Hereditary Hemochromatosis Phenotype Research Articles

The Allele Frequency of the HFE gene mutation H63D (rs1799945) and Its Relationship to a Hereditary Hemochromatosis Diagnosis in Metabolic Nutrition Students at Virginia Tech

Hereditary hemochromatosis (HH) is a disease that causes excess iron absorption from the diet. This excess iron can be stored in the liver, skin, heart, pancreas, and joints, and then can lead to other health conditions, as the human body has no way of actively excreting iron. The human hemochromatosis protein (HFE protein) is encoded by the HFE gene, and mutations in this gene can lead to a dysfunction of the protein resulting in HH or iron overload later in adulthood. The objective of this study was to analyze the mutant allele frequency and the penetrance of the H63D mutation (SNP rs1799945) of the HFE gene in a cohort of Virginia Tech students. This study had a total of 69 participants. Fifty-two participants provided saliva samples, genomic data from 23andMe®, and surveys with phenotypic information. Of these, 6 were genotyped using the RFLP technique and served as controls for genotype confirmation. An additional 17 participants provided saliva samples, but did not provide 23andMe® data; genomic DNA from these participants were genotyped using the RFLP technique. Our results showed that although none of the participants had been diagnosed with HH, the mutant allele frequency of this population was 13.04%. In conclusion, as HH is usually diagnosed in older adults, we could not identify any students with a phenotype of HH, even though we could detect the mutant allele. This data suggests that affordable and accessible genetic ancestry and health kits such as the 23andMe® kit, could provide an efficient way to identify, prevent, and manage HH and other genetic diseases before symptoms arise. KEYWORDS: Hereditary Hemochromatosis; Iron Absorption; Single Nucleotide Polymorphism; Restriction Fragment Length Polymorphism; College Student Population; 23andMe®; Population Analysis; Survey Results

Inherited Disorders of Iron Overload.

Dietary iron absorption and systemic iron traffic are tightly controlled by hepcidin, a liver-derived peptide hormone. Hepcidin inhibits iron entry into plasma by binding to and inactivating the iron exporter ferroportin in target cells, such as duodenal enterocytes and tissue macrophages. Hepcidin is induced in response to increased body iron stores to inhibit further iron absorption and prevent iron overload. The mechanism involves the BMP/SMAD signaling pathway, which triggers transcriptional hepcidin induction. Inactivating mutations in components of this pathway cause hepcidin deficiency, which allows inappropriately increased iron absorption and efflux into the bloodstream. This leads to hereditary hemochromatosis (HH), a genetically heterogenous autosomal recessive disorder of iron metabolism characterized by gradual buildup of unshielded non-transferrin bound iron (NTBI) in plasma and excessive iron deposition in tissue parenchymal cells. The predominant HH form is linked to mutations in the HFE gene and constitutes the most frequent genetic disorder in Caucasians. Other, more severe and rare variants are caused by inactivating mutations in HJV (hemojuvelin), HAMP (hepcidin) or TFR2 (transferrin receptor 2). Mutations in SLC40A1 (ferroportin) that cause hepcidin resistance recapitulate the biochemical phenotype of HH. However, ferroportin-related hemochromatosis is transmitted in an autosomal dominant manner. Loss-of-function ferroportin mutations lead to ferroportin disease, characterized by iron overload in macrophages and low transferrin saturation. Aceruloplasminemia and atransferrinemia are further inherited disorders of iron overload caused by deficiency in ceruloplasmin or transferrin, the plasma ferroxidase and iron carrier, respectively.

Targeted Next Generation Sequencing of the Five Hemochromatosis Genes in Italian Patients with Iron Overload and Non-Diagnostic First Level Genetic Test: A Pilot Study

