HAMP Mutations Research Articles

A novel hepcidin mutation.

The bioactive peptide hormone hepcidin-25 regulates iron levels by inhibiting iron transport to plasma via ferroportin. Hepcidin-25 is synthesized in the liver where the 84 amino acids pro-hepcidin is cleaved into the bioactive hepcidin-25. A patient admitted to the hospital presented with infertility and fatigue. Genomic DNA was purified from whole blood using the Maxwell 16 system (Promega). MLPA analysis was performed to detect large genomic rearrangements using the SALSA MLPA kit # P347, Hemochromatosis (MRC Holland, Holland). Plasma hepcidin measurements were performed using liquid chromatography/tandem mass spectrometry (LC-MS/MS). A novel HAMP mutation (homozygous one base deletion in c.215delG, p.Cys72Serfs*?) was detected. The deletion in nucleotide 215 causes a frameshift altering the predicted protein sequence from cysteine13 in mature peptide. Whether this leads to nonsense mediated decay of the mRNA or synthesis of an aberrant peptide in unknown, but bioactive hepcidin-25 was undetectable in plasma. The patient had massive iron overload with ferritin up to 8360µg/L. He was anaemic with a Hb at 7.0mmol/L (11.3g/dL) and suffered from hypogonadotropic hypogonadism with a total testosterone of 1.2nmol/l. Continued treatment with venesection and gonadotropins led to reduced fatigue, reduction in iron overload, a normalized Hb and improvement of semen quality. A novel hepcidin mutation was detected in a patient with massive iron overload, fatigue and hypogonadotropic hypogonadism.

S2568 DDD: Diagnostic Dilemma in Drug Induced Liver Injury: Adverse Reaction of Abiraterone in Treatment of Metastatic Prostate Cancer

INTRODUCTION: Drug-induced liver injury (DILI) is a benign and reversible form of liver injury that occurs as an adverse reaction to medication use. It is a leading cause of acute liver failure and requires prompt recognition and discontinuation of the offending agent to avoid development of chronic disease. We present a case of severe DILI secondary to Abiraterone use in a patient with prostate cancer. CASE DESCRIPTION/METHODS: A 67 year-old-male without known liver disease was seen for severely elevated liver enzymes, jaundice and abdominal pain. Pertinent history includes stage IV prostate cancer with metastasis to the bone and lymph nodes with initiation of Abiraterone four months prior. He denies excessive alcohol, acetaminophen or herbal supplement use. Initial liver panel results: AST 1668 (peak 2379), ALT 3093 (peak 3330), alk phos 185 (peak 361) and TB 14.3 (peak 29). INR was 1.40 (peak 1.65). While transplant center transfer was considered, given patient's stability and not meeting King's College Criteria for acute liver failure, this decision was deferred and he was placed on NAC for 48 hours. Extensive workup for other causes of elevated liver enzymes were negative including: Wilson’s disease, autoimmune hepatitis, HAV, HBV, HCV, EBV, HSV, CMV, alpha 1 antitrypsin deficiency, portal vasculature thrombosis and acetaminophen toxicity. Core needle biopsies revealed acute hepatitis with confluent and bridging necrosis as well as mild Kupffer cell siderosis with iron accumulation. Serum ferritin level was 28,441 and IgG4 was mildly elevated, which prompted concern for hemophagocytic lymphohistiocytosis(HLH), hemochromatosis and autoimmune cholangiopathy. Patient did not meet criteria for HLH, hemochromatosis types I-IV in the absence of HFE, HFE2, TFR2, HAMP and SLC40A1 gene mutations, nor did MRI or pathology staining reveal IgG4 cholangiopathy. LFTs normalized within four months and ferritin within six months. Abiraterone was not restarted. DISCUSSION: Abiraterone is an antiandrogen that inhibits CYP17. Although the mechanism of hepatic injury is unknown, Abiraterone has been shown to cause elevations in serum aminotransferase levels in up to 13% of patients and ALT elevations above 5x the ULN in 6% of patients, however only few documented cases are known in which patients displayed associated symptoms such as jaundice and no documentation of severe elevations in serum ferritin as seen in our patient. This case also highlights the diagnostic challenge in DILI.Figure 1.: A. Mild to moderate predominantly lymphocytic inflammation. B. Intact bile ducts.Figure 2.: Confluent necrosis with areas of bridging necrosis.Figure 3.: Iron stain with Kupffer cell siderosis.

