HFE Mutations Research Articles

Lower serum hepcidin and greater parenchymal iron in nonalcoholic fatty liver disease patients with C282Y HFE mutations

Hepcidin regulation is linked to both iron and inflammatory signals and may influence iron loading in nonalcoholic steatohepatitis (NASH). The aim of this study was to examine the relationships among HFE genotype, serum hepcidin level, hepatic iron deposition, and histology in nonalcoholic fatty liver disease (NAFLD). Single-nucleotide polymorphism genotyping for C282Y (rs1800562) and H63D (rs1799945) HFE mutations was performed in 786 adult subjects in the NASH Clinical Research Network (CRN). Clinical, histologic, and laboratory data were compared using nonparametric statistics and multivariate logistic regression. NAFLD patients with C282Y, but not H63D mutations, had lower median serum hepcidin levels (57 versus 65 ng/mL; P = 0.01) and higher mean hepatocellular (HC) iron grades (0.59 versus 0.28; P < 0.001), compared to wild-type (WT) subjects. Subjects with hepatic iron deposition had higher serum hepcidin levels than subjects without iron for all HFE genotypes (P < 0.0001). Hepcidin levels were highest among patients with mixed HC/reticuloendothelial system cell (RES) iron deposition. H63D mutations were associated with higher steatosis grades and NAFLD activity scores (odds ratio [OR], ≥1.4; 95% confidence interval [CI]: >1.0, ≤2.5; P ≤ 0.041), compared to WT, but not with either HC or RES iron. NAFLD patients with C282Y mutations had less ballooning or NASH (OR, ≤0.62; 95% CI: >0.39, <0.94; P ≤ 0.024), compared to WT subjects. The presence of C282Y mutations in patients with NAFLD is associated with greater HC iron deposition and decreased serum hepcidin levels, and there is a positive relationship between hepatic iron stores and serum hepcidin level across all HFE genotypes. These data suggest that body iron stores are the major determinant of hepcidin regulation in NAFLD, regardless of HFE genotype. A potential role for H63D mutations in NAFLD pathogenesis is possible through iron-independent mechanisms.

Hemochromatosis Gene Mutations: Prevalence and Effects on Pegylated-Interferon and Ribavirin Therapy Response in Chronic Hepatitis C in Sardinia

Considerable evidence suggests that iron could be a comorbid factor for liver injury in chronic hepatitis C (CHC). Elevated iron indices are frequently described in CHC and may impact negatively on the course of liver disease and on the response to interferon alfa therapy. The aim of this study was to evaluate the frequency of hemochromatosis gene mutations in Sardinian CHC patients, the association with iron overload and the impact on response to therapy. Sixty-nine CHC patients were enrolled. Iron indices, hepatic andviral parameters were detected. C282Y, H63D and S65C mutations were identified through a PCR. Liver biopsy was performed for hepatic fibrosis evaluation. All patients were treated for 6 months (viral genotype 2/3) or 12 months (viral genotype 1/4) with pegylated-interferon 180mcg once weekly and ribavirin 1000-1200mg/daily. Sustained virological response (SVR) was defined as undetectable HCV RNA 24 weeks after the end of treatment. HFE gene mutation was detected in 29 patients (42%). The presence of HFE mutations was significantly associated with elevated transferrin saturation (P<0.01). Hepatic fibrosis was more advanced in HFE mutation carriers (χ (2), P=0.04). Among mutation carriers 27.5% achieved responses at the end of treatment compared with 60% of non-carriers (P=0.005). Patients with HFE wildtype produced significant SVR compared with patients with HFE mutations (P=0.03). The literature shows discordant results about the prevalence, hepatic distribution and possible therapeutic implications of iron overload in chronic hepatitis C. Our findings shows that HFE gene mutations could favor, synergically with CHC and other genetic or acquired factors, the development of liver damage and could influence the outcome of interferon treatment with higher rate of non-response.