▪Background and Aim: Molecular diagnosis of HFE-related hereditary hemochromatosis (HH) is typically made by searching for the C282Y and H63D mutations (first level genetic test). However, in the Mediterranean area up to one third of patients with HH phenotype do not have the “diagnostic” genotypes (C282Y homozygosity, or C282Y/H63D compound heterozygosity). This pilot study was designed to develop a “second level” next generation sequencing (NGS)-based test for rapid and simultaneous analysis of the five HH genes (HFE, HFE2, HAMP, TFR2 and SLC40A1).Methodology: we studied 61 patients with relevant biochemical signs of iron overload (IO) and non-diagnostic first level genetic test suggesting a possible “non-HFE” HH. The five HH genes were captured by Halo-Plex™ technology, and then sequenced using aNGS platform (Illumina HiSeq 1000). Sequenced reads were aligned against human reference HG19 and analyzed by GoldenHelix™ software to annotate all the variants possibly involved in the disease.Results: In IO patients a large number of new non-synonymous variants (according to bioinformatics tools based on publicly available databases including the 1000-genomes project) were found. Many of them were relatively frequent and detected also in controls, thus being considered likely “non-pathogenic”, unless clearly enriched in patients. On the other hand, some rare variants (i.e. limited to a single or very few individuals), particularly in SCL40A1, TFR2, and HFE, were found exclusively in patients, and could be considered “potentially pathogenic”.Conclusions: The combination of the Halo-Plex™ approach with NGS platform and GoldenHelix™ algorithm appears a suitable approach for a better molecular characterization of patients with unexplained HH phenotype, and could represent a good option for second level genetic testing in referral centers. However, establishing the clinical relevance of NGS-detected “novel” genetic variants in a prevalently autosomal recessive disorder like HH remains a difficult task, requiring further functional studies and national/international collaborative efforts. DisclosuresNo relevant conflicts of interest to declare.

CYBRD1 as a modifier gene that modulates iron phenotype in HFE p.C282Y homozygous patients

Most patients with hereditary hemochromatosis in the Caucasian population are homozygous for the p.C282Y mutation in the HFE gene. The penetrance and expression of hereditary hemochromatosis differ largely among cases of homozygous p.C282Y. Genetic factors might be involved in addition to environmental factors. In the present study, we analyzed 50 candidate genes involved in iron metabolism and evaluated the association between 214 single nucleotide polymorphisms in these genes and three phenotypic outcomes of iron overload (serum ferritin, iron removed and transferrin saturation) in a large group of 296 p.C282Y homozygous Italians. Polymorphisms were tested for genetic association with each single outcome using linear regression models adjusted for age, sex and alcohol consumption. We found a series of 17 genetic variants located in different genes with possible additive effects on the studied outcomes. In order to evaluate whether the selected polymorphisms could provide a predictive signature for adverse phenotype, we re-evaluated data by dividing patients in two extreme phenotype classes based on the three phenotypic outcomes. We found that only a small improvement in prediction could be achieved by adding genetic information to clinical data. Among the selected polymorphisms, a significant association was observed between rs3806562, located in the 5'UTR of CYBRD1, and transferrin saturation. This variant belongs to the same haplotype block that contains the CYBRD1 polymorphism rs884409, found to be associated with serum ferritin in another population of p.C282Y homozygotes, and able to modulate promoter activity. A luciferase assay indicated that rs3806562 does not have a significant functional role, suggesting that it is a genetic marker linked to the putative genetic modifier rs884409. While our results support the hypothesis that polymorphisms in genes regulating iron metabolism may modulate penetrance of HFE-hereditary hemochromatosis, with emphasis on CYBRD1, they strengthen the notion that none of these polymorphisms alone is a major modifier of the phenotype of hereditary hemochromatosis.

Novel observations in hereditary hemochromatosis: potential implications for clinical strategies

Remarkable advances in understanding the pathogenesis of hereditary hemochromatosis have been made since 1996, the year in which the HFE gene, which is responsible for the vast majority of cases, was discovered.1 The recognition of the central role of the iron regulatory hormone hepcidin in the pathogenesis of hereditary hemochromatosis has been particularly significant. In addition to HFE, defects in four additional genes have been found to cause hereditary hemochromatosis: hepcidin, transferrin receptor 2 (TFR2), hemojuvelin (HJV), and ferroportin.2 Interestingly, these genes each encode for a protein that affects pathways influencing liver hepcidin synthesis or its interaction with the cellular iron export protein ferroportin. It is now clear that inadequate production of hepcidin or response to it is responsible for the excess dietary iron absorption and excess iron release from macrophages stores, and consequent hyperferremia, which characterize the classical hereditary hemochromatosis phenotype. For clinicians, the challenge is to diagnose hereditary hemochromatosis before this hyperferremia leads to irreversible tissue damage. At the same time the clinician must have ways to distinguish progressive heritable forms of iron overload from increasingly common acquired diseases with only moderately increased iron body stores, such as the metabolic syndrome. This challenge has raised interest in the possibility that recent bench top discoveries might lead to novel bedside laboratory tests. Two publications in this issue of Haematologica are relevant to this possibility.3,4 In one study, serum hepcidin levels were used to characterize the response of patients with HFE- or TFR2-associated hereditary hemochromatosis to an oral iron challenge.3 The other study identifed several clinically significant orphan mutations in the HFE gene.4 The contribution of each of these studies to a better understanding of the pathogenesis of hereditary hemochromatosis and the possible ways they might influence the future diagnostic work-up and monitoring of iron overload disorders are discussed.