Determination of mutations in iron regulating genes of beta thalassemia major patients of Khyber Pakhtunkhwa, Pakistan.

BackgroundHepcidin and hemochromatosis (HFE) are iron regulatory proteins that are encoded by HAMP and HFE genes. Mutation in either HAMP gene or HFE gene causes Hepcidin protein deficiency that can lead to iron overload in beta thalassemia patients. The aim of this research work was to study the presence of G71D mutation of HAMP gene and H63D mutation of HFE gene in beta thalassemia major and minor group to check the association of these mutations with serum ferritin level of beta thalassemia patients.MethodsThe study was conducted on 42 beta thalassemia major and 20 beta thalassemia minor samples along with 20 control samples. The genotyping of both mutations has done by ARM‐PCR technique with specific set of primers.ResultsSignificant effect of G71D and H63D mutations was observed on serum ferritin level of thalassemia major group. The risk allele of HAMP G71D and HFE H63D was found with high frequency (48% and 49%, respectively) in beta thalassemia major than in control group. High genotypic frequency of HAMP and HFE gene mutation gene mutation was observed in beta thalassemia major than beta thalassemia minor and control group (7% and 9%, respectively).ConclusionIt can be concluded that both HAMP and HFE gene mutations show high frequency in beta thalassemia major patients and mean significant association between mutations and high serum ferritin level of beta thalassemia major patients but the nonsignificant results of Odd ratios showed that both mutations do not act as major risk factor in beta thalassemia major.

Inherited iron overload disorders.

Hereditary iron overload includes several disorders characterized by iron accumulation in tissues, organs, or even single cells or subcellular compartments. They are determined by mutations in genes directly involved in hepcidin regulation, cellular iron uptake, management and export, iron transport and storage. Systemic forms are characterized by increased serum ferritin with or without high transferrin saturation, and with or without functional iron deficient anemia. Hemochromatosis includes five different genetic forms all characterized by high transferrin saturation and serum ferritin, but with different penetrance and expression. Mutations in HFE, HFE2, HAMP and TFR2 lead to inadequate or severely reduced hepcidin synthesis that, in turn, induces increased intestinal iron absorption and macrophage iron release leading to tissue iron overload. The severity of hepcidin down-regulation defines the severity of iron overload and clinical complications. Hemochromatosis type 4 is caused by dominant gain-of-function mutations of ferroportin preventing hepcidin-ferroportin binding and leading to hepcidin resistance. Ferroportin disease is due to loss-of-function mutation of SLC40A1 that impairs the iron export efficiency of ferroportin, causes iron retention in reticuloendothelial cell and hyperferritinemia with normal transferrin saturation. Aceruloplasminemia is caused by defective iron release from storage and lead to mild microcytic anemia, low serum iron, and iron retention in several organs including the brain, causing severe neurological manifestations. Atransferrinemia and DMT1 deficiency are characterized by iron deficient erythropoiesis, severe microcytic anemia with high transferrin saturation and parenchymal iron overload due to secondary hepcidin suppression. Diagnosis of the different forms of hereditary iron overload disorders involves a sequential strategy that combines clinical, imaging, biochemical, and genetic data. Management of iron overload relies on two main therapies: blood removal and iron chelators. Specific therapeutic options are indicated in patients with atransferrinemia, DMT1 deficiency and aceruloplasminemia.

Mechanisms, mishaps and manipulation of iron uptake

Mechanisms, mishaps and manipulation of iron uptake

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.

Adult onset hereditary hemochromatosis is associated with a novel recurrent Hemojuvelin (HJV) gene mutation in north Indians

Hereditary hemochromatosis (HH) is a rare disorder in Indians and is not associated with the common mutation Cys282Tyr in HFE gene found in Caucasians. Non-HFE HH can be associated with mutations in HJV, HAMP, TFR2 and SLC40A1 genes. Nineteen unrelated north Indian HH patients were detected after screening 258 chronic liver disease patients on the basis of increased transferrin saturation, ferritin levels >1000 ng/L and siderosis by Perl's stain on liver biopsy wherever available. Automated DNA sequencing was performed for the promoters and entire coding exons for HFE, HJV, HAMP, TFR2 and SLC40A1. A novel homozygous mutation at position p.Gly336Ter (c.1006 G>T) in exon 4 in HJV was identified in four adult unrelated patients. We encountered compound heterozygosity for p.Thr217Ile (c.650C>T) and p.His63Asp (c.187C>G) mutation of HFE gene in one patient. Two patients were compound heterozygous for two novel polymorphisms at c.−358 (G>A) and c.−36 (G>A) in 5′UTR of HJV gene. Our study shows a novel HJV gene mutation p.Gly336Ter as a recurrent mutation associated with HH in north Indians. Low index of suspicion, underlying nutritional iron deficiency and protective effect of menstrual blood loss may account for the late clinical presentation of juvenile HH.

