Maurizio Travi

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.

Parkin analysis in early onset Parkinson's disease

We analysed the parkin gene in a large consecutive series (146) of unrelated early onset Parkinsons disease (onset ?40 years of age) patients. Twelve cases (8.2%) had homozygous or compound heterozygous point mutations and/or exon rearrangements, while a single mutation was found in four subjects (2.7%). We identified eight exon rearrangements and nine point mutations, two of which were novel: c.735delT (p.C212/X224) and c.815C>G (p.C238W). Genotype-phenotype correlation revealed that parkin carriers had features similar to those of non-carrier early onset Parkinson disease patients.

Peptide-nucleic acid-mediated enriched polymerase chain reaction as a key point for non-invasive prenatal diagnosis of β-thalassemia

This study describes a novel approach to non-invasive pre-natal diagnosis of β-thalassemia based on microchip analysis of fetal DNA extracted from maternal plasma. The presence of fetal DNA in maternal plasma can be exploited to develop new procedures for non-invasive prenatal diagnosis. Tests to detect 7 frequent β-globin gene mutations in people of Mediterranean origin were applied to the analysis of maternal plasma in couples where parents carried different mutations. A mutant enrichment amplification protocol was optimized by using peptide nucleic acids (PNAs) to clamp maternal wild-type alleles. By this approach, 41 prenatal diagnoses were performed by microelectronic microchip analysis, with total concordance of results obtained on fetal DNA extracted from chorionic villi. Among these, 27/28 were also confirmed by direct sequencing and 4 by pyrosequencing.

Feasibility study for a microchip-based approach for noninvasive prenatal diagnosis of genetic diseases

Abstract: Fetal DNA in maternal plasma may represent a source of genetic material for prenatal noninvasive diagnosis of genetic diseases. We evaluated a cohort of physiological pregnancies to determine if fetal DNA can be retrieved at any gestational week in sufficient quantity to be analyzed with advanced mutation detection technologies. We performed fetal DNA quantification by real‐time polymerase chain reaction (PCR) on the SRY gene in 356 women sampled from 6 to 40 gestational weeks. Fetal DNA was retrieved at any week. All female fetuses were correctly identified. In 5 of 188 (2.6%) male‐bearing pregnancies, no amplification was obtained. For noninvasive testing, complete clearance of fetal DNA after delivery is mandatory. Long‐term persistence was not detected in women with previous sons or abortions. These findings confirm that maternal plasma may represent the optimal source of fetal genetic material. For noninvasive diagnosis of genetic diseases, we evaluated microchip technology. The detection limit for a minority allele determined by diluting a mutated DNA into a wild‐type plasma sample was 5 genome equivalents, indicating that the test might be applied to the identification of paternally inherited fetal alleles in maternal plasma. The addition of peptide nucleic acids (PNAs) to either the PCR reaction or the chip hybridization mixture allowed approximately 50% inhibition of wild‐type allele signals.

Simultaneous Mutations in the CLCNKB and SLC12A3 Genes in Two Siblings with Phenotypic Heterogeneity in Classic Bartter Syndrome

Two siblings (brother and sister) with renal tubular hypokalemic alkalosis underwent clinical, biochemical and molecular investigations. Although the biochemical findings were similar (including hypokalemia, metabolic alkalosis, hyperreninemia, hyperaldosteronism and normal blood pressure), the clinical findings were different: the boy, who also presented syndromic signs, developed glomerular proteinuria and renal biopsy revealed focal segmental glomerular sclerosis; the girl showed the typical signs of classic Bartter syndrome. As described in a previous paper, a heterozygous mutation (frameshift 2534delT) was demonstrated in the gene encoding the thiazide-sensitive NaCl co-transporter (SLC12A3) of the distal convoluted tubule; the second molecular analysis revealed a compound heterozygous mutation (A61D/V149E) in the CLCNKB chloride channel gene in both subjects, inherited in trans from the parents. The children were finally diagnosed as having classic Bartter syndrome. These cases represent the first report of the simultaneous presence of heterozygous and compound heterozygous mutations in the SLC12A3 and CLCNKB genes, both of which are involved in renal salt losing tubulopathies, and confirm previous observations regarding classic Bartter syndrome phenotype variability in the same kindred.

