Frequency of common HFE variants in the Saudi population: a high throughput molecular beacon-based study

Background  

Hereditary Hemochromatosis (HH) is an autosomal recessive disorder highlighted byiron-overload. Two popular mutations in HFE, p.C282Y and p.H63D, have been discovered and found to associate with HH in different ethnic backgrounds. p.C282Y and p.H63D diagnosis is usually made byrestriction enzyme analysis. However, the use of this technique is largelylimited to research laboratories because they are relativelyexpensive, time-consuming, and difficult to transform into a high throughput format.     

Prevalence of H63D,S65C and C282Y hereditary hemochromatosis gene mutations in Slovenian population by an improved high-throughput genotyping assay

Background  

Hereditary hemochromatosis (HH) is a common genetic disease characterized by excessive iron overload that leads to multi-organ failure. Although the most prevalent genotype in HH is homozygosity for C282Y mutation of the HFE gene, two additional mutations, H63D and S65C, appear to be associated with a milder form of HH. The aim of this study was to develop a high-throughput assay for HFE mutations screening based on TaqMan technology and to determine the frequencies of HFE mutations in the Slovenian population.     

DNA testing for haemochromatosis: diagnostic, predictive and screening implications

Since 1996, the identification of the HFE gene has enabled DNA testing for hereditary haemochromatosis (HH). The range of DNA testing available includes: (1) diagnostic, (2) predictive (also called presymptomatic testing) and (3) screening. Access to DNA testing has been facilitated by an Australian Medicare rebate, the first available for genetic disorders. Despite the availability of HFE DNA testing in HH, it remains necessary to interpret results in the context of the clinical picture. Traditional markers based on phenotype (transferrin ferritinsaturation, and liver biopsy) are still required in some circumstances. We report our experience with HFE DNA testing using a semi-automated approach, which allows multiplexing for the two common mutations (C282Y and H63D). Screening a cohort of beta-thalassaemia major and sickle cell anaemia patients of predominantly Mediterranean origin showed that these individuals do not have the common C282Y mutation. This excluded C282Y as a factor in the pathogenesis of iron overload in these haemoglobinopathies. It also showed that the C282Y mutation is of limited value when investigating HH in certain ethnic groups. An Australian family studied illustrated the relative contribution of C282Y and H63D in iron overload. A recently reported third mutation (S65C) in the HFE gene was detected in a low frequency in the populations tested.     

Iron‐overload and genotypic expression of HFE mutations H63D/C282Y and transferrin receptor Hin6I and BanI polymorphism in German patients with hereditary haemochromatosis

Gene variations of HFE, a HLA‐class I like molecule, are highly associated with hereditary haemochromatosis (HH). Functional as well as molecular studies of the HFE protein have indicated that the molecule is involved in iron metabolism and that the HFE gene variations observed among HH patients affect its interaction with the transferrin receptor (TfR). In the present study, we have therefore analysed the relationship between the HFE gene variants, C282Y and H63D, and body iron status among 85 German HH patients. In addition, two TfR gene polymorphism, TfR‐Hin6I and TfR‐BanI, were typed that have been reported to define ethnically distinct haplotypes. As controls we used 251/159 healthy German blood donors. Seventy‐eight (92%) patients were C292Y homozygous, the H63D mutation was present in five (6%) patients with none of the patients being H63D homozygous. Serum transferrin, transferrin saturation and liver iron content were determined prior to therapeutic intervention. Among C282Y homozygous patients serum ferritin levels (2294 ± 3174 vs. 463 ± 224 µg L^?1, P < 0.0001) and transferrin saturation (86 ± 18% vs. 62 ± 25%, P = 0.048) were elevated significantly compared with C282Y and/or H63D heterozygous patients. In addition, the liver iron content (291 ± 165 vs. 138 ± 95 µmol g^?1, P = 0.028) and liver iron index (6.4 ± 2.8 vs. 3.2 ± 2.3, P = 0.019) were increased among C282Y homozygotes compared with C282Y heterozygotes. In contrast, no difference was observed between patients and controls regarding the distribution of TfR‐Hin6I and TfR‐BanI alleles. These data indicate that the iron intake is higher among C282Y homozygous patients compared with C282Y heterozygous or C282Y/H63D compound heterozygous individuals and supports the functional role of the HFE protein in iron metabolism whereas the TfR gene variants seem to have no influence on iron uptake.     