Haemochromatosis.

Haemochromatosis is defined as systemic iron overload of genetic origin, caused by a reduction in the concentration of the iron regulatory hormone hepcidin, or a reduction in hepcidin-ferroportin binding. Hepcidin regulates the activity of ferroportin, which is the only identified cellular iron exporter. The most common form of haemochromatosis is due to homozygous mutations (specifically, the C282Y mutation) in HFE, which encodes hereditary haemochromatosis protein. Non-HFE forms of haemochromatosis due to mutations in HAMP, HJV or TFR2 are much rarer. Mutations in SLC40A1 (also known as FPN1; encoding ferroportin) that prevent hepcidin-ferroportin binding also cause haemochromatosis. Cellular iron excess in HFE and non-HFE forms of haemochromatosis is caused by increased concentrations of plasma iron, which can lead to the accumulation of iron in parenchymal cells, particularly hepatocytes, pancreatic cells and cardiomyocytes. Diagnosis is noninvasive and includes clinical examination, assessment of plasma iron parameters, imaging and genetic testing. The mainstay therapy is phlebotomy, although iron chelation can be used in some patients. Hepcidin supplementation might be an innovative future approach.

Biallelic Mutations in Hamp in Type 2b Hemochromatosis

▪IntroductionHereditary hemochromatosis (HH) is a group of genetic iron overload disorders caused by mutations in genes encoding HFE (HFE1), transferrin receptor 2 (TFR2), hepcidin (HAMP), hemojuvelin (HFE2) or ferroportin (SLC40A1), respectively. HH is classified into 4 different categories according to the gene causing the disease. Type1 is the most common form arising from homozygosity for HFE C282Y mutation at the homozygous state. Type2 HH includes type 2A caused by mutations in HJV and type 2B , caused by mutations in HAMP. Hepcidin is synthesized as a pro-peptide of 84amino acids, which is cleaved into peptides of 20, 22, and 25 amino acids, the last being the active form of hepcidin. It is established that hepcidin negatively regulates iron export by direct interaction with ferroportin. Mutations or deficiency in Hepcidin result in excessive iron absorption that can lead to massive iron overload. We report a patient with a severe iron overload related to an unusual genotype of two previously unreported HAMP variants in combination with the H63D HFE mutation.Case report:A 69 year old male patient was diagnosed to have a severe iron overload with serum ferritin (SF) 5076 µg/l , transferrin saturation (TS) 48% and liver iron concentration (LIC) 290 µmolFe/g. Factors contributing to the hyperferritinemia included a chronic daily consumption of alcohol (100 g /days with gGT : 150 UI/l). Genetic analysis revealed heterozygosity for the common HFE1 H63D mutation and two new mutations in HAMP: one located in the promoter ( -274 G>C ) and the other in the stop codon (c. 255 G>C , p. Ter85TyrextTer21) . After one year of phlebotomy therapy, 5.5 g iron was removed and serum ferritin dropped to 1700 µg/L. Phlebotomy therapy was interrupted when patient was lost to follow-up and one year later SF was 2730 µg/L. Hepcidin was measured using mass spectrophotometry before resuming the phlebotomy (27.6 ng/ml, nle : 1 to 20 ng/ml).Methods :In order to analyze the involvement of the two HAMP mutations in the iron overload, further DNA studies were performed including MLPA (MRC Holland , P347 Hemochromatosis probemix , version A2) and Next generation sequencing (NGS, MiseqTM, Illumina ). The iron regulatory gene panel included the following gene sets : HFE-1; HFE-2 ; TFR2; HAMP; SLC40A1; FTH1; TF; FTL; SLC11A2; TMPRSS6; HEPH; CP; ALAS2; BMP6;BMP4; SMAD4. The Hepcidin functional tests were performed by using HEK293T cells expressing FPN1. Subsequently, cells were grown with preconditioned supernatants obtained from cells transfected with hepcidinwt or hepcidin255C. Promoter mutation was tested using HepG2 cells lines. Linkage between the two mutations was tested by means of DDPCR (Bio-Rad, QX200™ Droplet Digital™). We used simultaneously two of hydrolysis probes with different labeling and results were analyzed by a Poisson statistical comparison of double labeled drops count versus simple labeled drop.Results of these studies showed that :1- No other significant mutation could be retrieved with the NGS analysis and MLPA analysis of HFE, HFE2, HAMP, TFR2 and SLC40A1 did not show any pathogenic copy number variant.2- The elongated hepcidin was unable to induce internalization of ferroportin.3- The elongated hepcidin did not display any dominant effect (ie: unability to suppress the effect of wt hepcidin)4- The functional study of the -274 G>C promoter mutation fails to show a reproducible and significant decreased function compared to wt5- The two mutations were located in transDiscussion and conclusion.The reason why in some HAMP-P individuals iron overload was comparable to that of wild-type patients could be due to the fact that they are heterozygous for the SNP and, therefore, show an incomplete penetrance or compensatory mechanisms. We report a patient in which the only iron metabolism related mutations are the Hamp:c.255 G>C (p. Ter85TyrextTer21 mutation) and an SNP in the promoter: Hamp:c.-274 G>C mutation. We show that the elongated hepcidin is a loss of function mutant. Unfortunately, we could not have access to other family members for the segregation study of the HAMP mutations. Serum Hepcidin level was determined when ferritin was 2730 µg/l and appears to be low according to iron parameters. Our observations suggest a hypothetical role of the HAMP-P -274 G>C SNP on promoter reactivity. From this set of data, we propose that the patient is affected by a new Hamp linked HH (HFE 2B) probably worsened by drinking habits. DisclosuresSaunier:Bio-Rad: Employment.

