S65C Mutations Research Articles

S3057 Iron Overload in an H63D Homozygote: Looking Beyond the Genotype

Introduction: Hereditary hemochromatosis (HH) is a genetic disorder in which excessive gastrointestinal absorption of iron results in iron-mediated damage to the liver and other organs. HH is inherited in an autosomal recessive fashion and is characterized by mutations in the HFE gene; however, due to low penetrance, many patients with two abnormal copies of HFE do not exhibit clinical iron overload. The most common pathologic mutations of HFE are C282Y and H63D. Over 90% of HH cases are due to homozygosity for C282Y, with the remainder attributed to C282Y/H63D compound heterozygosity, H63D homozygosity, or S65C mutation. We discuss a rare case of newly diagnosed HH in a patient with H63D homozygosity and family history of iron overload. Case Description/Methods: A healthy 67-year-old Caucasian male with history of hyperlipidemia presented for abnormal iron studies which were checked after he reported that his father used to require phlebotomy. He drinks two alcoholic beverages daily and denies any clinical complaints. Laboratory tests were notable for serum ferritin of 1240 ng/mL, transferrin saturation of 45.6%, hemoglobin 15.5 g/dL, ALT 91 units/L, and AST 43 units/L. Genetic testing revealed homozygosity for H63D. Due to his markedly elevated ferritin, liver biopsy was performed and showed diffusely increased iron deposition, predominantly in hepatocytes. Moderate steatosis and portal fibrosis (fibrosis stage 1/4) were also seen. He was diagnosed with HH and is scheduled for phlebotomy with goal ferritin less than 50 ng/mL. Discussion: Patients homozygous for H63D make up fewer than 10% of all HH cases and clinically significant iron overload is far less common. The likelihood of iron overload for a particular genotype is higher if a first-degree relative had iron overload which may explain our patient’s phenotype. Although our patient did not have cirrhosis on biopsy, his HH in addition to hepatic steatosis puts him at higher risk of liver disease. This risk can be mitigated with control of his ferritin via phlebotomy and modification of other risk factors. Our case highlights the importance of evaluating for HH and hepatic fibrosis in patients with family history, irrespective of genotype, if they present with phenotypic signs of iron overload.

S3055 Late Manifestation of Heterozygous H63D Mutation as Hereditary Hemochromatosis in 89-Year-Old Man

Introduction: Hereditary hemochromatosis (HH) is an autosomal recessive that usually affects middle-aged people [1]. In most patients, homozygous C282Y mutations in the HFE gene express the disease. But in some occurrences, compound heterozygous, including C282Y and H63D mutations, were also witnessed. We present an atypical case where heterozygous H63D manifested as HH in an 89-year-old man. Case Description/Methods: An 89-year-old Caucasian man of Italian descent presented with confusion, left-sided facial droop, and slurring of speech. Past medical history included end-stage renal disease on hemodialysis and atrial fibrillation. For the past few months before presentation, the patient has been complaining of increasingly tired with intermittent episodes of slurring speech, mainly after he receives dialysis. During the initial physical examination, the patient had confusion but was alert & oriented to self and time. There was mild abdominal distension and positive for asterixis, and the skin was bronze in color. Initial labs showed in table 1. The patient had persistent hypoglycemia and required intravenous glucose administration. Brain imaging was normal. CT scan of the abdomen, pelvis, and chest showed severe cardiomegaly and a moderate amount of ascites. Iron studies were abnormal. Ascites fluid analysis was consistent with cirrhosis. Although, an ultrasound right upper quadrant was negative for any cirrhotic morphology. MRI showed hemosiderosis. 2D Echo showed a severely dilated right ventricle. The patient empirically underwent two sessions of phlebotomy. Genetic testing confirmed heterozygous H63D mutation. The patient followed up for the phlebotomy sessions and demonstrated significant improvement. Discussion: This case highlights the consideration of HH in differential regardless of age if a patient presents with symptoms suspected of HH. Standard genetic testing includes testing C282Y and H63D mutations in the HFE gene on chromosome 6. Less frequently tested S65C, HFE4, TFR2-HHC, and FTH1 gene mutations also have the expression of HH in sporadic cases [2-3]. Previously, heterozygous C282Y carriers with iron overload are assumed to have other genetic changes or influence by environmental factors, such as alcohol or liver disease, that increase disease expression [2]. However, no such literature is available for heterozygous H63D carriers. Given the unusual age for phenotype expression and atypical disease, the presentation makes this unique case for learning the pathophysiology and genetics of HH.Figure 1.: A: MRI of liver showing decreased signal of the liver and spleen on in phase imaging is likely due to hemosiderosis. B: Two-dimensional transthoracic echocardiography showing severely dilated right ventricle and both atria. Moderate pulmonary hypertension was also noticed. Table 1. - Day 1 Day 2 Day 3 Day 4 Day 15 Day 42 Hemoglobin (g/dL) 15.6 14.8 15.1 13.8 13.6 12.2 AST (U/L) 85 102 135 125 109 71 ALT (U/L) 56 57 70 74 72 46 Bilirubin (mg/dL) 3.0 2.9 2.6 2.9 2.6 2.1 INR 1.3 1.4 1.4 1.4 1.2 1.2 Ammonia (µmol/L) 99 45 46 42 31 Platelets (10ˆ3/µL) 103 86 82 63 59 91 Ferritin (ng/ml) 1283 725 Iron (µg/dL) 187 130 Transferrin Saturation (%) 94 74 Total Iron-binding Capacity (µg/dL) 200 276