Impact of <Emphasis Type="Italic">HFE</Emphasis>genetic testing on clinical presentation of hereditary hemochromatosis: new epidemiological data

Background  

Hereditary hemochromatosis (HH) is a common inherited disorder of iron metabolism in Northern European populations. The discovery of a candidate gene in 1996 (HFE), and of its main mutation (C282Y), has radically altered the way to diagnose this disease. The aim of this study was to assess the impact of the HFE gene discovery on the clinical presentation and epidemiology of HH.     

Heteroduplex analysis for the three common HFE variants: methodology, reliability and analysis of over 5000 requests for testing

Objective

To describe the analysis of over 5300 patient samples for the HFE genotype.

Methods

Blood samples received from hospitals in England, Wales and Ireland were analysed with a single, multiplex PCR using heteroduplex generators for the C282Y, H63D and S65C variants of the HFE gene. PCR products labelled with fluorescent dyes were analysed by capillary electrophoresis. Genotype frequencies were analysed according to the reasons given for testing.

Results

Analysis of 400 samples sent in duplicate revealed one error that was associated with reporting rather than the methodology. Of 5327 samples received, 1122 were for family testing, 2470 for diagnostic testing and in 1735 cases no reason was given. Overall, homozygosity for C282Y was found in 14% of samples received for family testing and in 16% of the remaining samples. Clinical indications such as “liver disease” were of little predictive value for homozygosity for C282Y, but this increased if a raised serum ferritin concentration or transferrin saturation was indicated. When the diagnosis was iron overload, 39% of subjects tested were homozygous for C282Y. Compound heterozygosity (C282Y/H63D) was more frequent than in the general population but the frequency was not further increased in subjects for whom there was a diagnosis of iron overload. The frequencies of heterozygosity for H63D or S65C and homozygosity for H63D were not significantly increased in any group compared with the general population frequency.

Conclusion

These results demonstrate the reliability of the methodology and confirm the difficulty of identifying genetic haemochromatosis purely on the basis of clinical suspicion that haemochromatosis may be responsible for liver disease, diabetes or arthritis.Hereditary haemochromatosis (HH) is an autosomal recessive disorder in which there is iron accumulation as a result of increased dietary absorption. In the UK¹ over 90% of patients are homozygous for a single variant (C282Y) of the HFE gene.² Once diagnosed, HH is readily treatable by the means of venesection and, provided complications have not arisen, life expectancy is not reduced.^3,4,5 The allele frequency of HFE C282Y in people of northern European origin in the UK is about 8% with about one in seven people being heterozygous, and one in 150 being homozygous for the C282Y allele.⁶ The frequency, availability of a genetic test and an effective treatment have led to pressure to implement population screening⁷ although there were always concerns about the clinical penetrance of HFE mutations.⁸ These concerns were justified as it is now clear that although most men, and about 50% of women, who are homozygous for C282Y will show evidence of iron accumulation (a raised transferrin saturation) the clinical penetrance of homozygosity for C282Y is low.^9,10,11 Nevertheless, Cadet et al¹² found that 24% of adults from northern France had significant iron overload using a measure of “therapeutic penetrance” (removal of at least 5 g iron by venesection). This group may be considered to be at immediate risk of tissue damage from iron overload. Two other HFE variants are commonly found in European populations: H63D, with allele frequencies varying from 10% to 20%,¹³ and S65C with an allele frequency of 1–2%.¹⁴ These variants are not usually associated with significant iron loading in either the heterozygous or homozygous state but some compound heterozygotes with C282Y may accumulate iron.¹⁵ Detection of these variants, in addition to C282Y, is valuable in assessing their clinical significance. Here we describe a single, multiplex PCR using heteroduplex generators spanning the DNA sequence encoding amino acids 63, 65 and 282 to detect the three common HFE variants and the application of the multiplex analysis to the testing of over 5000 samples sent for diagnostic or family testing.     

Global prevalence of putative haemochromatosis mutations.

