YBCMD-01817; No. of pages: 6; 4C: Blood Cells, Molecules and Diseases xxx (2014) xxx–xxx
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A novel homozygous stop-codon mutation in human HFE responsible for nonsense-mediated mRNA decay Maria Carmela Padula a,⁎, Giuseppe Martelli a, Marilena Larocca a, Rocco Rossano a, Attilio Olivieri b a b
Department of Science, University of Basilicata, Viale dell’Ateneo Lucano, 85100 Potenza, Italy Clinic of Hematology, Hospital-University Company “Ospedali Riuniti di Ancona”, Via Conca, 60126 Ancona, Italy
a r t i c l e
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Article history: Submitted 14 February 2014 Revised 30 April 2014 Accepted 30 April 2014 Available online xxxx Communicated by M. Narla, DSc., 30 April 2014 Keywords: Hemochromatosis HFE Mutations Nonsense-mediated mRNA decay (NMD) Premature translation-termination codon (PTC)
a b s t r a c t HFE-hemochromatosis (HH) is an autosomal disease characterized by excessive iron absorption. Homozygotes for H63D variant, and still less H63D heterozygotes, generally do not express HH phenotype. The data collected in our previous study in the province of Matera (Basilicata, Italy) underlined that some H63D carriers showed altered iron metabolism, without additional factors. In this study, we selected a cohort of 10/22 H63D carriers with severe biochemical iron overload (BIO). Additional analysis was performed for studying HFE exons, exon–intron boundaries, and untranslated regions (UTRs) by performing DNA extraction, PCR amplification and sequencing. The results showed a novel substitution (NM_000410.3:c.847CNT) in a patient exon 4 (GenBankJQ478433); it introduces a premature stop-codon (PTC). RNA extraction and reverse-transcription were also performed. Quantitative real-time PCR was carried out for verifying if our aberrant mRNA is targeted for nonsense-mediated mRNA decay (NMD); we observed that patient HFE mRNA was expressed much less than calibrator, suggesting that the mutated HFE protein cannot play its role in iron metabolism regulation, resulting in proband BIO. Our finding is the first evidence of a variation responsible for a PTC in iron cycle genes. The genotype–phenotype correlation observed in our cases could be related to the additional mutation. © 2014 Elsevier Inc. All rights reserved.
Introduction HFE-hemochromatosis (HH; OMIM#235200) is a genetic disorder characterized by excess of iron absorption, which progressively leads to multi-organ failure. The main clinical consequences, related to the ferrotoxicity, are hepatic cirrhosis, hepatocellular carcinoma, cardiomyopathy, diabetes, arthritis and hypogonadism [1,2]. The early biochemical expression of HH is characterized by iron increase (particularly serum ferritin) and the diagnosis relies on the liver biopsy associated with evidence of increased iron content [1–3]. After the identification of the HFE gene (OMIM*613609) on short arm of chromosome 6 at location 6p22.2 [4], the genetic test has become the gold standard for diagnosis and it is now widely available. HFE coding region is formed by 1047 nucleotides that encode a 348 amino acid glycoprotein known as HFE. This transmembrane protein presents a structure very similar to the major histocompatibility
Abbreviations: β2M, beta-2-microglobulin; HH, HFE-hemochromatosis; MHC, major histocompatibility complex; NMD, nonsense-mediated mRNA decay; PTC, premature translation-termination codon; qPCR, quantitative real-time PCR; TFR, transferrin receptor; UTRs, untranslated regions. ⁎ Corresponding author. E-mail addresses: [email protected], [email protected] (M.C. Padula).
complex (MHC) class I molecules. It competes with transferrin for binding to the transferrin receptor (TFR; OMIM*90010) [5,6]. Two extracellular domains, α1 and α2, sit on the top of the α3 domain, which spans the cellular membrane and binds to the beta-2-microglobulin (β2M) protein (OMIM*109700). HFE protein is formed by a signal peptide, a topical extracellular domain, a transmembrane helical region and a cytoplasmic domain. It plays a role in the iron regulation by forming complexes with (a) TFR1 (OMIM*190010), in case of iron deficiency or (b) TFR2 (OMIM*604720), the TFR1 liver homologue, in case of iron overload. HFE/TfR2 complex activates a signaling cascade resulting in the upregulation of hepcidin (OMIM*606464), ferroportin degradation (OMIM*604653) and, consequently, a decreased dietary iron uptake [7–9]. HH clinical expression occurs earlier in male (around the age of 40) and later in females, because of the protective effects of menstrual blood loss and pregnancies [10]. HFE protein mutation leads to iron and organ damage in the liver, heart and pancreas [2]. Worldwide about 20 different mutations have been identified in HFE gene [11,12], but the main mutations are known as C282Y (rs1800562; exon 4, NM_000410.3: c.845GNA; NP_000401.1:p.Cys282Tyr) and H63D (rs1799945; exon 2, NM_000410.3:c.187CNG; NP_000401.1:p.His63Asp) [12,13]. C282Y mutation is responsible for the lack of HFE-β2M association [14]; H63D variant leads to the lack of HFE–TFR complex [6–15].
http://dx.doi.org/10.1016/j.bcmd.2014.04.010 1079-9796/© 2014 Elsevier Inc. All rights reserved.