Screening for hereditary haemochromatosis in patients undergoing knee arthroplasty : a retrospective cohort study of 2,035 patients.

AimsHereditary haemochromatosis is a genetic disorder that is caused by several known mutations in the human homeostatic iron regulator protein (HFE) gene. Abnormal accumulation of iron causes a joint disease that resembles osteoarthritis (OA), but appears at a relatively younger age and is accompanied by cirrhosis, diabetes, and injury to other organs. Increased serum transferrin saturation and ferritin levels are known markers of haemochromatosis with high positive predictive values.MethodsWe have retrospectively analyzed the iron studies of a cohort of 2,035 patients undergoing knee joint arthroplasty due to OA.ResultsNo patients had HFE gene C282Y, S65C, or H63D mutations testing. In total, 18 patients (2.96%) of the male cohort and 51 (3.58%) of the female cohort had pathologically increased ferritin levels that may be indicative of haemochromatosis. Seven patients (0.34%) had serum transferrin saturation above 45%.ConclusionThe awareness for the diagnosis of this disorder in Orthopaedics is low and needs improvement. Osteoarthritic patients undergoing knee arthroplasty should be routinely screened for haemochromatosis by iron studies and referred to genetic testing when needed.Level of evidence: Level III - Retrospective cohort study.Cite this article: Bone Jt Open 2021;2(12):1062–1066.

Chronic hepatitis B in children carried out of the hemochromatosis gene HFE

Purpose: to establish the frequency and clinical significance of mutant variations of the HFE gene polymorphism in chronic hepatitis B (CHB) in children with iron overload syndrome (IOS).Materials and methods: 60 children with chronic hepatitis B with iron overload syndrome (IOS) were examined. When distributing children into groups, we took into account the criteria we developed for assessing the degree of life expectancy in children with CHB: CST>0.5 – mild degree of life expectancy (43,3% of children), CST˃0.5 – mild degree of life expectancy (43,3% of children), CST˂0,5 – moderate severity of life expectancy (31,7% of children) and CST˂0,2 – severe degree of SPL (25,0%). Virological verification of HBV was performed by ELISA and PCR. Using PCR Real Time and molecular genetic analysis, HFE gene C282Y, H63D, S65C mutations were detected from amplified DNA using the PRONTO Hemochromatosis reagent kit (Israel). The transferrin saturation coefficient (CST) was calculated using the formula CST = sTfR / log10.Ft. Results:Results: The study of the hemochromatosis gene HFE showed that the overwhelming majority (84,0%) of children with CHB with IOS were carriers of heterozygous, phenotypically different, mutant types. And only 16,0% of sick children were homozites of the wild (normal) HFE gene. Analysis of the phenotypic polymorphism of the hemochromatosis gene HFE revealed the presence of three point heterozygous mutations: H63D, S65C and combined variations in H63D / S65C, the latter of which is associated with severe forms of CHB and severe IOS.Conclusion. Children with CHB with IOS are characterized by a high incidence of heterozygous mutations in the HFE gene, the phenotypic manifestations of which were S65C, H63D, H63D / S65C. The comparability of the heterozygous combined mutation H63D / S65C with severe forms of CHB and a severe degree of IOS gives grounds to consider this phenotype of the HFE gene as a factor in the progression of the disease.