The global prevalence of HFE and non-HFE hemochromatosis estimated from analysis of next-generation sequencing data

The prevalence of HFE-related hereditary hemochromatosis (HH) among European populations has been well studied. There are no prevalence data for atypical forms of HH caused by mutations in HFE2, HAMP, TFR2, or SLC40A1. The purpose of this study was to estimate the population prevalence of these non-HFE forms of HH. A list of HH pathogenic variants in publically available next-generation sequence (NGS) databases was compiled and allele frequencies were determined. Of 161 variants previously associated with HH, 43 were represented among the NGS data sets; an additional 40 unreported functional variants also were identified. The predicted prevalence of HFE HH and the p.Cys282Tyr mutation closely matched previous estimates from similar populations. Of the non-HFE forms of iron overload, TFR2-, HFE2-, and HAMP-related forms are predicted to be rare, with pathogenic allele frequencies in the range of 0.00007 to 0.0005. Significantly, SLC40A1 variants that have been previously associated with autosomal-dominant ferroportin disease were identified in several populations (pathogenic allele frequency 0.0004), being most prevalent among Africans. We have, for the first time, estimated the population prevalence of non-HFE HH. This methodology could be applied to estimate the population prevalence of a wide variety of genetic disorders.Genet Med 18 6, 618-626.

Exome Sequencing Identifies a GNPAT Variant Associated with Severe Iron Overload in HFE C282Y Homozygous Men with Extreme Phenotypes; Possible Role in Regulation of Hepcidin Expression