Please cite this article as: M.C. Padula, et al., Blood Cells Mol. Diseases (2014), http://dx.doi.org/10.1016/j.bcmd.2014.04.010
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M.C. Padula et al. / Blood Cells, Molecules and Diseases xxx (2014) xxx–xxx
The distribution of genotypes among iron loaded patients shows a prevalence of C282Y/C282Y subjects [16–19]. They are more than 90% in the UK, and more than 80% in Northern European countries; the percentage ranges from 60 to 83% in the USA [20] and it is equal to 64% in Italy [21]. A mild-moderate phenotype is associated to the compound heterozygosity condition (C28Y/H63D) [22]. H63D role in iron overload is controversial, but this condition less commonly develops iron overload [10,12,20] and is associated with extremely variable phenotypes [23]. In our previous study we firstly investigated the distribution of HH genotypes among iron overloaded patients belonging to the province of Matera (Basilicata, South Italy) based on the genetic testing describing a biochemical evidence of iron overload (serum ferritin value higher than 750 ng/ml) in H63D/wt individuals, in the absence of other factors related to iron overload [24]. The genetic test (Haemochromatosis Strip Assay StripAssayA by ViennaLab Diagnostic GmbH) covers 11 HFE gene mutations (V53M, V59M, H63D, H63H, S65C, Q127H, E168X, E168Q, W169X, C282Y, and Q283P), 4 transferrin receptor mutations (Y250X, E60X, M172K, and the AVA Q 594–597) and 2 ferroportin mutations (N144H and V162del). Routine clinical practice is affected by considering HH allelic heterogeneity and the existence of rare/private mutations, occasionally found in a small number of subjects [25]. Considering the spectrum of possible genetic variations and the need of early diagnosis, in order to optimize the treatment of some patients at risk of delayed appropriate treatment, we decided to evaluate the potential utility of additional genetic investigations. Therefore we retrospectively selected a cohort of patients with H63D heterozygosity and biochemical iron overload for additional studies. In this context we assessed the HFE mutational state in order to clarify the relationship between genotype and phenotype in a patient belonging to our cohort. Patient and methods We retrospectively identified a cohort of 22 patients with H63D heterozygosity and biochemical iron overload for additional studies. The patients were divided into two groups: the first one included 12 subjects with ferritin value lower than 750 ng/ml; within the second cluster resided 10 individuals whose ferritin level exceed 750 ng/ml (severe hyperferritinemia). We assessed the HFE mutational state in order to clarify the relationship between genotype and phenotype in this last cluster. The most significant data of the subjects belonging to the second group are reported in Table 1. About the methods, the first step of molecular biology consisted in (a) DNA isolation, (b) primer design, (c) PCR (polymerase chain reaction) amplification, (d) HFE fragment sequencing and (e) bioinformatics analysis. DNA extraction was carried out by using a commercial kit (Nuclear Laser Medicine S.r.l.) according to the manufacturer’s instructions. DNA was quantified by means of NanoDrop 1000 spectrophotometer (NanoDrop Technologies, Inc). On the basis of HFE RefSeq in NCBI
Table 1 The most significant data of the patients belonging to the “severe hyperferritinemia” group. Age, sex and serum ferritin value of the 10 subjects whose serum ferritin (SF) value is higher than 750 ng/ml. Patients
Age (years)
Sex
SF average value (ng/ml)
1 2 3 4 5 6 7 8 9 10
35 41 48 26 69 54 33 45 51 55
Male Male Male Male Male Male Female Male Male Male
1035 948 989 792 1230 957 922 875 1154 860
database, PCR primers were designed by means of NCBI Primer-Blast in order to detect HFE exons, exon–intron boundaries, and 5′ and 3′ untranslated regions (UTRs). A phase of the optimization of PCR components and conditions was carried out. For the amplification, 25 μl of PCR reaction was used: 1.5 μl MgCl2, each of dNTP 2 mM, 1 μl of specific primers, 0.4 U/μl of AmpliTaq Gold DNA polymerase in 10× PCR buffer (100 mM tris–HCl, pH 8.3, 500 mM KCl) (Roche Molecular Systems, Inc). The conditions of reaction were the following: (1) initial denaturation: 95 °C/7 min; (2) thermocycling: 94 °C/1 min; 58 °C/1 min; 72 °C/2 min (35 cycles); (3) final extension: 72 °C/10 min. Amplification products were analyzed by gel electrophoresis (1.5% agarose gel). The next step consisted in HFE fragments sequencing. The bioinformatics analysis allowed to confirm the similarity between our sequences and HFE RefSeq, by means of BlastN (NCBI database) [26]; a multiple alignment was performed between patient sequences and control sequences (three healthy controls were included in the study) to identify conserved/nonconserved nucleotides, by means of ClustalW2 (EMBL-EBI database) [27]. In addition the Mutation Surveyor software was employed for DNA variant analysis [28,29]. If a novel mutation was identified, it was confirmed by a second step of amplification. In addition, for assessing the HFE mRNA level, quantitative real-time PCR (qPCR) assay was carried out. First, the whole blood was collected by means of Tempus RNA tubes (Applied Biosystems). RNA isolation was achieved using Tempus Spin RNA Isolation Reagent Kit (Applied Biosystems), optimized in our laboratory. RNA was quantified using the NanoDrop Spectrophotometer 1000 (NanoDrop Technologies, Inc.) again. The reverse transcription of RNA into double-stranded cDNA was obtained by using the RETROscript Kit (Ambion), following the company instructions. The qPCR was carried out by means of the thermal cycler Chromo4 (Continuous Fluorescence Detector) and the Optical Monitor 2.03.5 software (MJ Research, Inc.). We employed the Power SYBR Green PCR Master Mix (Applied Biosystems) in the following volumes: 5 μl of Power SYBR Green PCR Master Mix (2 ×), 1.5 μl of forward primer 10 mM, 1.5 μl of reverse primer 10 mM, 1 μl of template. The protocol was (1) 95 °C/10 min, (2) 38 cycles of 95 °C/30 s and 64 °C/1 min, and (3) 72 °C/1 min. Melting curves were generated after 38 cycles by heating the sample up to 95 °C for 15 s followed by cooling down to 60 °C for 15 s and heating the samples to 95 °C for 15 s. We applied SYBR Green as reporter molecule, β-actin as housekeeping gene, three wild-type subjects as calibrators, two gene replicates and 2−ΔΔCt method [30] for data analysis, according to the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) [31]. After verifying that all gene amplification efficiency was comparable, we calculated the fold change (2−ΔΔCt). In detail, firstly we determined average Ct values for HFE replicates and for controls (calibrators) and, after normalization with housekeeping, we calculated ΔΔCt as ΔCt of target gene − ΔCt of calibrator gene and we used the data for deriving the fold change value. Results were expressed as mean ± standard deviation. Student's t test was employed in order to calculate the statistical significance (p b 0.05). PCR specificity was assessed by melting curve analysis of amplification products for confirming the occurrence of specific amplification peaks and the absence of primer dimer formation. Gel electrophoresis (2% agarose) was also performed in order to verify the presence of single gel bands of predicted size. Results HFE sequencing results underlined the presence of a variant not previously described in literature (GenBank Number: JQ478433) in 9/10 patients in the group of severe hyperferritinemia. We first identified this variation in the patient 1 of the cluster and we consequently extended the variant research to the whole group. For this reason the proband 1 was considered the case-model and we report about the subject, a 35-year-old Caucasian male coming from a small village of Basilicata, whose father (76 years old) showed severe