Mutation Analysis and Allele Frequencies of HFE Gene C282Y, H63D and S65D in Patients with Hyperferritinemia and Healthy Controls from a City in Northeastern Brazil

Hereditary hemochromatosis (HH) is a genetic disease caused by high iron absorption and deposition in several organs. This accumulation results in clinical disturbances such as cirrhosis, arthritis, cardiopathies, diabetes, sexual disorders and skin darkening. The C282Y mutation is well defined in hemochromatosis etiology. Two other mutations have been described, H63D and S65C, associated with a milder form of the disease. The aims of this study were to identify the H63D, S65C and C282Y genetic mutations in the hemochromatosis gene and assess the frequency of these mutations in the HFE protein gene in patients with hyperferritinemia which are sent to the DNA Center laboratory in Natal, Rio Grande do Norte state, in addition to evaluating the correlation between the H63D, C282Y and S65C mutations in hereditary hemochromatosis and serum ferritin. The biochemical dosages and molecular analyses are carried out using the enzymatic method and PCR with enzymatic restriction, respectively. Of the 300 patients investigated, 48.7% showed no mutation and 51.3% some type of mutation: 4.4 % exhibited C282Y heterozygous mutation; 2.7% C282Y homozygous mutation; 31.9% H63D heterozygous mutation; 8.1% H63D homozygous mutation; and 5.1%, heterozygous mutation in both genes. The S65C mutation was studied in 112 patients, 2.7% with heterozygous mutation (S65D/WT) and 1.8% with double heterozygosity (H63D/S65C). With regard to sex, we observed the highest number of cases with molecular changes in men compared to women in the two mutations evaluated. Since it is considered a public health problem and exhibits low toxicity with treatment, early genetic diagnosis of HH is recommended, especially in patients with elevated serum ferritin, thereby preventing severe clinical manifestations resulting from late diagnosis. Our findings show the importance of genetic studies in patients with suspected hereditary hemochromatosis due to the high incidence of this hereditary disease in our region. DisclosuresNo relevant conflicts of interest to declare.

HFE Gene Mutations and Iron Status in 100 Healthy Polish Children.

Iron participates in oxygen transport, energetic, metabolic, and immunologic processes. There are 2 main causes of iron overload: hereditary hemochromatosis which is a primary cause, is a metabolic disorder caused by mutations of genes that control iron metabolism and secondary hemochromatosis caused by multitransfusions, chronic hemolysis, and intake of iron rich food. The most common type of hereditary hemochromatosis is caused by HFE gene mutation. In this study, we analyzed iron metabolism in 100 healthy Polish children in relation to their HFE gene status. The wild-type HFE gene was predominant being observed in 60 children (60%). Twenty-five children (25%), presented with heterozygotic H63D mutation, and 15 children (15%), presented with other mutations (heterozygotic C282Y and S65C mutation, compound heterozygotes C282Y/S65C, C282Y/H63D, H63D homozygote). The mean concentration of iron, the level of ferritin, and transferrin saturation were statistically higher in the group of HFE variants compared with the wild-type group. H63D carriers presented with higher mean concentration of iron, ferritin levels, and transferrin saturation compared with the wild-type group. Male HFE carriers presented with higher iron concentration, transferrin saturation, and ferritin levels than females. This preliminary investigation demonstrates allelic impact on potential disease progression from childhood.