Hereditary hemochromatosis: Mutations in genes involved in iron homeostasis in Brazilian patients

Background p.C282Y mutation and rare variants in the HFE gene have been associated with hereditary hemochromatosis (HH). HH is also caused by mutations in other genes, such as the hemojuvelin ( HJV), hepcidin ( HAMP), transferrin receptor 2 ( TFR2) and ferroportin ( SLC40A1). The low rate homozygous p.C282Y mutation in Brazil is suggestive that mutations in non- HFE genes may be linked to HH phenotype. Aim To screen exon-by-exon DNA sequences of HFE, HJV, HAMP, TFR2 and SLC40A1 genes to characterize the molecular basis of HH in a sample of the Brazilian population. Materials and methods Fifty-one patients with primary iron overload (transferrin saturation ≥ 50% in females and ≥ 60% in males) were selected. Subsequent bidirectional DNA sequencing of HFE, HJV, HAMP, TFR2 and SLC40A1 exons was performed. Results Thirty-seven (72.5%) out of the 51 patients presented at least one HFE mutation. The most frequent genotype associated with HH was the homozygous p.C282Y mutation ( n = 11, 21.6%). In addition, heterozygous HFE p.S65C mutation was found in combination with p.H63D in two patients and homozygous HFE p.H63D was found in two patients as well. Sequencing in the HJV and HAMP genes revealed HJV p.E302K, HJV p.A310G, HJV p.G320V and HAMP p.R59G alterations. Molecular and clinical diagnosis of juvenile hemochromatosis (homozygous form for the HJV p.G320V) was described for the first time in Brazil. Three TFR2 polymorphisms (p.A75V, p.A617A and p.R752H) and six SLC40A1 polymorphisms (rs13008848, rs11568351, rs11568345, rs11568344, rs2304704, rs11568346) and the novel mutation SLC40A1 p.G204S were also found. Conclusions The HFE p.C282Y in homozygosity or in heterozygosity with p.H63D was the most frequent mutation associated with HH in this sample. The HJV p.E302K and HAMP p.R59G variants, and the novel SLC40A1 p.G204S mutation may also be linked to primary iron overload but their role in the pathophysiology of HH remain to be elucidated.

Low frequency of HFE gene mutations in Croatian patients suspected of having hereditary hemochromatosis

SummaryBackgroundHereditary hemochromatosis (HH) is a common autosomal recessive disorder in populations of European descent. It is characterized by a variable prevalence of mutations in the hemochromatosis gene (HFE) in different countries and a complex relationship between the HFE genotype and the HH phenotype. Genetic analysis has not been conducted in Croatian patients with iron overload. The aim of this study was to determine whether HFE mutations, C282Y, H63D, and S65C were correlated with clinical and biochemical parameters in Croatian patients with suspected HH.Material/MethodsClinical examination, biochemical analysis, and genotyping were performed in 175 patients suspected of having HH. The control group consisted of 350 healthy blood donors.ResultsAmong the patients, 20% had genotypes related to HH – 7.4% were homozygous for C282Y, 6.3% were compound heterozygous for C282Y and H63D, 5.7% were homozygous for H63D, and 0.6% was compound heterozygous for C282Y and S65C. The allelic frequencies were 14.6% for C282Y mutation, 23.7% for H63D mutation, and 1.4% for S65C mutation. A comparison of the clinical and laboratory profiles of patients revealed that C282Y homozygotes had higher frequencies of all clinical symptoms and higher levels of biochemical parameters than others. The C282Y/H63D compound heterozygotes and H63D homozygotes were found to be clinically important, despite the fact that they were associated with less severe disease.ConclusionsOur results show that HFE mutations are responsible for only about 20% of Croatian patients with suspected HH. Screening with biochemical methods and HFE genotyping may be not sufficient for diagnoses in the Croatian population.

