Iron overload (IO) in HFE-related hereditary haemochromatosis is associated with increased risk of liver cancer. This study aimed to investigate the role of other genes involved in hereditary IO among patients with hepatocellular carcinoma (HCC).

Patients with HCC diagnosed in our institution were included in this prospective study. Those with ferritin levels ≥300 μg/L (males) or ≥200 μg/L (females) and/or transferrin saturation ≥50% (males) or ≥45% (females) had liver iron concentration (LIC) evaluated by MRI. HFE C282Y and H63D mutations were screened. Genetic analyses of genes involved in hereditary IO (HFE, HJV/HFE2, HAMP, TFR2, SLC40A1, GNPAT) were performed in patients with increased LIC.

A total of 234 patients were included; 215 (92%) had common acquired risk factors of HCC (mainly alcoholism or chronic viral hepatitis). 119 patients had abnormal iron parameters. Twelve (5.1%) were C282Y homozygotes, three were compound C282Y/H63D heterozygotes. LIC was measured by MRI in 100 patients. Thirteen patients with a LIC>70 μmol/g were enrolled in further genetic analyses: two unrelated patients bore the HAMP:c.-153C>T mutation at the heterozygous state, which is associated with increased risk of IO and severe haemochromatosis. Specific haplotypes of SLC40A1 were also studied.

Additional genetic risk factors of IO were found in 18 patients (7.7%) among a large series of 234 HCC patients. Screening for IO and the associated at-risk genotypes in patients who have developed HCC, is useful for both determining etiologic diagnosis and enabling family screening and possibly primary prevention in relatives.

Haem carrier protein 1 (Hcpl) is a component of the haem-iron uptake pathway in the small intestine. Using quantitative real-time PCR, we examined the expression of Hcp1 and other intestinal iron-transporting proteins in male C57BL/6 mice with experimentally altered iron homeostasis. Intestinal Hcp1 mRNA content was not significantly changed by iron overload (600 mg/kg); however, it was increased to 170 % of controls 72 h after withdrawal of 0.7 ml of blood; the same treatment increased intestinal Cybrd1 mRNA to 900 % of controls. LPS treatment (1 mg/kg, 6 h) decreased intestinal Hcp1 mRNA content to 66 % of controls and Flvcr mRNA content to 65 % of controls, while Cybrd1 mRNA, Dmt1 mRNA and Fpn1 mRNA decreased to 6 %, 43 % and 32 %, respectively. In 129SvJ mice with targeted disruption of the hemojuvelin (Hfe2) gene, which display very low expression of liver hepcidin, Cybrd1 mRNA content increased to 1040 %, Dmt1 mRNA content to 200 % and Fpn1 mRNA to 150 % when compared to wild-type mice; changes in Hcp1, Abcg2 and Flver mRNA content were only minor. Overall, these results suggest that, during inflammation, the intestinal haem-iron uptake pathway is not as strongly transcriptionally downregulated as the non-haem iron uptake pathway. A decrease in circulating hepcidin increases the expression of proteins participating in non-haem iron uptake, but has no significant effect on Hcp1 mRNA content.

BACKGROUND

Next-generation sequencing of an iron metabolism gene panel could identify pathogenic mutations, improving on standard hemochromatosis genetic testing and providing a molecular diagnosis in patients with suspected iron overload.

METHODS

A next-generation sequencing panel of 15 genes with known roles in iron metabolism was constructed. A total of 190 patients were sequenced: 94 from a tertiary hemochromatosis clinic and 96 submitted for HFE testing with biochemical evidence of iron overload [elevated ferritin (>450 μg/L) or transferrin saturation (>55%)] obtained from a chart review.

RESULTS

From the hemochromatosis clinic cohort, six patients were diagnosed with non-HFE hemochromatosis due to homozygous hemojuvelin (HFE2) mutations. Ten additional heterozygous pathogenic mutations were observed. From the chart review cohort, a C-terminus ferritin light chain (FTL) frameshift mutation was observed consistent with neuroferritinopathy. Heterozygous deletion of HFE2 and four additional rare pathogenic or likely pathogenic heterozygous mutations were identified in seven patients.

