A form of inherited hyperferritinemia associated with bi-allelic pathogenic variants of STAB1.

Hyperferritinemia is a frequent finding in several conditions, both genetic and acquired. We previously studied eleven healthy subjects from eight different families presenting with unexplained hyperferritinemia. Their findings suggested the existence of an autosomal-recessive disorder. We carried out whole-exome sequencing to detect the genetic cause of hyperferritinemia. Immunohistochemistry and flow cytometry assays were performed on liver biopsies and monocyte-macrophages to confirm the pathogenic role of the identified candidate variants. Through a combined approach of whole-exome sequencing and homozygosity mapping, we found bi-allelic STAB1 variants in ten subjects from seven families. STAB1 encodes the multifunctional scavenger receptor stabilin-1. Immunohistochemistry and flow cytometry analyses showed absent or markedly reduced stabilin-1 in liver samples, monocytes, and monocyte-derived macrophages. Our findings show a strong association between otherwise unexplained hyperferritinemia and bi-allelic STAB1 mutations suggesting the existence of another genetic cause of hyperferritinemia without iron overload and an unexpected function of stabilin-1 in ferritin metabolism.

Hereditary Hyperferritinemia.

Ferritin is a ubiquitous protein that is present in most tissues as a cytosolic protein. The major and common role of ferritin is to bind Fe2+, oxidize it and sequester it in a safe form in the cell, and to release iron according to cellular needs. Ferritin is also present at a considerably low proportion in normal mammalian sera and is relatively iron poor compared to tissues. Serum ferritin might provide a useful and convenient method of assessing the status of iron storage, and its measurement has become a routine laboratory test. However, many additional factors, including inflammation, infection, metabolic abnormalities, and malignancy-all of which may elevate serum ferritin-complicate interpretation of this value. Despite this long history of clinical use, fundamental aspects of the biology of serum ferritin are still unclear. According to the high number of factors involved in regulation of ferritin synthesis, secretion, and uptake, and in its central role in iron metabolism, hyperferritinemia is a relatively common finding in clinical practice and is found in a large spectrum of conditions, both genetic and acquired, associated or not with iron overload. The diagnostic strategy to reveal the cause of hyperferritinemia includes family and personal medical history, biochemical and genetic tests, and evaluation of liver iron by direct or indirect methods. This review is focused on the forms of inherited hyperferritinemia with or without iron overload presenting with normal transferrin saturation, as well as a step-by-step approach to distinguish these forms to the acquired forms, common and rare, of isolated hyperferritinemia.

Intravenous iron therapy improves the hypercapnic ventilatory response and sleep disordered breathing in chronic heart failure.

AIMS: Intravenous iron therapy can improve symptoms in patients with heart failure, anaemia and iron deficiency. The mechanisms underlying such an improvement might involve chemoreflex sensing and nocturnal breathing patterns. METHODS AND RESULTS: Patients with heart failure, reduced left ventricular ejection fraction, anaemia (haemoglobin <13 g/dl in men; <12 g/dl in women) and iron deficiency (ferritin <100 or 100-299 µg/L with transferrin saturation <20%) were 2:1 randomized to patient-tailored intravenous ferric carboxymaltose dose or placebo. Chemoreflex sensitivity cardiorespiratory sleep study, symptom assessment and cardiopulmonary exercise test were performed before and 2 weeks after the last treatment dose. Fifty-eight patients (38 active arm/20 placebo arm) completed the study. Intravenous iron was associated with less severe symptoms, higher haemoglobin (12.5 ± 1.4 vs. 11.7 ± 1.0 mg/dl, p < 0.05) and improved haematinic parameters. Ferric carboxymaltose improved the central hypercapnic ventilatory response (-25.8%, p < 0.05 vs. placebo), without changes in peripheral chemosensitivity. In particular, the central hypercapnic ventilatory responses passed from 4.6 ± 6.5 to 2.9 ± 2.9 L/min/mmHg after ferric carboxymaltose and from 4.4 ± 4.6 to 4.6 ± 3.9 L/min/mmHg after placebo (ptreatment*condition  = 0.046). In patients presenting with sleep-related breathing disorder, apnoea-hypopnoea index was reduced with active treatment as compared to placebo (12 ± 11 vs. 19 ± 13 events/h, p < 0.05). After ferric carboxymaltose, but not after placebo, both peak oxygen uptake (VO2 ) increased (Δ1.1 ± 2.0 ml/kg/min, p < 0.05) and VO2 /workload slope was steeper (Δ0.67 ± 1.7 L/min/W, p < 0.01). CONCLUSIONS: Intravenous ferric carboxymaltose improves the hypercapnic ventilatory response and sleep-related breathing disorders in patients with heart failure, anaemia and iron deficiency. These newly described findings, along with improved oxygen delivery to exercising muscles, likely contribute to the favourable effects of ferric carboxymaltose in anaemic patients with heart failure.