Clinical significance of HFE gene mutations in patients with refractory anemia with ring sideroblasts (RARS).

e18556 Background: RARS is a subtype of myelodysplastic syndromes (MDS) defined by < 5% blasts and ≥15% ring sideroblasts (WHO 2008). Hereditary hemochromatosis is a disorder characterized by dysregulations in iron absorption, largely associated with C282Y and H63D mutations of the HFE gene. Iron levels are elevated in both disorders and pathophysiologic correlations were suggested. HFE gene mutations were previously found higher in MDS compared to controls (50% vs 36%) ( Nearman et al, Am J Hematol 2007). Methods: A total of 168 RARS patients’ data from 1994 to 2015 at Mayo Clinic were reviewed after appropriate IRB approval was obtained. All cases had their bone marrow slides reviewed at our center. We searched patients’ records retrospectively to Identify those tested for HFE gene (C282Y, H62D, S65C) mutations, done inside or outside our institution. Survival estimates were calculated using Kaplan-Meier curves. Results: Out of the 168 RARS patients, only 17 (10%) were tested for HFE gene mutations. Out of the 17 tested, 11 (65%) were found to have mutations; 2 of which (18%) had homozygous H63D mutation, 1 patient (9%) had double heterozygous H63D and C282Y mutations, 5 (45%) had only one H36D heterozygous mutation vs 3 patients (27%) with only one C282Y heterozygous mutation. Only one patient was tested for the additional S65C mutation and it was not detected. H63D mutation was present in a total of 8 patients (73%) vs C282Y mutation which was present in 4 patients (36%). Bone marrow iron stores were increased in all 17 tested patients, except one who had decreased stores, this patient had one heterozygous C282Y mutation. Median overall survival (mOS) was 117 months in the HFE mutated patients vs 75 months in the non-mutated (p = 0.6). Conclusions: Our study found the HFE gene, when tested, to be mutated in higher frequencies among patients with RARS compared to that reported in the general population (65% vs 36%), with H63D mutation in almost three quarters of all mutated patients. Although it did not reach statistical significance, the longer survival observed among HFE mutated patients compared to the wild-type raises the question whether testing for HFE gene mutations among patients with MDS-RARS should be further explored.

Novel Method Probe-based Real-Time PCR to Detect 2 Single-Nucleotide Polymorphisms Close to Each Other: HFE Hemochromatosis Gene Model.

Hereditary hemochromatosis is known as the most common genetic disorder among individuals of European genetic background. It is possible to find 2 mutations closely placed in the HFE gene (H63D and S65C) and this proximity can cause errors when genotyped by real-time polymerase chain reaction (PCR) genotyping assay. The aim of this study was to develop a hydrolysis probe-based PCR assay for detection of the H63D and S65C mutations without interference from on each other. Herein the study involved the standardization of an improvement of the real-time PCR 5' nuclease assay to detect the desired mutations close placed using a same probe system. The assay analytical properties performances were tested, including the primers selectivity and detection limits. Also, the interexaminer reproducibility and repeatability of assay were estimated in 30 blood samples. Others 153 results of samples were compared with reference method (PCR_RFLP) and the accordance of the results evaluated by Fleiss' κ method. The results of variation of interexaminer reproducibility and repeatability of assay were not statistically relevant (P<0.001). The comparison between the 2 methods by Fleiss' κ analysis showed that 5' nuclease assay identified the H63D and S65C haplotype as well as the reference method in all 153 tested samples. Our results showed that novel method probe-based real-time PCR were capable to detect 2 adjacent polymorphisms without errors in genotyping.