Please cite this article as: M.C. Padula, et al., Blood Cells Mol. Diseases (2014), http://dx.doi.org/10.1016/j.bcmd.2014.04.010
M.C. Padula et al. / Blood Cells, Molecules and Diseases xxx (2014) xxx–xxx Table 2 Clinical, biochemical and molecular data of our most significant clinical case. The variations have been labelled according to HGVS nomenclature. Proband data Gender Age at diagnosis (years) Clinical data Serum ferritin (ng/ml) Serum transferrin (mg/dl) Serum iron (μl/dl) GPT (ALT) (UI/l) GOT (AST) (UI/l) Hepatic iron content (μmol/g) Phlebotomies Total iron removed (g) Classical HFE mutation Novel HFE mutation
Type of the novel mutation
Male 35 Fatigue, joint and abdominal pains and jaundiced complexion 1035 ± 45 389 ± 16 151 ± 11 63 ± 8 42 ± 3 NA Yes 6.1 NM_000410.3:c.187CNG; rs1799945 NP_000401.1:p.His63Asp NM_000410.3:c.847CNT NP_000401.3:p.Gln283* Non-sense substitution
biochemical iron overload and hepatic damage. Other diseases have been ruled out within the family. He was heterozygous for H63D mutation at first genetic test and no variations in other gene related to the iron cycle (ferroportin and transferrin receptor) were found. The most significant clinical and laboratory characteristics of the proband are reported in Table 2. Laboratory testing showed BIO evidence and increased hepatic integrity enzymes. About the medical background no history of chronic liver disease, diabetes, alcohol abuse and other factors was reported. The patient underwent regular phlebotomies (at first one phlebotomy every 7–10 days) to normalize ferritin levels. (See Table 3.) In detail the nucleotide change CNT resides in HFE exon 4 at 847 position of RefSeq (NM_000410.3:c.847CNT) and is in homozygosity state (Table 2; Fig. 1). The sequence of primer pair used for amplification of the exon 4 was the subsequent: 5′–CTTCCTGGCAAGGGTAAACA-3′ (forward primer); 5′–CTCAGGCACTCCTCTCAACC-3′ (reverse primer). The variation is absent in homozygosity state in proband relatives during the family test. About the proband family, if the father showed the same genotype (and a comparable phenotype too), the proband’s brother and sister were found heterozygotes. The transition introduces a stop-codon that interrupts the reading frame, as the sequence prediction showed (Alamut 2.2e software, Interactive Biosoftware, France). A premature chain termination of translation could occur, resulting in truncated HFE protein at α3 domain level (NP_000401.1:p.Gln283*), whose secondary structure was predicted by means of Phyre2 tool (Protein Homology/Analogy Recognition Engine) [32]. However, the production of faulty proteins is prevented in eukaryotes by means of several surveillance mechanisms. A well-studied control mechanisms is the nonsense-mediated mRNA decay (NMD), which recognizes and degrades transcripts preventing a premature translation-termination codon (PTC) [33,34]. In order to check if
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our mRNA is targeted for NMD, we quantified mRNA levels in our patient and controls by applying real-time PCR. The ΔΔCt value for patient HFE gene was equal to 12.872 ± 0.785 (mean and standard deviation); a value equivalent to 0.034 ± 0.007 was obtained for control. The target-control difference was statistically significant (Student’s t test; p b 0.05); therefore HFE mRNA was expressed about 7000 times less than calibrator gene, that is the average control sample (fold change values were compared). We obtained single peak dissociation curves and single gel bands of predicted size; both confirmed the PCR specificity. In a few words, here we reported a novel substitution that generates PTC. As direct consequence an aberrant mRNA is produced. The mRNA is targeted for NMD surveillance mechanism affecting the normal production of the truncated HFE protein (Fig. 2). Discussion Here we selected a cohort of 22 H63D heterozygote patients, whose 10 subjects showed severe hyperferritinemia. Within this last cluster, 9/10 subjects showed a novel variation. The group is enough homogenous in terms of fold change (a dramatic reduction in the amount of HFE mRNA in the patients was observed in all cases) and number of phlebotomies (average number: 1 phlebotomy every 15 days). Here we reported about the clinical case of the first patient, a 35-year-old man considered as model. It should be observed that the clinical onset and laboratory alterations in young subjects are uncommon; therefore, the individuation of an underlying genetic disease, which could be successfully treated, represents an important opportunity. In addition, the clinical meaning of H63D status is partly unclear: literature and specific databases (dbSNP, LSDB, locus-specific database and OMIM database) describe this aspect as extremely variable (Table 2) and related to HH clinical phenotype if associated with other phenotypic modifiers, both environmental, e.g. diet, alcohol abuse, diabetes, inflammation and non-genetic factor such as sex and age [35]. In order to understand the genotype–phenotype correlation in our proband, we analyzed the crosslink between biochemical–clinical evidences and molecular data. Assuming a partial dominance of HFE wild-type allele, we characterized HFE coding region, exon–intron boundaries and UTRs. The novel c.847CNT change was found. At protein level the substitution corresponds to the p.Gln283* change. Another mutation described in literature involves the 283 position resulting in the p.Gln283Pro variant. This mutation was considered a C282Y-like causative allele: for being adjacent to the disulfide bridge formed by Cys225 and Cys282, the Q283P protein is retained in the endoplasmic reticulum, with structural and functional consequences similar to the C282Y mutation effects [36]; therefore, the Q283P variant represents a pathogenic variant (Table 2). In our case, the nucleotide change could result in a 283 amino acid protein, such as a truncated product. However the production of the faulty protein could be avoided due to the NMD surveillance
Table 3 HFE variations reported in dbSNP (LSDB, locus-specific database) and OMIM database and their clinical impact.
1 2 3 4 5 6 7 8 9 10
rs number
cDNA level
Protein level
Clinical significance
Reference
rs111033563 rs111033558 rs111033557 rs28934889 rs28934597 rs28934596 rs28934595 rs1800730 rs1800562 rs1799945
NM_000410.3:c.848ANC NM_000410.3:c.989GNT NM_000410.3:c.175GNA NM_000410.3:c.157GNA NM_000410.3:c.277GNC NM_000410.3:c.314TNC NM_000410.3:c.381ANC NM_000410.3:c.193ANT NM_000410.3:c.845GNA NM_000410.3:c.187CNG
NP_000401.1:p.Gln283Pro NP_000401.1:p.Arg330Met NP_000401.1:p.Val59Met NP_000401.1:p.Val53Met NP_000401.1:p.Gly93Arg NP_000401.1:p.Ile105Thr NP_000401.1:p.Gln127His NP_000401.1:p.Ser65Cys NP_000401.1:p.Cys282Tyr NP_000401.1:p.His63Asp
Pathogenic Pathogenic Non-Pathogenic Non-Pathogenic Pathogenic Pathogenic Pathogenic Pathogenic Pathogenic Variable
[53] [54] [54] [54] [55] [55] [54] [56] [4] [4]
Please cite this article as: M.C. Padula, et al., Blood Cells Mol. Diseases (2014), http://dx.doi.org/10.1016/j.bcmd.2014.04.010
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Fig. 1. From the PCR product electrophoresis to the sequencing of the proband and the controls HFE exon 4. About the agarose gel, the patient amplicon corresponds to the band of the line named “1”; the control amplicons resides into gel lines 2–4. A negative control (C−) was also utilized. Regarding to the sequencing results, we show the c.847CNT change is detectable in the proband and not in the consensus control sequence; this aspect is underlined both in alignment and electropherogram patient and controls.
