Mutation analysis of hepcidin and ferroportin genes in Italian prospective blood donors with iron overload.

Maintenance of iron balance is essential for humans and requires the coordinate regulation of iron transport into plasma from dietary sources in the duodenum, from recycled senescent red cells in macrophages, and from storage in hepatocytes. Hepcidin, a recently identified antimicrobial peptide produced in the liver, has been shown to play a central role in the homeostatic regulation of iron absorption and distribution [1]. It is a negative regulator of iron absorption in the small intestine and of iron release from macrophages engaged in the recycling of iron senescent erythrocytes [2]. The human hepcidin gene contains three exons that encode a 72-aa precursor (pro-hepcidin) with a characteristic furin cleavage site immediately N-terminal to the 25-aa major hepcidin species found in plasma and urine [3]. Recently, hepcidin has been shown to regulate iron homeostasis by interaction with ferroportin, an iron cellular exporter highly expressed in absorptive enterocytes, macrophages, hepatocytes, and placental cells [4].

Changes in erythropoiesis, iron metabolism and oxidative stress after half-marathon.

OBJECTIVE: In marathon runners changes in red blood cell count, haematocrit and haemoglobin in relation to haemodilution have been reported. Moreover, it has been hypothesized that strenuous exercise induces oxidant stress through several different mechanisms. This study investigated the haematological variables, iron status and oxidative indices before, immediately and 48 h after a race in 8 healthy trained males aged 33-44 years running a 21-km marathon in 79 +/- 3 min. METHODS: The haematological parameters were determined by standard procedures. Erythropoietin and soluble-transferrin receptor were evaluated immunoenzymatically. Nontransferrin-bound iron (NTBI) was assayed by high-performance liquid chromatography after nitrilotriacetic acid chelation. Malonyldialdehyde (MDA) concentration was assayed colorimetrically. RESULTS: The total number of reticulocytes rose significantly after the run with a significant increase in the high-RNA-content fraction (14 +/- 5, p < 0.0006). Erythropoietin rose by 26% (15.0 +/- 2.8 mU/ml, p < 0.004) and by 25% (14.9 +/- 2.13 mU/ml, p < 0.02) immediately and 48 h after the race, respectively. Serum iron and serum ferritin remained unchanged but NTBI and serum MDA increased significantly immediately after running (1.16 +/- 0.40 mmol/l, p < 0.0008; 0.76 +/- 0.16 mmol/l, p < 0.0001). Significant positive correlations at any time between MDA and polymorphonuclear neutrophils (p = 0.0005), MDA and NTBI (p = 0.0018), polymorphonuclear neutrophils and NTBI (p = 0.0008) and between lactate dehydrogenase and NTBI (p = 0.0212) were observed. CONCLUSIONS: The erythropoietic changes observed in marathon runners are the results of several interacting mechanisms that involve either the haemopoietic system per se or erythrocyte haemolysis and oxidative stress.

Exercise capacity in patients with beta-thalassaemia intermedia.

Thalassaemia intermedia patients can suffer fatigue and exercise capacity reduction, possibly because of anaemia, deconditioning and lack of exercise-induced haemoconcentration. We studied 21 beta-thalassaemia intermedia patients, 10 splenectomised (group A) and 11 not splenectomised (group B). Patients were evaluated by cardiopulmonary exercise test with blood sampling for haemoglobin and plasma protein measurements at rest and peak. During exercise, an isolated increase of haemoglobin suggested spleen contraction while a parallel increase of haemoglobin and proteins suggested fluid filtration through capillary wall. Groups were homogeneous for age and gender. Peak oxygen consumption (VO2) was 22.5 +/- 4.4 ml/min/kg (51 +/- 14%) and 24.3 +/- 7.0 (53 +/- 12%) in groups A and B respectively [not significant (NS)]. At rest, haemoglobin was 8.8 g/dl in both groups. Exercise-induced increment was 0.4 +/- 0.2 and 1.0 +/- 0.4 g/dl (P < 0.001) for haemoglobin and 4.0 +/- 3.0 and 5.0 +/- 4.0 g/l (NS) for proteins, in groups A and B respectively. Anaemia was the major cause of peak VO2 reduction (1097 +/- 260 ml/min). However, anaemia did not explain the entire exercise capacity reduction, suggesting the presence of muscular deconditioning. Exercise capacity is reduced in beta-thalassaemia intermedia because of anaemia and muscular deconditioning. Spleen contraction does not significantly influence exercise capacity although exercise-induced haemoconcentration was greater in patients with spleen.