Thalassaemia-like carriers not linked to the β-globin gene cluster

This study describes the largest series reported to date, of individuals belonging to unrelated families carrying a β‐thalassaemia‐like phenotype in whom the β‐globin gene was found to be structurally intact by sequence analysis. This genetic determinant appears haematologically heterogeneous, displaying either a silent β‐thalassaemia‐like phenotype or a typical β‐thalassaemia carrier‐like phenotype in different families. Compound heterozygosity for both β‐thalassaemia‐like determinant and typical β‐thalassaemia allele resulted either in thalassaemia intermedia or thalassaemia major. By linkage analysis both the silent and the typical β‐like determinants were found not to be linked to the β‐globin cluster. Sequence analysis of the hypersensitive site cores of locus control region and of the genes coding for the transcription factors erythroid Kruppel‐like factor and nuclear factor (erythroid‐derived 2) were normal. β‐globin mRNA levels determined by real‐time polymerase chain reaction were reduced in both types of β‐like carriers. These results indicate the existence of causative genetic determinants not yet molecularly defined, but most likely, resulting from either the reduction or loss of function of a gene coding for unknown transcriptional regulator(s) of the β‐globin gene. The knowledge of these rare β‐thalassaemia‐like determinants have implications for clinical and, especially, prenatal diagnosis of β‐thalassaemia.

Different approaches for noninvasive prenatal diagnosis of genetic diseases based on PNA-mediated enriched PCR

Abstract:  The aim of this work was to develop advanced and accessible protocols for noninvasive prenatal diagnosis of genetic diseases. We are evaluating different technologies for mutation detection, based on fluorescent probe hybridization of the amplified product and pyrosequencing, a technique that relies on the incorporation of nucleotides in a primer‐directed polymerase extension reaction. In a previous investigation, we have already proven that these approaches are sufficiently sensitive to detect a few copies of a minority‐mutated allele in the presence of an excess of wild‐type DNA, In this work, in order to further enhance the sensitivity, we have employed a mutant enrichment amplification strategy based on the use of peptide nucleic acids (PNAs). These DNA analogues bind wild‐type DNA, thus interfering with its amplification while still allowing the mutant DNA to become detectable. We have synthesized different PNAs, which are highly effective in clamping wild‐type DNA in the beta‐globin gene region, where four beta‐thalassemia mutations are located (IVSI.110, CD39, IVSI.1, IVSI.6) plus HbS. The fluorescence microchip readout allows us to monitor the extent of wild‐type allele inhibition, thus facilitating the assessment of the optimal PNA concentration.

A New De Novo Missense Mutation in Connexin 26 in a Sporadic Case of Nonsyndromic Deafness

Objectives: Mutations in the GJB2 gene, encoding Connexin 26, can cause nonsyndromic recessive deafness or dominant hearing loss (HL) with or without keratoderma. The objective was to perform a molecular evaluation to establish the inherited pattern of deafness in the sporadic cases afferent to our center.

Homozygous β‐thalassaemia resulting in the β‐thalassaemia carrier state phenotype

Summary. This paper describes the phenotypic manifestations of a very mild β‐thalassaemia mutation detected in several members of two families of Italian descent. The molecular defect, defined by denaturing gradient gel electrophoresis analysis and direct sequencing. consists of a C G substitution at position 844 of IVSII of the β‐globin gene within the consensus sequence of IVSII acceptor splice site. Heterozygotes for this mutation show a haematological phenotype ranging in severity from silent β‐thalassaemia to that of a mild β‐thalassaemia carrier silent β‐thalassaemia to that of a mild β‐thalassaemia carrier state, whereas homozygotes have the typical manifestations commonly resulting from heterozygosity for a β‐thalassaemia mutation. Compound heterozygotes for the IVSII nt844 (C G) mutation and a severe β‐thalassaemia mutation have the phenotype of thalassaemia intermedia.

Antenatal diagnosis of haemoglobinopathies by improved method of isoelectric focusing of haemoglobins

Summary. An improved method of isoelectric focusing (IEF) of haemoglobins, utilizing a spacer molecule to increase the separation between HbA and acetylated HbF, was carried out in parallel with carboxymethylcellulose (CMC) chromatography in 85 antenatal diagnoses for haemoglobinopathies (mainly thalassaemia). In 19 of 21 affected fetuses no HbA band was detectable on IEF while in two a very faint band was observed; in heterozygotes or normal fetuses a more obvious HbA band was present. Moreover, IEF was useful for the demonstration of HbLepore in two cases at risk for β‐thalassaemia/HbLepore and for interpreting a radioactive pre‐β globin peak on CMC chromatography. Pure fetal blood is needed for IEF. It can therefore be used for most cases in centres using fetoscopy for fetal blood sampling. About 80% of antenatal diagnosis of haemoglobinopathies may be carried out by IEF which is a very simple, rapid and inexpensive method.