Novel association to the proprotein convertase PCSK7 gene locus revealed by analysing soluble transferrin receptor (sTfR) levels

The level of body iron storage and the erythropoietic need for iron are indicated by the serum levels of ferritin and soluble transferrin receptor (sTfR), respectively. A meta-analysis of five genome-wide association studies on sTfR and ferritin revealed novel association to the PCSK7 and TMPRSS6 loci for sTfR and the HFE locus for both parameters. The PCSK7 association was the most significant (rs236918, P = 1.1 × 10E-27) suggesting that proprotein convertase 7, the gene product of PCSK7, may be involved in sTfR generation and/or iron homeostasis. Conditioning the sTfR analyses on transferrin saturation abolished the HFE signal and substantially diminished the TMPRSS6 signal while the PCSK7 association was unaffected, suggesting that the former may be mediated by transferrin saturation whereas the PCSK7-associated effect on sTfR generation appears to be more direct.

Clinical versus pre-clinical FE models for vertebral body strength predictions

The finite element analysis is an accepted method to predict vertebral body compressive strength. This study compares measurements obtained from in vitro tests with the ones from two different simulation models: clinical quantitative computer tomography (QCT) based homogenized finite element (hFE) models and pre-clinical high-resolution peripheral QCT-based (HR-pQCT) hFE models. About 37 vertebral body sections were prepared by removing end-plates and posterior elements, scanned with QCT (390/450μm voxel size) as well as HR-pQCT (82μm voxel size), and tested in compression up to failure. Non-linear viscous damage hFE models were created from QCT/HT-pQCT images and compared to experimental results based on stiffness and ultimate load. As expected, the predictability of QCT/HR-pQCT-based hFE models for both apparent stiffness (r2=0.685/0.801r2=0.685/0.801) and strength (r2=0.774/0.924r2=0.774/0.924) increased if a better image resolution was used. An analysis of the damage distribution showed similar damage locations for all cases. In conclusion, HR-pQCT-based hFE models increased the predictability considerably and do not need any tuning of input parameters. In contrast, QCT-based hFE models usually need some tuning but are clinically the only possible choice at the moment.

Naturally variant autosomal and sex-linked loci determine the severity of iron overload in β2-microglobulin-deficient mice

Hereditary hemochromatosis (HH) is a common chronic human genetic disorder whose hallmark is systemic iron overload. Homozygosity for a mutation in the MHC class I heavy chain paralogue gene HFE has been found to be a primary cause of HH. However, many individuals homozygous for the defective allele of HFE do not develop iron overload, raising the possibility that genetic variation in modifier loci contributes to the HH phenotype. Mice deficient in the product of the β2-microglobulin (β2M) class I light chain fail to express HFE and other MHC class I family proteins, and they have been found to manifest many characteristics of the HH phenotype. To determine whether natural genetic variation plays a role in controlling iron overload, we performed classical genetic analysis of the iron-loading phenotype in β2M-deficient mice in the context of different genetic backgrounds. Strain background was found to be a major determinant in iron loading. Sex played a role that was less than that of strain background but still significant. Resistance and susceptibility to iron overload segregated as complex genetic traits in F1 and back-cross progeny. These results suggest the existence of naturally variant autosomal and Y chromosome-linked modifier loci that, in the context of mice genetically predisposed by virtue of a β2M deficiency, can profoundly influence the severity of iron loading. These results thus provide a genetic explanation for some of the variability of the HH phenotype.

Lack of hepcidin gene expression and severe tissue iron overload in upstream stimulatory factor 2 (USF2) knockout mice

We previously reported the disruption of the murine gene encoding the transcription factor USF2 and its consequences on glucose-dependent gene regulation in the liver. We report here a peculiar phenotype of Usf2−/− mice that progressively develop multivisceral iron overload; plasma iron overcomes transferrin binding capacity, and nontransferrin-bound iron accumulates in various tissues including pancreas and heart. In contrast, the splenic iron content is strikingly lower in knockout animals than in controls. To identify genes that may account for the abnormalities of iron homeostasis in Usf2−/− mice, we used suppressive subtractive hybridization between livers from Usf2−/− and wild-type mice. We isolated a cDNA encoding a peptide, hepcidin (also referred to as LEAP-1, for liver-expressed antimicrobial peptide), that was very recently purified from human blood ultrafiltrate and from urine as a disulfide-bonded peptide exhibiting antimicrobial activity. Accumulation of iron in the liver has been recently reported to up-regulate hepcidin expression, whereas our data clearly show that a complete defect in hepcidin expression is responsible for progressive tissue iron overload. The striking similarity of the alterations in iron metabolism between HFE knockout mice, a murine model of hereditary hemochromatosis, and the Usf2−/− hepcidin-deficient mice suggests that hepcidin may function in the same regulatory pathway as HFE. We propose that hepcidin acts as a signaling molecule that is required in conjunction with HFE to regulate both intestinal iron absorption and iron storage in macrophages.