CONCLUSIONS

An iron metabolism gene panel provided a molecular diagnosis in six patients with non-HFE iron overload and is suitable for diagnostic purposes in exceptional cases in specialized clinics. Further research will be required to assess the modifier effect of rare heterozygous mutations in iron metabolism genes.

BACKGROUND

Type 2A hereditary haemochromatosis (type 2A HH) is a rare iron-loading disorder caused by mutations in the HFE2 gene, which encodes the HJV protein. We present characteristics, treatment and follow-up of subjects diagnosed with type 2A HH in the Netherlands to increase awareness of the disease and its treatment, and to define knowledge gaps.

METHODS

We collected clinical, biochemical and genetic data from seven patients (two female; five probands) from six families genetically diagnosed with type 2A HH at the Expertise Center for Iron Disorders, Radboud University Medical Centre between 2006 and 2016.

RESULTS

The five probands presented with heterogeneous complaints between the ages of 19 and 39. One of two patients with delayed clinical diagnosis developed hypogonadism and Y. enterocolitica sepsis. Diagnostic workup and follow-up varied. When assessed, elevated transferrin saturation (79-98%), ferritin (1400-6200 µg/l) and severely elevated liver iron levels were found, and in all subjects, phlebotomies were initiated. One subject was switched to erythrocytapheresis. Target ferritin levels varied. Despite long-term iron depletion, two subjects developed clinical complications. Sanger sequencing revealed two pathogenic HFE2 variants (homozygous or compound heterozygous) for the five families of Dutch descent and one new pathogenic variant in the family of non-Dutch descent.

CONCLUSIONS

Three genetic variants caused type 2A HH in six families. Clinical diagnosis was delayed in two subjects. We observed variance in presentation, workup, follow-up and treatment. We found new complications in long-term iron-depleted patients. We recommend research and guidelines for optimal workup, follow-up and treatment of type 2A HH.

METHODS

A 28 year young female presented to our hospital for further evaluation with recently diagnosed diabetes mellitus, hyperpigmentation of the skin, hepato- and splenomegaly, thrombocytopenia and an elevated transferrin saturation (96 %), but a negative test for HFE gene mutations such as C282Y and H63D.

RESULTS

Using the mini-laparascopic technique we diagnosed a smallnodular liver cirrhosis with an iron overload.

METHODS

This is the clinical presentation of one subtype of the so called Non-HFE-hemochromatosis, the juvenile hemochromatosis (HFE2). Other causes of primary and secondary iron overload have been ruled out. Different from the HFE-positive hemochromatosis (HFE 1) in which the gene defect is located on chromosome 6, the defect in HFE 2 is located on chromosome 1. The underlying genetic defect has been localized within recently identified HJV gene. Phlebotomy is the treatment of choice, to be performed until the ferritin level is lower than 50 microg/l.

CONCLUSIONS

If liver cirrhosis is present in hemochromatosis, the overall risk of developing heptatocellular carcinoma is 20 times higher than in the normal population. Therefore it is suggested to perform an ultrasound examination of the liver and an AFP-test every 6 months, whereas an MRI-scan should be performed once a year, as a basis for further treatment options.

Hereditary hemochromatosis (HH) is a group of inherited iron-overload disorders associated with pathogenic defects in the genes encoding hemochromatosis (HFE), hemojuvelin (HJV/HFE2), hepcidin (HAMP), transferrin receptor 2 (TfR2), and ferroportin (FPN1/SLC40A1) proteins, and the clinical features are well described. However, there have been only a few detailed reports of HH in Chinese populations. Thus, there is insufficient patient information for population-based analyses in Chinese populations or comparative studies among different ethical groups. In the current work, we describe eight Chinese cases of hereditary hemochromatosis. Gene sequencing results revealed eight mutations (five novel mutations) in HFE, HFE2, TfR2, and SLC40A1 genes in these Chinese HH patients. In addition, we used Polymorphism Phenotyping v2 (Polyphen), Sorting Intolerant From Tolerant (SIFT), and a sequence alignment program to predict the molecular consequences of missense mutations.

BACKGROUND

Considering that vitamin A deficiency modulates hepcidin expression and consequently affects iron metabolism, we evaluated the effect of vitamin A deficiency in the expression of genes involved in the hemojuvelin (HJV)-bone morphogenetic protein 6 (BMP6)-small mothers against decapentaplegic protein (SMAD) signaling pathway.