Angiotensin Type 2 and Mas Receptor Activation Prevents Myocardial Fibrosis and Hypertrophy through the Reduction of Inflammatory Cell Infiltration and Local Sympathetic Activity in Angiotensin II-Dependent Hypertension.

Compound 21 (C21), an AT2 receptor agonist, and Angiotensin 1-7 (Ang 1-7), through Mas receptor, play an important role in the modulation of the protective arm of the renin-angiotensin system. The aim of this study was to investigate in an experimental model of angiotensin II-dependent hypertension whether the activation of the potentially protective arm of the renin-angiotensin system, through AT2 or Mas receptor stimulation, counteracts the onset of myocardial fibrosis and hypertrophy, and whether these effects are mediated by inflammatory mechanism and/or sympathetic activation. Sprague Dawley rats (n = 67) were treated for 1 (n = 25) and 4 (n = 42) weeks and divided in the following groups: (a) Angiotensin II (Ang II, 200 ng/kg/min, osmotic minipumps, sub cutis); (b) Ang II+Compound 21 (C21, 0.3 mg/kg/day, intraperitoneal); (c) Ang II+Ang 1-7 (576 µg/kg/day, intraperitoneal); (d) Ang II+Losartan (50 mg/kg/day, per os); (e) control group (physiological saline, sub cutis). Systolic blood pressure was measured by tail cuff method and, at the end of the experimental period, the rats were euthanized and the heart was excised to evaluate myocardial fibrosis, hypertrophy, inflammatory cell infiltration and tyrosine hydroxylase expression, used as marker of sympathetic activity. Ang II caused a significant increase of blood pressure, myocardial interstitial and perivascular fibrosis and myocardial hypertrophy, as compared to control groups. C21 or Ang 1-7 administration did not modify the increase in blood pressure in Ang II treated rats, but both prevented the development of myocardial fibrosis and hypertrophy. Treatment with losartan blocked the onset of hypertension and myocardial fibrosis and hypertrophy in Ang II treated rats. Activation of AT2 receptors or Mas receptors prevents the onset of myocardial fibrosis and hypertrophy in Ang II-dependent hypertension through the reduction of myocardial inflammatory cell infiltration and tyrosine hydroxylase expression. Unlike what happens in case of treatment with losartan, the antifibrotic and antihypertrophic effects that follow the activation of the AT2 or Mas receptors are independent on the modulation of blood pressure.

Prolonged exposure to welding fumes as a novel cause of systemic iron overload.

BACKGROUND & AIMS: Inhalation of welding fume may cause pulmonary disease known as welder's lung. At our centre we came across a number of welders with systemic iron overload and prolonged occupational history and we aimed at characterizing this novel clinical form of iron overload. METHODS: After exclusion of other known causes of iron overload, 20 welders were fully evaluated for working history, hepatic, metabolic and iron status. MRI iron assessment was performed in 19 patients and liver biopsy in 12. We included 40 HFE-HH patients and 24 healthy controls for comparison. RESULTS: 75% of patients showed lung HRCT alterations; 90% had s-FERR > 1000 ng/mL and 60% had TSAT > 45%. Liver iron overload was mild in 8 and moderate-severe in 12. The median iron removed was 7.8 g. Welders showed significantly lower TSAT and higher SIS and SIS/TIS ratio than HFE-HH patients. Serum hepcidin was significantly higher in welders than in HFE-HH patients and healthy controls. At liver biopsy, 50% showed liver fibrosis that was mild in four, and moderate-severe in two. Liver staging correlated with liver iron overload. CONCLUSIONS: Welders with prolonged fume exposure can develop severe liver iron overload. The mechanism of liver iron accumulation is quite different to that of HFE-HH suggesting that reticuloendothelial cells may be the initial site of deposition. We recommend routine measurement of serum iron indices in welders to provide adequate diagnosis and therapy, and the inclusion of prolonged welding fume exposure in the list of acquired causes of hyperferritinemia and iron overload.