Next-generation sequencing of iron-metabolism related genes in Portuguese patients with iron overload: novel pathogenic genetic variants

Objective: In Southern European countries up to one-third of the patients with hereditary hemochromatosis (HH) do not present the common HFE risk genotype. In order to investigate the molecular basis of these cases we have designed a gene panel for rapid and simultaneous analysis of 6 HH-related genes (HFE, TFR2, HJV, HAMP, SLC40A1 and FTL) by next-generation sequencing (NGS). Materials and Methods: Eighty-eight iron overload Portuguese patients, negative for the common HFE mutations, were analysed. A TruSeq Custom Amplicon kit (TSCA, by Illumina) was designed in order to generate 97 amplicons covering exons, intron/exon junctions and UTRs of the mentioned genes with a cumulative target sequence of 12115bp. Amplicons were sequenced in the MiSeq instrument (IIlumina) using 250bp paired-end reads. Sequences were aligned against human genome reference hg19 using alignment and variant caller algorithms in the MiSeq reporter software. Novel variants were validated by Sanger sequencing and their pathogenic significance were assessed by in silico studies. Results: We found a total of 55 different genetic variants. These include novel pathogenic missense and splicing variants (in HFE and TFR2), a very rare variant in IRE of FTL, a variant that originates a novel translation initiation codon in the HAMP gene, among others. Conclusion: The merging of TSCA methodology and NGS technology appears to be an appropriate tool for simultaneous and fast analysis of HH-related genes in a large number of samples. However, establishing the clinical relevance of NGS-detected variants for HH development remains a hard-working task, requiring further functional studies.

Ferroportin Disease (Haemochromatosis-type IV): a case report

Introdution: Haemochromatosis-type IV, the ferroportin disease, is characterized by an autosomal-dominant transmission and early iron accumulation in macrophages. It is caused by mutations in the transmembrane iron exporter protein ferroportin1 (SLC40A1 gene). In form A (classic), ferroportin loss of function mutants are unable to export iron from cells leading to cellular iron accumulation with decreased availability of iron for serum transferrin (TS). We present a Portuguese rare clinical case of HH-IV. Materials and Methods: A 41-year-old woman with hyperferritinemia and normal TS. Causes of hyperferritinemia (inflammation, chronic alcohol consumption, metabolic syndrome, cell necrosis, non-alcoholic fatty liver disease and aceruloplasminemia) were assessed. Liver iron, evaluated by magnetic resonance imaging (MRI) was carried out. Screening for mutation in HFE and SCL40A1 genes were performed by Sanger sequencing. Baseline: Ferritin:708ng/ml; TS: 27%; MRI:85µmol/g; Hb:13,6g/dl. Therapy: weekly 450ml Therapeutic Phlebotomies (TP) until ferritin≤50ng/ml. Results: Hyperferritinemia comorbidities and common genetic mutations for haemochromatosis were negative. However, sequencing of the patient SLC40A1 gene has revealed the presence in heterozygosity of the variant c.238G>A; p.Gly80Ser. Due to low tolerance to TP, we adopted smaller phlebotomies every three weeks. Conclusion: This patient has a rare autosomal-dominant Ferroportin disease due to a mutated ferroportin which is predicted to be defective in iron cellular export. In agreement, she presents hyperferritinemia, with normal TS and liver iron overload. The genotype/phenotype association allowed to diagnosis this rare FD case. Although a mild form A, we decided to start TP. Her father also has been treated for iron overload.