Food iron fortification, high meat intake and HFE gene in a healthy adult male population from Argentina (804.25)

Wheat flour fortification with iron (Fe) is mandatory in Argentina since 2002, although feeding habits are characterized by high bovine meat intake‐ (54.1 kg/per capita/y, FAO 2009). To estimate heme and nonheme Fe intake, 87 male blood donors (19‐65 y) attending the Hospital de Clínicas, Universidad de Buenos Aires, Argentina, (2012‐2013) were enrolled in the study. A 135‐item food‐frequency questionnaire including bovine meat, poultry, fish, pork, processed meat, cereal products and bakery products prepared with wheat flour fortified with Fe (30 mg Fe/Kg) was administered. Daily Fe intake was calculated according to ARGENFOODS and USDA National Nutrient Database on Standard Reference. Genotyping for the C282Y, H63D and S65C mutations in the HFE gene was performed (PCR‐RFLP) in samples (n=40) collected with EDTA. Dietary Fe intake (mg/d) was: Mean ± SD (range): Total Fe (T): 24.2±12.5 (8.9‐63); Heme Fe (H): 2.26±1.45 (0.00‐6.84); Nonheme Fe (NonH): 22± 11.8 (7.4 ‐59.8); Fe‐fortified wheat flour (Fe‐WF): 10.5 ±6.9 (2.6‐44). Fe intake was higher (111% ‐ 788%) than RDA (8 mg/d, FNB); Fe‐WF was 47.7% of NonH. Heterozygosity and homozygosity for H63D mutation was 15% and 2.5%, respectively; C282Y nor S65C occurred. These preliminary results alert about potential diet‐related effects of Fe accumulation in a population most probably unaware of any family history of Fe overload.Grant Funding Source: Supported by Universidad de Buenos Aires 20720120200004BA, Argentina

Analysis of HFE genes C282Y, H63D, and S65D in patients with hyperferritinemia from northeastern Brazil.

Hereditary hemochromatosis (HH) is a genetic disease caused by the high absorption and deposition of iron in several organs. This accumulation results in several clinical complications such as cirrhosis, arthritis, cardiopathies, diabetes, sexual disorders, and skin darkening. The H63D and C282Y mutations are well defined in the HH etiology. The objective of this article is identification of the H63D and C282Y mutations in the HFE protein gene and the frequency assessment of these mutations in patients with persistent increase of serum ferritin in patients from Natal City from state of Rio Grande do Norte, located in northeastern Brazil. Of the 299 patients studied for C282Y and H63D, 48.49% showed absence of mutation and 51.51% showed some sort of mutation: heterozygous C282Y mutation in 4.35% patients, homozygous C282Y mutation in 2.67% patients, heterozygous H63D mutation in 31.44% patients, homozygous H63D mutation in 8.03% patients, and heterozygous for the mutation in both genes (C282Y/H63D) in 5.02% patients. The S65C mutation was studied in 112 patients and heterozygous mutation (S65D/WT) in 2.67% of patients and double mutation (H63D/S65C) in 1.78% of patients were observed. Due to the high prevalence of hemochromatosis, its genetic diagnosis has become a challenge, especially in the high-risk group.