Global Sequencing for the Molecular Background of Hereditary Hemochomatosis In Brazilian Patients

AbstractAbstract 5146 INTRODUTION:Most hereditary hemochromatosis (HH) patients are homozygous for the p.C282Y mutation in the HFE gene. But rare HFE variants have been shown to be associated with HH. In addition, four main types of non-HFE HH are caused by mutations in the hemojuvelin (HJV), hepcidin (HAMP), transferrin receptor 2 (TFR2) and ferroportin (SLC40A1) genes. The main aim of this study was to screen for HFE, HJV, HAMP, TFR2 and SLC40A1 mutations and to investigate their relationship with HH. MATERIAL E METHODS:Fifty-one Brazilian patients with primary iron overload (transferrin saturation > 50% in females and 60% in males) were eligible. Subsequent bidirectional sequencing for each exon of HFE, HJV, HAMP, TFR2 and SLC40A1 genes was performed. The effect of HFE p.V256I novel mutations on protein structure was analyzed by in silico molecular dynamic and free energy calculations. RESULTS:Thirty-seven (72.5%) out of the 51 patients presented at least one HFE mutation. The most frequent genotype associated with HH was the homozygous p.C282Y mutation (n = 11, 21.6%). In addition, the p.S65C mutation was found in heterozygosis with p.H63D in two patients and the homozygous genotype for the p.H63D was found in two patients. One novel mutation (p.V256I) was indentified in heterozygosis with the p.H63D mutation. In silico modeling analysis of protein behavior suggested that the p.V256I mutation does not reduce the binding affinity between HFE and β2-microglobulin (β2M) in the same way the p.C282Y mutation does compared with the native HFE protein. Sequencing HJV revealed one patient presenting Juvenile hemochromatosis (JH) (homozygous genotype for the HJV p.G320V mutation); two patients carrying heterozygous genotype for the p.E302K mutation; and one patient with heterozygosis p.A310G polymorphism. Sequencing HAMP revealed one patient carrying p.P48G novel mutation in the heterozygous form. Three and five non-pathogenic polymorphisms were observed in the TFR2 and SLC40A1 genes, respectively. Sequencing SLC40A1 also identified one patient with homozygous genotype for the p.R561G described mutation; and one patient with homozygous genotype for the p.G204S novel mutation. CONCLUSION:The HFE p.C282Y in homozygosis or in heterozygosis with p.H63D was the most frequent mutation associated with HH in our sample population. The novel HFE p.V256I mutation could not be implicated in the molecular basis of the HH phenotype. Two described mutations, HJV p.E302K and SLC40A1 p.R561G, could have functional consequences according to previously studies contributing to HH phenotype. Two novel mutations, HAMP p.R48G and SLC40A1 p.G204S, may be implicated with iron overload in these patients, but further studies are need to explain their impact on proteins. Disclosures:No relevant conflicts of interest to declare.

Hemojuvelin and Hepcidin Genes Sequencing in Brazilian Patients with Primary Iron Overload

most hereditary hemochromatosis (HH) patients are homozygous for the p.C282Y mutation in the HFE gene. Some studies reported that HH phenotypic expression could be modulated by genetic factors such as HJV and HAMP gene mutations. the aims of this study were to identify HJV and HAMP mutations and to analyze their impact on HH phenotype in non-p.C282Y homozygous individuals. Twenty-four Brazilian patients with primary iron overload and non-p.C282Y homozygous genotype (transferrin saturation >50% in women and >60% in men and absence of secondary causes) were selected. Subsequent bidirectional sequencing of the HJV and HAMP exons was performed. sequencing revealed a substitution in heterozygosis, c.929C > G, which corresponds to p.A310G polymorphism in HJV exon 4 (rs7540883). In the same gene, in another individual, an IVS1-36C > G intronic variant was detected in heterozygosis. In the HAMP gene, an IVS3 + 42G > A intronic variant was identified. There were six (25.0%) patients carrying a heterozygous genotype for the HFE p.C282Y and nine (37.5%) patients carrying a heterozygous genotype for the HFE p.H63D. HJV p.A310G polymorphism and two intronic variants were found, but none of these alterations were associated with digenic inheritance with the HFE gene. Our data indicate that HJV and HAMP functional mutations are not frequent in these patients.