Fig. 2. The effect of the novel variation at DNA, RNA and protein level. The novel c.CNT substitution generates a PTC in HFE exon 4, responsible for the production of an aberrant mRNA. The mRNA is targeted for NMD surveillance mechanism. As consequence the normal production of the truncated protein is affected. Both the protein primary structure (the missed region is reported in black and underlined) and the secondary structure prediction (conducted by means of Phyre2 software) are shown.
Please cite this article as: M.C. Padula, et al., Blood Cells Mol. Diseases (2014), http://dx.doi.org/10.1016/j.bcmd.2014.04.010
M.C. Padula et al. / Blood Cells, Molecules and Diseases xxx (2014) xxx–xxx
mechanisms. Our c.CNT substitution directly generates a stop-codon, namely PTC, responsible for an aberrant HFE mRNA. PTCs can derive either from mutations at DNA level (e.g. frame-shift or nonsense mutations) or from alternative splicing mechanisms that leads to the production of mRNA isoforms with truncated reading frames. Consequently the nonsense transcripts would result in the production of Cterminally truncated proteins with potentially negative effects [34,37]. It was estimated that about 30% of all known disease-associated mutations generate a PTC-containing mRNA [33,38]. β-Thalassemia is one of the first genetic disorder where the role of NMD as phenotypic modifier was reported 20 years ago: the majority of β-globin gene mutations leads to a PTC in the first or second exon of the three-exon gene [33,39,40]. Nowadays a large number of inherited genetic disorders arising from generation of PTC were identified: SOX10, rhodopsin, receptor tyrosine kinase-like orphan receptor 2, cone-rod homeobox, coagulation factor X, dystrophin, and cystic fibrosis genes are example of genes targeted for NMD [33]. In addition, some evidences of NMD role in the control of wild-type gene expression were reported: NMD pathway also regulates the level of many physiological mRNAs involved in different cellular processes, such as DNA repair and metabolism [33,41–43]. Very interestingly, PTCs occur more frequently in genes belonging to the immunoglobulin superfamily than in other genes; in addition the steady-state mRNA level is highly reduced by NMD [33,44,45]. HFE gene belongs to the immunoglobulin superfamily and is very similar to the MHC class I molecules [4] and so, a candidate for NMD process. Considering this last aspect and considering that the phenotypic severity of diseases caused by non-sense mutations can be predicted by quantifying the reduction amount of mRNA level [40], qPCR was carried out. The quantification of gene expression results showed a minimal HFE expression compared to the normal expression. This aspect is consistent with the NMD role in preventing the gain-of-function effects of truncated protein that could result if non-sense transcripts were stable. So, in our case, NMD pathway acts in the control of HFE gene expression by strongly reducing the transcript level and by affecting the levels of HFE protein. Previous studies reported in literature a few non-sense mutations in human HFE gene [17,46–49]. About the crosslink between non-sense mutation and NMD in HFE gene, Pointon and collaborators [49] identified a single nucleotide deletion (c.del478) in HFE exon 3 that causes a frameshift and introduces a PTC leading to mRNA degradation by NMD pathway. Afterward, Martins’ investigations demonstrated that the expression of physiological human HFE transcripts, specifically those species with 3′-end cleavage and polyadenylation at exon 7, is down-regulated by the NMD mechanism. In addition, the same group showed that NMD and alternative polyadenylation may act coordinately to control HFE mRNA levels, possibly varying its protein expression according to the physiological cellular requirements [50]. This aspect suggests the significance of NMD mechanism in determination of both wild and mutant human HFE mRNA level. In fact, the present paper reports the first evidence of a point substitution that directly generates PTC and its ability to commit the HFE mRNA to NMD and affect the formation of the truncated protein. As regard the functional implication of our finding, we supposed that the formation of the iron sensor and signal transduction effector complex should have been involved, consequently inducing the dysregulation of systemic iron homeostasis. In fact, considering that the primary HFE role is the control of hepcidin expression [51,52], it is known that in mutated or absent HFE, hepcidin production is inappropriately low, when related to the overall body iron status and, as direct consequence, ferroportin activity is not attenuated [9,51,52]. As final effect of this chain reaction, the iron accumulation is promoted; this aspect is consistent with the biochemical pattern of iron overload (hyperferritinemia in particular) of our proband. In general, the biochemical iron overload in our clinical cases could be related to a variant not reported in literature, and our data explain,
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at molecular level, the pathologic iron state in our cases who resulted to be only apparently H63D carriers at first genetic test. Moreover our data emphasize the importance of HFE gene sequencing in diagnosis and management of HH: when heterozygous subjects show biochemical iron overload with alteration of transaminase levels, the routine genetic test-based approach could be insufficient; in these cases HFE sequencing is very important prior to study other iron genes because additional mutations in this gene may play a clinical impact in heterozygotes. Additional molecular investigations should be requested, in order to explain the absence of correlation between genotype and clinical phenotype. In these cases we suggest to follow our three steps experimental design: (1) additional factors (causes of secondary iron overload, such as chronic liver disease, diabetes, alcohol abuse, etc.) must be ruled out; (2) the absence of the compound heterozygosity condition (C282Y/H63D) must be checked; (3) HFE sequencing to identify unknown HFE variants, as reported here, must be performed. Finally our results may provide a response to some questions arising from the apparent lack of correlation between genotype and phenotype observed in clinical practice [18,23,25]. The individuation of novel mutations could extend the panel of the common HFE mutations and contribute to improve both the patient diagnosis and therapy. Finally, as our novel, private variant concurs to the altered iron state, the possibility to assess its presence in the genome could allow an earlier diagnosis and, consequently, an earlier iron depletion therapy. This aspect is very important in terms of quality of life: a specific dietary regime and an early therapy could avoid the adverse outcomes of the disease. Since we analyzed a small group belonging to a small Italian region, in order to strengthen the significance of our de novo mutation, further investigations will consist in the screening of all iron loaded subjects, in the same area and in different geographic areas (epidemiologic implementation).