HIF1A: A Putative Modifier of Hemochromatosis.

HFE-related hereditary hemochromatosis (HH) is characterized by marked phenotypic heterogeneity. Homozygosity for p.C282Y is a low penetrance genotype suggesting that the HFE-HH is a multifactorial disease resulting from a complex interaction involving a major gene defect, genetic background and environmental factors. We performed a targeted NGS-based gene panel to identify new candidate modifiers by using an extreme phenotype sampling study based on serum ferritin and iron removed/age ratio. We found an increased prevalence of the HIF1A p.Phe582Ser and p.Ala588Thr variants in patients with a severe iron and clinical phenotype. Accordingly, Huh-7 cells transfected with both variants showed significantly lower HAMP promoter activity by luciferase assay. The qRT-PCR assays showed a downregulation of hepcidin and an upregulation of the HIF1A target genes (VEGF, HMOX, FUR, TMPRSS6) in cells transfected with the HIF1A-P582S vector. We identified mutations in other genes (e.g., Serpina1) that might have some relevance in single cases in aggravating or mitigating disease manifestation. In conclusion, the present study identified HIF1A as a possible modifier of the HFE-HH phenotype cooperating with the genetic defect in downregulating hepcidin synthesis. In addition, this study highlights that an NGS-based approach could broaden our knowledge and help in characterizing the genetic complexity of HFE-HH patients with a severe phenotype expression.

Inherited iron overload disorders.

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

Hepcidin regulation in a mouse model of acute hypoxia.

OBJECTIVE: During hypoxia, hepcidin expression is inhibited to allow iron mobilization to sustain erythropoietic expansion. We analyzed molecular mechanisms underlying hypoxia-induced hepcidin inhibition in an in vivo model of acute hypoxia. METHODS: Mice were kept under normal or hypoxic conditions for 6 hours and 15 hours and treated with α-PDGF-BB antibody or PDGF-BB receptor inhibitor. Blood, liver, spleen, and bone marrow were collected to extract RNA and protein or to quantify EPO and PDGF-BB. mRNA and protein levels were assessed by RT-PCR and Western blot. RESULTS: Hepcidin was strongly inhibited at 15 hours, and this downregulation followed erythropoiesis activation and upregulation of several growth factors. PDGF-BB, erythroferrone, GDF15, and TWSG1 were upregulated by hypoxia in the bone marrow, but not in spleen or liver. Inactivation of PDGF-BB or its receptor suppressed the hypoxia-induced hepcidin inhibition. CONCLUSION: Spleen and liver are not involved in the early stages of hypoxia-induced hepcidin downregulation. Our data support the role of PDGF-BB and probably also of erythroferrone in the recruitment of iron for erythropoiesis in the hypoxia setting. The rapid normalization of all the erythroid factors against persistent hepcidin suppression suggests that other signals are involved that should be clarified in future studies.