▪Variability in the severity of iron overload among homozygotes for the HFE C282Y polymorphism is one of the major unsolved problems in our understanding of hereditary hemochromatosis (HH). We previously conducted exome sequencing of DNA from 35 HFE C282Y male homozygotes with either markedly increased iron stores (n=22; cases) or normal to mildly increased iron stores (n=13; controls) to identify rare and common causal variants associated with variability of disease expression in HH. The 35 participants, residents of the U.S., Canada, and Australia, reported little or no alcohol consumption. Criteria for HFE C282Y homozygotes with increased iron stores included serum ferritin >1000 µg/L at diagnosis and either (a) hepatic iron concentration >236 µmol/g dry weight (reference range 0-36 µmol/g) or (b) mobilized body iron >10 g by quantitative phlebotomy. Criteria for HFE C282Y homozygotes with normal or mildly elevated iron stores included (a) serum ferritin <300 µg/L or either (a) age ≥40 y with ≤2.5 g iron removed by phlebotomy to achieve serum ferritin <50 µg/L, or (b) age ≥50 y with ≤3.0 g iron removed by phlebotomy. After quality control filtering, sequencing data included 82,068 single nucleotide variants and 1,403 insertions/deletions (indels); 10,337 genes were tested for a difference between cases and controls. We identified the polymorphism GNPAT p.D519G (1556A>G; exon 11; chromosome 1q42; rs11558492) as the most significantly different variant between cases and controls (p=0.033 by the likelihood ratio test after correction for multiple comparisons). In a principal components analysis of ancestry, all 35 study participants were clustered closely together within a larger group of Europeans. Mean (SD) ages at presentation were 53 (11.5) y and 57 (10.0) y for cases and controls, respectively. Median serum ferritin was 2391 µg/L in cases and 302 μg/L in controls. The median transferrin saturation (96%) was greater in cases than controls (70%).Sixteen of 22 case participants had polymorphism GNPAT p.D519G (rs11558492) (15 heterozygotes, 1 homozygote); no control participant had this polymorphism. The homozygous case presented at age 26 y with severe iron overload but no cirrhosis. One GNPAT p.D519G heterozygote presented at age 36 y with severe iron overload and cirrhosis. GNPAT p.D519G is common among people of European descent (allele frequency 20.6%) and might interact with aberrant HFE to increase the risk of hepatic iron overload. More recently, we compared the allele frequencies of GNPAT p.D519G in the present 22 cases and 13 controls with that of 4300 European Americans in the NHLBI Exome Sequencing Project Exome Variant Server. The allele frequency in cases was greater than that of European Americans (38.6% vs. 20.6%, respectively; p = 0.0076). The allele frequency in controls was significantly lower (0% vs. 20.6%, p = 0.0054). Next, to determine whether other known mutations influenced iron phenotypes, the exome data were used to screen for mutations in HAMP, HJV, TFR2, FPN1, and TMPRSS6. One case participant was heterozygous for HJV p.G320V; he was among the six case participants who did not have GNPAT p.D519G. No other known or probable mutation that would possibly explain differences in expression between cases and controls was found.To examine functional consequences of GNPAT deficiency, we now have performed siRNA-based knockdown of GNPAT in the human liver cell line HepG2/C3A. GNPAT was efficiently knocked down by its siRNA by ~85% compared to control siRNA as assayed by qPCR. This knockdown resulted in a >17-fold decrease in HAMP mRNA expression. mRNA expression of two genes coordinately regulated with HAMP, ID1 (inhibitor of DNA binding protein 1) and SMAD7 (SMAD family member 7), was similarly decreased, as was expression of phospho-SMAD 1/5/8, suggesting that GNPAT knockdown affects the baseline activity of the bone morphogenetic protein 6 (BMP6)-SMAD pathway.Our data indicate that GNPAT p.D519G is associated with a high-iron phenotype in male HFE C282Y homozygotes and may participate in hepcidin regulation, thereby modifying severity of iron overload. The results identify GNPAT as a candidate gene for expanded studies to examine its function in regulating iron absorption and metabolism and to identify newly-diagnosed C282Y homozygotes whose risk for development of severe iron overload is great. DisclosuresNo relevant conflicts of interest to declare.

Clinical Testing of Five Hereditary Hemochromatosis-Related Genes: Preliminary Evidence for the Benefit of Next Generation Sequencing

