Maurizio Sampietro

PRENATAL SEX DETERMINATION BY DNA AMPLIFICATION FROM MATERNAL PERIPHERAL BLOOD

The polymerase chain reaction was used to amplify a Y-specific repeat sequence in peripheral blood DNA samples from 19 pregnant women who had a gestational age of 9 to 41 weeks. Y-specific sequences were amplified from all 12 women who bore a male fetus but in none of 7 women who bore a female fetus. With stringent precautions against contamination, this technique may assist prenatal diagnosis of sex-linked genetic disorders.

Heterogeneity of Hemochromatosis in Italy

BACKGROUND & AIMS Patients with hemochromatosis show variable phenotype expression. We evaluated the frequency of hemochromatosis gene (HFE) mutations and the contribution of HFE genotype, ancestral haplotype, ethnic background, and additional factors (alcohol intake, hepatitis viruses, and beta-thalassemia trait) to the severity of iron overload in a large series of Italian patients with a hemochromatosis phenotype. METHODS HFE genotype was studied in 188 patients. Phenotype evaluation was available in 153 men and 20 women and was based mainly on iron removed. HFE genotype was determined by a polymerase chain reaction restriction assay and ancestral haplotype through D6S265 and D6S105 microsatellite analysis. RESULTS The frequency of C282Y homozygotes was 64%, with a decreasing gradient from north to south. C282Y homozygotes showed more severe iron overload than the other HFE genotypes. In the same group, ancestral haplotype was associated with a more severe phenotype. Additional factors may favor the development of a relatively mild hemochromatosis phenotype in patients nonhomozygous for the C282Y mutation. CONCLUSIONS Hemochromatosis in Italy is a nonhomogenous disorder in which genetic and acquired factors are involved. In patients with a single or no HFE mutation, further studies will enable a differentiation between true genetic disorders and interactions between genetic and acquired factors.

Hyperferritinemia, iron overload, and multiple metabolic alterations identify patients at risk for nonalcoholic steatohepatitis

OBJECTIVE:The aim of this study was to define in patients with hyperferritinemia and normal transferrin saturation the relationships among hyperferritinemia, iron overload, HFE gene mutations, the presence of metabolic alterations, and nonalcoholic steatohepatitis (NASH).METHODS:Forty patients with increased serum ferritin, resistant to dietary restriction and normal transferrin saturation, 90 with ultrasonographic evidence of hepatic steatosis, and 60 obligate heterozygotes for hemochromatosis, all negative for alcohol abuse, hepatitis virus infections, and inflammation were studied. Transferrin saturation, serum ferritin, uric acid, lipids, glucose tolerance, insulin resistance, HFE gene mutations, liver histology, and hepatic iron concentration were analyzed.RESULTS:Of the 40 patients with hyperferritinemia, 29 (72%) had biochemical metabolic abnormalities, 18 of the 26 examined (69%) had insulin resistance, 26 (65%) had the presence of one of the two HFE gene mutations (normal controls, 33 of 128 [26%], p < 0.0001), and all had increased liver iron concentration. Thirty-one patients (77%) had histology compatible with NASH. At univariate analysis, NASH was significantly associated with the presence of metabolic alterations, the C282Y mutation, and severity of fibrosis. At multivariate analysis, NASH was associated with the coexistence of multiple metabolic alterations (odds ratio = 5.2, 95% CI = 0.95–28.7). The risk of having NASH augmented in the presence of higher values of ferritin and liver iron concentration. Among the 90 patients with ultrasonographic evidence of hepatic steatosis, 24 (27%) had increased serum ferritin with normal transferrin saturation, but only six remained hyperferritinemic after dietary restriction.CONCLUSION:Increased ferritin with normal transferrin saturation is frequently found in patients with hepatic steatosis, but it reflects iron overload only in those patients in whom it persists despite an appropriate diet. The simultaneous disorder of iron and glucose and/or lipid metabolism, in most of the cases associated with insulin resistance, is responsible for persistent hyperferritinemia and identifies patients at risk for NASH.