GNPAT rs11558492 is not a Major Modifier of Iron Status: Study of Italian Hemochromatosis Patients and Blood Donors

ABSTRACT Background and Aim. HFE-related Hemochromatosis (HH) is characterized by marked phenotype heterogeneity, probably due to the combined action of acquired and genetic factors. Among them, GNPATrs11558492 was proposed as genetic modifier of iron status, but results are still controversial. To shed light on these discrepancies, we genotyped 298 Italian p.C282Y homozygotes and 169 healthy controls. Material and methods. Allele and genotype frequencies were analysed and compared with those reported in Exorne Variant Server (EVS). To explore the role of rs11558492 as a potential modifier of iron status, serum ferritin (SF), liver iron concentration (LIC) and iron removed (IR) were studied according to allele and genotype frequencies. In addition, the effect of the SNP on liver fibrosis was examined comparing patients with absent/mild-moderate fibrosis to those with severe fibrosis-cirrhosis. Results. GNPAT rs11558492 minor allele (G) frequency (MAF) was 20.3% in HFE- HH, 17.2% in controls and 20.6% in EVS database. Genotype frequencies were 64% and 69.2% (AA), 31.2% and 27.2% (AG), 4.8% and 3.6% (GG) in HFE-HH and controls, respectively. No significant differences were found comparing genotype and allele frequencies even selecting subgroups of only-males with extreme phenotypes and low alcohol intake. SF, IR and LIC levels did not significantly differ according to rs11558492 genotypes. Also, MAF did not differ between patients with absent/mild fibrosis and severe fibrosis/cirrhosis. Conclusions. Our findings indicate that GNPAT rs11558492 is not a major modifier of iron status and is not associated with liver fibrosis in HFE- HH patients.(AU)

GNPAT rs11558492 is not a Major Modifier of Iron Status: Study of Italian Hemochromatosis Patients and Blood Donors.

BACKGROUND AND AIM: HFE-related Hemochromatosis (HH) is characterized by marked phenotype heterogeneity, probably due to the combined action of acquired and genetic factors. Among them, GNPAT rs11558492 was proposed as genetic modifier of iron status, but results are still controversial. To shed light on these discrepancies, we genotyped 298 Italian p.C282Y homozygotes and 169 healthy controls. MATERIAL AND METHODS: Allele and genotype frequencies were analysed and compared with those reported in Exome Variant Server (EVS). To explore the role of rs11558492 as a potential modifier of iron status, serum ferritin (SF), liver iron concentration (LIC) and iron removed (IR) were studied according to allele and genotype frequencies. In addition, the effect of the SNP on liver fibrosis was examined comparing patients with absent/mild-moderate fibrosis to those with severe fibrosis-cirrhosis. RESULTS: GNPAT rs11558492 minor allele (G) frequency (MAF) was 20.3% in HFE-HH, 17.2% in controls and 20.6% in EVS database. Genotype frequencies were 64% and 69.2% (AA), 31.2% and 27.2% (AG), 4.8% and 3.6% (GG) in HFE-HH and controls, respectively. No significant differences were found comparing genotype and allele frequencies even selecting subgroups of only-males with extreme phenotypes and low alcohol intake. SF, IR and LIC levels did not significantly differ according to rs11558492 genotypes. Also, MAF did not differ between patients with absent/mild fibrosis and severe fibrosis/cirrhosis. CONCLUSIONS: Our findings indicate that GNPAT rs11558492 is not a major modifier of iron status and is not associated with liver fibrosis in HFE-HH patients.

Unexplained isolated hyperferritinemia without iron overload.

Although hyperferritinemia may be reflective of elevated total body iron stores, there are conditions in which ferritin levels are disproportionately elevated relative to iron status. Autosomal dominant forms of hyperferritinemia due to mutations in the L-ferritin IRE or in A helix of L-ferritin gene have been described, however cases of isolated hyperferritinemia still remain unsolved. We describe 12 Italian subjects with unexplained isolated hyperferritinemia (UIH). Four probands have affected siblings, but no affected parents or offspring. Sequencing analyses did not identify casual mutations in ferritin gene or IRE regions. These patients had normal levels of intracellular ferritin protein and mRNA in peripheral blood cells excluding pathological ferritin production at transcriptional and post-transcriptional level. In contrast with individuals with benign hyperferritinemia caused by mutations affecting the ferritin A helix, low rather than high glycosylation of serum ferritin was observed in our UIH subjects compared with controls. These findings suggest that subjects with UIH have a previously undescribed form of hyperferritinemia possibly attributable to increased cellular ferritin secretion and/or decreased serum ferritin clearance. The cause remains to be defined and we can only speculate the existence of mutations in gene/s not directly implicated in iron metabolism that could affect ferritin turnover including ferritin secretion.