HFE gene mutations in patients with primary iron overload: Is there a significant improvement in molecular diagnosis yield with HFE sequencing?

Rare HFE variants have been shown to be associated with hereditary hemochromatosis (HH), an iron overload disease. The low frequency of the HFE p.C282Y mutation in HH-affected Brazilian patients may suggest that other HFE-related mutations may also be implicated in the pathogenesis of HH in this population. The main aim was to screen for new HFE mutations in Brazilian individuals with primary iron overload and to investigate their relationship with HH. Fifty Brazilian patients with primary iron overload (transferrin saturation > 50% in females and 60% in males) were selected. Subsequent bidirectional sequencing for each HFE exon was performed. The effect of HFE mutations on protein structure were analyzed by molecular dynamics simulation and free binding energy calculations. p.C282Y in homozygosis or in heterozygosis with p.H63D were the most frequent genotypic combinations associated with HH in our sample population (present in 17 individuals, 34%). Thirty-six (72.0%) out of the 50 individuals presented at least one HFE mutation. The most frequent genotype associated with HH was the homozygous p.C282Y mutation ( n = 11, 22.0%). One novel mutation (p.V256I) was indentified in heterozygosis with the p.H63D mutation. In silico modeling analysis of protein behavior indicated that the p.V256I mutation does not reduce the binding affinity between HFE and β2-microglobulin (β2M) in the same way the p.C282Y mutation does compared with the native HFE protein. In conclusion, screening of HFE through direct sequencing, as compared to p.C282Y/p.H63D genotyping, was not able to increase the molecular diagnosis yield of HH. The novel p.V256I mutation could not be implicated in the molecular basis of the HH phenotype, although its role cannot be completely excluded in HH-phenotype development. Our molecular modeling analysis can help in the analysis of novel, previously undescribed, HFE mutations.

Analysis of HFE And Non-HFE Gene Mutations in Brazilian Patients with Hemochromatosis

BACKGROUNDApproximately one-half of Brazilian patients with hereditary hemochromatosis (HH) are neither homozygous for the C282Y mutation nor compound heterozygous for the H63D and C282Y mutations that are associated with HH in Caucasians. Other mutations have been described in the HFE gene as well as in genes involved in iron metabolism, such as transferrin receptor 2 (TfR2) and ferroportin 1 (SCL40A1).AIMSTo evaluate the role of HFE, TfR2 and SCL40A1 mutations in Brazilian subjects with HH.PATIENTS AND METHODSNineteen male subjects (median age 42 [range: 20–72] years) with HH were evaluated using the Haemochromatosis StripAssay A®. This assay is capable of detecting twelve HFE mutations, which are V53M, V59M, H63D, H63H, S65C, Q127H, P160delC, E168Q, E168X, W169X, C282Y and Q283, four TfR2 mutations, which are E60X, M172K, Y250X, AVAQ594-597del, and two SCL40A1 mutations, which are N144H and V162del.RESULTSIn our cohort, nine (47%) patients were homozygous for the C282Y mutation, two (11%) were heterozygous for the H63D mutation, and one each (5%) was either heterozygous for C282Y or compound heterozygous for C282Y and H63D. No other mutations in the HFE, TfR2 or SCL40A1 genes were observed in the studied patients.CONCLUSIONSOne-third of Brazilian subjects with the classical phenotype of HH do not carry HFE or other mutations that are currently associated with the disease in Caucasians. This observation suggests a role for other yet unknown mutations in the aforementioned genes or in other genes involved in iron homeostasis in the pathogenesis of HH in Brazil.

What regulates the iron regulator?