▪IntroductionHereditary hemochromatosis (HH) is a genetic form of iron overload. In cases of excessive iron deposition, serious clinical manifestations may occur, such as liver damage, cardiomyopathy, diabetes, and arthritis. First described in 1996, the HFE gene leads to autosomal recessive HH with reduced penetrance. In other words, two mutations in the HFE gene need to be present in a patient in order to develop symptoms of HFE-related HH, but not all patients with two mutations are affected. In the last 15 years, 4 additional genes were discovered that cause HH: HAMP (hepcidin), HFE2 (hemojuvelin), SLC40A1 (ferroportin), and TFR2 (transferrin receptor 2). HAMP, HFE2 and TFR2 mutations are inherited in a recessive pattern, whereas SLC40A1 mutations are inherited in a dominant pattern. HAMP and HFE2 mutations cause a severe, early-onset form of HH. There is some evidence that sequence changes in HAMP, HFE2 and TFR2 may interact with homozygous HFE mutations, causing a more severe phenotype. Current HH testing guidelines only exist for the most common HFE mutations (C282Y and H63D), with no specific recommendations regarding full gene sequencing for any of the HH genes. Recent research suggests that sequential sequencing may be beneficial in patients who test negative for the most common HFE mutations, exhibit a more severe or early-onset phenotype compared to what is normally seen in HFE-related HH, and/or are of non-Caucasian ethnicity. Next Generation Sequencing (NGS) is a new high-throughput sequencing technology that allows testing of multiple genes concurrently and can detect rare and novel HH-causing mutations that are not typically assayed using targeted methods. However, sequencing can also identify sequence changes known as variants of uncertain significance (VUS) - changes that have not yet been characterized as disease-causing or benign. This abstract summarizes the results of clinical NGS for the five HH-related genes, and shows preliminary evidence as to its’ increased diagnostic yield for HH diagnosis.MethodsPatients were referred for clinical full gene sequencing of HFE, HAMP, HFE2, SLC40A1, and/or TFR2 using Next Generation Sequencing (Illumina MiSeq). Results from patients with a clinical indication of iron overload or HH who were tested from 9/2013 - 7/2014 were reviewed. The diagnostic yield of sequencing for all five HH genes was determined. Patients who only had sequencing for a subset of the five genes were analyzed separately. Patients who had testing for a familial mutation were excluded from the review.ResultsIn total, 56 patients underwent HH-related NGS. Thirty-five (62.5%) were males and 21 (37.5%) were females. Ages ranged from 3-77yrs (avg. 40.9yrs). Fifty-one percent were Caucasian, 9% Hispanic, 4% African American, 16% Asian, and 20% not specified.Forty-one patients were tested for all five genes. HH-causing mutations were found in 9 patients: 5 (12.2%) were c.187C>G (p.H63D) HFE homozygous, 1 (2.4%) was c.845G>A (p.C282Y) HFE homozygous, and 3 (7.3%) had mutations in non-HFE genes: SLC40A1 c.430A>T (p.N144Y) heterozygous, SLC40A1 c.533G>A (p.R178Q) heterozygous, and HFE2 c.959G>T (p.R178Q) homozygous. Nine patients (22%) were heterozygous carriers of an HFE mutation. One or more VUSes were found in 5 patients (12.2%). In 18 patients (43.9%), no pathogenic mutations or VUSes were found. There were 15 additional patients who only had sequencing of 1-3 of the available genes. Results for those patients consisted of 1 H63D HFE homozygote, 3 HFE heterozygotes (2 H63D and 1 C282Y) and 1 VUS.ConclusionsThe new sequencing technology of NGS makes it possible to test multiple genes at the same time. In this sample, sequencing of HFE, HAMP, HFE2, SLC40A1, and TFR2 genes resulted in an additional diagnostic yield compared to HFE C282Y and H63D testing alone. In patients who have a genetic explanation for their HH, management can be personalized based on genotype-phenotype correlation (e.g. N144Y SLC40A1 mutations may lead to reduced phlebotomy tolerance) and at-risk family members can be screened. In addition, all patients in this sample with non-HFE positive results were reportedly Caucasian, highlighting the benefit of sequencing regardless of ethnic background. This preliminary study is an important step toward gaining a better understanding of the genetics of HH. Ultimately, NGS data may make it possible to update current clinical guidelines for HH. DisclosuresRabideau:Invitae: Employment, Equity Ownership.

Hereditary hypotransferrinemia can lead to elevated transferrin saturation and, when associated to HFE or HAMP mutations, to iron overload.

As our understanding of iron metabolism improves through the more accurate description of iron metabolism actors, new causes of iron overload are identified. We, here, report 16 cases of hereditary hypotransferrinemia related to 4 previously undescribed TF (transferrin) mutations (p.Val221Gly, p.Arg609Trp, p.Glu370Lys, p.Tyr533X and p.Cys421Arg). We show that, besides increasing serum transferrin saturation without iron overload, hypotransferrinemia, when associated to mutations in HFE or HAMP or to acquired factors, can lead to clinically relevant iron burden. These cases emphasize the usefulness of serum transferrin determination in the diagnostic evaluation of iron overload and the importance for clinicians to be aware of this syndrome.

Variable age of onset and clinical severity in transferrin receptor 2 related haemochromatosis: novel observations

Variable age of onset and clinical severity in transferrin receptor 2 related haemochromatosis: novel observations

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

Sideroblastic anemia, iron overload, andALAS2R452S in African-American males: Phenotype and genotype features of five unrelated patients

Sideroblastic anemia, iron overload, and<i>ALAS2</i>R452S in African-American males: Phenotype and genotype features of five unrelated patients

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.