METHODS

Male Wistar rats were treated: control AIN-93G diet (CT), vitamin A-deficient diet (VAD), iron-deficient diet (FeD), vitamin A- and iron-deficient diet (VAFeD), or 12 mg all-trans retinoic acid (atRA)/kg diet.

RESULTS

Vitamin A deficiency (VAD) increased hepatic Bmp6 and Hfe2 mRNA levels and down-regulated hepatic Hamp, Smad7, Rarα, and intestinal Fpn1 mRNA levels compared with the control. The FeD rats showed lower hepatic Hamp, Bmp6, and Smad7 mRNA levels compared with those of the control, while in the VAFeD rats only Hamp and Smad7 mRNA levels were lower than those of the control. The VAFeD diet up-regulated intestinal Dmt1 mRNA levels in relation to those of the control. The replacement of retinyl ester by atRA did not restore hepatic Hamp mRNA levels; however, the hepatic Hfe2, Bmp6, and Smad7 mRNA levels were similar to the control. The atRA rats showed an increase of hepatic Rarα mRNA levels and a reduction of intestinal Dmt1 mRNA and Fpn1 levels compared with those of the control.

CONCLUSIONS

The HJV-BMP6-SMAD signaling pathway that normally activates the expression of hepcidin in iron deficiency is impaired by vitamin A deficiency despite increased expression of liver Bmp6 and Hfe2 mRNA levels and decreased expression of Smad7 mRNA. This response may be associated to the systemic iron deficiency and spleen iron retention promoted by vitamin A deficiency.

p.Cys282Tyr (C282Y) homozygosity explains most cases of HFE-related hemochromatosis, but a significant number of patients presenting with typical type I hemochromatosis phenotype remain unexplained. We sought to describe the clinical relevance of rare HFE variants in non-C282Y homozygotes. Patients referred for hemochromatosis to the National Reference Centre for Rare Iron Overload Diseases from 2004 to 2010 were studied. Sequencing was performed for coding region and intronic flanking sequences of HFE, HAMP, HFE2, TFR2, and SLC40A1. Nine private HFE variants were identified in 13 of 206 unrelated patients. Among those, five have not been previously described: p.Leu270Argfs*4, p.Ala271Valfs*25, p.Tyr52*, p.Lys166Asn, and p.Asp141Tyr. Our results show that rare HFE variants are identified more frequently than variants in the other genes associated with iron overload. Rare HFE variants are therefore the most frequent cause of hemochromatosis in non-C282Y homozygote HFE patients. Am. J. Hematol. 91:1202-1205, 2016. © 2016 Wiley Periodicals, Inc.

Celecoxib is an inhibitor of cyclooxygenase-2, a gene that is often aberrantly expressed in the lung squamous cell carcinoma (LSQCC). The present study aims to provide novel insight into chemoprevention by celecoxib treatment. The human LSQCC cell line SK‑MES‑1 was treated with or without celecoxib and RNA‑sequencing (RNA‑seq) was performed on the Illumina HiSeq 2000 platform. Expression levels of genes or long non‑coding RNAs (lncRNAs) were calculated by Cufflinks software. Subsequently, differentially expressed genes (DEGs) and differentially expressed lncRNAs (DE‑LNRs) between the two groups were selected using the limma package and LNCipedia 3.0, respectively; followed by co‑expression analysis based on their expression correlation coefficient (CC). Enrichment analysis for the DEGs and co‑expressed DE‑LNRs were performed. Protein‑protein interaction (PPI) network analysis for DEGs was performed using STRING database. A set of 317 DEGs and 25 DE‑LNRs were identified between celecoxib‑treated and non‑treated cell lines. A total of 12 pathways were enriched by the DEGs, including 'protein processing in endoplasmic reticulum' for activating transcription factor 4 (ATF4), 'mammalian target of rapamycin (mTOR) signaling pathway' for vascular endothelial growth factor A (VEGFA) and 'ECM‑receptor interaction' for fibronectin 1 (FN1). Genes such as VEGFA, ATF4 and FN1 were highlighted in the PPI network. VEGFA was linked with lnc‑AP000769.1‑2:10 (CC= ‑0.99227), whereas ATF4 and FN1 were closely correlated with lnc‑HFE2‑2:1 (CC=0.996159 and ‑0.98714, respectively). lncRNAs were also enriched in pathways such as 'mTOR signaling pathway' for lnc‑HFE2‑2:1. Several important molecules were identified in celecoxib‑treated LSQCC cell lines, such as VEGFA, ATF4, FN1, lnc‑AP000769.1‑2:10 and lnc‑HFE2‑2:1, which may enhance the anti‑cancer effects of celecoxib on LSQCC.