HFE MUTATIONS AND IRON OVERLOAD IN PATIENTS WITH ALCOHOLIC LIVER DISEASE

Alcoholic liver disease (ALD) is generally associated with iron overload, which may contribute to its pathogenesis, through increased oxidative stress and cellular damage. There are conflicting reports in literature about hemochromatosis (HFE) gene mutations and the severity of liver disease in alcoholic patients. To compare the prevalence of mutations in the hemochromatosis (HFE) gene between patients with ALD and healthy controls; to assess the relation of HFE mutations with liver iron stores and liver disease severity. Liver biopsy specimens were obtained from 63 ALD patients (during routine treatment) and 52 healthy controls (during elective cholecystectomy). All individuals underwent routine liver function tests and HFE genotyping (to detect wild-type sequences and C282Y, H63D, S65C, E168Q, E168X, V59M, H63H, P160delC, Q127H, Q283P, V53M and W164X mutations). Associations between HFE mutations and risk of excessive liver iron stores, abnormal serum ferritin, liver fibrosis, or necroinflammatory activity were assessed by multivariate logistic regression analysis. ALD patients had significantly higher serum ferritin and transferrin saturation than controls (both P<0.05), but the distribution of HFE mutations was similar between the two groups. For ALD patients, the odds ratio for having at least one HFE mutation and excessive liver iron stores was 17.23 (95% confidence interval (CI): 2.09-142.34, P = 0.008). However, the presence of at least one HFE mutation was not associated with an increased risk of liver fibrosis or necroinflammatory activity. Active alcohol ingestion showed the strongest association to increased serum ferritin (OR = 8.87, 95% CI: 2.11-34.78, P = 0.003). s ALD patients do not present with a differential profile of HFE mutations from healthy controls. In ALD patients, however, the presence of at least one HFE mutation increases the risk of having excessive liver iron stores but has no detectable effects on liver disease activity or severity.

Role of Magnetic Ressonance Imaging – T2* in Hereditary Hemochromatosis.

AbstractAbstract 2096 Introduction:Hereditary hemochromatosis (HH) is an autossomic recessive disorder characterized by increased iron absorption. Magnetic resonance imaging – T2* (MRI-T2*) has become a reliable and robust methodology to directly assess the iron burden, with better results in transfusional hemosiderosis compared to indirect methods, such as serum ferritin and transferrin saturation (TS). However, little is known about its role in HH. Objectives:Describe the demographic profile of HH type 1 patients as to the type of the HFE mutation and correlate laboratory parameters to MRI-T2*results. Methods:We collected data from patients with a positive HFE gene mutation who performed abdominal and/or cardiac MRI-T2* in our institution from 2004 to 2011. Images retrieved from the digital archive were analyzed by two blinded independent radiologists using the Thalassemia-Tools software (Cardiovascular Imaging Solutions, London, UK). Laboratory data available within 6 months before or after the MRI study were analyzed using the t-Student test, Exact Fisher's test analysis and multivariate analyses. Results:We analyzed 81 patients, 76 (93%) males and 5 (6.2%) females, with a median age of 48 years (21–80). Liver, pancreatic and splenic MRI-T2*values and LIC calculation were performed in 80 patients, and cardiac T2* assessment in 57 patients. The inter-observer T2* variation coefficient was 5%. Serum ferritin was abnormal in 70 patients (90.9%), while TS was abnormal in 34% of the tests.In our study sample, the H63D mutation was present in 70 patients (86.4%): 11 (13.6%) were homozygous, 59 (72.8%) heterozygous and 7 (8.6%) double heterozygous for C282Y/H63D. Only three patients (3.7%) were homozygous and 6 (7.4%) were heterozygous only for the C282Y mutation. The S65C mutation was detected in heterozygous state in 2 (2.5%) of cases.Two out 57 cases had a positive T2* result and were classified as light cardiac overload (T2*:18.98 e 19.14 ms). Both had the H63D mutation (1 homozygous and 1 heterozygous). Thirty seven out of 80 patients (46.3%) had liver overload in abdominal MRI (T2*: 3.8–11.4ms), being 33 (41.3%) light overload and four (5%) moderate overload (T2*:1.8–3.8ms). We found that 77.8% of patients with liver overload were C282Y carriers, of which 57.2% had double mutation and 40.3% had H63D mutation in hetero or homozigosity. Pancreatic overload was found in 20 patients (25.1%), while 30 patients (37.5%) had splenic overload. There was a slight correlation (r: 0.365) between liver T2* and splenic T2* (p=0.001).The presence of C282Y and H63D mutations was statistically associated with a higher frequency of abnormal liver T2* (p=0.017 and p=0.042, respectively). The H63D mutation was associated with iron accumulation in the liver (p=0,037) and homozygous carriers showed higher levels of liver overload (p=0,038). Conclusion:In our study, serum ferritin was a better surrogate marker for iron overload than ST. In addition, up to 40.3% of patients with H63D mutation had evidence of hepatic iron overload by MRI. These findings differ from the currente literature. The higher RMI positivity might be due to a higher sensitivity to detect lower levels of organic iron. Despite the lack of a control group and laboratory tests or MRI in all the cases studied, our results suggest that RMI-T2* is a promising methodology to guide the therapeutic management of HH patients. The clinical impact of this finding must be investigated in further studies. Disclosures:No relevant conflicts of interest to declare.