Early in my career, we were pretty ignorant about iron (we did not pump it yet), although we did know a few things. We were aware that the daily intake of iron is (should be) about 10 mg and that 1–2 mg of this iron was absorbed. Iron traveled in the blood bound to a carrier protein called “transferrin” and was stored in various places, such as the reticulo-endothelial system (RES) and bone marrow. As a matter of fact, the court-of-last-appeals for iron deficiency was the staining of bone marrow for iron with “Prussian blue”. We were aware that 200 billion erythrocytes were produced daily and that this production would take about 20 mg elemental iron, more than was absorbed. Thus, active recycling had to take place. Total body iron stores were known to be about 5 g and that half this amount resided in the erythrocytes. We were aware of hereditary hemochromatosis (HH), a favorite examination issue by oral examiners who generally had never ever seen a case either. And we knew about the poor Bantus and their ironcontaining cooking-pots. That was about it, although the iron storage mechanism and the way to measure it, namely ferritin, were introduced about 10 years later. The rest was a mystery, including the other common anemia, termed the “anemia of chronic disease”, that not even our smartest professors understood. Fortunately, all that ignorance is behind us, or at any rate, much of it is. Hepcidin (also known as HFE2) is a hepatic iron regulatory hormone that maintains systemic iron homeostasis. Hepcidin is made by the liver and secreted into the blood stream, where it binds to the iron exporter ferroportin that is located on macrophages, hepatocytes, and intestinal enterocytes. Hepcidin causes ferroportin to be internalized and proteolyzed (chewed up). As a result, hepcidin reduces gastrointestinal iron absorption and macrophage-mediated iron recycling. If hepcidin is so critical to iron-metabolic regulation, then the regulation of hepcidin is critical for our understanding how this very basic portion of human physiology works. In this issue, Casanovas et al. shed light on this question [1]. As is now commonly the case, the understanding of a rare Mendelian disease, HH, led the way. We now know that HH can be caused by mutations in a membrane protein encoded by a gene that is similar to those coding MHC class I-type proteins. The protein product associates with beta2-microglobulin (beta2M) and is called “HFE” although I am uncertain why. HFE is thought to regulate iron absorption by influencing the interaction of the transferrin receptor with transferrin. The HH phenotype is also produced by mutations in transferrin receptor 2 (TfR2), hemojuvelin (HJV), or hepcidin itself. Mutations in any of these proteins results in increased duodenal iron absorption, increased macrophage iron release, increased serum iron levels, and liver iron overload. HJV is a member of the “repulsive guidance molecule” (RGM) family, which also includes the bone-morphogenetic protein (BMP) coreceptors RGMA and DRAGON (RGMB). Babitt et al. [2] reported that HJV is a BMP coreceptor and that HJV mutants associated with the HH phenotype have impaired BMP signaling ability. They showed that BMP upregulates hepatocyte hepcidin expression, a process enhanced by HJV, and that is blunted in hepcidin genedeleted hepatocytes. Their data suggested a mechanism by which hepcidin mutations cause hemochromatosis, namely HJV dysfunction decreases BMP signaling, thereby lowering hepcidin expression. Babbitt et al. [3] next showed that BMP-2 administration increases hepcidin expression and J Mol Med (2009) 87:447–449 DOI 10.1007/s00109-009-0453-4

Simultaneous detection of the C282Y, H63D and S65C mutations in the hemochromatosis gene using quenched-FRET real-time PCR

Hereditary hemochromatosis (HH) is a common autosomal disorder of iron metabolism mainly affecting Caucasian populations. Three recurrent disease-associated mutations have been detected in the hemochromatosis gene (HFE): C282Y, H63D, and S65C. Although HH phenotype has been associated with all three mutations, C282Y is considered the most relevant mutation responsible for hemochromatosis. Clinical complications of HH include cirrhosis of the liver, congestive cardiac failure and cardiac arrhythmias, endocrine pancreatic disease, which can be prevented by early diagnosis and treatment. Therefore, a reliable genotyping method is required for presymptomatic diagnosis. We describe the simultaneous detection of the C282Y, H63D and S65C mutations in the hemochromatosis gene by real-time PCR followed by melting curve analysis using fluorescence resonance energy transfer (FRET) probes. The acceptor fluorophore may be replaced by a quencher, increasing multiplex possibilities. Real-time PCR results were compared to the results of sequencing and conventional PCR followed by restriction digestion and detection by agarose gel electrophoresis (PCR-RFLP). Genotypes from 80 individuals obtained both by the conventional PCR-RFLP method and quenched-FRET real-time PCR were in full agreement. Sequencing also confirmed the results obtained by the new method, which proved to be an accurate, rapid and cost-effective diagnostic assay. Our findings demonstrate the usefulness of real-time PCR for the simultaneous detection of mutations in the HFE gene, which allows a reduction of a significant amount of time in sample processing compared to the PCR-RFLP method, eliminates the use of toxic reagents, reduces the risk of contamination in the laboratory, and enables full process automation.