Haemochromatosis is a genetic disease associated with progressive iron overload, and is common among populations of northern European origin. HLA-H is a recently reported candidate gene for this condition. Two mutations have been identified, a substitution of cysteine for tyrosine at amino acid 282 (C282Y, nucleotide 845) and of histidine for aspartate at amino acid 63 (H63D, nucleotide 187). Over 90% of UK haemochromatosis patients are homozygous for the C282Y mutation. We have examined 5956 chromosomes (2978 people) for the presence of HLA-H C282Y and H63D by PCR followed by restriction enzyme analysis. We have found world wide allele frequencies of 1.9% for C282Y and 8.1% for H63D. The highest frequencies were 10% for C282Y in 90 Irish chromosomes and 30.4% for H63D in 56 Basque chromosomes. C282Y was most frequent in northern European populations and absent from 1042 African chromosomes, 484 Asian chromosomes, and 644 Australasian chromosomes. The distribution of the C282Y mutation coincides with that of populations in which haemochromatosis has been reported and is consistent with the theory of a north European origin for the mutation. The H63D polymorphism is more widely distributed and its connection with haemochromatosis remains unclear.     

Highly sensitivity adhesion molecules detection in hereditary haemochromatosis patients reveals altered expression

Several abnormalities in the immune status of patients with hereditary haemochromatosis (HH) have been reported, suggesting an imbalance in their immune function. This may include persistent production of, or exposure to, altered immune signalling contributing to the pathogenesis of this disorder. Adhesion molecules L-, E- and P-Selectin, intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) are some of the major regulators of the immune processes and altered levels of these proteins have been found in pathological states including cardiovascular diseases, arthritis and liver cancer. The aim of this study was to assess L-, E- and P-Selectin, ICAM-1 and VCAM-1 expression in patients with HH and correlate these results with HFE mutation status and iron indexes. A total of 139 subjects were diagnosed with HH (C282Y homozygotes = 87, C282Y/H63D = 26 heterozygotes, H63D homozygoyes = 26), 27 healthy control subjects with no HFE mutation (N/N), 18 normal subjects heterozygous for the H63D mutation served as age-sex-matched controls. We observed a significant decrease in L-selectin (P = 0.0002) and increased E-selectin and ICAM-1 (P = 0.0006 and P = 0.0059) expression in HH patients compared with healthy controls. This study observes for the first time that an altered adhesion molecules profile occurs in patients with HH that is associated with specific HFE genetic component for iron overload, suggesting that differential expression of adhesion molecules may play a role in the pathogenesis of HH.     

HFE蛋白与遗传性血色病

遗传性血色病(hereditary haemochromatosis,HH)是一种遗传性铁代谢疾病。发病遍及全球,以白种人发病较多,北欧人群发病率可高达1/200。大约1/10的白色人种是HFE突变基因携带者[1]。国内对HH的发病率尚无确切统计数字,但全国各地均有病例报道[2]。HH主要特征为小肠铁吸收过量增加,逐渐在肝、心、胰和其它内分泌器官的实质细胞沉积,造成器官功能障碍、肝硬化、心力衰竭、糖尿病、垂体功能减退和关节疾病等。此种疾病首次报道於1865年,当时认为HH是糖尿病的一种特殊病例。尔后将这类疾病称为色素性肝硬化(pigment cirrho-sis),或古铜色糖…     

Determination of gene frequencies for two common haemochromatosis mutations in the Danish population by a novel polymerase chain reaction with sequence-specific primers

Abstract: Hereditary haemochromatosis (HH), a condition of abnormal iron metabolism which leads to iron overload and organ damage, previously known as bronze diabetes or idiopathic haemochromatosis, is the most common disease-producing genetic disorder among Europeans. Two mutations, C282Y and H63D, are described for the candidate gene, HFE, reported as being responsible for the disease. Since molecular testing of these mutations will be of value in early diagnosis of haemochromatosis, the aim of this study was to develop a simple, fast and inexpensive technique for the determination of the polymorphism in the HFE gene on a large scale. We designed sequence-specific primers for polymerase chain reaction (PCR-SSP) and tested 200 randomly selected healthy Danes and found the result completely comparable to results obtained by a previously described method, PCR-RFLP. The gene frequencies in the Danish population are similar to reported results for the White population, with a frequency of 0.068 for the C282Y mutation and a frequency of 0.128 for the H63D mutation.     