Hepatitis C as a Rare Cause of Acute Liver Failure

Background: Acute liver failure (ALF) is defined as coagulopathy (INR>1.5) and encephalopathy in a patient without pre-existing liver disease. The typical interval from onset of symptoms to onset of encephalopathy is 1 to 2 weeks. Common causes include Acetaminophen (most common), drug-induced, viral (Hepatitis B and A) and autoimmune hepatitis. In up to 15% cases, the etiology may remain indeterminate. In contrast, acute Hepatitis C infection is generally asymptomatic and ALF at presentation is extremely rare. Case Report: We present the case of a 41-year-old African American male with the complaint of his eyes being yellow for 4 days. His wife reported irritability and insomnia. Past medical history included hypertension and end stage renal disease on hemodialysis. He reported no alcohol use, no recent travel and sexual contact with wife only. He reported using acetaminophen occasionally and last use was 2 tablets 1 week back. The rest of his medications did not include any known hepatotoxic agents. On exam, his vitals were stable. He had icterus and yellowish discoloration of the oral mucous membranes. Abdominal exam showed nontender hepatomegaly. Liver function tests showed ALT 1689 U/L, AST >2000 U/L and Alkaline Phosphatase 432 U/L. Total Bilirubin was 14.7 mg/dl with direct fraction of 7.5 mg/dl. INR was 1.68 with Albumin of 2.8. Hepatitis Panel showed HepA IgM negative, HepBsAg negative, HepBs Ab >1000 mIU/ml and HepC Ab positive (negative 4 months back). Acetaminophen level was negative. Iron panel showed ferritin 49,595 ng/ml, iron saturation 104%, TIBC 146 mcg/dl and Iron 152 mcg/dl suggestive of iron overload which was not seen 1 month back. Hepatitis C viral load was 51 million with genotype 1a. Ultrasound and MRI/MRCP did not show any liver masses or biliary dilation. There was hemosiderin deposition in the liver and spleen. Liver biopsy was done after which patient developed life threatening intra-abdominal bleeding and died after 2 days. Biopsy showed extensive ballooning degeneration of hepatocytes, hepatocellular apoptosis with reticulin framework collapse consistent with acute viral hepatitis and increased iron deposition. Hereditory hemochromatosis panel was negative for C282Y, H63D or S65C mutations. Conclusion: This report suggests Hepatitis C infection to be the cause of ALF in this patient. Hepatic iron accumulation is observed in advanced stages of HCV infection and iron overload may exacerbate inflammation and fibrosis in chronic HCV. However, it has not been described in acute viral hepatitis and may portend a worse prognosis. Acute hepatitis C should be considered in the evaluation of acute liver failure and the association with iron overload needs to be further studied.

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.

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.

An association study of HFE gene mutation with idiopathic male infertility in the Chinese Han population

Mutations in the haemochromatosis gene (HFE) influence iron status in the general population of Northern Europe, and excess iron is associated with the impairment of spermatogenesis. The aim of this study is to investigate the association between three mutations (C282Y, H63D and S65C) in the HFE gene with idiopathic male infertility in the Chinese Han population. Two groups of Chinese men were recruited: 444 infertile men (including 169 with idiopathic azoospermia) and 423 controls with proven fertility. The HFE gene was detected using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. The experimental results demonstrated that no C282Y or S65C mutations were detected. Idiopathic male infertility was not significantly associated with heterozygous H63D mutation (odds ratio=0.801, 95% confidence interval=0.452-1.421, χ(2)=0.577, P=0.448). The H63D mutation frequency did not correlate significantly with the serum luteinizing hormone (LH), follicle-stimulating hormone (FSH) and testosterone (T) levels in infertile men (P=0.896, P=0.404 and P=0.05, respectively). Our data suggest that the HFE H63D mutation is not associated with idiopathic male reproductive dysfunction.