A desregulação da homeostase do ferro: base molecular e patologias associadas

A homeostase do ferro requer um rigoroso processo de regulação, uma vez que este é um elemento essencial para alguns dos mecanismos celulares básicos mas, quando se encontra em excesso, origina profundos danos celulares e falha de órgãos. Dado que o organismo humano não possui um mecanismo ativo de excreção de ferro, é essencial que a sua homeostase seja estabelecida através de uma estreita comunicação entre os locais de absorção, utilização e armazenamento. Esta interligação é conseguida, essencialmente, através da ação de uma hormona circulante, a hepcidina. A hepcidina é sintetizada ao nível dos hepatócitos do fígado, sendo a sua expressão aumentada pelos níveis de ferro e inflamação e suprimida pela eritropoiese e hipoxia. A hepcidina regula negativamente a absorção duodenal do ferro proveniente da alimentação, a libertação pelos macrófagos do ferro resultante da fagocitose dos glóbulos vermelhos senescentes, assim como a libertação do ferro armazenado nos hepatócitos. A hemocromatose hereditátria (HH) do tipo 1 é uma doença de transmissão autossómica recessiva associada a mutações no gene HFE (p.Cys282Tyr e p.His63Asp). É a patologia humana mais comum de sobrecarga primária em ferro, apresenta penetrância incompleta, e é um dos distúrbios genéticos mais frequentes em caucasianos de ascendência Norte-Europeia. Na hemocromatose, apesar de haver um excesso de ferro no organismo, este facto não é refletido no nível de expressão da hormona hepcidina (cujos níveis deveriam aumentar). Pelo contrário, o nível de expressão da hepcidina encontra-se diminuído o que perpetua a constante absorção do ferro a nível duodenal. Os sintomas associados à doença iniciam-se geralmente na meia-idade e começam por consistir em sintomas gerais de fadiga e dores articulares. No entanto, a progressiva acumulação do ferro em vários órgãos (tais como fígado, coração e pâncreas) provoca aí graves danos, tais como cirrose, carcinoma hepatocelular, cardiomiopatias e diabetes. Para além da HH do tipo 1, podem ocorrer outros tipos de hemocromatose por mutações noutros genes relacionados com o metabolismo do ferro (tais como TFR2, HJV, HAMP, SLC40A1, etc). Mutações em genes como HAMP e HJV associam-se a hemocromatoses mais graves, de início ainda na juventude (hemocromatose juvenil). A implementação no nosso laboratório da nova metodologia de Next-Generation Sequencing permitiu-nos realizar a pesquisa de variantes simultaneamente em 6 genes relacionados com o metabolismo do ferro, em 88 doentes com fenótipo de hemocromatose hereditária não-clássica. Foram identificadas 54 variantes diferentes sendo algumas delas novas. Estudos in silico e estudos funcionais in vitro (em linhas celulares) permitiram-nos comprovar a patogenicidade de algumas das variantes novas e compreender os mecanismos moleculares subjacentes ao desenvolvimento da sobrecarga em ferro. Pelo contrário, no lado oposto do espetro das patologias relacionadas com o ferro, encontram-se as anemias por falta de ferro (anemias ferropénicas). A Organização Mundial de Saúde define anemia quando os níveis de hemoglobina no sangue são menores do que 12 g/dL na Mulher e 13 g/dL no Homem. A hemoglobina é a proteína existente nos glóbulos vermelhos do sangue, responsável pelo transporte de oxigénio no organismo, e cuja molécula é um tetrâmero formado por 4 cadeias polipeptídicas (as globinas) e 4 grupos heme que contêm 4 átomos de ferro. A falta de ferro impede que se formem as moléculas de hemoglobina a níveis normais em cerca de 20% da população portuguesa e isso é devido a carências alimentares ou a dificuldades na absorção do ferro proveniente da alimentação. Entre os fatores genéticos moduladores desta última situação parecem estar algumas variantes polimórficas no gene TMPRSS6, codificante da proteína Matriptase-2, um dos agentes envolvidos na regulação da expressão da hepcidina. Por outro lado, mutações neste gene dão origem a anemias ferropénicas graves, refratárias ao tratamento oral com ferro (Iron Refractory Iron Deficiency Anaemia - IRIDA). As Hemoglobinopatias são outro tipo de anemia hereditária. Estas não estão relacionadas com o défice de ferro mas sim com defeitos nas cadeias globínicas, constituintes da hemoglobina (α2β2). As hemoglobinopatias que estão relacionadas com um problema quantitativo, ou seja quando há ausência ou diminuição de síntese de uma cadeia globínica, denominam-se talassémias: beta-talassémia, alfa-talassémia, delta-talassémia, etc, consoante o gene afetado. Por outro lado, quando o problema é de carácter qualitativo, ou seja ocorre a síntese de uma cadeia globínica estruturalmente anómala, esta é denominada uma variante de hemoglobina. Enquadra-se neste último grupo a Anemia das Células Falciformes ou Drepanocitose. As hemoglobinopatias são das patologias genéticas mais frequentes no mundo, sendo que nalguns locais são um grave problema de saúde pública. Em Portugal foram realizados estudos epidemiológicos que permitiram determinar a frequência de portadores na população e foi implementado um programa de prevenção.

Recommendations for Genetic Testing to Reduce the Incidence of Anthracycline-induced Cardiotoxicity

