[Show abstract][Hide abstract] ABSTRACT: Mutations in the GJB2 gene, which encodes the gap-junction protein connexin 26, are the most common cause of nonsyndromic hearing loss (NSHL) and account for about 32% of cases. We analyzed 734 patients and identified mutations in 474/1468 chromosomes. Thirty-six different mutations and five polymorphisms were found in 269 NSHL subjects. Our data confirm 35delG as the most frequent GJB2 mutation in the Italian population, accounting for about 68% of all the mutated GJB2 alleles analyzed. We also identified two novel variants: the V156I mutation and the C>A change at nucleotide 684 in the 3'UTR of the gene. The GJB6 gene deletion, del(GJB6-D13S1830), which can cause HL in combination with GJB2 mutations in trans, was identified in three patients, while the del(GJB6-D13S1854) was not observed in our cohort of patients. We collected audiometric data from 200 patients with biallelic DFNB1 mutations or with dominant mutation in GJB2 to determine the degree of HL to correlate the genotypes with the audiological phenotypes.
[Show abstract][Hide abstract] ABSTRACT: 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 beta-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.
[Show abstract][Hide abstract] ABSTRACT: We analysed the parkin gene in a large consecutive series (146) of unrelated early onset Parkinson's 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.
Parkinsonism & Related Disorders 01/2008; 14(4):326-33. · 3.27 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
The subject was a 2-year-old Italian girl with nonsyndromic early onset HL. We performed DNA sequencing of the GJB2 gene and deletion analysis of the GJB6 gene in all family members.
Direct sequencing of the gene showed a heterozygous C-->G transition at nucleotide 172 resulting in a proline to alanine amino acid substitution at codon 58 (P58A). The analyses indicate that the P58A mutation appeared de novo in the proband with a possible dominant effect.
This mutation occurs in the first extracellular domain (EC1), which seems to be very important for connexon-connexon interaction and for the control of voltage gating of the channel. The de novo occurrence of an EC1 mutation in a sporadic case of deafness is consistent with the assumption that P58A can cause dominant HL.
The Laryngoscope 06/2007; 117(5):821-4. · 1.98 Impact Factor
[Show abstract][Hide abstract] 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.
Annals of the New York Academy of Sciences 10/2006; 1075:137-43. · 4.38 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This study describes the largest series reported to date, of individuals belonging to unrelated families carrying a beta-thalassaemia-like phenotype in whom the beta-globin gene was found to be structurally intact by sequence analysis. This genetic determinant appears haematologically heterogeneous, displaying either a silent beta-thalassaemia-like phenotype or a typical beta-thalassaemia carrier-like phenotype in different families. Compound heterozygosity for both beta-thalassaemia-like determinant and typical beta-thalassaemia allele resulted either in thalassaemia intermedia or thalassaemia major. By linkage analysis both the silent and the typical beta-like determinants were found not to be linked to the beta-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. beta-globin mRNA levels determined by real-time polymerase chain reaction were reduced in both types of beta-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 beta-globin gene. The knowledge of these rare beta-thalassaemia-like determinants have implications for clinical and, especially, prenatal diagnosis of beta-thalassaemia.
British Journal of Haematology 04/2006; 132(5):640-50. · 4.94 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Pediatric Research 01/2006; 58(6):1269-73. · 2.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To report a multi-technical approach to Duchenne muscular dystrophy (DMD) mutation testing through carrier analysis, in the prenatal diagnosis of a male foetus without a known mutation segregating in the family and with inconclusive results of linkage analysis.
Haplotype analysis with the DMD region markers for assigning the carrier status of the mother and for prenatal diagnosis of foetal DNA; semiquantitative multiplex analysis of maternal and foetal DNA for the promoter and for 34 exons of the DMD gene; sequencing analysis of the maternal and foetal DNA for confirmation of the results.
Because of an intragenic recombination of the DMD gene in foetal DNA, haplotype analysis gave inconclusive results. Semiquantitative PCR analysis displayed a pattern compatible with a heterozygous exon 60 mutation in the mother's DNA, while foetal DNA showed a normal migration pattern. Sequencing analysis confirmed the presence of a novel 7 base-pair deletion in exon 60 of the DMD gene in the mother and excluded the deletion in the foetus.