Juvenile hemochromatosis (JH), type 2A hemochromatosis, is a rare autosomal recessive disorder of systemic iron overload due to homozygous mutations of HJV (HFE2), which encodes hemojuvelin, an essential regulator of the hepcidin expression, causing liver fibrosis, diabetes, and heart failure before 30 years of age, often with fatal outcomes. We report two Japanese sisters of 37 and 52 years of age, with JH, who showed the same homozygous HJV I281T mutation and hepcidin deficiency and who both responded well to phlebotomy on an outpatient basis. When all reported cases of JH with homozygous HJV mutations in the relevant literature were reviewed, we found-for the first time-that JH developed in females and males at a ratio of 3:2, with no age difference in the two groups. Furthermore, we found that the age of onset of JH may depend on the types of HJV mutations. In comparison to patients with the most common G320V/G320V mutation, JH developed earlier in patients with L101P/L101P or R385X/R385X mutations and later in patients with I281T/I281T mutations.

Keywords: hemochromatosis; hemojuvelin; hepcidin.

Hemojuvelin, also known as RGMc, is encoded by hfe2 gene that plays an important role in iron homeostasis. hfe2 is specifically expressed in the notochord, developing somite and skeletal muscles during development. The molecular regulation of hfe2 expression is, however, not clear. We reported here the characterization of hfe2 gene expression and the regulation of its tissue-specific expression in zebrafish embryos. We demonstrated that the 6 kb 5'-flanking sequence upstream of the ATG start codon in the zebrafish hfe2 gene could direct GFP specific expression in the notochord, somites, and skeletal muscle of zebrafish embryos, recapitulating the expression pattern of the endogenous gene. However, the Tg(hfe2:gfp) transgene is also expressed in the liver of fish embryos, which did not mimic the expression of the endogenous hfe2 at the early stage. Nevertheless, the Tg(hfe2:gfp) transgenic zebrafish provides a useful model to study liver development. Treating Tg(hfe2:gfp) transgenic zebrafish embryos with valproic acid, a liver development inhibitor, significantly inhibited GFP expression in zebrafish. Together, these data indicate that the tissue specific expression of hfe2 in the notochord, somites and muscles is regulated by regulatory elements within the 6 kb 5'-flanking sequence of the hfe2 gene. Moreover, the Tg(hfe2:gfp) transgenic zebrafish line provides a useful model system for analyzing liver development in zebrafish.

OBJECTIVE

To report novel mutations SEC23B gene in congenital dyserythropoietic anemia (CDA).

METHODS

By direct sequencing method, we sequenced CDAN1 and SEC23B genes in a Chinese CDA II patient, presented with chronic fatigue and dark urine, as well as his family members. Serum hepcidin was assayed by mass spectrometry.

RESULTS

We found a c.71G>A mutation and a c.74C> A mutation in the patient. In addition, a heterozygous c.55A>G mutation of HFE2 gene was found in some family members. The level of serum hepcidin of the patient was below the detection limit (<1 nmol/L).

CONCLUSIONS

Contrary with what have been reported previously in the Europe, especially in the Italy, the gene mutations identified in this case was different and novel. The two novel mutations contribute to the diagnosis of CDAII and are the first report in East Asian CDAII patients.

OBJECTIVE

Despite the obligate iron loss from blood donation, some donors present with hyperferritinaemia that can result from a wide range of acute and chronic conditions including hereditary haemochromatosis (HH). The objective of our study was to investigate the causes of hyperferritinaemia in the blood donor population and explore the value of extensive HH mutational analyses.