AIM Anthracycline-induced cardiotoxicity (ACT) occurs in 57% of treated patients and remains an important limitation of anthracycline-based chemotherapy. In various genetic association studies, potential genetic risk markers for ACT have been identified. Therefore, we developed evidence-based clinical practice recommendations for pharmacogenomic testing to further individualize therapy based on ACT risk. METHODS We followed a standard guideline development process; including a systematic literature search, evidence synthesis and critical appraisal, and the development of clinical practice recommendations with an international expert group. RESULTS RARG rs2229774, SLC28A3 rs7853758 and UGT1A6 rs17863783 variants currently have the strongest and the most consistent evidence for association with ACT. Genetic variants in ABCC1, ABCC2, ABCC5, ABCB1, ABCB4, CBR3, RAC2, NCF4, CYBA, GSTP1, CAT, SULT2B1, POR, HAS3, SLC22A7, SCL22A17, HFE and NOS3 have also been associated with ACT, but require additional validation. We recommend pharmacogenomic testing for the RARG rs2229774 (S427L), SLC28A3 rs7853758 (L461L) and UGT1A6*4 rs17863783 (V209V) variants in childhood cancer patients with an indication for doxorubicin or daunorubicin therapy (Level B - moderate). Based on an overall risk stratification, taking into account genetic and clinical risk factors, we recommend a number of management options including increased frequency of echocardiogram monitoring, follow-up, as well as therapeutic options within the current standard of clinical practice. CONCLUSIONS Existing evidence demonstrates that genetic factors have the potential to improve the discrimination between individuals at higher and lower risk of ACT. Genetic testing may therefore support both patient care decisions and evidence development for an improved prevention of ACT.

Des schwæbischen Ritters Georg von Ehingen Reisen nach der Ritterschaft.

The original ed., Augsburg, 1600, has title: Itinerarium, das ist: Historische Beschreibung, weylund Herrn Georgen von Ehingen Raisenn nach der Ritterschafft, vor 150 Jaren, in X. vnderschidliche Königreich verbracht ... Neben beygefügten Contrafacturn, deren Potentaten vnd Könige, an welcher Höfe obgedachter Ritter sich begeben ...

Systemic regulation of intestinal iron absorption

The intestinal absorption of the essential trace element iron and its mobilization from storage sites in the body are controlled by systemic signals that reflect tissue iron requirements. Recent advances have indicated that the liver-derived peptide hepcidin plays a central role in this process by repressing iron release from intestinal enterocytes, macrophages and other body cells. When iron requirements are increased, hepcidin levels decline and more iron enters the plasma. It has been proposed that the level of circulating diferric transferrin, which reflects tissue iron levels, acts as a signal to alter hepcidin expression. In the liver, the proteins HFE, transferrin receptor 2 and hemojuvelin may be involved in mediating this signal as disruption of each of these molecules decreases hepcidin expression. Patients carrying mutations in these molecules or in hepcidin itself develop systemic iron loading (or hemochromatosis) due to their inability to down regulate iron absorption. Hepcidin is also responsible for the decreased plasma iron or hypoferremia that accompanies inflammation and various chronic diseases as its expression is stimulated by pro-inflammatory cytokines such as interleukin 6. The mechanisms underlying the regulation of hepcidin expression and how it acts on cells to control iron release are key areas of ongoing research.

Establishment of a PCR and restriction enzyme assay for the detection of the hemochromatosis gene in normal subjects and diabetic patients

The purpose of this study was to establish a polymerase chain reaction (PCR)-restriction enzyme assay for detecting the hereditary hemochromatosis (HHC) mutation, C282Y, in gestational and gestational diabetic subjects in South Florida. DNA samples from 43 gestational subjects were amplified by PCR, digested with RsaI, and analyzed by electrophoresis. An allelic frequency of 2.33%, or 4.65% heterozygosity, was observed. The assay is successful and applicable to future studies on HHC and gestational diabetes. ^

Characterization of hepcidin response to holotransferrin in novel recombinant TfR1 HepG2 cells

Hepcidin is the key regulator of systemic iron homeostasis. The iron-sensing mechanisms and the role of intracellular iron in modulating hepatic hepcidin secretion are unclear. Therefore, we created a novel cell line, recombinant-TfR1 HepG2,expressing iron-response-element-independent TFRC mRNA to promote cellular iron overload and examined the effect of excess holotransferrin (5 g/L) on cell-surface TfR1, iron content, hepcidin secretion and mRNA expressions of TFRC, HAMP, SLC40A1,HFE and TFR2. Results showed that the recombinant cells exceeded levels of cell surface TfR1 in wild-type cells under basal (2.8-fold; p<0.03) and holotransferrin supplemented conditions for 24 h and 48 h (4.4- and 7.5-fold, respectively; p<0.01). Also, these cells showed higher intracellular iron content than wild-type cells under basal (3-fold; p<0.03) and holotransferrin-supplemented conditions (6.6-fold at 4 h; p<0.01). However, hepcidin secretion was not higher than wild-type cells. Moreover, holotransferrin treatment to recombinant cells did not elevate HAMP responses compared to untreated or wild-type cells. In conclusion, increased intracellular iron content in recombinant cells did not increase hepcidin responses compared to wild-type cells, resembling hemochromatosis. Furthermore, TFR2 expression altered within 4 h of treatment, while HFE expression altered later at 24 h and 48 h, suggesting that TFR2 may function prior to HFE in HAMP regulation.