Spectrum and haplotypes of the HFE hemochromatosis gene in Iran: H63D in beta-thalassemia major and the first E277K homozygous.

We present the molecular analysis of HFE gene in 400 Southwest Iranian individuals. We have studied 43 newborn, selected for the presence of HbBart's at birth, 203 normal adult and 154 transfused patients affected with beta-thalassemia. Mutation analysis consisted of amplification and direct sequencing using two different pairs of forward and reverse primers. The C282Y and S65C mutations were not found. The H63D mutation was present with an allele frequency of 0.10 in newborns, 0.082 in normal adults and 0.080 in the beta-thalassemia major populations, respectively. No differences were found between normal adults and thalassemia major patients suggesting that this mutation does not increase mortality in beta-thalassemia. The H63D mutation was found associated with haplotype VI in 41% of the chromosomes. Other haplotypes were found suggesting a multicentric origin rather than a single mutation of European origin. While sequencing exon 4, a G --> A transition was found in the proximity of the C282Y mutation. The effect of this single base substitution (E277K) previously reported in an Asian individual and now found in homozygous form in a young transfused and chelated homozygous beta-thalassemia patient is not yet known.

Clinical and histological characterization of liver disease in patients with transfusion-dependent beta-thalassemia. A multicenter study of 117 cases.

BACKGROUND AND OBJECTIVES: Updated information on liver disease in transfusion-dependent beta-thalassemia is lacking. We conducted a multicenter study within the Cooleycare Group to describe the clinical and histopathological features of liver disease in currently treated thalassemics. DESIGN AND METHODS: Two-hundred and three thalassemics with laboratory signs of liver disease were eligible. Liver biopsy was performed in the 129 (63.5%) who consented (age 26+/-7 years). Biological samples were sent to the central laboratory. RESULTS: Anti-hepatitis C virus (HCV) antibodies were found in 118 patients (91%), 85 (72%) of whom were viremic. Ninety-one patients (70%) had abnormal aminotransferase concentrations. In the 117 liver biopsies that met the criteria for evaluation (88%), the median Ishak's necroinflammatory and fibrosis scores were 4 (range, 0-9) and 2 (range, 0-6), respectively. Significant fibrosis (score >or=3) was found in 53 (45%); 9 (8%) had cirrhosis. At multivariate analysis, necroinflammation was related to HCV viremia, and fibrosis to increased serum aminotransferases, higher iron stores (including serum ferritin, Deugnier's total iron score, and liver iron content) and male gender (p<0.05). In HCV-RNA negative subjects, the median total iron score was 27 (range, 0-52). Iron accumulated in both mesenchymal cells and hepatocytes, and the presence of a lobular gradient was interpreted to indicate intestinal hyperabsorption. INTERPRETATION AND CONCLUSIONS: Transfusion-dependent thalassemics have mild liver necroinflammation, mainly attributable to HCV infection. Significant fibrosis is frequent, and its progression is mostly influenced by iron overload which, with current therapy regimens, may be attributable to both erythrocyte catabolism and iron hyperabsorption.

Cytotoxic T-lymphocyte antigen-4 A49G polymorphism is associated with susceptibility to and severity of alcoholic liver disease in Italian patients.

AIMS: To determine whether the functional A49G polymorphism of cytotoxic T-lymphocyte antigen-4 (CTLA-4), a T-cell surface molecule that modulates T-lymphocyte activation and influences the risk of developing alcohol-induced autoantibodies, plays a role in susceptibility to alcoholic liver disease (ALD) and influences disease severity in Italian alcohol abusers. METHODS: One hundred and eighty-three patients with chronic ALD (61 cirrhosis), 115 end-stage HCV cirrhosis, 102 non-alcoholic fatty liver disease (NAFLD), 93 healthy subjects and 43 heavy drinkers without liver disease were studied. CTLA-4 gene polymorphism was analysed by restriction analysis. RESULTS: The frequency of the CTLA-4 polymorphism was higher in patients with ALD than in patients with HCV chronic hepatitis and NAFLD, healthy subjects (P < 0.0001), and heavy drinkers without liver disease (P = 0.02). In patients with ALD, homozygosity for the CTLA-4 polymorphic allele (G/G genotype) was more represented in subjects with cirrhosis (P = 0.047), and independently associated with the risk of cirrhosis (OR 3.5; P = 0.03). CONCLUSIONS: The CTLA-4 polymorphic G allele, probably by interfering with the immune response, may confer susceptibility to ALD and, in homozygous state, to alcoholic cirrhosis.