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A novel homozygous stop-codon mutation in human HFE responsible for nonsense-mediated mRNA decay Maria Carmela Padula a,⁎, Giuseppe Martelli a, Marilena Larocca a, Rocco Rossano a, Attilio Olivieri b a b
Department of Science, University of Basilicata, Viale dell’Ateneo Lucano, 85100 Potenza, Italy Clinic of Hematology, Hospital-University Company “Ospedali Riuniti di Ancona”, Via Conca, 60126 Ancona, Italy
a r t i c l e
i n f o
Article history: Submitted 14 February 2014 Revised 30 April 2014 Accepted 30 April 2014 Available online xxxx Communicated by M. Narla, DSc., 30 April 2014 Keywords: Hemochromatosis HFE Mutations Nonsense-mediated mRNA decay (NMD) Premature translation-termination codon (PTC)
a b s t r a c t HFE-hemochromatosis (HH) is an autosomal disease characterized by excessive iron absorption. Homozygotes for H63D variant, and still less H63D heterozygotes, generally do not express HH phenotype. The data collected in our previous study in the province of Matera (Basilicata, Italy) underlined that some H63D carriers showed altered iron metabolism, without additional factors. In this study, we selected a cohort of 10/22 H63D carriers with severe biochemical iron overload (BIO). Additional analysis was performed for studying HFE exons, exon–intron boundaries, and untranslated regions (UTRs) by performing DNA extraction, PCR amplification and sequencing. The results showed a novel substitution (NM_000410.3:c.847CNT) in a patient exon 4 (GenBankJQ478433); it introduces a premature stop-codon (PTC). RNA extraction and reverse-transcription were also performed. Quantitative real-time PCR was carried out for verifying if our aberrant mRNA is targeted for nonsense-mediated mRNA decay (NMD); we observed that patient HFE mRNA was expressed much less than calibrator, suggesting that the mutated HFE protein cannot play its role in iron metabolism regulation, resulting in proband BIO. Our finding is the first evidence of a variation responsible for a PTC in iron cycle genes. The genotype–phenotype correlation observed in our cases could be related to the additional mutation. © 2014 Elsevier Inc. All rights reserved.
Introduction HFE-hemochromatosis (HH; OMIM#235200) is a genetic disorder characterized by excess of iron absorption, which progressively leads to multi-organ failure. The main clinical consequences, related to the ferrotoxicity, are hepatic cirrhosis, hepatocellular carcinoma, cardiomyopathy, diabetes, arthritis and hypogonadism [1,2]. The early biochemical expression of HH is characterized by iron increase (particularly serum ferritin) and the diagnosis relies on the liver biopsy associated with evidence of increased iron content [1–3]. After the identification of the HFE gene (OMIM*613609) on short arm of chromosome 6 at location 6p22.2 [4], the genetic test has become the gold standard for diagnosis and it is now widely available. HFE coding region is formed by 1047 nucleotides that encode a 348 amino acid glycoprotein known as HFE. This transmembrane protein presents a structure very similar to the major histocompatibility
Abbreviations: β2M, beta-2-microglobulin; HH, HFE-hemochromatosis; MHC, major histocompatibility complex; NMD, nonsense-mediated mRNA decay; PTC, premature translation-termination codon; qPCR, quantitative real-time PCR; TFR, transferrin receptor; UTRs, untranslated regions. ⁎ Corresponding author. E-mail addresses: [email protected], [email protected] (M.C. Padula).
complex (MHC) class I molecules. It competes with transferrin for binding to the transferrin receptor (TFR; OMIM*90010) [5,6]. Two extracellular domains, α1 and α2, sit on the top of the α3 domain, which spans the cellular membrane and binds to the beta-2-microglobulin (β2M) protein (OMIM*109700). HFE protein is formed by a signal peptide, a topical extracellular domain, a transmembrane helical region and a cytoplasmic domain. It plays a role in the iron regulation by forming complexes with (a) TFR1 (OMIM*190010), in case of iron deficiency or (b) TFR2 (OMIM*604720), the TFR1 liver homologue, in case of iron overload. HFE/TfR2 complex activates a signaling cascade resulting in the upregulation of hepcidin (OMIM*606464), ferroportin degradation (OMIM*604653) and, consequently, a decreased dietary iron uptake [7–9]. HH clinical expression occurs earlier in male (around the age of 40) and later in females, because of the protective effects of menstrual blood loss and pregnancies [10]. HFE protein mutation leads to iron and organ damage in the liver, heart and pancreas [2]. Worldwide about 20 different mutations have been identified in HFE gene [11,12], but the main mutations are known as C282Y (rs1800562; exon 4, NM_000410.3: c.845GNA; NP_000401.1:p.Cys282Tyr) and H63D (rs1799945; exon 2, NM_000410.3:c.187CNG; NP_000401.1:p.His63Asp) [12,13]. C282Y mutation is responsible for the lack of HFE-β2M association [14]; H63D variant leads to the lack of HFE–TFR complex [6–15].
http://dx.doi.org/10.1016/j.bcmd.2014.04.010 1079-9796/© 2014 Elsevier Inc. All rights reserved.