Effect of the A736V TMPRSS6 polymorphism on the penetrance and clinical expression of hereditary hemochromatosis

Hereditary hemochromatosis (HH) is most frequently related to homozygosity for the p.C282Y HFE mutation (C282Y(+/+)), hampering hepcidin induction in response to iron. The rs855791 polymorphism, encoding for the p.A736V variant of TMPRSS6 regulating hepcidin, influences iron status in the population. The aim of this study was to assess the influence of rs855791 on the penetrance and clinical expression of HH. We retrospectively considered 315 HH patients (163 C282Y(+/+), and 152 with other HFE genotypes) evaluated at the time of diagnosis, and 271 healthy controls with normal iron parameters, residents of Northern Italy; TMPRSS6 genotype was assessed by allele specific polymerase chain reaction. The p.736V variant determining higher hepcidin release was under-represented in the patients (p=0.0023), independently of the presence of the C282Y(+/+) genotype, and the p.736V/V genotype protected from HH independently of age and sex (OR of HH for p.736A/A: 2.57, 1.3-4.1 and for p.736A/V: 1.84, 1.1-3.2). In the 96 C282Y(+/+) male patients without chronic viral hepatitis and alcohol abuse, the "high hepcidin" p.736V allele was negatively associated with cirrhosis independently of age, ferritin, ALT levels, and alcohol intake (OR 3.93, 95% C.I. 1.17-14.61 for the p.736A variant), and with the cumulative incidence of hepatocellular carcinoma (17% p.736A/A, 4% p.736A/V, 0 p.736V/V, p=0.05). The p.A736V TMPRSS6 polymorphism is likely a modifier of HH expression. Additional studies are warranted to validate these findings in other cohorts and test their potential relevance for the clinical management of HH patients.

Common local founder effects for Wilson's disease and hereditary hemochromatosis; mutation studies of a large family

Wilson's disease (WND) and hereditary hemochromatosis (HH) are two metal loading diseases of copper and iron, respectively, and are both recessively inherited. In central Sweden, where HH is common, 9 Wilson kindred (14 members) were identified. Aims of the study were to test whether nine WND families shared a common origin, a common mutation and if carrying HFE mutations affected their phenotype Results. The nine families were traced through 13 generations to a common founder origin in the mid-seventeenth century. Despite identity of descent, four different ATP7B mutations appeared with homozygosity in four, with two different mutations, W779X and T977M. There were three compound heterozygotes, W779X/T977M, R1319X/H1069Q and one T977M combined with a new, previously not described mutation, probably of Finnish origin. The founder family also included 26 descendant kindred (55 members) with HH as shown by HFE mutations. This admixture coincided with a migration out of the original parish into hemochromatosis-rich localities. One WND patient had iron overload (serum ferritin 672 µg/l and raised liver enzymes), but lacked HFE mutations. In another family with serious hemochromatosis (two sons dying from bronze diabetes), the coinheritance of congenital spherocytosis was probably the cause rather than an additional effect of WND. Conclusions. WND though a rare disease may become aggregated like HH in certain areas due to local founder effects. Despite extensive pedigree studies leading back to the local founder family, the authors were unable to find a single defining mutation of the ATP7B gene.

Clinical cofactors and hepatic fibrosis in hereditary hemochromatosis: The role of diabetes mellitus

The risk of hepatic fibrosis and cirrhosis in hereditary hemochromatosis relates to the degree of iron loading, but iron alone does not explain the variability in disease penetrance. This study sought to identify clinical cofactors that increase the risk of progressive liver disease. We identified 291 patients from our database who were homozygous for the C282Y mutation in HFE and had undergone a liver biopsy with quantification of hepatic iron concentration (HIC) and fibrosis staging. Data were collected from a retrospective chart review, including age, gender, alcohol consumption, medical therapy, smoking history, metabolic risk factors, mobilizable iron, and laboratory results. Male gender, excess alcohol consumption, HIC, and the presence of diabetes were independently associated with increasing fibrosis stage in multivariate analysis. Of these, the presence of diabetes showed the strongest association (odds ratio, 7.32; P = 0.03). The presence of steatosis was associated with higher fibrosis scores, but this was of borderline statistical significance. Risk factors for hepatic steatosis were male gender, impaired glucose tolerance, and increased body mass index. The presence of diabetes was associated with more severe hepatic fibrosis independent of iron loading, male gender, and alcohol consumption. The mechanism for this association is unknown and deserves further evaluation; however, it is possible that diabetes produces an additional hepatic oxidative injury from hyperglycemia. Thus, management of such cofactors in patients with hemochromatosis is important to reduce the risk of liver injury and fibrosis.