Proprotein convertase 7 rs236918 associated with liver fibrosis in Italian patients with HFE-related hemochromatosis.

BACKGROUND AND AIM: p.Cys282Tyr homozygosity is the prevalent genotype in (HFE)-related Hereditary Hemochromatosis with low penetrance and variable expression. However, liver cirrhosis and hepatocellular carcinoma remain the main causes of mortality in these patients. Detection of genetic modifiers identifying patients at risk for liver damage would be relevant for their clinical management. We evaluated proprotein convertase 7 (PCSK7) rs236918 as genetic marker of risk of liver fibrosis in an Italian cohort of p.Cys282Tyr homozygotes. METHODS: Liver fibrosis was histologically assessed by Ishak score. We evaluated PCSK7 alleles and genotypes frequencies according to single or grouped staging scores: absent/mild fibrosis (stage: 0-2), moderate (stage: 3-4), and severe fibrosis/cirrhosis (stage: 5-6). Single nucleotide polymorphism genotyping was performed by restriction fragment length polymorphism or Taqman 5'-nuclease assays. RESULTS: The rs236918 allele C frequency increased from stages 0-2 to 5-6 (7.1% vs 13.6%, vs 21.9%, P = 0.003). The wild-type genotype was significantly more frequent in the absent/mild fibrosis group (54.2%) compared with only 17% in patients with severe fibrosis/cirrhosis. At univariate proportional odds model, patients with GC + CC genotypes were 2.77 times (P = 0.0018) more likely to have worse liver staging scores than wild-type patients. In the adjusted analysis, odds ratio was 2.37 (P = 0.0218), and 2.56 (P = 0.0233) when the analysis was restricted to males. An exploratory mediation analysis suggested a direct effect of genotype on severe fibrosis/cirrhosis (odds ratio = 3.11, P = 0.0157), and a mild non-significant indirect effect mediated through iron accounting for 28%. CONCLUSIONS: These findings confirm that PCSK7 rs236918 C allele is a risk factor for cirrhosis development in Italian patients with HFE-Hemochromatosis.

Simultaneous liver iron and fat measures by magnetic resonance imaging in patients with hyperferritinemia.

OBJECTIVE: Hyperferritinemia is frequent in chronic liver diseases of any cause, but the extent to which ferritin truly reflects iron stores is variable. In these patients, both liver iron and fat are found in variable amount and association. Liver biopsy is often required to quantify liver fat and iron, but sampling variability and invasiveness limit its use. We aimed to assess single breath-hold multiecho magnetic resonance imaging (MRI) for the simultaneous lipid and iron quantification in patients with hyperferritinemia. MATERIAL AND METHODS: We compared MRI results for both iron and fat with their respective gold standards - liver iron concentration and computer-assisted image analysis for steatosis on biopsy. We prospectively studied 67 patients with hyperferritinemia and other 10 consecutive patients were used for validation. We estimated two linear calibration equations for the prediction of iron and fat based on MRI. The agreement between MRI and biopsy was evaluated. RESULTS: MRI showed good performances in both the training and validation samples. MRI information was almost completely in line with that obtained from liver biopsy. CONCLUSION: Single breath-hold multiecho MRI is an accurate method to obtain a valuable measure of both liver iron and steatosis in patients with hyperferritinemia.

Genome-wide association study identifies TF as a significant modifier gene of iron metabolism in HFE hemochromatosis.