METHODS

Forty-nine consecutive donors (f = 6, m = 43) were included prospectively from the Capital Regional Blood Center. Inclusion criteria were a single ferritin value >1000 μg/l or repeated hyperferritinaemia with at least one value >500 μg/l. All donors were questioned about their medical history and underwent a physical examination, biochemical investigations and next-generation sequencing of HH-related genes, including the HFE gene, the haemojuvelin gene (HFE2/HJV), the hepcidin gene (HAMP), the ferroportin 1 gene (SLC40A1) and the transferrin receptor 2 gene (TFR2).

RESULTS

Forty of 49 donors were mutation positive with a combined 69 mutations, 54 of which were located in the HFE gene. There were 11 mutations in the TFR2 gene, two mutations in the HFE2 gene and two mutations in the HAMP gene. Only four donors had apparent alternative causes of hyperferritinaemia.

CONCLUSIONS

HH-related mutations were the most frequent cause of hyperferritinaemia in a Danish blood donor population, and it appears that several different HH-genotypes can contribute to hyperferritinaemia. HH screening in blood donors with high ferritin levels could be warranted. HH-related iron overload should not in itself result in donor ineligibility.

The reaction of Fe2(pdt)(CO)6 with two equivalents of Ph2PC6H4NH2 (PNH2) affords the amido hydride HFe2(pdt)(CO)2(PNH2)(PNH) {[H1H]0, pdt2- = CH2(CH2S-)2}. Isolated intermediates in this conversion include Fe2(pdt)(CO)5-(κ1-PNH2) and Fe2(pdt)(CO)4(κ2-PNH2). X-ray crystallographic analysis of [H1H]0 shows that the chelating amino/amido-phosphine ligands occupy trans-dibasal positions. The 31P NMR spectrum indicates that [H1H]0 undergoes rapid proton exchange between the amido and amine centers. No exchange was observed for the hydride. Protonation of [H1H]0 gives [HFe2(pdt)(CO)2(PNH2)2]+ ([H21H]+), which contains two equivalent amino-phosphine ligands. Single-crystal X-ray crystallographic analysis of [H21H]+ also reveals hydrogen bonds between the exo amine protons with a THF molecule and BF4. Deprotonation of [H1H]0 with potassium tert-butoxide gave [HFe2(pdt)(CO)2(PNH)2]- ([1H]-), which was characterized spectroscopically. The complex has time-averaged C2 symmetry with two amido-phosphine ligands. FTIR spectroscopic measurements show that υCO shifts by approximately 20 cm-1 in the series [1H]-, [H1H]0, and [H21H]+. These shifts are comparable to those seen for the S-protonation of the (NC)2(CO)Fe-(μ-Scys)2Ni(Scys)2 site in the [NiFe]-hydrogenases.[1].

Objective: Aimed to construct an immune-related risk signature and nomogram predicting endometrial cancer (EC) prognosis.

Methods: An immune-related risk signature in EC was constructed using the least absolute shrinkage and selection operator regression analysis based on The Cancer Genome Atlas and Gene Expression Omnibus databases. A nomogram integrating the immune-related genes and the clinicopathological characteristics was established and validated using the Kaplan-Meier survival curve and receiver operating characteristic (ROC) curve to predict the overall survival (OS) of EC patients. The Estimation of STromal and Immune cells in MAlignant Tumor tissues using Expression data (ESTIMATE) R tool was used to explore the immune and stromal scores.

Results: CCL17, CTLA4, GPI, HDGF, HFE2, ICOS, IFNG, IL21R, KAL1, NR3C1, S100A2, and S100A9 were used in developing an immune-related risk signature evaluation model. The Kaplan-Meier curve indicated that patients in the low-risk group had better OS (p<0.001). The area under the ROC curve (AUC) values of this model were 0.737, 0.764, and 0.782 for the 3-, 5-, and 7-year OS, respectively. A nomogram integrating the immune-related risk model and clinical features could accurately predict the OS (AUC=0.772, 0.786, and 0.817 at 3-, 5-, and 7-year OS, respectively). The 4 immune cell scores were lower in the high-risk group. Forkhead box P3 (FOXP3) and basic leucine zipper ATF-like transcription factor (BATF) showed a potential significant role in the immune-related risk signature.

Conclusion: Twelve immune-related genes signature and nomogram for assessing the OS of patients with EC had a good practical value.

Keywords: BATF; Endometrial Cancer; FOXP3; Immune Signature; Nomogram; Overall Survival.