The diagnosis and management of non-alcoholic fatty liver disease: Practice Guideline by the American Association for the Study of Liver Diseases, American College of Gastroenterology, and the American Gastroenterological Association

These recommendations are based on the following: (1) a formal review and analysis of the recently published world literature on the topic [Medline search up to June 2011]; (2) the American College of Physicians’ Manual for Assessing Health Practices and Designing Practice Guidelines; (3) guideline policies of the three societies approving this document; and (4) the experience of the authors and independent reviewers with regards to NAFLD. Intended for use by physicians and allied health professionals, these recommendations suggest preferred approaches to the diagnostic, therapeutic and preventive aspects of care. They are intended to be flexible and adjustable for individual patients. Specific recommendations are evidence-based wherever possible, and when such evidence is not available or inconsistent, recommendations are made based on the consensus opinion of the authors. To best characterize the evidence cited in support of the recommendations, the AASLD Practice Guidelines Committee has adopted the classification used by the Grading of Recommendation Assessment, Development, and Evaluation (GRADE) workgroup with minor modifications (Table 1). The strength of recommendations in the GRADE system is classified as strong (1) or weak (2). The quality of evidence supporting strong or weak recommendations is designated by one of three levels: high (A), moderate (B) or low-quality (C). This is a practice guideline for clinicians rather than a review article and interested readers can refer to several comprehensive reviews published recently.

The functional significance of E277K and V295A HFE mutations

Hereditary haemochromatosis (HH) is an autosomal recessive disorder characterized by excessive intestinal iron absorption resulting in increased pathological body iron stores. It is typically associated with homozygosity for the c.845G>A (p.C282Y) mutation in the HFE gene. However, other HFE alterations have been reported in affected individuals but their association with the disease is unclear. This study analysed the functional consequences of two HFE mutations, c.829G>A (p.E277K) and c.884T>C (p.V295A). Firstly, it was shown that c.829G>A affects the HFE splicing by diminishing the full length HFE and ivs4_66bp inclusion transcript levels, while increasing the amount of exon 4 skipping transcript. Immunofluorescent techniques showed that the HFE_E277K protein had a diffuse distribution (similar to HFE_C282Y) while HFE_V295A presented at the cell surface and perinuclear compartments (resembling HFE_wt). Immunoprecipitation assays revealed a decreased association of HFE_E277K and HFE_V295A with both β2-microglobulin (B2M; 38 ± 7% and 66 ± 8%, respectively) and transferrin receptor (TFRC, also termed TFR1) (58 ± 2% and 49 ± 16%, respectively). Herein, we prove that both mutations partially abrogate HFE association with B2M and TFRC, crucial for its correct processing and cell surface presentation. Although E277K has a more deleterious effect than V295A, we propose that both mutations may play a role in the development of hereditary haemochromatosis.

Clinical impact of HFE mutations in Japanese patients with chronic hepatitis C

HFE mutations, a common cause of hereditary hemochromatosis (HH), are reportedly associated with hepatic iron overload, severe liver fibrosis, and good response to interferon treatment in European patients with chronic hepatitis C (CHC). HH shows ethnicity-based differences and little is known about the effects of HH mutations on CHC in the Japanese. Thus, the aim of this study was to clarify the clinical influence of HFE mutations in Japanese CHC patients. In a total of 251 patients with CHC, we analyzed the frequencies of H63D and S65C mutations in the HFE gene, and the influence of these mutations on clinical parameters and response to pegylated-interferon-alpha 2b (PEG-IFN) plus ribavirin therapy. Fourteen patients (5.6%) carried the H63D mutation; all were heterozygotes. No S65C mutations were found. Only hemoglobin levels in the H63D heterozygotes were higher than in wild-type patients. Eleven of 14 H63D heterozygotes achieved sustained virological response (SVR). On univariate analysis, factors associated with SVR were interleukin 28B (IL28B) polymorphism, age, hepatitis C virus (HCV) genotype, HCV viral load, white blood cell count, stage of fibrosis and H63D mutation. All patients with both TT genotype in IL28B (rs8099917) and H63D mutation in HFE (n = 10) achieved SVR. The H63D mutation has little impact on the clinical characteristics of CHC, but is related to favorable response to PEG-IFN plus ribavirin therapy, particularly in patients with the TT allele in IL28B.