Distribution of HFE C282Y and H63D mutations in the Balearic Islands (NE Spain)

The HFE gene contains two main missense mutations: C282Y and H63D. Individuals with these mutations carry a risk of developing hereditary haemochromatosis (HH). The common form of this disease is due to homozygosity for the C282Y mutation. Population studies have shown the variation of the prevalence of these mutations in different countries and ethnic groups. The purposes of this current study were to determine the prevalence of the C282Y and H63D mutations in the Balearic Islands and the genotypic characterization of patients diagnosed with HH, as well as those with iron overload and liver diseases. A total of 1330 Balearic chromosomes were analyzed. The results showed that the populations of the Balearic Islands were not homogeneous. No C282Y carriers were observed in a group of descendants of Majorcan Jews (Chuetas) and the frequency was very low in Minorca (1.2%) in comparison with the other islands of Majorca (4.7%) and Ibiza (6.5%). The carrier frequency of the H63D mutation was similar in the three islands and very high (43.1%) in the descendants of Majorcan Jews. The study of patients was carried out in 129 individuals. The homozygous C282Y genotype was the principal one involved in hereditary haemochromatosis (90%), whereas the other HH patients were C282Y/H63D compound heterozygous and H63D homozygous.     

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.

     

The role of HFE mutations on iron metabolism in beta-thalassemia carriers

Hereditary hemochromatosis (HH) is an autosomal recessive disorder of iron metabolism characterized by increased iron absorption and progressive storage resulting in organ damage. HFE gene mutations C282Y and H63D are responsible for the majority of HH cases. A third HFE mutation, S65C, has been associated with the development of a mild form of hemochromatosis. The beta-thalassemia trait is characterized by mild, ineffective erythropoiesis that can induce excess iron absorption and ultimately lead to iron overload. The aim of this study was to evaluate the effect of genetic markers (HFE mutations C282Y, H63D, and S65C) on the iron status of beta-thalassemia carriers. A total of 101 individuals heterozygous for beta-thalassemia and 101 normal control individuals were studied. The allelic frequencies of C282Y (1.5 versus 3.5%), H63D (15.3 versus 18.3%), and S65C (1.0 versus 1.5%) did not differ significantly between beta-thalassemia carriers and normal controls. Serum iron (P=0.029) and transferrin saturation (P=0.009) were increased in beta-thalassemia carriers heterozygous for H63D mutation. The number of subjects carrying C282Y or S65C mutations was too low to conclude their effect on the iron status. These results suggest that the beta-thalassemia trait tends to be aggravated with the coinheritance of H63D mutation, even when present in heterozygosity.     

Reverse cascade screening of newborns for hereditary haemochromatosis: a model for other late onset diseases?

Background: Genetic testing can determine those at risk for hereditary haemochromatosis (HH) caused by HFE mutations before the onset of symptoms. However, there is no optimum screening strategy, mainly owing to the variable penetrance in those who are homozygous for the HFE Cys282Tyr (C282Y) mutation. The objective of this study was to identify the majority of individuals at serious risk of developing HFE haemochromatosis before they developed life threatening complications.     

Digenic inheritance of mutations in HAMP and HFE results in different types of haemochromatosis

Haemochromatosis (HH) is a clinically and genetically heterogeneous disease caused by inappropriate iron absorption. Most HH patients are homozygous for the C282Y mutation in the HFE gene. However, penetrance of the C282Y mutation is incomplete, and other genetic factors may well affect the HH phenotype. Ferroportin and TFR2 mutations also cause HH, and two HAMP mutations have recently been reported that causes juvenile haemochromatosis (JH) in the homozygous state. Here, we report evidence for digenic inheritance of HH. We have detected two new HAMP mutations in two different families, in which there is concordance between severity of iron overload and heterozygosity for HAMP mutations when present with the HFE C282Y mutation. In family A, the proband has a JH phenotype and is heterozygous for C282Y and a novel HAMP mutation Met50del IVS2+1(-G). This is a four nucleotide ATGG deletion which causes a frameshift. The proband's unaffected mother is also heterozygous for Met50del IVS2+1(-G), but lacks the C282Y mutation and is heterozygous for the HFE H63D mutation. Met50del IVS2+1(-G) was absent from 642 control chromosomes. In family B, a second novel, less severe HAMP mutation, G71D, was identified. This was detected in the general population at an allele frequency of 0.3%. We propose that the phenotype of C282Y heterozygotes and homozygotes may be modified by heterozygosity for mutations which disrupt the function of hepcidin in iron homeostasis, with the severity of iron overload corresponding to the severity of the HAMP mutation.     

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.     