BACKGROUND & AIMS: Hereditary hemochromatosis (HH) is the most common form of genetic iron loading disease. It is mainly related to the homozygous C282Y/C282Y mutation in the HFE gene that is, however, a necessary but not a sufficient condition to develop clinical and even biochemical HH. This suggests that modifier genes are likely involved in the expressivity of the disease. Our aim was to identify such modifier genes. METHODS: We performed a genome-wide association study (GWAS) using DNA collected from 474 unrelated C282Y homozygotes. Associations were examined for both quantitative iron burden indices and clinical outcomes with 534,213 single nucleotide polymorphisms (SNP) genotypes, with replication analyses in an independent sample of 748 C282Y homozygotes from four different European centres. RESULTS: One SNP met genome-wide statistical significance for association with transferrin concentration (rs3811647, GWAS p value of 7×10(-9) and replication p value of 5×10(-13)). This SNP, located within intron 11 of the TF gene, had a pleiotropic effect on serum iron (GWAS p value of 4.9×10(-6) and replication p value of 3.2×10(-6)). Both serum transferrin and iron levels were associated with serum ferritin levels, amount of iron removed and global clinical stage (p<0.01). Serum iron levels were also associated with fibrosis stage (p<0.0001). CONCLUSIONS: This GWAS, the largest one performed so far in unselected HFE-associated HH (HFE-HH) patients, identified the rs3811647 polymorphism in the TF gene as the only SNP significantly associated with iron metabolism through serum transferrin and iron levels. Because these two outcomes were clearly associated with the biochemical and clinical expression of the disease, an indirect link between the rs3811647 polymorphism and the phenotypic presentation of HFE-HH is likely.

CYBRD1 as a modifier gene that modulates iron phenotype in HFE p.C282Y homozygous patients.

BACKGROUND: Most patients with hereditary hemochromatosis in the Caucasian population are homozygous for the p.C282Y mutation in the HFE gene. The penetrance and expression of hereditary hemochromatosis differ largely among cases of homozygous p.C282Y. Genetic factors might be involved in addition to environmental factors. DESIGN AND METHODS: In the present study, we analyzed 50 candidate genes involved in iron metabolism and evaluated the association between 214 single nucleotide polymorphisms in these genes and three phenotypic outcomes of iron overload (serum ferritin, iron removed and transferrin saturation) in a large group of 296 p.C282Y homozygous Italians. Polymorphisms were tested for genetic association with each single outcome using linear regression models adjusted for age, sex and alcohol consumption. RESULTS: We found a series of 17 genetic variants located in different genes with possible additive effects on the studied outcomes. In order to evaluate whether the selected polymorphisms could provide a predictive signature for adverse phenotype, we re-evaluated data by dividing patients in two extreme phenotype classes based on the three phenotypic outcomes. We found that only a small improvement in prediction could be achieved by adding genetic information to clinical data. Among the selected polymorphisms, a significant association was observed between rs3806562, located in the 5'UTR of CYBRD1, and transferrin saturation. This variant belongs to the same haplotype block that contains the CYBRD1 polymorphism rs884409, found to be associated with serum ferritin in another population of p.C282Y homozygotes, and able to modulate promoter activity. A luciferase assay indicated that rs3806562 does not have a significant functional role, suggesting that it is a genetic marker linked to the putative genetic modifier rs884409. CONCLUSIONS: While our results support the hypothesis that polymorphisms in genes regulating iron metabolism may modulate penetrance of HFE-hereditary hemochromatosis, with emphasis on CYBRD1, they strengthen the notion that none of these polymorphisms alone is a major modifier of the phenotype of hereditary hemochromatosis.

Patatin-like phospholipase domain containing-3 gene I148M polymorphism, steatosis, and liver damage in hereditary hemochromatosis.