Hemochromatoza HFE u dzieci – problem niedoceniany

IntroductionHemochromatosis is a disease of iron overload. There are primary – inherited diseases leading to increased intestinal absorption and secondary caused by multi transfusions or iron pills and iron rich food intake. Hereditary hemochromatosis is a metabolic disorder caused by mutations of genes that control iron metabolism. Mutations in HFE gene localized on the short arm of chromosome 6 are responsible for autosomal recessive hemochromatosis type 1. The disease symptoms are observed in adult males. The aim of the study was to analyse iron metabolism in 41 children. In chosen cases HFE mutations were determined. ResultsHFE mutations were documented in 15 cases. ConclusionsIron metabolism tests are indispensable in the diagnosis of its elevated status. In chosen cases molecular tests are essential.

Effect of ambiguous hemochromatosis gene test results on physician utilization.

Genetic test results may be available to greater numbers of people through genetic screening projects and other means. The effects of widespread genetic testing and notification of genetic test results, particularly added costs through increased physician utilization, have not been clearly established. A primary care-based cohort of 20,306 participants (Hemochromatosis and Iron Overload Study, Ontario site) were tested for the C282Y and H63D mutations of the HFE gene and for abnormal serum ferritin (SF) and transferrin saturation levels. The primary outcome variable was the total number of physician claims per patient after genetic test notification by mail. Multiple Poisson regression was used to adjust for age, sex, baseline SF, diagnoses of arthritis, diabetes, heart failure and impotence, self-rated health, and the number of claims during the 12 months before notification of results. The reference group had no HFE mutations (wild type) and normal transferrin saturation/SF values. Participants with an ambiguous hemochromatosis gene test and normal iron levels had statistically significantly higher average physician utilization of 3.0%. Participants with HFE mutations (excluding C282Y homozygotes) and elevated iron values showed a 6% increase in physician utilization. The health effects, if any, of increased utilization in heterozygotes or those with mild ferritin elevations are unknown but are unlikely to be large at the population level. Ambiguous genetic test results are associated with increased physician service use and should be considered when assessing the complete societal costs of widespread genetic testing.

Metals and the liver

Hereditary liver diseases resulting in copper and iron overload may cause significant morbidity and mortality if not diagnosed and treated early. The goal of this review is to highlight the key publications on genetics, diagnosis and management of hemochromatosis and Wilson disease over the past 18 months. Several recent advancements have been made in the genetic diagnosis of hemochromatosis and Wilson disease. Uncommon HFE mutations resulting in phenotypic hemochromatosis among C282Y heterozygotes have been identified from HFE gene sequencing. A serum ferritin less than 1000 μg/l in C282Y homozygotes was found to be associated with milder symptoms of hemochromatosis. Deferasirox was shown to reduce iron overload in patients with hemochromatosis and may be an option for patients who cannot tolerate phlebotomy. There was found to be evidence of genotype and phenotype correlation in Wilson disease, which can be diagnosed by genetic sequencing. A modified diagnostic guideline has been developed for children with Wilson disease with mild liver disease that increases the sensitivity and specificity of diagnosis. Also treatment with copper chelating agents has less hepatic treatment failures when compared with zinc monotherapy. Advancements in diagnosis of hemochromatosis and Wilson disease may lead to earlier diagnosis and treatment with resulting decrease in morbidity and mortality.

