Rute Martins

Non-classical hereditary hemochromatosis in Portugal: novel mutations identified in iron metabolism-related genes

The most frequent genotype associated with Hereditary hemochromatosis is the homozygosity for C282Y, a common HFE mutation. However, other mutations in HFE, transferrin receptor 2 (TFR2), hemojuvelin (HJV) and hepcidin (HAMP) genes, have also been reported in association with this pathology. A mutational analysis of these genes was carried out in 215 Portuguese iron-overloaded individuals previously characterized as non-C282Y or non-H63D homozygous and non-compound heterozygous. The aim was to determine the influence of these genes in the development of iron overload phenotypes in our population. Regarding HFE, some known mutations were found, as S65C and E277K. In addition, three novel missense mutations (L46W, D129N and Y230F) and one nonsense mutation (Y138X) were identified. In TFR2, besides the I238M polymorphism and the rare IVS5 −9T→A mutation, a novel missense mutation was detected (F280L). Concerning HAMP, the deleterious mutation 5’UTR −25G→A was found once, associated with Juvenile Hemochromatosis. In HJV, the A310G polymorphism, the novel E275E silent alteration and the novel putative splicing mutation (IVS2 +395C→G) were identified. In conclusion, only a few number of mutations which can be linked to iron overload was found, revealing their modest contribution for the development of this phenotype in our population, and suggesting that their screening in routine diagnosis is not cost-effective.

The role of HFE mutations on iron metabolism in beta-thalassemia carriers

AbstractHereditary hemochromatosis (HH) is an autosomal recessive disorder of iron metabolism characterized by increased iron absorption and progressive storage resulting in organ damage. HFE gene mutations C282Y and H63D are responsible for the majority of HH cases. A third HFE mutation, S65C, has been associated with the development of a mild form of hemochromatosis. The beta-thalassemia trait is characterized by mild, ineffective erythropoiesis that can induce excess iron absorption and ultimately lead to iron overload. The aim of this study was to evaluate the effect of genetic markers (HFE mutations C282Y, H63D, and S65C) on the iron status of beta-thalassemia carriers. A total of 101 individuals heterozygous for beta-thalassemia and 101 normal control individuals were studied. The allelic frequencies of C282Y (1.5 versus 3.5%), H63D (15.3 versus 18.3%), and S65C (1.0 versus 1.5%) did not differ significantly between beta-thalassemia carriers and normal controls. Serum iron (P=0.029) and transferrin saturation (P=0.009) were increased in beta-thalassemia carriers heterozygous for H63D mutation. The number of subjects carrying C282Y or S65C mutations was too low to conclude their effect on the iron status. These results suggest that the beta-thalassemia trait tends to be aggravated with the coinheritance of H63D mutation, even when present in heterozygosity.

Differential HFE Gene Expression Is Regulated by Alternative Splicing in Human Tissues

Background The pathophysiology of HFE-derived Hereditary Hemochromatosis and the function of HFE protein in iron homeostasis remain uncertain. Also, the role of alternative splicing in HFE gene expression regulation and the possible function of the corresponding protein isoforms are still unknown. The aim of this study was to gain insights into the physiological significance of these alternative HFE variants. Methodology/Principal Findings Alternatively spliced HFE transcripts in diverse human tissues were identified by RT-PCR, cloning and sequencing. Total HFE transcripts, as well as two alternative splicing transcripts were quantified using a real-time PCR methodology. Intracellular localization, trafficking and protein association of GFP-tagged HFE protein variants were analysed in transiently transfected HepG2 cells by immunoprecipitation and immunofluorescence assays. Alternatively spliced HFE transcripts present both level- and tissue-specificity. Concerning the exon 2 skipping and intron 4 inclusion transcripts, the liver presents the lowest relative level, while duodenum presents one of the highest amounts. The protein resulting from exon 2 skipping transcript is unable to associate with β2M and TfR1 and reveals an ER retention. Conversely, the intron 4 inclusion transcript gives rise to a truncated, soluble protein (sHFE) that is mostly secreted by cells to the medium in association with β2M. Conclusions/Significance HFE gene post-transcriptional regulation is clearly affected by a tissue-dependent alternative splicing mechanism. Among the corresponding proteins, a sHFE isoform stands out, which upon being secreted into the bloodstream, may act in remote tissues. It could be either an agonist or antagonist of the full length HFE, through hepcidin expression regulation in the liver or by controlling dietary iron absorption in the duodenum.