Unique frequencies of HFE gene variants in Roma/Gypsies

The aim of this study was to assess the frequencies of three hemochromatosis gene (HFE) mutations in ethnic Roma/Gypsies in Slovakia. A cohort of 367 individuals representing general population and not preselected for health status was genotyped by TaqMan real-time PCR assay for C282Y, H63D and S65C mutations in HFE gene. A unique genetic profile was revealed: C282Y is found in the highest frequency of all Central European countries (4.90%), while the frequency of H63D mutation (4.09%) is lower than any reported in Europe so far. S65C mutation was not present in the cohort. These mutation frequencies can be explained rather by gene influx and genetic isolation than by genetic inheritance from a former Roma/Gypsy homeland.

Prevalence of C282Y, H63D, and S65C mutations in hereditary HFE-hemochromatosis gene in Lithuanian population

HFE-hemochromatosis is a common autosomal recessive disease caused by HFE gene mutations and characterized as iron overload and failure of different organs. The aim of this study was to determine the prevalence of C282Y (c.845 G>A), H63D (c.187 C>G), and S65C (c.193A>T) alleles of HFE gene in the Lithuanian population. One thousand and eleven healthy blood donors of Lithuanian nationality were examined in four different ethnic Lithuanian regions to determine HFE gene alleles and genotype frequencies. The samples of DNA were analyzed for the presence of restriction fragment length polymorphism and validated by DNA sequencing. Among 1,011 blood donors tested, the frequency of C282Y, H63D, and S65C alleles were 2.6%, 15.9%, and 1.9%, respectively. One third of the tested subjects (n = 336) had at least one of the C282Y or H63D HFE gene mutations. The screening of Lithuanian blood donors has detected 13 (1.3%) subjects with a genotype C282Y/C282Y or C282Y/H63D responsible for the development of HFE-hemochromatosis. The prevalence of C282Y mutation was significantly higher among the inhabitants of Zemaitija (Somogitia) at the Baltic Sea area (5.9%) in comparison to the regions of continental part of Lithuania (2.4% in Dzukija, 2.3% in Aukstaitija, and 2% in Suvalkija, p < 0.05). These data support the hypothesis that the p.C282Y mutation originated from Scandinavia and spread with the Vikings along the Baltic Sea coast. The first epidemiological investigation of HFE gene mutations in ethnic Lithuanians showed that the frequencies of H63D, C282Y, and S65C of HFE gene alleles are similar to the other North-Eastern Europeans, especially in the Baltic region (Estonia, Latvia), Poland, and part of Russia (Moscow region).

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

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

Role of HFE gene mutations on developing iron overload in beta thalassaemia carriers in Egypt

A case-control study aimed to determine the prevalence of C282Y, H63D and S65C mutations of the HFE gene in beta-thalassaemia carriers and investigate their influence on iron absorption. A total of 41 beta-thalassaemia carriers and 40 control subjects without haemoglobinopathies were screened for the C282Y, H63D and S65C mutations by polymerase chain reaction-restriction fragment-length polymorphism. The iron status in these subjects was studied and correlated with the HFE gene mutations. H63D, S65C and C282Y allele frequencies were 30.5%, 13.4% and 7.3% respectively in beta-thalassaemia carriers and 10.0%, 2.5% and 0.0% respectively in the control group. Compound heterozygosis was found in 10 carriers (24.4%). The transferrin saturation level was high in compound heterozygote cases. Our study has shown that the HFEgene mutations are common in Egypt among beta-thalassaemia carriers compared with normal controls.