Congenital Erythropoietic Porphyria Due to Co-Inheritance of GATA1 and UROS Gene Mutations

AbstractAbstract 3208The Congenital Erythropoietic Porphyria (CEP) is a rare form of Chronic Porphyria characterized by severe photosensivity. Secondary infections of cutaneous lesions may lead to scarring, deformities and disfigurement of the light-exposed parts of the body such as hands, ears, nose, and eyelids. Erythrodontia, osteodystrophia, combining osteolysis and osteoporosis, hypercellular bone marrow, hemolytic anemia, hypersplenism, splenomegaly and red-coloured urine are present in almost all patients. Generally, CEP is an autosomic recessive disease caused by mutations in homozygosis or compound heterozygosis in UROS gene coding for the fourth enzyme of the heme biosynthesis pathway. At present a new form of CEP with an X-linked inheritance associated with R216W mutation in the GATA 1 gene has been described only in one French-English family by Philips JD et al. GATA1 is a key zinc finger transcription factor that coordinates hematopoietic cell differentiation. We described a Turkish family where a severe CEP phenotype segregates with thalassemia and thrombocytopenia. The propositus is an 4 year old boy who presented photosensitive bullous dermatosis, splenomegaly, short stature and pallor. His mother was chronically anemic (Hb 10.9 g/dL) and thrombocytopenic (96×109/L) known as chronic immune thrombocytopenic purpura, whereas the father and the brother were asymptomatic. At birth, the boy had hypochromic, microcytic anemia, thrombocytopenia and thrombasthenia. Reticulocyte counts slightly increased. At 2 months he was transfused, splenomegaly and heart murmur were detected on physical examination. At this point his Hb level was 6,9 g/dl, WBC 7,400×109/L, platelets 64–106×109/L, MCV 70 flL, RDW 28%. The patient's peripheral-blood smear revealed microcytic, hypochromic red cells, target cells, basophilic stippling, and rare nucleated red cells, findings compatible with thalassemia but DNA analysis for beta and alpha thalassemia was normal. A bone marrow aspirate revealed a hypercellular marrow and erythroid hyperplasia. Dyserythropoiesis was noted with nuclear budding, nuclear bridging, and occasional multinucleation. Megakaryocytes were decreased in number, Hb electrophoresis revealed increased levels of fetal hemoglobin (HbA2 2%; Hb F 68%). At 3 years liver MRI detected iron overload. Ferritin level was 329ng/dL. Because of some skin lesions on his body he was then tested with urine and plasma analysis and the results were consistent with the diagnosis of congenital erythropoietic porphyria. Urine total porphyrins were 5414 nmol/mmol creatinin (N<35) with an excess of isomer I of both Uroporphyrins and Coproporhyrins. Total plasma porhyrins were 312 nmol/L (N<10). We analyzed the UROS gene and we identified a new mutation c.338A>T (D113V) in heterozygosis in the propositus, in his father and in his brother. On the basis of hematological status we decided to analyze also GATA1 gene and the mutation c.646C>T (R216W) has been detected in the child and on one allele of his mother who presents a balanced pattern of X-inactivation. The patient is similar to the case of association of CEP with an hematologic phenotype of beta-thalassemia intermedia. Markedly elevated Hb F levels, and thrombocytopenia result from mutations in the transacting factor GATA-1 presented by Philips JD et al. This patient additionally has a severe liver iron overload. A newborn sibling died at birth because of sepsis and neonatal hepatitis. Liver iron overload was also detected in the sibling's autopsy. All components of family were heterozygous for the H63D mutation in HFE gene but no other HAMP, HJV and SLC40A1 mutations were found. The patient started a regular transfusion program and Deferasirox was given for hemosiderosis after Desferroxamine uncompliance. This is the first report of the association of GATA1 and UROS gene mutations that could explain the very severe hematological phenotype. 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.

HAMP promoter mutation nc.-153C&gt;T in non p.C282Y homozygous patients with iron overload

In a recent paper, Island and colleagues[1][1] described a heterozygous hepcidin ( HAMP ) promoter mutation, nc.- 153C>T, which in association with HFE p.C282Y homozygosity appeared to lead to very severe iron overload (IO). They demonstrated in vitro that this HAMP mutation decreases