The Hemochromatosis Gene affects the Age of Onset of sporadic Alzheimer's Disease.

In the present study we analysed the genotype of HFE, the gene involved in hemochromatosis, in 107 patients with sporadic late-onset AD and in 99 age-matched non-demented controls. We observed that patients carrying the mutant HFE-H63D allele had a mean age at onset of 71.7 +/- 6.0 years versus 76.6 +/- 5.8 years of those who were homozygous for the wild-type allele (p = 0.001). The frequency of the HFE-H63D mutation was highest (0.22) in the patients aged <70 years at the time of disease onset, whereas it was 0.12 in those with disease onset at an age of 70-80 years, and 0.04 in those aged more than 80 years. The APOE genotype did not significantly modify the effect of HFE on age at onset. We conclude that mild disturbances of iron homeostasis associated with a common genetic determinant may interact with other pathogenic mechanisms involved in AD. HFE mutations may anticipate AD clinical presentation in susceptible individuals.

Association of thalassaemia intermedia with a beta-globin gene haplotype

We have identified 14 Asian patients with homozygous β° thalassaemia who had a mild clinical disorder related to an augmented production of haemoglobin F. None of their parents had an elevated level of Hb F. Restriction fragment length polymorphism analysis of the β‐globin cluster of these patients and a control group of Asian thalassaemia major patients showed that 6/14 of the thalassaemia intermedia patients were homozgyous for a particular 5′β‐globin haplotype (−+−++), in contrast to 1/42 of the thalassaemia major patients. Furthermore, the —+—++β haplotype is also associated with amelioration of disease severity in β thalassaemia in an Italian population. This β haplotype is linked to a DNA sequence variation 5′ (at position — 158) to the cγ globin gene which can be detected by the presence (+) of an Xmn I restriction enzyme site. The possible role of the Xmn I‐γ polymorphism in relation to this variant HPFH is discussed. We conclude that much of the observed clinical variability of β thalassaemia can now be explained by the inheritance of β thalassaemia chromosomes with different propensities for fetal haemoglobin production.

The expression of uridine diphosphate glucuronosyltransferase gene is a major determinant of bilirubin level in heterozygous β‐thalassaemia and in glucose‐6‐phosphate dehydrogenase deficiency

We evaluated the effect of Gilberts syndrome, the most common defect of bilirubin conjugation, on the bilirubin levels of subjects with inherited haematological disorders which cause increased bilirubin production. 57 patients heterozygous for β‐thalassaemia, 21 with G6PD deficiency and 44 controls were examined by typing the TATA‐box in the promoter of the gene uridine diphosphate glucuronosyltransferase 1A. Nearly 80% of patients with increased bilirubin levels were heterozygous or homozygous for the UGT1A TA(7) variant associated with Gilberts syndrome. These findings indicate that Gilberts syndrome accounts for a large proportion of the variability of bilirubin levels in β‐thalassaemia and G6PD deficiency.

Immunochemical estimation of haemoglobin types in red blood cells by FACS analysis.

Summary. A fixation and permeabilization procedure using formaldehyde and acetone has been developed which allows immunostaining of intracellular haemoglobin for fluorescence activated cell sorter (FACS) analysis of erythrocytes. The treatment preserves antigenicity and light‐scattering properties. Validation of the method was given by the correlation of F cell number in adults determined by FACS analysis with that assessed by microscopic examination of cell smears, and by the direct relationship between β chain synthesis and intensity of β chain/Hb A immunofluorescence within fetal erythrocyte samples known to vary in their β chain/Hb A content. The procedure is rapid, non‐subjective and sensitive, and makes analysis of haemoglobin content, type and distribution amongst red cell populations possible.

