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.

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.

Tyrosinase-catalyzed grafting of sericin peptides onto chitosan and production of protein-polysaccharide bioconjugates.

The capability of Agaricus bisporus tyrosinase to catalyze the oxidation of tyrosine residues of silk sericin was studied under homogeneous reaction conditions, by using sericin peptides purified from industrial wastewater as the substrate. Tyrosinase was able to oxidize about 57% of sericin-bound tyrosine residues. The reaction rate was higher than with silk fibroin, but lower than with other silk-derived model peptides, i.e. tryptic and chymotryptic soluble peptide fractions of silk fibroin, suggesting that the size and the molecular conformation of the substrate influenced the kinetics of the reaction. The concentration of tyrosine in oxidized sericin samples decreased gradually with increasing the enzyme-to-substrate ratio. The average molecular weight of sericin peptides significantly increased by oxidation, indicating that cross-linking occurred via self-condensation of o-quinones and/or coupling with the free amine groups of lysine and, probably, with sulfhydryl groups of cysteine. The high temperature shift of the main thermal transitions observed in the differential scanning calorimetry curves confirmed the formation of peptide species with higher molecular weight and higher thermal stability. Fourier transform-infrared spectra of oxidized sericin samples showed slight changes related to the loss of tyrosine and formation of oxidation products. Oxidized sericin peptides were able to undergo non-enzymatic coupling with chitosan. Infrared spectra provided clear evidence of the formation of sericin-chitosan bioconjugates under homogeneous reaction conditions. Spectral changes in the NH stretching region seem to support the formation of bioconjugates via the Michael addition mechanism.

Wet spinning of Bombyx mori silk fibroin dissolved in N-methyl morpholine N-oxide and properties of regenerated fibres.

Silk fibroin (SF) was dissolved in N-methyl morpholine N-oxide (NMMO) at a polymer concentration of 13% (w/w); thermal and rheological solution properties were characterized. The melting/crystallization behaviour of NMMO was influenced by SF presence. Melting of NMMO hydrate decreased to 71 degrees C and a cold crystallization peak appeared at 35 degrees C on heating. None crystallization occurred on cooling. Quenching at a temperature of 50 degrees C or higher did not induce any crystallization on heating. Viscosity of SF-NMMO solutions decreased as a function of temperature. At 75 degrees C, viscosity remained constant for 360 min. SF-NMMO dope was spun by using a lab-scale wet spinning line. The extruded filament was coagulated in an ethanol bath. Regenerated SF fibres were collected at different draw ratios and their morphological, physical, and mechanical properties were characterized. Fibre diameters ranged from 133 to 19mum, cross-section was regularly circular, and surface was generally smooth, with a very fine granular aspect. Birefringence increased with increasing the draw ratio, especially when take up and post-spinning draw were coupled. FT-IR spectra and DSC thermograms confirmed that SF fibres crystallized into Silk II structure. The IR crystallinity index did not change as a function of drawing. Regenerated SF fibres undrawn or drawn only during the coagulation step showed the mechanical behaviour typical of a brittle material. However, when both take up and post-spinning draw were applied, fibres displayed a ductile-stable behaviour. Typical values of the mechanical parameters of regenerated SF fibres were: E=8.7 GPa, sigma(b)=120 MPa and epsilon(b)=35%.

Iron accumulation in chronic hepatitis C: relation of hepatic iron distribution, HFE genotype, and disease course.

The aim of the present study was to describe the histopathologic features of hepatic iron accumulation in patients with chronic hepatitis C (CH-C) infection, the relation between HFE mutations and hepatic iron location and among iron distribution, HFE, and hepatic damage. We studied 206 patients with CH-C infection. Of 101 patients with hemosiderin deposits, 90.1% had iron deposits in hepatocytes (alone or with sinusoidal and/or portal involvement). The hepatic iron score increased significantly as iron accumulation involved sinusoidal and portal tract compartments and according to HFE genotypes. Severe fibrosis and cirrhosis were associated more markedly with the presence of hemosiderin iron in the 3 hepatic compartments, HFE mutations, and high alcohol intake. We suggest that part of the iron accumulation in CH-C infection derives from increased iron absorption and release from storage cells and that the amount and distribution of hepatic iron deposits is related to hepatic damage. HFE mutations favor both processes, but other factors, genetic or acquired, are involved.

Type 3 hemochromatosis and beta-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 beta-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 Rappaport's 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 beta-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.

Prevalence of C282Y and E168X HFE mutations in an Italian population of Northern European ancestry.

BACKGROUND AND OBJECTIVES: In Italy, the prevalence of C282Y is lower than in Northern European countries. We hypothesized a higher prevalence of C282Y in Northern than in Southern Italian populations. We previously identified a nonsense mutation (E168X) in hemochromatosis probands originating from a region in the north-west of Italy. We aimed to define the prevalence of C282Y and E168X in that region and the origin of the E168X mutation by haplotype analysis. DESIGN AND METHODS: Six-hundred and six blood donors were investigated for C282Y, H63D, S65C and E168X mutations by polymerase chain reaction (PCR)-restriction assays. Three hundred were also tested for rare HFE and TFR2 mutations by reverse-hybridization test strips. D6S265, D6S105 and D6S1281 microsatellites were analyzed to define E168X 6p-associated haplotypes. RESULTS: One C282Y homozygote, thirteen C282Y/ H63D compound heterozygotes, four E168X heterozygotes and three E168X/H63D compound heterozygotes were found. The allele frequencies of C282Y, H63D, S65C, and E168X were 4.7%, 14.9%, 0.74% and 0.58%, respectively. INTERPRETATION AND CONCLUSIONS: The prevalence of C282Y in the region investigated was much higher than that previously reported in Italy. This finding is probably due to the heavy Celtic component of this north-western population and suggests that in populations of Northern Italian descent screening studies for hemochromatosis could be cost-effective. The prevalence of E168X in this region, although low, suggests that the mutation probably originated here many years ago and its frequency increased as a result of a local founder effect. Given its severity, we suggest that the E168X mutation should be searched for in all hemochromatosis patients of Northern ancestry with an incomplete HFE genotype.