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The distribution of genotypes among iron loaded patients shows a prevalence of C282Y/C282Y subjects [16–19]. They are more than 90% in the UK, and more than 80% in Northern European countries; the percentage ranges from 60 to 83% in the USA [20] and it is equal to 64% in Italy [21]. A mild-moderate phenotype is associated to the compound heterozygosity condition (C28Y/H63D) [22]. H63D role in iron overload is controversial, but this condition less commonly develops iron overload [10,12,20] and is associated with extremely variable phenotypes [23]. In our previous study we firstly investigated the distribution of HH genotypes among iron overloaded patients belonging to the province of Matera (Basilicata, South Italy) based on the genetic testing describing a biochemical evidence of iron overload (serum ferritin value higher than 750 ng/ml) in H63D/wt individuals, in the absence of other factors related to iron overload [24]. The genetic test (Haemochromatosis Strip Assay StripAssayA by ViennaLab Diagnostic GmbH) covers 11 HFE gene mutations (V53M, V59M, H63D, H63H, S65C, Q127H, E168X, E168Q, W169X, C282Y, and Q283P), 4 transferrin receptor mutations (Y250X, E60X, M172K, and the AVA Q 594–597) and 2 ferroportin mutations (N144H and V162del). Routine clinical practice is affected by considering HH allelic heterogeneity and the existence of rare/private mutations, occasionally found in a small number of subjects [25]. Considering the spectrum of possible genetic variations and the need of early diagnosis, in order to optimize the treatment of some patients at risk of delayed appropriate treatment, we decided to evaluate the potential utility of additional genetic investigations. Therefore we retrospectively selected a cohort of patients with H63D heterozygosity and biochemical iron overload for additional studies. In this context we assessed the HFE mutational state in order to clarify the relationship between genotype and phenotype in a patient belonging to our cohort. Patient and methods We retrospectively identified a cohort of 22 patients with H63D heterozygosity and biochemical iron overload for additional studies. The patients were divided into two groups: the first one included 12 subjects with ferritin value lower than 750 ng/ml; within the second cluster resided 10 individuals whose ferritin level exceed 750 ng/ml (severe hyperferritinemia). We assessed the HFE mutational state in order to clarify the relationship between genotype and phenotype in this last cluster. The most significant data of the subjects belonging to the second group are reported in Table 1. About the methods, the first step of molecular biology consisted in (a) DNA isolation, (b) primer design, (c) PCR (polymerase chain reaction) amplification, (d) HFE fragment sequencing and (e) bioinformatics analysis. DNA extraction was carried out by using a commercial kit (Nuclear Laser Medicine S.r.l.) according to the manufacturer’s instructions. DNA was quantified by means of NanoDrop 1000 spectrophotometer (NanoDrop Technologies, Inc). On the basis of HFE RefSeq in NCBI
Table 1 The most significant data of the patients belonging to the “severe hyperferritinemia” group. Age, sex and serum ferritin value of the 10 subjects whose serum ferritin (SF) value is higher than 750 ng/ml. Patients
Age (years)
Sex
SF average value (ng/ml)
1 2 3 4 5 6 7 8 9 10
35 41 48 26 69 54 33 45 51 55
Male Male Male Male Male Male Female Male Male Male
1035 948 989 792 1230 957 922 875 1154 860
database, PCR primers were designed by means of NCBI Primer-Blast in order to detect HFE exons, exon–intron boundaries, and 5′ and 3′ untranslated regions (UTRs). A phase of the optimization of PCR components and conditions was carried out. For the amplification, 25 μl of PCR reaction was used: 1.5 μl MgCl2, each of dNTP 2 mM, 1 μl of specific primers, 0.4 U/μl of AmpliTaq Gold DNA polymerase in 10× PCR buffer (100 mM tris–HCl, pH 8.3, 500 mM KCl) (Roche Molecular Systems, Inc). The conditions of reaction were the following: (1) initial denaturation: 95 °C/7 min; (2) thermocycling: 94 °C/1 min; 58 °C/1 min; 72 °C/2 min (35 cycles); (3) final extension: 72 °C/10 min. Amplification products were analyzed by gel electrophoresis (1.5% agarose gel). The next step consisted in HFE fragments sequencing. The bioinformatics analysis allowed to confirm the similarity between our sequences and HFE RefSeq, by means of BlastN (NCBI database) [26]; a multiple alignment was performed between patient sequences and control sequences (three healthy controls were included in the study) to identify conserved/nonconserved nucleotides, by means of ClustalW2 (EMBL-EBI database) [27]. In addition the Mutation Surveyor software was employed for DNA variant analysis [28,29]. If a novel mutation was identified, it was confirmed by a second step of amplification. In addition, for assessing the HFE mRNA level, quantitative real-time PCR (qPCR) assay was carried out. First, the whole blood was collected by means of Tempus RNA tubes (Applied Biosystems). RNA isolation was achieved using Tempus Spin RNA Isolation Reagent Kit (Applied Biosystems), optimized in our laboratory. RNA was quantified using the NanoDrop Spectrophotometer 1000 (NanoDrop Technologies, Inc.) again. The reverse transcription of RNA into double-stranded cDNA was obtained by using the RETROscript Kit (Ambion), following the company instructions. The qPCR was carried out by means of the thermal cycler Chromo4 (Continuous Fluorescence Detector) and the Optical Monitor 2.03.5 software (MJ Research, Inc.). We employed the Power SYBR Green PCR Master Mix (Applied Biosystems) in the following volumes: 5 μl of Power SYBR Green PCR Master Mix (2 ×), 1.5 μl of forward primer 10 mM, 1.5 μl of reverse primer 10 mM, 1 μl of template. The protocol was (1) 95 °C/10 min, (2) 38 cycles of 95 °C/30 s and 64 °C/1 min, and (3) 72 °C/1 min. Melting curves were generated after 38 cycles by heating the sample up to 95 °C for 15 s followed by cooling down to 60 °C for 15 s and heating the samples to 95 °C for 15 s. We applied SYBR Green as reporter molecule, β-actin as housekeeping gene, three wild-type subjects as calibrators, two gene replicates and 2−ΔΔCt method [30] for data analysis, according to the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) [31]. After verifying that all gene amplification efficiency was comparable, we calculated the fold change (2−ΔΔCt). In detail, firstly we determined average Ct values for HFE replicates and for controls (calibrators) and, after normalization with housekeeping, we calculated ΔΔCt as ΔCt of target gene − ΔCt of calibrator gene and we used the data for deriving the fold change value. Results were expressed as mean ± standard deviation. Student's t test was employed in order to calculate the statistical significance (p b 0.05). PCR specificity was assessed by melting curve analysis of amplification products for confirming the occurrence of specific amplification peaks and the absence of primer dimer formation. Gel electrophoresis (2% agarose) was also performed in order to verify the presence of single gel bands of predicted size. Results HFE sequencing results underlined the presence of a variant not previously described in literature (GenBank Number: JQ478433) in 9/10 patients in the group of severe hyperferritinemia. We first identified this variation in the patient 1 of the cluster and we consequently extended the variant research to the whole group. For this reason the proband 1 was considered the case-model and we report about the subject, a 35-year-old Caucasian male coming from a small village of Basilicata, whose father (76 years old) showed severe