Prenatal sex determination from maternal peripheral blood using the polymerase chain reaction

We have investigated the use of a nested polymerase chain reaction assay for the detection of a fetal-specific Y-chromosomal sequence (DYS14) from DNA extracted from unsorted maternal peripheral blood. Serial dilutions of male DNA into female cord blood DNA indicated that the assay could detect an equivalent of a single male cell in 300000 female cells. The assay exhibited absolute specificity for male DNA with no amplification from a DNA panel obtained from 10 female cord blood samples. When used on DNA extracted from unsorted peripheral blood from a series of pregnant women, the predictive values of a positive test for a male fetus were 86%, 67% and 87% in the first, second and third trimesters, respectively. We have also demonstrated that retesting the samples allows the detection of a proportion of male-bearing pregnancies with a high degree of accuracy, in that all 15 women who gave positive signals in two consecutive amplifications had male fetuses. We have also applied the test at 8 weeks post-partum to eight women who had previously delivered male babies; no Y-specific signal could be detected in any of them, suggesting that most women have cleared their circulation of fetal cells by 8 weeks after parturition.

Haemochromatosis in patients with beta-thalassaemia trait.

Severe iron overload has been reported in patients with the β‐thalassaemia trait. Studies performed before the discovery of the haemochromatosis gene (HFE) have yielded conflicting results: some suggest that iron overload might arise from the interaction of the β‐thalassaemia trait with heterozygosity for haemochromatosis, some with homozygosity for haemochromatosis and others that it was unrelated to haemochromatosis. We have studied the clinical phenotype, iron indices and HFE genotypes of 22 unrelated patients with the β‐thalassaemia trait and haemochromatosis, the inheritance of chromosome 6p and 1q haplotypes in families of non‐homozygous C282Y probands and serum measures of iron status in relatives heterozygous for C282Y with or without the β‐thalassaemia trait. We demonstrate that the β‐thalassaemia trait aggravates the clinical picture of C282Y homozygotes, favouring higher rates of iron accumulation and the development of severe iron‐related complications. We suggest that the coexistence of the β‐thalassaemia trait might also increase the risk of iron overload in patients with HFE genotypes at a mild risk of haemochromatosis. Our findings do not support the hypothesis that the association of the β‐thalassaemia trait with a single C282Y or H63D allele might lead to iron overload and suggest that other non‐HFE‐related inherited factors are present in haemochromatosis patients with incomplete HFE genotypes.

Liver iron influences the response to interferon alpha therapy in chronic hepatitis C

Objective: To define whether there is any relation between the iron status of patients with hepatitis C virus (HCV) chronic liver disease and their response to interferon therapy. Design: To evaluate the long‐term response to 1 year of interferon therapy with addition of phlebotomies after 3 months of treatment if at that time alanine aminotransferase (ALT) had not normalized in a group of patients with HCV‐positive chronic liver disease whose iron status had been characterized. Setting: A northern Italian hospital. Participants: Fifty‐eight anti‐HCV‐positive patients (four HCV‐RNA negative) with biopsy proven chronic hepatitis and no evidence of iron overload as indicated by normal transferrin saturation at the time of enrolment in the study. Intervention: Three times a week intramuscular injection of alpha interferon 3MU for 1 year with addition of phlebotomies (350ml/week) till iron depletion if after 3 months of interferon therapy ALT had not normalized. Results: A long‐term response was observed in 19 of the 52 patients who completed the treatment, four HCV‐RNA negative and 15 positive. The four RNA‐negative and seven of the 15 RNA‐positive long‐term responders had been treated with interferon alone, and the other eight also with phlebotomies. At univariate analysis only HCV genotype, gammaglutamyltranspeptidase and liver iron concentration were significantly associated with response whereas sinusoidal iron deposition was of borderline significance. No association was found with sex, age, duration of disease, histology, Knodell score, transferrin saturation %, serum ferritin, hepatocytic iron score, and portal iron score. HCV‐RNA serum levels, measured in 29 patients, did not correlate with response. At multivariate analysis liver iron concentration was still significant and one unit reduction of liver iron concentration (natural logarithm transformed) was associated with 2.95 odds ratio of response. Conclusion: These results indicate that iron in the liver is more closely related to response to interferon than the other variables considered, including HCV characteristics.