of inheriting HFE mutations and developing iron overload can be estimated in family members of a proband with HFE mutations. Many C282Y homozygotes will not have clinical iron overload. The risk is highest in men and their C282Y homozygous brothers and significantly lower in homozygous women. Iron overload is uncommon in compound heterozygotes and H63D homozygotes.     

Population screening for hemochromatosis by PCR using sequence-specific primers

Over 90% of patients with hemochromatosis in the United Kingdom are homozygous for the C282Y mutation on the HFE gene. The Centers for Disease Control (CDC) in the United States has recommended that adults should be screened for HFE mutations to identify susceptible individuals before onset of disease. The aim of this study was to evaluate the polymerase chain reaction using sequence-specific primers (PCR-SSP) as a method of large-scale population screening for the common HFE gene mutations, H63D and C282Y. A total of 10,583 consenting blood donors were tested using nonautomated procedures. Three alleles, termed HFE-1, -2, and -3, were detected with phenotype frequencies of 94.56%, 28.33%, and 15.79%, respectively, and gene frequencies of 0.76421, 0.15342, and 0.08237, respectively. All donors identified as homozygous for the C282Y mutation or heterozygous for both the H63D and C282Y mutations were confirmed by heterduplex analysis and/or PCR-SSP. The number of technical failures that affected the identification of donors homozygous for the C282Y mutation was 390 giving an overall repeat rate 3.7%, although this fell to 1% over the last quarter of the study. This study demonstrates that PCR-SSP may be used for large-scale population screening for the C282Y genotype associated with hemochromatosis.     

The effects of wild-type and mutant HFE expression upon cellular iron uptake in transfected human embryonic kidney cells

In order to measure the effects of HFE (haemochromatosis) upon iron uptake, stable expression of wild-type and C282Y, H63D and S65C mutant HFE cDNA was established in HEK 293 cells. Control cells were transfected with empty vector. Expression of HFE mRNA and protein was detected in the cell lines transfected with HFE cDNA, but not in the control cell line. The ferritin concentration in wild-type cells cultured in 40 microM ferric ammonium citrate was 69% of that in control cells and 81% of that in C282Y cells. The ferritin concentration in H63D cells was intermediate between wild-type and C282Y and the ferritin concentration in S65C cells was similar to wild-type cells. Uptake of transferrin-iron in wild-type, C282Y and control cells was measured over 45 min. The Hill coefficients for transferrin-iron uptake were similar. The V(max) for transferrin-iron uptake in wild-type cells was 59.5% of control cells and 69.5% of C282Y cells. Estimates of K(m) were 232 nM for wild-type cells, 338 nM for C282Y cells and 570 nM for controls. Transferrin receptor levels were lowered, but not significantly, in the HFE transfected cells. The results show that HFE reduces transferrin-iron uptake, probably as an uncompetitive inhibitor.     

CCR2 and CCR5 genes polymorphisms in women with cervical lesions from Pernambuco,Northeast Region of Brazil: a case-control study

Polymorphisms in chemokine receptors play an important role in the progression of cervical intraepithelial neoplasia (CIN) to cervical cancer (CC). Our study examined the association of CCR2-64I (rs1799864) andCCR5-Δ32 (rs333) polymorphisms with susceptibility to develop cervical lesion (CIN and CC) in a Brazilian population. The genotyping of 139 women with cervical lesions and 151 women without cervical lesions for the CCR2-64I and CCR5-Δ32 polymorphisms were performed using polymerase chain reaction-restriction fragment length polymorphism. The individuals carrying heterozygous or homozygous genotypes (GA+AA) for CCR2-64I polymorphisms seem to be at lower risk for cervical lesion [odds ratio (OR) = 0.37, p = 0.0008)]. The same was observed for the A allele (OR = 0.39, p = 0.0002), while no association was detected (p > 0.05) with CCR5-Δ32 polymorphism. Regarding the human papillomavirus (HPV) type, patients carrying the CCR2-64Ipolymorphism were protected against infection by HPV type 16 (OR = 0.35, p = 0.0184). In summary, our study showed a protective effect ofCCR2-64I rs1799864 polymorphism against the development of cervical lesions (CIN and CC) and in the susceptibility of HPV 16 infection.