Please cite this article as: M.C. Padula, et al., Blood Cells Mol. Diseases (2014), http://dx.doi.org/10.1016/j.bcmd.2014.04.010
M.C. Padula et al. / Blood Cells, Molecules and Diseases xxx (2014) xxx–xxx Table 2 Clinical, biochemical and molecular data of our most significant clinical case. The variations have been labelled according to HGVS nomenclature. Proband data Gender Age at diagnosis (years) Clinical data Serum ferritin (ng/ml) Serum transferrin (mg/dl) Serum iron (μl/dl) GPT (ALT) (UI/l) GOT (AST) (UI/l) Hepatic iron content (μmol/g) Phlebotomies Total iron removed (g) Classical HFE mutation Novel HFE mutation
Type of the novel mutation
Male 35 Fatigue, joint and abdominal pains and jaundiced complexion 1035 ± 45 389 ± 16 151 ± 11 63 ± 8 42 ± 3 NA Yes 6.1 NM_000410.3:c.187CNG; rs1799945 NP_000401.1:p.His63Asp NM_000410.3:c.847CNT NP_000401.3:p.Gln283* Non-sense substitution
biochemical iron overload and hepatic damage. Other diseases have been ruled out within the family. He was heterozygous for H63D mutation at first genetic test and no variations in other gene related to the iron cycle (ferroportin and transferrin receptor) were found. The most significant clinical and laboratory characteristics of the proband are reported in Table 2. Laboratory testing showed BIO evidence and increased hepatic integrity enzymes. About the medical background no history of chronic liver disease, diabetes, alcohol abuse and other factors was reported. The patient underwent regular phlebotomies (at first one phlebotomy every 7–10 days) to normalize ferritin levels. (See Table 3.) In detail the nucleotide change CNT resides in HFE exon 4 at 847 position of RefSeq (NM_000410.3:c.847CNT) and is in homozygosity state (Table 2; Fig. 1). The sequence of primer pair used for amplification of the exon 4 was the subsequent: 5′–CTTCCTGGCAAGGGTAAACA-3′ (forward primer); 5′–CTCAGGCACTCCTCTCAACC-3′ (reverse primer). The variation is absent in homozygosity state in proband relatives during the family test. About the proband family, if the father showed the same genotype (and a comparable phenotype too), the proband’s brother and sister were found heterozygotes. The transition introduces a stop-codon that interrupts the reading frame, as the sequence prediction showed (Alamut 2.2e software, Interactive Biosoftware, France). A premature chain termination of translation could occur, resulting in truncated HFE protein at α3 domain level (NP_000401.1:p.Gln283*), whose secondary structure was predicted by means of Phyre2 tool (Protein Homology/Analogy Recognition Engine) [32]. However, the production of faulty proteins is prevented in eukaryotes by means of several surveillance mechanisms. A well-studied control mechanisms is the nonsense-mediated mRNA decay (NMD), which recognizes and degrades transcripts preventing a premature translation-termination codon (PTC) [33,34]. In order to check if
3
our mRNA is targeted for NMD, we quantified mRNA levels in our patient and controls by applying real-time PCR. The ΔΔCt value for patient HFE gene was equal to 12.872 ± 0.785 (mean and standard deviation); a value equivalent to 0.034 ± 0.007 was obtained for control. The target-control difference was statistically significant (Student’s t test; p b 0.05); therefore HFE mRNA was expressed about 7000 times less than calibrator gene, that is the average control sample (fold change values were compared). We obtained single peak dissociation curves and single gel bands of predicted size; both confirmed the PCR specificity. In a few words, here we reported a novel substitution that generates PTC. As direct consequence an aberrant mRNA is produced. The mRNA is targeted for NMD surveillance mechanism affecting the normal production of the truncated HFE protein (Fig. 2). Discussion Here we selected a cohort of 22 H63D heterozygote patients, whose 10 subjects showed severe hyperferritinemia. Within this last cluster, 9/10 subjects showed a novel variation. The group is enough homogenous in terms of fold change (a dramatic reduction in the amount of HFE mRNA in the patients was observed in all cases) and number of phlebotomies (average number: 1 phlebotomy every 15 days). Here we reported about the clinical case of the first patient, a 35-year-old man considered as model. It should be observed that the clinical onset and laboratory alterations in young subjects are uncommon; therefore, the individuation of an underlying genetic disease, which could be successfully treated, represents an important opportunity. In addition, the clinical meaning of H63D status is partly unclear: literature and specific databases (dbSNP, LSDB, locus-specific database and OMIM database) describe this aspect as extremely variable (Table 2) and related to HH clinical phenotype if associated with other phenotypic modifiers, both environmental, e.g. diet, alcohol abuse, diabetes, inflammation and non-genetic factor such as sex and age [35]. In order to understand the genotype–phenotype correlation in our proband, we analyzed the crosslink between biochemical–clinical evidences and molecular data. Assuming a partial dominance of HFE wild-type allele, we characterized HFE coding region, exon–intron boundaries and UTRs. The novel c.847CNT change was found. At protein level the substitution corresponds to the p.Gln283* change. Another mutation described in literature involves the 283 position resulting in the p.Gln283Pro variant. This mutation was considered a C282Y-like causative allele: for being adjacent to the disulfide bridge formed by Cys225 and Cys282, the Q283P protein is retained in the endoplasmic reticulum, with structural and functional consequences similar to the C282Y mutation effects [36]; therefore, the Q283P variant represents a pathogenic variant (Table 2). In our case, the nucleotide change could result in a 283 amino acid protein, such as a truncated product. However the production of the faulty protein could be avoided due to the NMD surveillance
Table 3 HFE variations reported in dbSNP (LSDB, locus-specific database) and OMIM database and their clinical impact.
1 2 3 4 5 6 7 8 9 10
rs number
cDNA level
Protein level
Clinical significance
Reference
rs111033563 rs111033558 rs111033557 rs28934889 rs28934597 rs28934596 rs28934595 rs1800730 rs1800562 rs1799945
NM_000410.3:c.848ANC NM_000410.3:c.989GNT NM_000410.3:c.175GNA NM_000410.3:c.157GNA NM_000410.3:c.277GNC NM_000410.3:c.314TNC NM_000410.3:c.381ANC NM_000410.3:c.193ANT NM_000410.3:c.845GNA NM_000410.3:c.187CNG
NP_000401.1:p.Gln283Pro NP_000401.1:p.Arg330Met NP_000401.1:p.Val59Met NP_000401.1:p.Val53Met NP_000401.1:p.Gly93Arg NP_000401.1:p.Ile105Thr NP_000401.1:p.Gln127His NP_000401.1:p.Ser65Cys NP_000401.1:p.Cys282Tyr NP_000401.1:p.His63Asp
Pathogenic Pathogenic Non-Pathogenic Non-Pathogenic Pathogenic Pathogenic Pathogenic Pathogenic Pathogenic Variable
[53] [54] [54] [54] [55] [55] [54] [56] [4] [4]
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Fig. 1. From the PCR product electrophoresis to the sequencing of the proband and the controls HFE exon 4. About the agarose gel, the patient amplicon corresponds to the band of the line named “1”; the control amplicons resides into gel lines 2–4. A negative control (C−) was also utilized. Regarding to the sequencing results, we show the c.847CNT change is detectable in the proband and not in the consensus control sequence; this aspect is underlined both in alignment and electropherogram patient and controls.