The kinetically robust hydride [t-HFe2(Me2pdt)(CO)2(dppv)2]+ ([t-H1]+) (Me2pdt2- = Me2C(CH2S-)2; dppv = cis-1,2-C2H2(PPh2)2) and related derivatives were prepared with 57Fe enrichment for characterization by NMR, FT-IR, and NRVS. The experimental results were rationalized using DFT molecular modeling and spectral simulations. The spectroscopic analysis was aimed at supporting assignments of Fe-H vibrational spectra as they relate to recent measurements on [FeFe]-hydrogenase enzymes. The combination of bulky Me2pdt2- and dppv ligands stabilizes the terminal hydride with respect to its isomerization to the 5-16 kcal/mol more stable bridging hydride ([μ-H1]+) with t1/2(313.3 K) = 19.3 min. In agreement with the nOe experiments, the calculations predict that one methyl group in [t-H1]+ interacts with the hydride with a computed CH···HFe distance of 1.7 Å. Although [t-H571]+ exhibits multiple NRVS features in the 720-800 cm-1 region containing the bending Fe-H modes, the deuterated [t-D571]+ sample exhibits a unique Fe-D/CO band at ∼600 cm-1. In contrast, the NRVS spectra for [μ-H571]+ exhibit weaker bands near 670-700 cm-1 produced by the Fe-H-Fe wagging modes coupled to Me2pdt2- and dppv motions.

Hereditary hemochromatosis (HH) is an inherited iron overload. The most common form of HH is type 1 hereditary hemochromatosis (HFE-related), which is associated with mutation of the HFE gene located on chromosome 6 and inherited in an autosomal recessive pattern. Type 2 hereditary hemochromatosis or juvenile hemochromatosis is less frequent autosomal recessive disease that results from mutations in the HJV gene on chromosome 1 (type2a) or the HAMP gene on chromosome19 (type2b). Mutation of type 2 transferrin receptor gene and mutation of the ferroportin gene result in hemochromatosis type 3 and hemochromatosis type 4, respectively. Juvenile hemochromatosis is characterized by an early onset of excess accumulation of iron in various organs. It could affect the liver, heart, pancreas and joints, resulting in arthropathy. Most juvenile hemochromatosis cases exhibit severe symptoms due to early onset. Cardiac and hypogonadism are the dominating features of the disease. Prevalence of arthropathy in juvenile hemochromatosis is higher than classic HH. Early diagnosis and intervention of juvenile hemochromatosis may prevent irreversible organ damage. The diagnosis can be made based on laboratory testing (of increased transferrin saturation, serum iron and ferritin levels), liver biopsy, imaging or genotype. According to international guidelines, treatment of HH is indicated when serum ferritin concentrations are above the upper limit of normal. We report two sisters who presented to the rheumatology clinic with arthralgia, which was subsequently found to have a homozygous mutation variant of unknown significance in the HFE2 gene: c.497A>G;p.(His166Arg) and has been treated with deferasirox (Exjade®). Musculoskeletal symptoms completely resolved in both patients in two months and remained so for one year on treatment.

Keywords: HFE gene; arthritis; deferasirox; hereditary hemochromatosis.

Hereditary hemochromatosis (HH) is an iron metabolism disease clinically characterized by excessive iron deposition in parenchymal organs such as liver, heart, pancreas, and joints. It is caused by mutations in at least five different genes. HFE hemochromatosis is the most common type of hemochromatosis, while non-HFE related hemochromatosis are rare cases. Here, we describe six new patients of non-HFE related HH from five different families. Two families (Family 1 and 2) have novel nonsense mutations in the HFE2 gene have novel nonsense mutations (p.Arg63Ter and Asp36ThrfsTer96). Three families have mutations in the TFR2 gene, one case has one previously unreported mutation (Family A-p.Asp680Tyr) and two cases have known pathogenic mutations (Family B and D-p.Trp781Ter and p.Gln672Ter respectively). Clinical, biochemical, and genetic data are discussed in all these cases. These rare cases of non-HFE related hereditary hemochromatosis highlight the importance of an earlier molecular diagnosis in a specialized center to prevent serious clinical complications.

Keywords: HFE related hereditary hemochromatosis; HFE2 gene; TFR2 gene; homozygous; iron overload; missense; non-HFE related hereditary hemochromatosis; nonsense.