Mutations of the hemochromatosis gene in Italian candidate blood donors with increased transferrin saturation.

The aim of this study was to analyze the role of HFE mutations in blood donors with iron parameters suggesting iron overload, taking into account the regional distribution of HFE mutations in Italy. We studied 5880 subjects undergoing evaluation for blood donation eligibility, from different areas of Italy. Abnormal iron parameters were defined as transferrin saturation (TS) >50% or >45% and serum ferritin (SF) >300 or >250 microg/ml in males and females, respectively. Subjects with increased TS and/or SF were re-tested and typed for HFE mutations C282Y and H63D. A total of 548 individuals had increased iron parameters at first testing. In total, 179/548 were available for retesting, and in 109 increased TS and/or SF were confirmed. Increased TS was confirmed in 25 individuals, among whom three were C282Y homozygotes and six were compound heterozygotes for C282Y and H63D. Increased TS was more frequent in northern Italy than in southern regions. In individuals with increased TS and/or SF, the frequency of C282Y and H63D was 0.13 and 0.21 in northern-Italy versus 0.05 and 0.45 in southern Italy (P=0.004 for H63D). Nine out of 10 individuals carrying hemochromatosis-associated genotypes (including compound heterozygosity for C282Y and H63D) originated from northern regions. Among controls, the allelic frequencies of C282Y and H63D were 0.037 and 0.16 in the northern regions and 0.015 and 0.16 in the southern regions. In conclusion, over one-third of individuals with persistently altered TS carried hemochromatosis-associated genotypes, confirming that a diagnostic approach based on TS and genotyping of selected cases may represent a viable screening procedure.

Analysis of HFE and TFR2 mutations in selected blood donors with biochemical parameters of iron overload.

BACKGROUND AND OBJECTIVES: Hereditary hemochromatosis is a recessive condition characterized by iron accumulation in several organs, followed by organ damage and failure. The disorder is prevalently due to C282Y and H63D mutations in the HFE gene, but additional HFE and TFR2 mutations have been reported. Early iron overload may be assessed by biochemical parameters such as increased transferrin saturation and serum ferritin. DESIGN AND METHODS: Taking advantage of the collection of 178 DNA samples selected for increased transferrin saturation (>50% in males and >45% in females) from a previous large scale screening of Italian blood donors, we simultaneously assessed the presence of 14 hemochromatosis-associated molecular defects (11 of HFE and 3 of TFR2) by a reverse hybridization-based strip assay. RESULTS: In the series studied the overall C282Y allele frequency was 9% and that of the H63D and S65C was 22.2% and 1.4%, respectively. One rare HFE allele (E168Q), but no TFR2 mutation was detected. When checked at a second examination, transferrin saturation was significantly higher in C282Y homozygotes, H63D/ C282Y compound heterozygotes and H63D homozygotes as compared to wild-type subjects (p<0.05). INTERPRETATION AND CONCLUSIONS: Our results confirm previous findings on C282Y and H63D mutations in Italy, show that the C282Y allele frequency is enriched in samples selected for altered iron parameters, and that a few rare genotypes are present in Northern Italy. None of the known TFR2 mutations was identified in this series confirming the preliminary indication of their rare occurrence. Subjects with hemochromatosis-associated genotypes show a persistently higher mean transferrin saturation than do those with wild type genotypes.

Sustained response to combination therapy in patients with chronic hepatitis C who failed to respond to interferon.

BACKGROUND/AIMS: The best treatment for chronic hepatitis C patients who do not respond to interferon is still unknown. Reported rates of response to treatment vary as the result of heterogeneous definitions of non-responders and small study size. METHODS: One hundred nineteen hepatitis C virus (HCV) RNA-positive non-responders to high-dose interferon monotherapy received alpha-interferon, 5 MU tiw plus oral ribavirin, 1000-1200 mg/day for 48 weeks (Group A, n=74) or alpha-interferon, 5 MU daily for 4 weeks, followed by 5 MU tiw plus oral ribavirin, 1000-1200 mg/day for 44 weeks (Group B, n=45) according to the Institution where they were followed. Persistently normal alanine aminotransferase and negative HCV RNA up to 72 weeks from treatment onset defined a sustained response. RESULTS: Eighteen patients discontinued treatment (13 developed anemia, two mucositis, one granulocytopenia; two were dropouts), none for serious adverse events. There were 24 (20%) sustained responders, with similar final response rates in Groups A and B. Sustained response was more frequent in patients aged

Peripheral lymphocytes and intracellular cytokines in C282Y homozygous hemochromatosis patients.