Simultaneous detection of the C282Y, H63D and S65C mutations in the hemochromatosis gene using quenched-FRET real-time PCR

Hereditary hemochromatosis (HH) is a common autosomal disorder of iron metabolism mainly affecting Caucasian populations. Three recurrent disease-associated mutations have been detected in the hemochromatosis gene (HFE): C282Y, H63D, and S65C. Although HH phenotype has been associated with all three mutations, C282Y is considered the most relevant mutation responsible for hemochromatosis. Clinical complications of HH include cirrhosis of the liver, congestive cardiac failure and cardiac arrhythmias, endocrine pancreatic disease, which can be prevented by early diagnosis and treatment. Therefore, a reliable genotyping method is required for presymptomatic diagnosis. We describe the simultaneous detection of the C282Y, H63D and S65C mutations in the hemochromatosis gene by real-time PCR followed by melting curve analysis using fluorescence resonance energy transfer (FRET) probes. The acceptor fluorophore may be replaced by a quencher, increasing multiplex possibilities. Real-time PCR results were compared to the results of sequencing and conventional PCR followed by restriction digestion and detection by agarose gel electrophoresis (PCR-RFLP). Genotypes from 80 individuals obtained both by the conventional PCR-RFLP method and quenched-FRET real-time PCR were in full agreement. Sequencing also confirmed the results obtained by the new method, which proved to be an accurate, rapid and cost-effective diagnostic assay. Our findings demonstrate the usefulness of real-time PCR for the simultaneous detection of mutations in the HFE gene, which allows a reduction of a significant amount of time in sample processing compared to the PCR-RFLP method, eliminates the use of toxic reagents, reduces the risk of contamination in the laboratory, and enables full process automation.

Hfe Acts in Hepatocytes to Prevent Hemochromatosis

Hereditary hemochromatosis (HH) is a prevalent, potentially fatal disorder of iron metabolism hallmarked by intestinal hyperabsorption of iron, hyperferremia, and hepatic iron overload. In both humans and mice, type I HH is associated with mutations in the broadly expressed HFE/Hfe gene. To identify where Hfe acts to prevent HH, we generated mice with tissue-specific Hfe ablations. This work demonstrates that local Hfe expression in hepatocytes serves to maintain physiological iron homeostasis, answering a long-standing question in medicine and explaining earlier clinical observations.

Chronic inflammation does not appear to modify the homozygous hereditary hemochromatosis phenotype

The mechanism of excessive iron storage in patients with hereditary hemochromatosis caused by mutations of the HFE gene seems to be a failure to up-regulate hepcidin in the face of increased body iron. Since the cytokines IL-1 and IL-6 stimulate hepcidin transcription in the absence of HFE, chronic inflammatory states might counteract the effect of HFE mutations. We measured the pre-phlebotomy plasma levels of C reactive protein (CRP) and of interleukin 6 (IL-6) in homozygotes for the C282Y mutation of HFE. There was no difference in these levels in subjects with high iron stores than in those with low iron stores, suggesting that the phenotypic differences between such homozygotes is not appreciably affected by ongoing chronic inflammation.

Expression of hepcidin is down-regulated in TfR2 mutant mice manifesting a phenotype of hereditary hemochromatosis

AbstractTransferrin receptor 2 (TfR2) is a membrane glycoprotein that mediates cellular iron uptake from holotransferrin. Homozygous mutations of this gene cause one form of hereditary hemochromatosis in humans. We recently reported that homozygous TfR2(Y245X) mutant mice, which correspond to the TfR2(Y250X) mutation in humans, showed a phenotype similar to hereditary hemochromatosis. In this study, we further analyzed the phenotype as well as iron-related gene expression in these mice by comparing the TfR2-mutant and wild-type siblings. Northern blot analyses showed that the levels of expression of hepcidin mRNA in the liver were generally lower, whereas those of duodenal DMT1, the main transporter for uptake of dietary iron, were higher in the TfR2-mutant mice as compared to the wild-type siblings. Expression of hepcidin mRNA in the TfR2 mutant mice remained low even after intraperitoneal iron loading. In isolated hepatocytes from both wild-type and TfR2 mutant mice, interleukin-6 and lipopolysaccharide each induced expression of hepcidin mRNA. These results suggest that up-regulation of hepcidin expression by inflammatory stimuli is independent of TfR2 and that TfR2 is upstream of hepcidin in the regulatory pathway of body iron homeostasis. (Blood. 2005;105:376-381)

