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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.

Aceruloplasminemia is an ultra-rare and fatal autosomal recessive disease with a long lasting neurological disabling period of life caused by mutations in ceruloplasmin gene. Disease phenotype is heterogeneous and variably characterized by iron-restricted erythropoiesis and microcytic anemia, hyperferritinemia with tissue iron accumulation in liver, pancreas and brain, diabetes, retinopathy and neurodegeneration. Although most heterozygotes are asymptomatic, they might present with significant neurological symptoms at some point in their lives. We report here a patient with hyperferritinemia and severe depressive disorder, harbouring two mutations in ceruloplasmin in the compound heterozygous state (p.Pro477Leu and p.Gly895Ala). Both mutations are classified as deleterious in silico, but in vitro functional study partially confirmed it. Our findings suggest that the two mutations cooperate in inducing low ceruloplasmin production in the range observed in aceruloplasminemia heterozygotes and raise the question whether this might increase patient's susceptibility to neurologic manifestations.

Background. Several inherited diseases cause hyperferritinemia with or without iron overload. Differential diagnosis is complex and requires an extensive work-up. Currently, a clinical-guided approach to genetic tests is performed based on gene-by-gene sequencing. Although rea-sonable, this approach is expensive and time-consuming and Next Generation Sequencing (NGS) technology may provide cheaper and quicker large-scale DNA sequencing. Methods. We analysed 36 patients with non-HFE-related hyperferritinemia. Liver iron concentration was measured in 33 by magnetic resonance. A panel of 25 iron related genes was designed using SureDesign software. Custom libraries were generated and then sequenced using Ion Torrent PGM. Results. We identified six novel mutations in SLC40A1, three novel and one known mutation in TFR2, one known mutation and a de-novo deletion in HJV, and a novel mutation in HAMP in ten patients. In silico analyses supported the pathogenic role of the mutations. Conclusions. Our results support the use of an NGS-based panel in selected patients with hyperferritinemia in a tertiary center for iron metabolism disorders. However, 26 out of 36 patients did not show genetic variants that can individually explain hyperferritinemia and/or iron overload suggesting the existence of other genetic defects or gene-gene and gene-environment interactions needing further studies.

Introduction: Aceruloplasminemia is an ultra-rare hereditary disorder characterized by iron-restricted microcytic anemia and tissue iron overload associated with diabetes, retinal and progressive neurological degeneration. We describe genotypes and phenotypes at diagnosis, and disease evolution of seven Italian patients. Methods: Anagraphical, biochemical, genetic, clinical and instrumental data were collected at diagnosis and during a long-term follow-up. Mutations, ferroxidase activity and Western Blot analysis of ceruloplasmin were performed according to standard protocols. Results: Three mutations were already described (p.Phe217Ser, deletions of exon 11 and 12), p.Ile991Thr is a very rare variant, p.Cys338Ser and IVS6+1G > A were novel mutations. In silico analyses suggested they were highly likely or likely to be damaging. At diagnosis, 100% had microcytosis, 86% had mild-moderate anemia, low serum iron and high serum ferritin. Four (57%) had type 1 diabetes or glucose intolerance, 3/7 had neurological manifestations, and only one had early diabetic retinopathy. All but one underwent iron chelation therapy requiring temporary discontinuation because of anemia worsening. At the end of follow-up, three patients aggravated and 2 developed neurological symptoms; only two patients were free of neurological manifestations and showed mild or absent brain iron. Conclusion: Aceruloplasminemia phenotypes ranged from classical characterized by progressive neurologic derangement to milder in which signs of systemic iron overload prevailed over brain iron accumulation. Within this large heterogeneity, microcytosis with or without anemia, low serum iron and high serum ferritin were the early hallmarks of the disease. Therapeutic approaches other than iron chelation should be explored to reduce morbidity and improve life expectancy

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

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.

Hepcidin is the main regulator of systemic iron homeostasis, acting by inhibiting macrophages iron release and intestinal iron absorption. During hypoxia hepcidin expression is inhibited to allow iron requirement to bone marrow to sustain erythropoietic expansion, but mechanisms of hypoxia-induced hepcidin down-regulation are partially unknown. Our aim was to improve our knowledge on hypoxia modulation of hepatic hepcidin, investigating the role of erythroid and iron-related pathways using in vitro and in vivo studies. We firstly studied the effect of hypoxia on healthy volunteers living at high altitude for 24h and 72h, confirming that hypoxia is a strong inhibitor of hepcidin production and that this inhibition followed EPO induction. Indeed s-hepcidin was strictly down-regulated at 72h of hypoxia while EPO levels were already markedly increased at 24h slightly decreasing thereafter. We then analyzed the effect of sera of these subjects on hepcidin expression in HuH-7 cells using both luciferase assay and RT-PCR. HAMP wild-type promoter activity decreased after exposure to hypoxic sera, suggesting that circulating factors present in sera may modulate hepcidin expression. However, analysis of the transcripts gave less homogenous results. In fact only a subgroup of hypoxic sera significantly inhibited hepcidin. These sera also induced a significant ID1 down-regulation indicating that inhibitory factor(s) seems to act through the SMADs pathway. Our results also suggest that a cellular model cannot reproduce what really happens in vivo that might depend to complex events involving both circulating and tissue factors. Therefore, we took advantages from a mouse model. For hypoxia challenge, C57BL/6 mice were housed in a hypoxic chamber at 10% O2 for 6 and 15h. mRNA levels of erythroid genes were assessed in liver, spleen and bone marrow by RT-PCR. Hamp1 levels were slightly increased after 6h of hypoxia and strongly down-regulated after 15h. After 6h we already observed

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.