Molecular diagnosis of HFE mutations in routine laboratories. Results of a survey from reference laboratories in France

HFE-related hemochromatosis (HFE hemochromatosis) or type 1 hemochromatosis is an autosomal recessive disease characterized by progressive iron overload usually expressed in adulthood. The HFE gene, located on the short arm of chromosome 6 (6p21.3), encodes a protein that plays a crucial role in iron metabolism by modulating hepcidin synthesis in the liver. Homozygosity for the p.Cys282Tyr mutation accounts for nearly 80% of cases of hemochromatosis in France. Genetic testing is the key investigation to confirm the diagnosis of HFE hemochromatosis. A survey on routine practices was carried out among the eight reference laboratories of the French national network on genetic iron disorders. The main findings from this survey are as follows: 1) the p.Cys282Tyr mutation must be searched for as an initial step to establish the diagnosis of HFE hemochromatosis. This is in agreement with the recommendations of the French Health Authority (HAS) published in 2005. In these recommendations, homozygosity for the p.Cys282Tyr mutation with at least elevated transferrin saturation, is considered the only genotype that confirms of the diagnosis of HFE hemochromatosis; 2) in combination with the p.Cys282Tyr mutation (compound heterozygous genotypes), the p.Ser65Cys and the p.His63Asp variants may contribute to the occurrence of mild iron overload; 3) family screening is mandatory following the detection of homozygous individuals for the p.Cys282Tyr mutation.

Hepatitis C, porphyria cutanea tarda and liver iron: an update

Porphyria cutanea tarda (PCT) is the most common form of porphyria across the world. Unlike other forms of porphyria, which are inborn errors of metabolism, PCT is usually an acquired liver disease caused by exogenous factors, chief among which are excess alcohol intake, iron overload, chronic hepatitis C, oestrogen therapy and cigarette smoking. The pathogenesis of PCT is complex and varied, but hereditary or acquired factors that lead to hepatic iron loading and increased oxidative stress are of central importance. Iron loading is usually only mild or moderate in degree [less than that associated with full-blown haemochromatosis (HFE)] and is usually acquired and/or mutations in HFE. Among acquired factors are excessive alcohol intake and chronic hepatitis C infection, which, like mutations in HFE, decrease hepcidin production by hepatocytes. The decrease in hepcidin leads to increased iron absorption from the gut. In the liver, iron loading and increased oxidative stress leads to the formation of non-porphyrin inhibitor(s) of uroporphyrinogen decarboxylase and to oxidation of porphyrinogens to porphyrins. The treatment of choice of active PCT is iron reduction by phlebotomy and maintenance of a mildly iron-reduced state without anaemia. Low-dose antimalarials (cinchona alkaloids) are also useful as additional therapy or as alternative therapy for active PCT in those without haemochromatosis or chronic hepatitis C. In this review, we provide an update of PCT with special emphasis upon the important role often played by the hepatitis C virus.

Abstract 3599: Expression and function of HFE in mammary gland iron homeostasis and its relevance to breast cancer

Abstract Emerging evidences on the role of iron-regulatory proteins in tumor growth provide new insights into the role of iron in cancer. Iron, though essential for normal cellular function, is toxic if present in excess. Hemochromatosis is an autosomal recessive disease of iron overload linked to mutations in HFE gene [Histocompatability leukocyte antigen class I-like protein involved in iron (FE) homeostasis]. HFE interacts with transferrin receptors, TfR1 and TfR2, and inhibits cellular iron uptake. The prevalence of the disease is very high with homozygosity incidence of ∼1 in 300 and heterozygosity incidence of ∼1 in 10. The disease manifests with liver cirrhosis/cancer, cardiomyopathy, diabetes, and arthritis. Several studies have shown increased prevalence of HFE mutations in breast cancer patients across all races. There is also evidence of increased levels of iron in breast cancer tissue. Although excessive iron and oxidative stress have been postulated to play a role in breast cancer, the expression/function of HFE in mammary gland iron homeostasis and how mutations in HFE affect breast cancer progression have not been studied directly. Here we demonstrate the expression of HFE mRNA in mouse mammary gland and primary mammary epithelial cells. The mRNA was absent in HFE-/- mice. Immunohistochemistry showed localization of HFE protein in luminal and myoepithelial cells. Similar staining was also seen in primary mammary epithelial cells from wild type mice but not from HFE-/- mice. We then examined the physiological role of HFE in mammary gland. Ferritin is a cytoplasmic protein involved in iron storage, and increase in intracellular iron levels results in increased level of ferritin. Recently, ferritin has also been shown to promote angiogenesis. Western blot and immunohistochemical analysis revealed that mammary tissue from HFE-/- mice had increased ferritin levels in an age-dependent manner. Thus, for the first time we show that HFE-/- mice have increased iron accumulation in the mammary gland. Immunostaining with antibody against 4-hydroxynonenal, one of the byproducts of lipid peroxidation, showed that HFE-/- mammary tissue have oxidative stress mediated by iron accumulation. MTT assay and thymidine incorporation showed that primary mammary epithelial cells from HFE-/- mice proliferate faster than the cells from HFE+/+ mice. Increase in histone deacetylase (HDAC) activity has been implicated in many cancers. We found that mammary tissue from HFE-/- mice has increased HDAC activity compared to wild type tissue. This shows that functional loss of HFE in mammary tissue can also promote cell cycle progression via epigenetic mechanisms. Based on these data, we hypothesize that tissue iron is an important modifier of breast cancer progression and that mutations in HFE (heterozygosity as well as homozygosity) may enhance the incidence and/or progression of breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3599. doi:1538-7445.AM2012-3599

