Sara Pelucchi

Modulation of hepcidin production during hypoxia-induced erythropoiesis in humans in vivo: data from the HIGHCARE project

Iron is tightly connected to oxygen homeostasis and erythropoiesis. Our aim was to better understand how hypoxia regulates iron acquisition for erythropoiesis in humans, a topic relevant to common hypoxia-related disorders. Forty-seven healthy volunteers participated in the HIGHCARE project. Blood samples were collected at sea level and after acute and chronic exposure to high altitude (3400-5400 m above sea level). We investigated the modifications in hematocrit, serum iron indices, erythropoietin, markers of erythropoietic activity, interleukin-6, and serum hepcidin. Hepcidin decreased within 40 hours after acute hypoxia exposure (P < .05) at 3400 m, reaching the lowest level at 5400 m (80% reduction). Erythropoietin significantly increased (P < .001) within 16 hours after hypoxia exposure followed by a marked erythropoietic response supported by the increased iron supply. Growth differentiation factor-15 progressively increased during the study period. Serum ferritin showed a very rapid decrease, suggesting the existence of hypoxia-dependent mechanism(s) regulating storage iron mobilization. The strong correlation between serum ferritin and hepcidin at each point during the study indicates that iron itself or the kinetics of iron use in response to hypoxia may signal hepcidin down-regulation. The combined and significant changes in other variables probably contribute to the suppression of hepcidin in this setting.

Hepcidin and iron-related gene expression in subjects with dysmetabolic hepatic iron overload

BACKGROUND/AIMS Many patients with hepatic iron overload do not have identifiable mutations and often present with metabolic disorders and hepatic steatosis. Since the pathophysiology of Dysmetabolic Hepatic Iron Overload (DHIO) is still obscure, the aim of this study was to evaluate, in these patients, possible alterations in iron-related molecule expression. METHODS Iron-related gene mRNA levels were determined by quantitative-PCR in liver biopsies of subjects with NAFLD without iron overload and patients with HFE-hemochromatosis, beta-thalassemia major and DHIO. Urinary hepcidin was measured by immunoblotting. RESULTS No alterations in mRNA expression of either iron transporters or exporters were found in DHIO. mRNA and urinary hepcidin levels normalized for the amount of iron overload showed a significantly lower ratio than in controls, although not as low as in hemochromatosis or beta-thalassemia. Differently from what observed in hemochromatosis, hepcidin mRNA did not correlate with urinary hepcidin. CONCLUSIONS Patients with DHIO show appropriate regulation of mRNAs encoding proteins involved in iron uptake and efflux but dysregulation of hepcidin production. The relatively elevated urinary hepcidin can explain the iron phenotype in DHIO (more macrophage iron retention and low/normal transferrin saturation).

A time course of hepcidin response to iron challenge in patients with HFE and TFR2 hemochromatosis

Background Inadequate hepcidin production leads to iron overload in nearly all types of hemochromatosis. We explored the acute response of hepcidin to iron challenge in 25 patients with HFE-hemochromatosis, in two with TFR2-hemochromatosis and in 13 controls. Sixteen patients (10 C282Y/C282Y homozygotes, 6 C282Y/H63D compound heterozygotes) had increased iron stores, while nine (6 C282Y/C282Y homozygotes, 3 C282Y/H63D compound heterozygotes) were studied after phlebotomy-induced normalization of iron stores. Design and Methods We analyzed serum iron, transferrin saturation, and serum hepcidin by both enzyme-linked immunosorbent assay and mass-spectrometry at baseline, and 4, 8, 12 and 24 hours after a single 65-mg dose of oral iron. Results Serum iron and transferrin saturation significantly increased at 4 hours and returned to baseline values at 8–12 hours in all groups, except in the iron-normalized patients who showed the highest and longest increase of both parameters. The level of hepcidin increased significantly at 4 hours and returned to baseline at 24 hours in controls and in the C282Y/H63D compound heterozygotes at diagnosis. The hepcidin response was smaller in C282Y-homozygotes than in controls, barely detectable in the patients with iron-depleted HFE-hemochromatosis and absent in those with TFR2-hemochromatosis. Conclusions Our results are consistent with a scenario in which TFR2 plays a prominent and HFE a contributory role in the hepcidin response to a dose of oral iron. In iron-normalized patients with HFE hemochromatosis, both the low baseline hepcidin level and the weak response to iron contribute to hyperabsorption of iron.