Semiquantitative PCR results allowed the DMD mutation detection in the mother and the exclusion in the foetus, showing its crucial importance in prenatal diagnosis in those cases where linkage analysis is not conclusive.
[Show abstract][Hide abstract] 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.
Annals of the New York Academy of Sciences 07/2004; 1022:105-12. · 4.38 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: beta-Thalassemia is one of the most common genetic diseases in humans. We developed an automated electronic microchip for fast and reliable detection of the nine most frequent mutations accounting for >95% of the beta-thalassemia alleles in the Mediterranean area.
We developed a microchip-based assay to identify the nine most frequent mutations (cd39C>T, IVS1-110G>A, IVS1-1G>A, IVS1-6T>C, IVS2-745C>G, cd6delA, -87C>G, IVS2-1G>A, and cd8delAA) by use of the Nanogen Workstation. The biotinylated amplicon was electronically addressed on the chip to selected pads, where it remained embedded through interaction with streptavidin in the permeation layer. The DNA at each test site was then hybridized to a mixture of fluorescently labeled wild-type or mutant probes.
Assays conditions were established based on the analysis of 700 DNA samples from compound heterozygotes or homozygotes for the nine mutations. The assays were blindly validated on 250 DNA samples previously genotyped by other methods, with complete concordance of results. Alternative multiplexed formats were explored: the combination of multiplex PCR with multiple addressing and/or hybridization allowed analysis of all nine mutations in the same sample on one test site of the chip.
The open flexible platform can be designed by the user according to the local prevalence of mutations in each geographic area and can be rapidly extended to include the remaining mutations causing beta-thalassemia in other regions of the world.
[Show abstract][Hide abstract] ABSTRACT: The behavior of different sieving polymers for unambiguous determination of point mutations in genomic DNA, based on electrophoresis in thin capillaries, is evaluated. High melters from thalassemia patients are separated by exploiting the principle of denaturing gradient gel electrophoresis, in fact, of its variant utilizing temperature gradients (TGGE), along the migration path, encompassing the melting points of both homo- and heteroduplex, polymerase chain reaction (PCR)-amplified DNA fragments. Unlike TGGE, where the temperature gradient exists along the separation space, the denaturing temperature gradient in the fused-silica capillaries is time-programmed, so as to reach the Tm's of all species under analysis prior to electrophoretic transport past the detector window. The DNA fragments are injected in a capillary maintained (by combined chemical and thermal means) just below the expected Tm values. The deltaT applied is rather minute (1-1.5 degrees C) and the temperature gradient quite shallow (e.g., 0.05 degrees C/min). The denaturing thermal gradient is generated internally, via Joule heat produced by voltage ramps. This method is applied to the analysis of the most common point mutations in thalassemias, characterized by being high melters (in the temperature range of 60-62 degrees C) in presence of 6 M urea. Point mutants are fully resolved into a spectrum of four bands only when poly(N-acryloylaminopropanol) and hydroxyethylcellulose are used. However, the former offers the best separation capability at such high temperatures.
[Show abstract][Hide abstract] ABSTRACT: In order to verify the genetic factors influencing the clinical expression of beta-thalassemia we have studied 292 Italian patients, 165 with thalassemia intermedia and 127 with thalassemia major. The beta-globin gene mutations were defined in all cases. The number of alpha-globin genes and the integrity of specific control regions of the beta-globin cluster--gamma promoters and beta-Locus Control Region (beta-LCR)--were studied in selected cases. Homozygosity for mild mutations (group I) accounts for 24% of the intermedia patients and it is not represented among major patients. Forty-four percent of intermedia patients had combinations of mild/severe (group II) mutations and 32% had homozygosity or double heterozygosity for severe mutations (group III). Seventy-six percent of patients with thalassemia major were classified in group III and 24% in group II. Deletion type-alpha3.7 thalassemia, assessed in a part of the cases, was found in 5% of thalassemia major and 19.5% of intermedia patients in groups II and III. Structural analysis of gamma promoters and beta-LCR HS2 and HS4 regions, carried out in order to look for alterations associated with Hb F increase, did not reveal new mutations. Only rare polymorphic changes were observed at the HS2 and HS4 level. The -158G gamma C T change was found with an increased incidence in intermedia patients in groups II and III. A subset of 10 beta-thalassemia heterozygotes with mild intermedia phenotype resulted from coinheritance of a triplicated alpha-locus. We have been unable to find a molecular basis for the benign clinical course in approximately 20% of patients with thalassemia intermedia. Other genetic or acquired factors must be hypothesized which ameliorate the clinical condition.