HFE gene mutations analysis in Basque hereditary haemochromatosis patients and controls

C282Y/C282Y genotype is the prevalent genotype in Hereditary Haemochromatosis (HH), however, other genotypes have been associated with the disease. The objective of our study was to analyse the frequency of the three main mutations of HFE gene in HH patients and controls from the Basque population with differential genetic characteristics. Thirty five HH patients and 116 controls were screened for C282Y, H63D and S65C mutations using a PCR-RFLP technique. The association of HLA-A and-B alleles and HFE mutations was also studied in Basque controls. The frequency of C282Y homozygotes in the group of patients was only 57%. The rest of the patients presented heterogeneous genotypes, including compound heterozygotes: 11% of them were C282Y/H63D; and 2.85% were H63D/S65C. H63D or S65C heterozygotes had a frequency of 11% and 2.85 respectively and 5.71% patients lacked any mutation The high frequency of H63D in the healthy Basque population is confirmed in this study. A considerable incidence of S65C is observed either in controls and in HH (3%) or in iron overloaded patients. The peculiar genetic characteristics of Basques could explain the heterogeneity of genotypes in HH patients of this group. Further studies should be carried out to confirm these findings although the implication of other genetic or external factors in the development of HH is suggested.     

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.     

Iron genes, iron load and risk of Alzheimer's disease

Background

Compound heterozygotes of the haemochromatosis gene (HFE) variants, H63D and C282Y, have raised transferrin saturation compared with that in the wild type. In the cohort of the Oxford Project To Investigate Memory and Ageing (OPTIMA), bicarriers of the HFE C282Y and the transferrin C2 gene variants are at five times greater risk of developing Alzheimer''s disease; the addition of HFE H63D may raise the risk still further.

Objective

To investigate transferrin saturation by HFE and transferrin genotype among people without dementia–that is, controls and those with mild cognitive impairment (MCI)—and also among those with Alzheimer''s disease.

Methods

Serum iron status and genotype were examined of 177 patients with Alzheimer''s disease, 69 patients with MCI and 197 controls from the OPTIMA cohort.

Results

Although each of these variants alone had relatively little effect on iron status, the combination of either HFE C282Y and HFE H63D or of HFE C282Y and transferrin C2 markedly raised transferrin saturation in those without dementia, but had little effect in those with mature Alzheimer''s disease.

Conclusions

These combinations may raise the risk for Alzheimer''s disease, owing to higher iron loads and therefore oxidative stress in the preclinical phase. If replicated, these findings will have implications for the prevention of Alzheimer''s disease.The effects of the haemochromatosis gene (HFE) variants, H63D and C282Y, on serum iron status of a healthy human population were first examined in 1998¹ and have been extensively studied since. Several large studies^2,3,4,5,6 have obtained broadly consistent results. For instance, Jackson et al³ summarised their results for transferrin saturation as follows: C282Y homozygotes >compound heterozygotes (C282Y and H63D) >H63D homozygotes >C282Y heterozygotes >H63D heterozygotes >wild type. However, to our knowledge, the influence on iron status of the combination of HFE and transferrin variants has not been studied, either in healthy controls or in those with Alzheimer''s disease.We previously reported⁷ that in the cohort of the Oxford Project To Investigate Memory and Ageing (OPTIMA), carriers of either the HFE C282Y or transferrin C2 variants alone had no effect on risk for Alzheimer''s disease; however, bicarriers of the two alleles, whether heterozygotes or homozygotes, were at five times greater risk for Alzheimer''s disease. The addition of the third variant, HFE H63D, seemed to increase the risk still further.Alzheimer''s disease has a long preclinical phase.⁸ One of the early events in the development of the disease is thought to be oxidative stress,^9,10 due crucially to the actions of redox‐active iron.^11,12 The above results therefore suggested that combinations of the HFE C282Y, HFE H63D and transferrin C2 alleles might contribute to higher iron loads during the preclinical phase of Alzheimer''s disease. We therefore investigated transferrin saturation by genotype among those without dementia—that is, controls and patients with mild cognitive impairment (MCI)—as well as among patients with Alzheimer''s disease, in the OPTIMA cohort. We examined combinations of the HFE H63D, HFE C282Y and transferrin C2 alleles.

Key points

• Bicarriers of the haemochromatosis gene (HFE) C282Y and the transferrin C2 variants may have higher iron loads than those of the wild type, as has previously been shown for compound heterozygotes of HFE H63D and HFE C282Y.
• The association of the combination of HFE C282Y and transferrin C2 with increased risk for Alzheimer''s disease, as previously reported, may therefore be due to oxidative stress in the preclinical phase.
• If confirmed, these results have implications for the prevention of Alzheimer''s disease in the 6% of northern Europeans who carry one or both combinations, as iron overload is a treatable condition.