Measurement of serum hepcidin-25 levels as a potential test for diagnosing hemochromatosis and related disorders

BackgroundIron overload syndromes include a wide spectrum of genetic and acquired conditions. Recent studies suggest suppressed hepcidin synthesis in the liver to be the molecular basis of hemochromatosis. However, a liver with acquired iron overload synthesizes an adequate amount of hepcidin. Thus, hepcidin could function as a biochemical marker for differential diagnosis of iron overload syndromes.MethodsWe measured serum iron parameters and hepcidin-25 levels followed by sequencing HFE, HJV, HAMP, TFR2, and SLC40A1 genes in 13 Japanese patients with iron overload syndromes. In addition, we performed direct measurement of serum hepcidin-25 levels using liquid chromatography–tandem mass spectrometry in 3 Japanese patients with aceruloplasminemia and 4 Italians with HFE hemochromatosis.ResultsOne patient with HJV hemochromatosis, 2 with TFR2 hemochromatosis, and 3 with ferroportin disease were found among the 13 Japanese patients. The remaining 7 Japanese patients showed no evidence for genetic basis of iron overload syndrome. As far as the serum hepcidin-25 was concerned, seven patients with hemochromatosis and 3 with aceruloplasminemia showed markedly decreased serum hepcidin-25 levels. In contrast, 3 patients with ferroportin disease and 7 with secondary iron overload syndromes showed serum hepcidin levels parallel to their hyperferritinemia. Patients with iron overload syndromes were divided into 2 phenotypes presenting as low and high hepcidinemia. These were then associated with their genotypes.ConclusionDetermining serum hepcidin-25 levels may aid differential diagnosis of iron overload syndromes prior to genetic analysis.

Expression of hepcidin and other iron-related genes in type 3 hemochromatosis due to a novel mutation in transferrin receptor-2

This brief report describes the decreased hepatic and urinary expression of hepcidin in type 3 hemochromatosis. Transferrin receptor-2 (TFR2) regulates hepatic hepcidin secretion and when mutated causes type-3 hemochromatosis. No functional study is available in humans. We studied a 47 year-old woman with hemochromatosis. TFR2 DNA and its hepatic transcript were directly sequenced. Hepatic expression of hepcidin and other iron-related genes were measured by qRT-PCR. Urinary hepcidin was measured at baseline and after an oral iron challenge (ferrous sulfate, 65 mg) by SELDI-TOF-MS. A novel homozygous TFR2 mutation was identified in the splicing donor site of intron 4 (c.614+4 A>G) causing exon 4 skipping. Hepcidin and hemojuvelin expression were markedly reduced. Urinary hepcidin was lower than normal and further decreased after iron challenge. This is the first description of iron-related gene expression profiles in a TFR2 mutated patient. The decreased hepatic and urinary expression of hepcidin and lack of acute response to iron challenge confirms the primary role of TFR2 in iron homeostasis.

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.

Self-Forgiveness in Romantic Relationships: It Matters to Both of Us

This study investigates self-forgiveness for real hurts committed against the partner in a romantic relationship (N = 168 couples). Using a dyadic perspective, we evaluated whether offender self-forgiveness, conceived as a bidimensional construct distinct from self-excusing, was uniquely related to both own and partner relationship satisfaction. For both males and females, offending partners were more satisfied with their romantic relationship to the extent that they had more positive and less negative sentiment and thoughts toward themselves, whereas victimized partners were more satisfied with the relationship when the offending partner had less negative sentiment and thoughts (but not more positive ones) toward himself/herself. The implications of these findings for understanding self-forgiveness and its pro-relationship effects in romantic couples are discussed.

Novel mutations of the ferroportin gene (SLC40A1) : analysis of 56 consecutive patients with unexplained iron overload

The aim of this study was to search for SLC40A1 mutations in iron overloaded patients, which tested negative for HFE mutations and other iron‐related genes. After a careful differential diagnosis, we selected 56 patients with unexplained iron overload whose phenotype could suggest the ferroportin disease. Iron overload was assessed by liver biopsy or by superconducting quantum interference device. SLC40A1 exons and intron–exon boundaries were amplified by polymerase chain reaction and sequenced. We also evaluated the presence of the insulin‐resistance hepatic iron overload and of non‐alcoholic fatty liver disease. Iron status was assessed in 44 families. We identified two novel mutations (D157N and V72F) at the heterozygous state in two probands. Phenotype heterogeneity was observed in both families, suggesting variable penetrance and expression. Including the two affected ones, 25 of the 44 families (57%) available for the iron study had one or more relatives with increased serum iron indices. Our findings not only suggest that the presence of major alterations of serum iron parameters in probands’ relatives is a main criteria to improve the power of the genetic testing for ferroportin disease but also indicate that a number of patients exists in which the etiology of iron overload remains still undefined.

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.

Type 3 hemochromatosis and β‐thalassemia trait

Type 3 hemochromatosis is a rare autosomal recessive disorder due to mutations of the TFR2 gene. We describe clinical, biochemical and histopathologic findings of a patient with type 3 hemochromatosis at presentation and during a follow‐up of more than 20 yr and we evaluate the effect of an associated β‐thalassemia trait on phenotypic expression. At the age of 33 yr the patient showed a marked iron overload and severe iron‐related complications. After removal of 26 g of iron by subcutaneous deferoxamine infusion a marked clinical improvement was observed. Liver biopsies, performed at the age of 34 and 49 yr, indicate that in type 3 hemochromatosis there is a progressive hepatocellular iron accumulation from Rappaports zone 1–3 and that iron loading in sinusoidal and portal macrophages occurs only in the more advanced stage. As observed in HFE hemochromatosis, the β‐thalassemia trait seems to aggravate the clinical picture of patients lacking TFR2, favoring higher rates of iron accumulation probably by activation of the erythroid iron regulator.