Non-Coding Keratin Variants Associate with Liver Fibrosis Progression in Patients with Hemochromatosis

BackgroundKeratins 8 and 18 (K8/K18) are intermediate filament proteins that protect the liver from various forms of injury. Exonic K8/K18 variants associate with adverse outcome in acute liver failure and with liver fibrosis progression in patients with chronic hepatitis C infection or primary biliary cirrhosis. Given the association of K8/K18 variants with end-stage liver disease and progression in several chronic liver disorders, we studied the importance of keratin variants in patients with hemochromatosis.MethodsThe entire K8/K18 exonic regions were analyzed in 162 hemochromatosis patients carrying homozygous C282Y HFE (hemochromatosis gene) mutations. 234 liver-healthy subjects were used as controls. Exonic regions were PCR-amplified and analyzed using denaturing high-performance liquid chromatography and DNA sequencing. Previously-generated transgenic mice overexpressing K8 G62C were studied for their susceptibility to iron overload. Susceptibility to iron toxicity of primary hepatocytes that express K8 wild-type and G62C was also assessed.ResultsWe identified amino-acid-altering keratin heterozygous variants in 10 of 162 hemochromatosis patients (6.2%) and non-coding heterozygous variants in 6 additional patients (3.7%). Two novel K8 variants (Q169E/R275W) were found. K8 R341H was the most common amino-acid altering variant (4 patients), and exclusively associated with an intronic KRT8 IVS7+10delC deletion. Intronic, but not amino-acid-altering variants associated with the development of liver fibrosis. In mice, or ex vivo, the K8 G62C variant did not affect iron-accumulation in response to iron-rich diet or the extent of iron-induced hepatocellular injury.ConclusionIn patients with hemochromatosis, intronic but not exonic K8/K18 variants associate with liver fibrosis development.

Control of iron metabolism – Lessons from neonatal hemochromatosis

When hepatologists begin their investigations at the scene of liver damage, iron is certainly on the list of ‘‘usual suspects’’ to be rounded up. Hemochromatosis, a well-known cause of chronic liver disease, is the most prevalent genetic disorder in adults [1]. Serum iron parameters are therefore included in most guidelines on how to investigate patients with elevated transaminases or chronic liver disease [2]. The hemochromatosis (HFE) genotype is an important piece of evidence in patients with liver disease and elevated transferrin saturation with hyperferritinemia, because homozygosity for the C282Y allele of HFE is considered sufficient to diagnose hemochromatosis under these circumstances [1]. Compound heterozygosity for C282Y and H63D has been almost removed from the watch-list and is currently considered a mere risk factor for liver diseases – sufficient to cause hemochromatosis or even cirrhosis only in combination with other health burdens. Hence, a partner in crime for HFE mutation must be found in patients with liver disease and C282Y/H63D compound heterozygosity [3]. If there is evidence for iron overload in the absence of hemochromatosis-associated HFE genotypes and the hepatopathologist finds iron confined to hepatocytes, sharp investigators are well advised to search for the culprit in the genes encoding transferrin receptor 2 (TFR2) and ferroportin (SLC40A1) or even to hunt for combined mutations in these genes if a patient presents with severe iron overload or early-onset disease [4]. If the patient is adolescent or before the 3rd decade of life and presents with heart failure or hypogonadism in addition to – often overlooked – liver disease, other genes will be committed in custody. Mutations in HJV and HAMP, which respectively encode hemojuvelin and hepcidin, will be sufficient to identify juvenile hemochromatosis [5]. Through the power of genetics, iron-related diseases are now subject to close scrutiny. In addition, genetic studies have greatly advanced our understanding of hemochromatosis. This progress is reflected in the proposed re-classification of human iron