Background. The effects of multimodality treatment on the survival of patients with esophageal carcinoma are unclear. The authors performed a prospective, Phase II study to assess the long-term results of chemotherapy plus radiotherapy (RT) on patients with esophageal squamous cell carcinoma. Methods. Of 106 consecutive patients who were recruited between 1985 and 1992, 101 patients were evaluable. Cisplatin (100 mg/m2 per day) on Day 1 and fluorouracil (1000 mg/m2 per day) on Days 1-4 were given for two cycles, with concomitant RT (30 grays [Gy] in 15 fractions) over 19 days. Patients with potentially resectable tumors were then assessed for curative surgery; the other patients received two more courses of chemotherapy and further RT (20 Gy in 10 fractions). Results. Of 40 patients who were candidates for surgery, 32 patients underwent surgery, and 24 patients had complete resection; 8 patients (25%) had no residual tumor in the specimen, and 12 patients (37%) had microscopic foci only. Surgical mortality was high (22%). Of 61 nonsurgical patients, 37 patients (61%) achieved complete clinical remission, and 14 patients (23%) achieved partial remission. The median survival for the entire series was 15 months (range, 1-136 months). The overall survival rate was 22% at 5 years and 12% at 10 years. At 10 years, freedom from disease progression was similar in the two groups (24%), whereas the median survival (22 months vs. 12 months) and the overall survival rates (17% vs. 9%) were better in nonsurgical patients compared with surgical patients, respectively, probably in relation to high surgical mortality. The larynx was preserved in 28% of 32 patients with cervical disease sites, with a 10-year disease free survival rate of 31%. Three deaths were attributed to nonsurgical treatments. Conclusions. Careful multidisciplinary pretreatment evaluation can identify patients who are ineligible for surgery without compromising long-term results. For patients with inoperable dis

From February 1985 to June 1993, 173 consecutive, previously untreated patients with small cell lung cancer received individualized treatment tailored to disease extent. Almost all patients (14 of 16) with stage I and II disease and 30 patients with operable stage III disease were submitted to surgery preceded or followed by chemotherapy. Chest irradiation and prophylactic brain radiotherapy (in complete responders) were administered at the end of treatment in 42 of 44 cases. Patients with inoperable limited disease received chemotherapy followed by radiotherapy in 67 of 71 cases, while chemotherapy alone or followed by radiotherapy in sites of either initially bulky or residual disease was administered to 58 patients with extensive disease. The overall response rate was 77% (complete response, 45%; partial response, 32%). Complete responses were documented more frequently in limited disease than in extensive disease (57% v 22%; P < .001). The 2- and 5-year freedom from progression rates (24% and 16%, respectively), as well as overall survival rates (31% and 16%, respectively) were significantly affected by disease extent. No patient with extensive disease was progression free and alive at 2 years, while more than half of stage I and II patients were disease free and alive at 5 years. This retrospective analysis performed on a large number of consecutive, nonrandomized patients suggests that, at least in patients with limited disease, it is possible to achieve favorable long-term results using treatment tailored to disease extent. Nonetheless, the disappointing results commonly achieved in the treatment of small cell lung cancer strongly support the need for either prospective, randomized studies to confirm recently reported improved results or new pilot studies with investigation of entirely innovative approaches

Platinum microquantities were determined in plasma of patients affected by lung carcinoma during treatment with radiotherapy (RT) and concurrent low-dose continuous infusion of cis-dichlorodiammineplatinum(II) (CDDP). RT was given at 50 Gy in continuous course; CDDP was continuously infused at 4 mg/m(2) daily for 100 h/week for 5 weeks, and the infusions were separated by 68 h of rest. The percentage of free drug versus total drug in plasma was about 3%. It did not vary with therapy duration and was not significantly different from that found in 5-day continuous infusions at much higher daily doses. Nevertheless, maximal values of free Pt in plasma were very low and agreed with the low level of CDDP toxicity encountered on the present administration schedule.

Hepcidin transcription is strongly down-regulated under hypoxic conditions, however whether hypoxia inhibits hepcidin directly or indirectly is still unknown. We investigated the time course of hypoxia-mediated hepcidin down-regulation in vivo in healthy volunteers exposed to hypobaric hypoxia at high altitude and, based on the hypothesis that circulating factors are implicated in hepcidin inhibition, we analyzed the effect of sera of these volunteers exposed to normoxia and hypoxia on hepcidin expression in Huh-7 cell lines. Hypoxia led to a significant hepcidin down-regulation in vivo that was almost complete within 72h of exposure and followed erythropoietin induction. This delay in hepcidin down-regulation suggests the existence of soluble factor/s regulating hepcidin production. We then stimulated HuH-7 cells with normoxic and hypoxic sera to analyze the effects of sera on hepcidin regulation. Hypoxic sera had a significant inhibitory effect on hepcidin promoter activity assessed by a luciferase assay, although the amount of such decrease was not as relevant as that observed in vivo. Cellular mRNA analysis showed that a number of volunteers' sera inhibited hepcidin expression, concurrently with ID1 inhibition, suggesting that inhibitory factor(s) may act through the SMAD-pathway.

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.