Alternative polyadenylation and nonsense-mediated decay coordinately regulate the human HFE mRNA levels

Nonsense-mediated decay (NMD) is an mRNA surveillance pathway that selectively recognizes and degrades defective mRNAs carrying premature translation-termination codons. However, several studies have shown that NMD also targets physiological transcripts that encode full-length proteins, modulating their expression. Indeed, some features of physiological mRNAs can render them NMD-sensitive. Human HFE is a MHC class I protein mainly expressed in the liver that, when mutated, can cause hereditary hemochromatosis, a common genetic disorder of iron metabolism. The HFE gene structure comprises seven exons; although the sixth exon is 1056 base pairs (bp) long, only the first 41 bp encode for amino acids. Thus, the remaining downstream 1015 bp sequence corresponds to the HFE 3′ untranslated region (UTR), along with exon seven. Therefore, this 3′ UTR encompasses an exon/exon junction, a feature that can make the corresponding physiological transcript NMD-sensitive. Here, we demonstrate that in UPF1-depleted or in cycloheximide-treated HeLa and HepG2 cells the HFE transcripts are clearly upregulated, meaning that the physiological HFE mRNA is in fact an NMD-target. This role of NMD in controlling the HFE expression levels was further confirmed in HeLa cells transiently expressing the HFE human gene. Besides, we show, by 3′-RACE analysis in several human tissues that HFE mRNA expression results from alternative cleavage and polyadenylation at four different sites – two were previously described and two are novel polyadenylation sites: one located at exon six, which confers NMD-resistance to the corresponding transcripts, and another located at exon seven. In addition, we show that the amount of HFE mRNA isoforms resulting from cleavage and polyadenylation at exon seven, although present in both cell lines, is higher in HepG2 cells. These results reveal that NMD and alternative polyadenylation may act coordinately to control HFE mRNA levels, possibly varying its protein expression according to the physiological cellular requirements.

The functional significance of E277K and V295A HFE mutations.

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

HFE gene mutations are extremely rare in Western sub-Saharan Africa.

Hereditary hemochromatosis (HH) is a common autosomal recessive disorder of iron metabolism among Caucasian populations of Northern European descent [1]. The gene implicated in the disease (HFE) was discovered in 1996, and the C282Y mutation was identified as the main cause of the disease development [2]. H63D and S65C are other HFE missense mutations and are implicated in a mild form of HH [2, 3]. The high frequency of the C282Y mutation (5–10%) in Northern Europeans and its gradient towards the South (1– 5%) suggested that it had a Nordic origin [4]. On the other hand, the H63D mutation has a more general distribution, being spread worldwide [4–8]. The several haplotypes described associated with H63D in non-Caucasian populations disclose a multicentric origin for this mutation [4, 9, 10]. The geographical distribution of S65C has been less extensively analyzed than that of C282Y or H63D [7, 11, 12]. Studies regarding the prevalence of these HFE gene mutations in African populations are uncommon [4, 13–17]. Therefore, the main purpose of this study was to analyze the frequency of C282Y, H63D and S65C mutations in a group of 143 unrelated individuals (286 chromosomes), all with a Western sub-Saharan African origin (mainly from the ex-Portuguese colonies in Africa, Angola, Sao Tome and Principe, and Cape Verde). Moreover, these frequencies were compared with those described for populations from North Africa (Morocco and Algeria) and South Africa (Khoisan population). The origin of the positive H63D samples was investigated by HFE haplotype analysis. Total genomic DNAwas isolated from peripheral blood leukocytes by a salting-out procedure and HFE gene mutations (C282Y, H63D and S65C) were analyzed by a PCR restriction method (restriction fragment length polymorphism, RFLP). Fisher’s exact test was used to compare the allelic frequencies of the mutations, and the level of significance was set at 5%. The 95% confidence intervals for the allelic frequencies were calculated by the binomial exact method [18]. All statistical analyses were performed using the SPSS v11.0 software. The results obtained for the allelic frequencies of the C282Y, H63D and S65C mutations (and the respective 95% confidence intervals) are shown in Table 1. C282Y and S65C mutations were absent in all individuals studied, which resembles the results published for a South African population (Khoisan) [15]. Moreover, the frequency obtained for the C282Y mutation did not differ significantly from those described in the North African populations presented in Table 1 (p>0.05). The H63D allele was present in seven individuals, resulting in an overall allelic frequency of 2.45%. This frequency was significantly lower when compared with the North African population (p<0.05) [16]. The present study provides evidence that the HH-related mutations C282Y, H63D and S65C are extremely rare in the Western sub-Saharan African population. In prior studies, the C282Y mutation was not found in native individuals from Algeria (Mzab), Ethiopia, Kenya, Senegal, Gambia, Nigeria, Ghana, Zambia and South Africa [13–15, 19]. Similarly, in this study, the C282Y mutation was not found in individuals from Angola, Sao Tome and Principe, and Cape Verde. Therefore, its presence in people of North African ancestry [16] can be explained by the fact that these individuals have a different ethnic background than black African people or can be related to a European admixture. Curiously, the C282Y mutation was also found in R. Martins . I. Picanço . L. Romão . P. Faustino (*) Laboratório de Biologia Molecular, Instituto Nacional de Saúde Dr. Ricardo Jorge (INSA), Centro de Genética Humana, Lisbon, Portugal e-mail: paula.faustino@insa.min-saude.pt Tel.: +351-217519234 Fax: +351-217526410