HFE Gene Mutations, Iron Overload and Cryptogenic Liver Cirrhosis

Dear Editor, The diagnosis of cryptogenic cirrhosis is an exclusion diagnosis. It has become far less frequent over the last decades, but it still effects a significant number of patients. Many previously unknown chronic liver disease entities were described in the second half of the last century, including chronic viral hepatitis B, C and D, along with refined criteria for diagnosing autoimmune hepatitis and insights into the pathogenesis and disease progression of fatty liver disease [1]. Although hemochromatosis, and its pathophysiology were not understood until cloning of the HFE-gene [2], it has been known as a chronic liver disease for a long time, although it may be under-recognized due to the lack of specific symptoms and thus contribute to cases of cryptogenic cirrhosis. Jowkar et al. [3] described the frequency of the most common disease-causing mutations of the HFE-gene (C282Y and H63D) in Iranian patients with cryptogenic cirrhosis and healthy controls. They did not observe any C282Y mutations and only heterozygous H63D mutations were found in about one quarter of both healthy and cirrhotic patients. As the C282Y mutation is most likely to be of northern European origin, the low frequency found in the present report is not surprising. The H63D mutation however, was present heterozygous in almost one quarter of the patients without any of these patients carrying homozygous mutations, this is surprising and much higher than would be expected [4][5]. As there were no differences between healthy controls and cirrhotic patients, these numbers most likely reflect the allele frequency in the general Iranian population and were described for the first time in the current report. The authors present results for patients with and without iron overload. Unfortunately, the numbers of patients diagnosed with iron overload are not reported. Therefore, the relative proportion of patients carrying the H63D mutation cannot be calculated and the impact of iron overload on cryptogenic cirrhosis cannot be deduced. Furthermore, iron overload in the paper by Jowkar et al. has been defined by transferrin saturation. It would be valuable to know serum-ferritin levels in order to get further insights into the state of iron metabolism. Classic HFE related hemochromatosis, as demonstrated by the authors, is extremely unlikely in this population. If iron overload were indeed present it would be worthwhile testing for other hemochromatosis associated mutations, namely transferrin receptor 2 (tfr2) and ferroportin (fpn) [6]. In conclusion, Jowkar et al. confirmed the low frequency of hemochromatosis causing HFE mutations in the Iranian population for the first time. Further studies are still needed to evaluate the impact of hereditary iron metabolism disorders on cryptogenic cirrhosis in Iran.

The prognostic role of hemochromatosis H63D allele in allogeneic hematopoietic stem cell transplantation

Iron overload is considered as a significant cause of morbidity and mortality in hematopoietic stem cell transplant (HSCT) recipients. The presence of hemochromatosis gene (HFE) mutations might exacerbate iron toxicity in the post-transplant setting. This prospective study was planned to evaluate the genetic spectrum of HFE mutations in Turkish patients undergoing HSCT and the impact of HFE genotype on transplant morbidity and mortality. HFE genotypes of 102 patients [median age, 27.5 years (16-64 years); male/female, 73:29], who underwent allogeneic HSCT, were analyzed. Twenty-two patients were heterozygous and 1 patient was homozygous for the H63D mutation, while the C282Y mutation was observed in none of our patients. The frequency of invasive fungal infections (IFI) was significantly higher in H63D-mutated patients (p=0.004). H63D mutation was identified as an independent risk factor for the development of IFI (p=0.006, OR=0.554, SE=0.208), without an impact on overall survival and transplant-related mortality. The multifactorial iron-overloaded state in HSCT recipients might affect the phenotypic expression of HFE mutations and alter the severity of clinical presentation. The impact of HFE genotype on iron parameters and transplant-related morbidity and mortality should be validated with further studies.