BACKGROUND/AIMS: Several abnormalities in the immune status of hereditary hemochromatosis patients have been reported. We evaluated the peripheral blood lymphocytes phenotype and cytokine profile of CD8(+) and CD4(+) T cells in C282Y homozygous hereditary hemochromatosis patients compared to control subjects. METHODS: Peripheral blood lymphocytes from 17 asymptomatic patients and 14 control subjects were analyzed. We determined the distribution of lymphocyte subsets and investigated at single-cell level by flow-cytometry the potential of cytokines production. The frequency of cytokine (interferon gamma, tumor necrosis factor alpha, interleukin 2 (IL-2), IL-4, IL-5, IL-10 and IL-13) producing cells was assessed in total T-lymphocytes, CD3(+)CD8(+) and CD3(+)CD4(+) subsets. RESULTS: The patients studied showed a significant decrease of total lymphocyte count, T CD4(+)CD3(+), CD28(+), CD8(+)CD28(+) lymphocytes and natural killer (NK) CD56(+)CD16(+)CD3(-) cells. The reduction of CD28(+) and CD8(+)CD28(+) lymphocyte count was inversely related to transferrin saturation index. An increase in the ability of T-cells to produce all the cytokines studied and a major increase in IL-4 and IL-10 production in the CD3(+)CD8(+) subset was found. Our results demonstrate that activated Th1 and Th2 lymphocytes coexist in the peripheral blood of hereditary hemochromatosis patients and that T-cytotoxic (Tc) 2 subset is more expanded than in control population. CONCLUSIONS: The association of a decreased number of T CD8(+) cytotoxic lymphocytes and NK cells, and the development of Tc2 cells in asymptomatic C282Y homozygous patients represents an imbalance in their immune function that might contribute to the high incidence of hepatocarcinoma.

Iron reduction and sustained response to interferon-alpha therapy in patients with chronic hepatitis C: results of an Italian multicenter randomized study.

OBJECTIVES: It has been suggested that iron depletion improves the response to interferon in patients with chronic hepatitis C. We aimed to evaluate whether iron reduction by phlebotomy before interferon improves the rate of virological sustained response in previously untreated noncirrhotic patients. METHODS: One hundred fourteen hepatitis C virus (HCV) RNA positive patients with hepatic iron concentrations of > or =700 microg/g dry wt (men) and > or =500 microg/g dry wt (women), stratified according to HCV genotype and gamma-glutamyltransferase values, were randomly allocated to interferon alone (6 MU three times a week) (group A) or to phlebotomy until iron depletion followed by interferon (6 MU three times a week) (group B). After 4 months dosage was reduced to 3 MU three times a week for another 8 months. RESULTS: Virological sustained response was observed in 25 patients (22%), nine (15.8%, 95% CI = 7.5-27.9) of group A and 16 (28.1%, 95% CI = 17.0-41.6) of group B. At univariate analysis the variables associated with the response were HCV genotypes 2-3, normal gamma-glutamyltransferase, higher levels of baseline ALT, normal ALT values, and negativity for HCV-RNA at the 3rd month of therapy. At multivariate analysis, genotype and ALT levels at enrollment maintained their association with the response. A trend toward a better response to interferon was observed in patients who received phlebotomy (odds ratio = 2.32, 95% CI = 0.96-6.24, p = 0.082). Patients with hepatic iron concentration of < or = 1100 microg/g dry wt had a trend toward a higher rate of virological sustained response (p = 0.059) when submitted to treatment B. CONCLUSION: Iron removal by phlebotomy is able to improve the rate of response to interferon, especially in patients with lower hepatic iron deposits; it could be useful as adjuvant therapy to new therapeutic modalities.

Levels of uroporphyrinogen decarboxylase (URO-D) in erythrocytes of Italian porphyria cutanea tarda patients.