American Journal of Hematology 03/1995; 48(2):82-7. · 4.00 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This paper describes the phenotypic manifestations of a very mild beta-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 beta-globin gene within the consensus sequence of the IVSII acceptor splice site. Heterozygotes for this mutation show a haematological phenotype ranging in severity from silent beta-thalassaemia to that of a mild beta-thalassaemia carrier state, whereas homozygotes have the typical manifestations commonly resulting from heterozygosity for a beta-thalassaemia mutation. Compound heterozygotes for the IVSII nt844 (C-->G) mutation and a severe beta-thalassaemia mutation have the phenotype of thalassaemia intermedia. This paper indicates that the presence of borderline red blood cell indices or HbA2 values should make one suspect the presence of a very mild or silent beta-thalassaemia.
British Journal of Haematology 12/1994; 88(3):562-5. · 4.94 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: . 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.This paper indicates that the presence of borderline red blood cell indices or HbA2 values should make one suspect the presence of a very mild or silent β-thalassaemia.
British Journal of Haematology 01/1994; 88(3):562-565. · 4.94 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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 300,000 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 postpartum 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.
Human Genetics 02/1993; 90(5):483-8. · 4.63 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Transabdominal chorionic villus sampling (TA-CVS) was attempted in 328 high-risk pregnancies at 6-7 weeks of gestation. Sampling was feasible in 97.7 per cent of cases; chorionic tissue specimens of more than 10 mg were obtained in 94.4 per cent of cases at the first needle insertion and in 100 per cent after a second attempt. Fetal karyotyping succeeded in 99.4 per cent of cases, while no diagnostic failures were reported in enzymatic and DNA analyses. Fetal loss rate in the first 4 weeks after CVS was significantly higher than in the later CVS series (7.2 vs. 2.5 per cent), but 50 per cent of losses were observed within 2 weeks in cases of inviable aneuploidies. A high incidence of severe limb abnormalities (1.6 per cent) was detected in pregnancies intended to continue, confirming the aetiological role of early CVS. Unclear visualization of the placental limits and poor control of the needle path are thought to be the main reasons for the vascular disruption of the chorionic plate, and thereby hypoxic embryo tissue damage. A better selection of cases, together with high-resolution vaginal ultrasound visualization, and analytical techniques requiring a minimal amount of tissue should avoid any teratogenic effect of early CVS.
[Show abstract][Hide abstract] ABSTRACT: Transabdominal chorionic villus sampling (TA-CVS) was attempted in 328 high-risk pregnancies at 6–7 weeks of gestation. Sampling was feasible in 97.7 per cent of cases; chorionic tissue specimens of more than 10 mg were obtained in 94.4 per cent ofcases at the first needle insertion and in 100 per cent after a second attempt. Fetal karyotyping succeeded in 99.4 per cent of cases, while no diagnostic failures were reported in enzymatic and DNA analyses. Fetal loss rate in the first 4 weeks after CVS was significantly higher than in the later CVS series (7.2 vs. 2.5 per cent), but 50 per cent of losses were observed within 2 weeks in cases of inviable aneuploidies. A high incidence of severe limb abnormalities (1.6 per cent) was detected in pregnancies intended to continue, confirming the aetiological role of early CVS. Unclear visualization of the placental limits and poor control of the needle path are thought to be the main reasons for the vascular disruption of the chorionic plate, and thereby hypoxic embryo tissue damage. A better selection of cases, together with high-resolution vaginal ultrasound visualization, and analytical techniques requiring a minimal amount of tissue should avoid any teratogenic effect of early CVS.