Fig. 2. The effect of the novel variation at DNA, RNA and protein level. The novel c.CNT substitution generates a PTC in HFE exon 4, responsible for the production of an aberrant mRNA. The mRNA is targeted for NMD surveillance mechanism. As consequence the normal production of the truncated protein is affected. Both the protein primary structure (the missed region is reported in black and underlined) and the secondary structure prediction (conducted by means of Phyre2 software) are shown.
Please cite this article as: M.C. Padula, et al., Blood Cells Mol. Diseases (2014), http://dx.doi.org/10.1016/j.bcmd.2014.04.010
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mechanisms. Our c.CNT substitution directly generates a stop-codon, namely PTC, responsible for an aberrant HFE mRNA. PTCs can derive either from mutations at DNA level (e.g. frame-shift or nonsense mutations) or from alternative splicing mechanisms that leads to the production of mRNA isoforms with truncated reading frames. Consequently the nonsense transcripts would result in the production of Cterminally truncated proteins with potentially negative effects [34,37]. It was estimated that about 30% of all known disease-associated mutations generate a PTC-containing mRNA [33,38]. β-Thalassemia is one of the first genetic disorder where the role of NMD as phenotypic modifier was reported 20 years ago: the majority of β-globin gene mutations leads to a PTC in the first or second exon of the three-exon gene [33,39,40]. Nowadays a large number of inherited genetic disorders arising from generation of PTC were identified: SOX10, rhodopsin, receptor tyrosine kinase-like orphan receptor 2, cone-rod homeobox, coagulation factor X, dystrophin, and cystic fibrosis genes are example of genes targeted for NMD [33]. In addition, some evidences of NMD role in the control of wild-type gene expression were reported: NMD pathway also regulates the level of many physiological mRNAs involved in different cellular processes, such as DNA repair and metabolism [33,41–43]. Very interestingly, PTCs occur more frequently in genes belonging to the immunoglobulin superfamily than in other genes; in addition the steady-state mRNA level is highly reduced by NMD [33,44,45]. HFE gene belongs to the immunoglobulin superfamily and is very similar to the MHC class I molecules [4] and so, a candidate for NMD process. Considering this last aspect and considering that the phenotypic severity of diseases caused by non-sense mutations can be predicted by quantifying the reduction amount of mRNA level [40], qPCR was carried out. The quantification of gene expression results showed a minimal HFE expression compared to the normal expression. This aspect is consistent with the NMD role in preventing the gain-of-function effects of truncated protein that could result if non-sense transcripts were stable. So, in our case, NMD pathway acts in the control of HFE gene expression by strongly reducing the transcript level and by affecting the levels of HFE protein. Previous studies reported in literature a few non-sense mutations in human HFE gene [17,46–49]. About the crosslink between non-sense mutation and NMD in HFE gene, Pointon and collaborators [49] identified a single nucleotide deletion (c.del478) in HFE exon 3 that causes a frameshift and introduces a PTC leading to mRNA degradation by NMD pathway. Afterward, Martins’ investigations demonstrated that the expression of physiological human HFE transcripts, specifically those species with 3′-end cleavage and polyadenylation at exon 7, is down-regulated by the NMD mechanism. In addition, the same group showed that NMD and alternative polyadenylation may act coordinately to control HFE mRNA levels, possibly varying its protein expression according to the physiological cellular requirements [50]. This aspect suggests the significance of NMD mechanism in determination of both wild and mutant human HFE mRNA level. In fact, the present paper reports the first evidence of a point substitution that directly generates PTC and its ability to commit the HFE mRNA to NMD and affect the formation of the truncated protein. As regard the functional implication of our finding, we supposed that the formation of the iron sensor and signal transduction effector complex should have been involved, consequently inducing the dysregulation of systemic iron homeostasis. In fact, considering that the primary HFE role is the control of hepcidin expression [51,52], it is known that in mutated or absent HFE, hepcidin production is inappropriately low, when related to the overall body iron status and, as direct consequence, ferroportin activity is not attenuated [9,51,52]. As final effect of this chain reaction, the iron accumulation is promoted; this aspect is consistent with the biochemical pattern of iron overload (hyperferritinemia in particular) of our proband. In general, the biochemical iron overload in our clinical cases could be related to a variant not reported in literature, and our data explain,
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at molecular level, the pathologic iron state in our cases who resulted to be only apparently H63D carriers at first genetic test. Moreover our data emphasize the importance of HFE gene sequencing in diagnosis and management of HH: when heterozygous subjects show biochemical iron overload with alteration of transaminase levels, the routine genetic test-based approach could be insufficient; in these cases HFE sequencing is very important prior to study other iron genes because additional mutations in this gene may play a clinical impact in heterozygotes. Additional molecular investigations should be requested, in order to explain the absence of correlation between genotype and clinical phenotype. In these cases we suggest to follow our three steps experimental design: (1) additional factors (causes of secondary iron overload, such as chronic liver disease, diabetes, alcohol abuse, etc.) must be ruled out; (2) the absence of the compound heterozygosity condition (C282Y/H63D) must be checked; (3) HFE sequencing to identify unknown HFE variants, as reported here, must be performed. Finally our results may provide a response to some questions arising from the apparent lack of correlation between genotype and phenotype observed in clinical practice [18,23,25]. The individuation of novel mutations could extend the panel of the common HFE mutations and contribute to improve both the patient diagnosis and therapy. Finally, as our novel, private variant concurs to the altered iron state, the possibility to assess its presence in the genome could allow an earlier diagnosis and, consequently, an earlier iron depletion therapy. This aspect is very important in terms of quality of life: a specific dietary regime and an early therapy could avoid the adverse outcomes of the disease. Since we analyzed a small group belonging to a small Italian region, in order to strengthen the significance of our de novo mutation, further investigations will consist in the screening of all iron loaded subjects, in the same area and in different geographic areas (epidemiologic implementation).
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