DMT1 genetic variability is not responsible for phenotype variability in hereditary hemochromatosis

Background/aims. Homozygosity for a cysteine to tyrosine translocation at position 282 within the HFE gene (C282Y) is responsible for over 90% of hereditary hemochromatosis (HH) in Celtic populations. Determining those C282Y homozygotes at greatest risk for iron overload is a major clinical concern as only a small percentage will develop clinically significant iron overload. Divalent metal transport protein (DMT1) on the apical surface of duodenal enterocytes is recognised as the major iron import protein. We investigated whether genetic variability within the DMT1 gene may partly explain the phenotypic variability seen amongst a group of C282Y homozygotes with iron overload. Methods. One hundred and one unrelated C282Y homozygotes and 103 C282Y negative controls were analysed for the presence of four specific mutations/polymorphisms within the DMT1 gene ( 1245T/C, 1303C/A, IVS4 + 44C/A, IVS15Ex16-16C/G) using standard PCR techniques. Hepatic iron deposition was determined in 32 HH patients following Perls Prussian blue staining (0–4+). Estimations of the haplotype frequencies were performed utilising the program Arlequin version 2. Results. There were no significant differences in the allele frequencies of the IVS4 + 44C/A, 1303C/A, 1254T/C and IVS15Ex16-16C/G polymorphisms in the patient cohort compared to those observed in the control cohort. The commonest haplotypes identified were CCTC: IVS4C + 44C, 1303C, 1254T, IVS15ex16-16C; ACCC: IVS4C + 44A, 1303C, 1254C, IVS15ex16-16C and ACTG: IVS4C + 44A, 1303C, 1254T, IVS15ex16-16G. Similarly, there were no significant differences in the frequencies of these three haplotypes in the patient cohorts (regardless of the degree of hepatic iron deposition) compared to the control cohort. Conclusions. Polymorphisms within DMT1 gene do not influence penetrance of the HH phenotype.

Mechanisms of iron accumulation in hereditary hemochromatosis.

Hereditary hemochromatosis (HH) is a common inborn error of iron metabolism characterized by excess dietary iron absorption and iron deposition in several tissues. Clinical consequences include hepatic failure, hepatocellular carcinoma, diabetes, cardiac failure, impotence, and arthritis. Despite the discovery of the mutation underlying most cases of HH, considerable uncertainty exists in the mechanism by which the normal gene product, HFE, regulates iron homeostasis. Knockout of the HFE gene clearly confers the HH phenotype on mice. However, studies on HFE expressed in cultured cells have not yet clarified the mechanism by which HFE mutations lead to increased dietary iron absorption. Recent discoveries suggest other genes, including a second transferrin receptor and the circulating peptide hepcidin, participate in a shared pathway with HFE in regulation of iron absorption. This review summarizes our current understanding of the relationship between iron stores and absorption and presents models to explain the dysregulated iron homeostasis in HH.

Naturally variant autosomal and sex-linked loci determine the severity of iron overload in beta 2-microglobulin-deficient mice.

Hereditary hemochromatosis (HH) is a common chronic human genetic disorder whose hallmark is systemic iron overload. Homozygosity for a mutation in the MHC class I heavy chain paralogue gene HFE has been found to be a primary cause of HH. However, many individuals homozygous for the defective allele of HFE do not develop iron overload, raising the possibility that genetic variation in modifier loci contributes to the HH phenotype. Mice deficient in the product of the beta(2)-microglobulin (beta(2)M) class I light chain fail to express HFE and other MHC class I family proteins, and they have been found to manifest many characteristics of the HH phenotype. To determine whether natural genetic variation plays a role in controlling iron overload, we performed classical genetic analysis of the iron-loading phenotype in beta(2)M-deficient mice in the context of different genetic backgrounds. Strain background was found to be a major determinant in iron loading. Sex played a role that was less than that of strain background but still significant. Resistance and susceptibility to iron overload segregated as complex genetic traits in F(1) and back-cross progeny. These results suggest the existence of naturally variant autosomal and Y chromosome-linked modifier loci that, in the context of mice genetically predisposed by virtue of a beta(2)M deficiency, can profoundly influence the severity of iron loading. These results thus provide a genetic explanation for some of the variability of the HH phenotype.