AIM: To investigate whether the patatin-like phospholipase domain containing-3 gene (PNPLA3) I148M polymorphism is associated with steatosis, fibrosis stage, and cirrhosis in hereditary hemochromatosis (HH). METHODS: We studied 174 consecutive unrelated homozygous for the C282Y HFE mutation of HH (C282Y+/+ HH) patients from Northern Italy, for whom the presence of cirrhosis could be determined based on histological or clinical criteria, without excessive alcohol intake (< 30/20 g/d in males or females) or hepatitis B virus and hepatitis C virus viral hepatitis. Steatosis was evaluated in 123 patients by histology (n = 100) or ultrasound (n = 23). The PNPLA3 rs738409 single nucleotide polymorphism, encoding for the p.148M protein variant, was genotyped by a Taqman assay (assay on demand, Applied Biosystems). The association of the PNPLA3 I148M protein variant (p.I148M) with steatosis, fibrosis stage, and cirrhosis was evaluated by logistic regression analysis. RESULTS: PNPLA3 genotype was not associated with metabolic parameters, including body mass index (BMI), the presence of diabetes, and lipid levels, but the presence of the p.148M variant at risk was independently associated with steatosis [odds ratio (OR) 1.84 per p.148M allele, 95% confidence interval (CI): 1.05-3.31; P = 0.037], independently of BMI and alanine aminotransaminase (ALT) levels. The p.148M variant was also associated with higher aspartate aminotransferase (P = 0.0014) and ALT levels (P = 0.017) at diagnosis, independently of BMI and the severity of iron overload. In patients with liver biopsy, the 148M variant was independently associated with the severity (stage) of fibrosis (estimated coefficient 0.56 ± 0.27, P = 0.041). In the overall series of patients, the p.148M variant was associated with cirrhosis in lean (P = 0.049), but not in overweight patients (P = not significant). At logistic regression analysis, cirrhosis was associated with BMI ≥ 25 (OR 1.82, 95% CI: 1.02-3.55), ferritin > 1000 ng/mL at diagnosis (OR 19.3, 95% CI: 5.3-125), and with the G allele in patients with BMI < 25 (OR 3.26, 95% CI: 1.3-10.3). CONCLUSION: The PNPLA3 I148M polymorphism may represent a permissive factor for fibrosis progression in patients with C282Y+/+ HH.

Hepcidin expression in iron overload diseases is variably modulated by circulating factors.

Hepcidin is a regulatory hormone that plays a major role in controlling body iron homeostasis. Circulating factors (holotransferrin, cytokines, erythroid regulators) might variably contribute to hepcidin modulation in different pathological conditions. There are few studies analysing the relationship between hepcidin transcript and related protein expression profiles in humans. Our aims were: a. to measure hepcidin expression at either hepatic, serum and urinary level in three paradigmatic iron overload conditions (hemochromatosis, thalassemia and dysmetabolic iron overload syndrome) and in controls; b. to measure mRNA hepcidin expression in two different hepatic cell lines (HepG2 and Huh-7) exposed to patients and controls sera to assess whether circulating factors could influence hepcidin transcription in different pathological conditions. Our findings suggest that hepcidin assays reflect hepatic hepcidin production, but also indicate that correlation is not ideal, likely due to methodological limits and to several post-trascriptional events. In vitro study showed that THAL sera down-regulated, HFE-HH and C-NAFLD sera up-regulated hepcidin synthesis. HAMP mRNA expression in Huh-7 cells exposed to sera form C-Donors, HFE-HH and THAL reproduced, at lower level, the results observed in HepG2, suggesting the important but not critical role of HFE in hepcidin regulation.

Hepcidin response to acute iron intake and chronic iron loading in dysmetabolic iron overload syndrome.

BACKGROUND: The pathogenesis of dysmetabolic iron overload syndrome (DIOS) is still unclear. Hepcidin is the key regulator of iron homeostasis controlling iron absorption and macrophage release. AIM: To investigate hepcidin regulation by iron in DIOS. METHODS: We analysed urinary hepcidin at baseline and 24 h after a 65 mg oral iron dose in 24 patients at diagnosis and after iron depletion (n = 13) and compared data with those previously observed in 23 healthy controls. Serum iron indices, liver histology and metabolic data were available for all patients. RESULTS: At diagnosis, hepcidin values were significantly higher than in controls (P < 0.001). After iron depletion, hepcidin levels decreased to normal values in all patients. At baseline, a significant response of hepcidin to iron challenge was observed only in the subgroup with lower basal hepcidin concentration (P = 0.007). In iron-depleted patients, urinary hepcidin significantly increased after oral iron test (P = 0 .006). CONCLUSIONS: Ours findings suggest that in DIOS, the progression of iron accumulation is counteracted by the increase in hepcidin production and progressive reduction of iron absorption, explaining why these patients develop a mild-moderate iron overload that tends to a plateau.