Porphyria cutanea tarda (PCT) is a human metabolic disorder due to the acquired or genetic impairment of uroporphyrinogen decarboxylase (URO-D) activity, the fifth enzyme of the heme biosynthetic pathway. A classification of inherited and non-inherited forms is based on the enzyme activity levels in red blood cells (RBC). Clinical manifestations of PCT are often precipitated by triggering factors such as alcohol, drug abuse, estrogens, virus infections, hepatotoxic chemicals and hepatic siderosis. We measured URO-D activity in RBC from a large sample of Italian PCT patients in order to define the enzyme activity distribution and to attempt a correlation among activity, risk factors and clinical outcome. Three classes of patients with low, normal and over-normal URO-D activity were defined according to control values. Low URO-D levels were present in 25.8% of patients, suggesting the familial form of PCT (type II). In this group, the outcome of PCT seems to be less influenced by risk factors. Patients with over-normal URO-D activity in RBC deserve further investigation.

Red blood cell antioxidant and iron status in alcoholic and nonalcoholic cirrhosis.

BACKGROUND: Iron overload has been reported in alcoholic liver cirrhosis but it remains to be established whether iron is involved in inducing oxidative damage to erythrocytes in alcoholic cirrhosis. The aim of this study was to assess oxidative damage and red cell indicators of antioxidant defences in alcoholics with mild-to-severe liver cirrhosis, taking into account the iron status. MATERIALS AND METHODS: Twenty-nine patients with alcoholic liver cirrhosis (AC) and 27 with nonalcoholic cirrhosis (NAC) were studied. Serum lipid peroxides (LPO) were assayed by a colourimetric method. Serum-free malonyldialdehyde (MDA) was assayed by selected ion monitoring in positive chemical ionization; serum 4-hydroxy-2(E)-nonenal (4-HNE) was determined by a colorimetric method. Reduced (GSH) and oxidized glutathione (GSSG), adenine and pyridine cofactors were assayed in whole blood extracts by HPLC. Hexose-monophosphate shunt (HMPS), glycolytic pathway (EMP) and antioxidant enzyme activities were determined by standard methods. Iron status was evaluated by standard clinical chemistry and by histological grading of liver iron. Nontransferrin-bound iron (NTBI) was measured in serum by HPLC. RESULTS: GSH progressively decreased with increasing severity of liver involvement in AC and NAC. MDA, 4-HNE and NTBI were significantly higher in AC serum. Stimulation of red cell HMPS and reducing potential, in terms of NADPH production, were more pronounced in AC. CONCLUSIONS: These results suggest that NTBI is more important than the decrease of antioxidant defences in inducing lipid peroxidation. NTBI may play a catalytic role in free radical reactions in the presence of cellular reductants such as NADPH.

Tumor necrosis factor alpha promoter polymorphisms and insulin resistance in nonalcoholic fatty liver disease.

BACKGROUND & AIMS: Nonalcoholic fatty liver disease, which can range from fatty liver alone to nonalcoholic steatohepatitis and cirrhosis, is related to insulin resistance. Tumor necrosis factor alpha (TNF-alpha) may induce insulin resistance, and polymorphisms of its promoter have been associated with an increased release of this cytokine. We analyzed (1) the prevalence of insulin resistance, (2) the prevalence of the 238 and 308 TNF-alpha polymorphisms, and (3) the relationship among TNF-alpha polymorphisms, insulin resistance, and the occurrence of steatohepatitis in 99 patients with nonalcoholic fatty liver diagnosed by ultrasonography and confirmed by histologic analysis in the 53 who underwent biopsy. METHODS: Insulin resistance was evaluated by the homeostatic metabolic assessment insulin resistance indices and TNF-alpha polymorphisms by polymerase chain reaction and restriction fragment length polymorphism analysis. RESULTS: Insulin resistance was detected in almost all of the patients and was more severe in those with steatohepatitis. The prevalence of the 238, but not of the 308, TNF-alpha polymorphism was higher in subjects with nonalcoholic fatty liver than in controls (31% vs. 15%; P < 0.0001), and patients positive for TNF-alpha polymorphisms had higher insulin resistance indices, a higher prevalence of impaired glucose tolerance, and a lower number of associated risk factors for steatosis. CONCLUSIONS: TNF-alpha polymorphisms could represent a susceptibility genotype for insulin resistance, nonalcoholic fatty liver, and steatohepatitis.