HFE gene mutations an Apulian population: allele frequencies.

Hereditary hemochromatosis (HH) is an autosomal recessive trait regarding iron metabolism frequently found in Caucasian populations. The C282Y mutation of the HFE gene, held responsible for HH, has been identified as the major genetic basis for the phenotypic expression of HH whereas two additional mutations of the HFE H63D and S65C gene appear to be associated with a milder form of HH. A high allele frequency of C282Y and H63D has been reported in Northern European populations. In Italy, the overall allele frequency was 0.5% for the C282Y mutation, 12.6% for the H63D mutation and 1.1% for the S65C mutation. In this study, we evaluated the allele frequency of the three principal HFE mutations (C282Y, H63D, S65C) together with eight additional mutations (V53M, H63H, Q127H, E168Q, E168stop, W169stop, V59M, Q238P) in 500 healthy Apulian subjects. No subject homozygous for the C282Y mutation was found while 3% of subjects were heterozygous for this mutation. Heterozygosity and homozygosity for the H63D mutation were 26 and 1%, respectively. Only five subjects were heterozygous for the S65C mutation. Overall, the allele frequency was 1.5% for C282Y, 14% for H63D, 0.5% for S65C and 0% for the other mutations. The transferrin saturation (TS) was significantly higher in subjects heterozygous for the H63D mutations with respect to subjects with a normal genotype, though all were within the normal range. No statistically significant difference in the allele frequency was noted in the Apulian population compared to that in Northern and Southern Italy.

Analysis of HLA-DRB1*-A* and -B* alleles in prenatal diagnosis for determination of maternal contamination in fetal DNA.

During chorionic villi sampling for prenatal diagnosis with molecular biology techniques, contamination by maternal decidua frequently occurs and can lead to misinterpretation of the test results. To avoid such problems, we present a new method for appraising maternal contamination of fetal DNA, based on genomic typing of the highly variable human leukocyte antigen (HLA) locus-DRB1*, locus A* and locus B* regions by genetic amplification with sequence-specific primers and PCR. Fetal DNA samples obtained for beta-thalassemia diagnosis were analysed after artificial contamination with increasing maternal DNA concentrations ranging from 0.5 to 10% (0.5, 1, 3, 5 and 10%). The approach was found to be rapid, specific, reproducible and highly sensitive and permits recognition of 1-3% contamination by maternal DNA concentrations. The system currently used for detecting maternal DNA contamination in fetal samples is the analysis of polymorphic loci by variable number of tandem repeats and/or short tandem repeats. We propose that the analysis of HLA alleles may provide a valid alternative or complement to this system.

Genotype and phenotype correlation in glucose-6-phosphate dehydrogenase deficiency.

BACKGROUND AND OBJECTIVES: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common erythrocytic enzymatic disorder in Italy and is characterized by wide clinical, biochemical and molecular variability. We studied the clinical and hematologic data from 54 G6PD-deficient, unrelated males from the Apulia region. DESIGN AND METHODS: Analyses for enzymatic activity, G6PD electrophoresis and molecular typing were performed on all subjects. Thirty-nine subjects (72.2%) showed a severe G6PD deficiency (<10% residual enzymatic activity) and 15 subjects (27.8%) a moderate deficiency (10--60% residual activity). RESULTS: The Mediterranean variant was found in 48.2% of cases, the Seattle variant in 33.3%, the A- variant in 7.45% and the Montalbano variant in 3.7%; the variant was not identified in four subjects. Thirty-two patients (59.2%) were asymptomatic; of these, 37.04% demonstrated acute hemolytic crises induced mainly by ingestion of fava beans and 3.7% had had neonatal jaundice. Acute hemolytic anemia was found in 53.8% of subjects with the Mediterranean variant, in 5.5% with the Seattle variant, in 100% with the A-variant and 0% with the Montalbano variant. INTERPRETATION AND CONCLUSIONS: Enzymatic activity was shown to be a poor predictive parameter of acute hemolytic crises and was not correlated with clinical features. Subjects with Mediterranean or A- variants had a more severe clinical phenotype which was not related to enzymatic activity. The Seattle, and probably the Montalbano, variant appears to have a milder clinical expression.

Prenatal diagnosis of beta-thalassaemia using fetal erythroblasts enriched from maternal blood by a novel gradient.

We have assessed a new technique for the isolation of fetal erythroblasts from maternal blood for the non-invasive prenatal diagnosis of pregnancies at risk of beta-thalassaemia. This method relies on the separation of erythroblasts from maternal nucleated cells by a novel step gradient and high speed centrifugation. In four of the six cases examined, single erythroblasts were identified by immunohistochemistry for zeta (zeta) globin. These were individually micromanipulated and analysed by single cell polymerase chain reaction (PCR) and subsequent sequencing of the region of beta-globin locus where the mutations most common to the region of Puglia, Italy, are clustered. In each of the four instances where fetal erythroblasts were identified by antibody staining, the fetal beta-globin genotype was correctly determined. To date, this represents the largest series of non-invasive prenatal diagnoses performed for this haemoglobinopathy.