Validation of an Unlabeled Probe Melting Analysis Assay Combined with High-Throughput Extractions for Genotyping of the Most Common Variants inHFE-Associated Hereditary Hemochromatosis, C282Y, H63D, and S65C

Hereditary hemochromatosis is an inherited disorder of iron metabolism, characterized by high absorption of iron by the gastrointestinal tract leading to a toxic accumulation of iron in various organs and impaired organ function. Three variants in the HFE gene (p.C282Y, p.H63D, and p.S65C) are commonly associated with the development of the disease. Of these, p.C282Y homozygotes are at the highest risk. Compound heterozygotes of p.C282Y along with p.H63D or p.S65C have reduced penetrance. Furthermore, p.H63D homozygotes are not at an increased risk and little is known about the risk associated with homozygocity for p.S65C. Our current clinical assay for the three common HFE variants utilizes the LightCycler platform and paired probes employing fluorescent resonance energy transfer. To increase throughput and decrease costs, we developed a method whereby automated extraction was combined with unlabeled probes and differential melt profiles to detect these variants using the LightCycler 480 instrument. Using this approach, 43 samples extracted with three different extraction platforms were correctly genotyped. These data demonstrate that the newly developed assay to genotype the HFE mutations p.C282Y, p.H63D, and p.S65C, combined with high-throughput extraction platforms, is accurate and reproducible and represents an alternative to previously described tests.

Iron Overload and HFE Mutations: Are They Relevant in Cryptogenic Cirrhosis?

Dear Editor, Hereditary hemochromatosis (HH) is the most frequent genetic disease in populations of European origin. The HH gene was cloned by Feder et al. in 1996, and 2 major mutations were discovered: C282Y and H63D. Geographical differences with mutation frequencies have been published [1][2] with a decreasing gradient of occurrence in Europe from north to south. HH leads to liver iron overload and raised liver iron concentration (LIC) is associated with liver fibrosis. When the LIC reaches 60µmol/g, or approximately twice the upper limit of the normal range (36µmol/g), activation of stellate cells appears. These cells are principally responsible for the beginning of liver fibrosis and fibrosis [3]. This process can lead to hepatic cirrhosis at the end stage. So it is of interest to ask ourselves, for those patients with cirrhosis but without any known culprit as the cause, whether iron surcharge and mutations of the HFE gene that may induce this iron overload could play a relevant role in the etiopathogenesis of cryptogenic cirrhosis. Jowkar et al. [4] have recently published a prospective study to investigate the probable association of HFE gene mutations in a group of 100 patients with cryptogenic cirrhosis and compared the results with a control group of 50 normal, unrelated healthy individuals. No homozygotes for C282Y and H63D mutations were found, and only 22% of the patients and 28% of controls (P > 0.05) were heterozygous for the H63D mutation. Most of the cirrhotic patients and controls with this mutation did not have liver iron overload. Few studies have investigated the frequency of HFE gene mutations in patients with chronic liver diseases and cirrhosis. The results from the Jowkar et al. [4] study are similar to those published from Indian and Turkish patients [5][6]. More recently, Sikorska et al. [7] studied iron overload and HFE gene mutations in Polish patients with liver cirrhosis (61 patients), and they were compared with a control group of 42 consecutive patients subjected to liver biopsy because of chronic liver disease. They concluded that iron disorders are frequently detected in patients with liver cirrhosis, but without significant association with HFE gene mutations. Another recent study from India [8] tried to find the association of common HFE mutations and primary iron overload in liver cirrhosis. Of the 496 patients (242 cryptogenic cirrhosis) included in the study, only 13 had iron overload and only two were H63D heterozygous. The overall frequency of the H63D allele in patients and controls was not significantly different (P < 0.17). Jowkar et al. [4], with their interesting study developed in Iran, seem to reinforce the theory that cryptogenic cirrhosis is not associated with iron overload, nor with major HFE mutations. It also appears that there are no geographical differences, since their results are similar to those published in Poland, Europe.