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S Rossetti

Publications and source records attributed to S Rossetti.

At least 37 records · Page 2Linked to original sources
PubMed1997

Detection of mutations in human genes by a new rapid method: cleavage fragment length polymorphism analysis (CFLPA).

Cleavage fragment length polymorphism analysis with silver staining visualization (CFLPA-SS) was used for the detection of mutations previously detected by single strand conformation (SSCA) or heteroduplex analyses (HA); in order to assess this new method for mutation screening. The analysed mutations include single nucleotide transitions, transversions, a deletion and a duplication in the following genes: CFTR (cystic fibrosis transmembrane conductance regulator), COL4A5 (collagen type 4 alpha 5 chain), PKD1 (polycystic kidney disease 1), and FGFR3 (fibroblast growth factor receptor 3). Peripheral blood leukocyte genomic DNA was isolated, amplified by polymerase chain reaction (PCR), and then cleaved by Cleavase I enzyme at different temperatures. Electrophoresis of the fragments on denaturing polyacrylamide gel was followed by silver staining for 1 min. All 13 mutations investigated were reproducibly detected. CFLPA-SS proved to be a reliable method for mutation detection and more rapid than SSCA and HA.

Achondroplasia↗
PubMed1997

Rapid DNA-based prenatal diagnosis by genetic linkage in three families with Alport's syndrome.

Alport's syndrome (AS) is a clinically and genetically heterogeneous progressive inherited glomerulonephritis characterized by hematuria, sensorineural hearing loss, ocular lesions, and specific alterations of the glomerular basement membrane. Typically, AS shows an X-linked dominant pattern of inheritance, with mutations affecting the collagen type IV alpha5 chain gene (COL4A5) at Xq22. Rarely, AS is caused in some families by mutations of the COL4A3/A4 genes on chromosome 2q, showing an autosomal recessive transmission. Very few families have been described with possible autosomal dominant AS, but no mutations in any of the COL4 genes have been found. We describe three unrelated families affected with a severe AS phenotype in which DNA-based prenatal diagnosis by linkage analysis was made in fetuses at risk for the disease. In two families, the pedigree structure and the clinical picture were consistent with typical X-linked dominant AS. In these families, autosomal inheritance was also ruled out molecularly. In one family, despite careful clinical and molecular evaluation, the mode of transmission could not be firmly established. We used tightly linked and intragenic COL4A5 markers, as well as COL4A3/A4-linked markers. A chromosome Y-specific marker for fetal sex determination was simultaneously used. In all the families, before the fetal analysis, the putative at-risk X haplotype was identified with high diagnostic accuracy. We diagnosed a healthy male fetus in one family, and female but carrier fetuses in the other two kindreds, who decided not to terminate their pregnancies. We used rapid nonisotopic polymerase chain reaction-based methods, and the results were available within 2 to 3 days. The genetic results significantly affected the reproductive decisions of the parents. This report illustrates the application of genetic linkage analysis as an additional tool for molecular diagnosis in AS, and also addresses the issue of the attitudes of the families toward prenatal testing. To our knowledge, prenatal diagnosis of AS using a genetic linkage approach has not been previously reported.

Adult↗
PubMed1997

Phenotypic and phylogenetic description of an Italian isolate of "Microthrix parvicella".

"Microthrix parvicella" strain RN1 was isolated from an activated sludge treatment plant in Italy using micromanipulation techniques. The strain grows as thin unbranchedfilaments which are Gram-positive with Neisser-positive granules. The isolate was characterized by analysis of the 16S rDNA which was amplified directly from cell biomass by the polymerase chain reaction and sequenced. "Microthrix parvicella" strain RN1 presents a very high similarity (100%) with another "M. parvicella" strain recently isolated in Australia, suggesting that this micro-organism, a novel, deep branching member of the actinomycetes subphylum, is the same causing the common events of bulking and foaming phenomena in activated sludge treatment plants throughout the world.

DNA, Bacterial↗
PubMed1997

The characterization and description of representatives of 'G' bacteria from activated sludge plants.

The name Tetracoccus cechii is proposed for two strains of the tetrad arranged cocci, previously known as 'G' bacteria, which were isolated from laboratory scale activated sludge plants in the Czech Republic and in Italy. They were morphologically, phenotypically and phylogenetically characterized and found to comprise a novel lineage in the alpha-3 group of the proteobacterial phylum in the domain Bacteria. The strains are Gram-negative and produce intracellular inclusions of poly-beta-hydroxybutyrate. Although commonly seen in activated sludge mixed liquor as cocci 1-2 microns in diameter, arranged in tetrads, in pure culture they can also grow in amorphous aggregations and the cells are generally more variable in their size and shape with coccobacilli as well as cocci being present. They are not able to grow phototrophically, nor can they reduce nitrate beyond nitrite nor grow anaerobically. The closest phylogenetic neighbours of T. cechii are Rhodobacter sphaeroides and R. capsulatus which are 93% similar by 16S rDNA comparison. Tetracoccus cechii is oxidase- and catalase-positive, non-motile and has an optimal growth temperature between 25 degrees and 35 degrees C. The 16S rRNA of T. cechii has a 21 nucleotide deletion in the V9 region (Escherichia coli positions 1258-1278) and this feature is a unique molecular synapomorphy in the alpha-3 group.

Base Sequence↗
PubMed1996

Autosomal dominant polycystic kidney disease (ADPKD) in an Italian family carrying a novel nonsense mutation and two missense changes in exons 44 and 45 of the PKD1 Gene.

Sixty-seven Italian patients with autosomal dominant polycystic kidney disease (ADPKD) were screened for mutations in the 3' unique region of the PKD1 gene, using heteroduplex DNA analysis. Novel aberrant bands were detected in 3 patients from the same family. DNA sequencing showed a C to T transition in exon 44 (C12269T), resulting in a premature stop codon (R4020X), predicted to impair the synthesis of the putative intracytoplasmic C-terminus tail of the PKD1 protein, polycystin. The mutation also generates a novel DdeI restriction site, and the abnormal restriction pattern was observed both on genomic DNA and on cDNA from the affected relatives, indicating that this is indeed the pathogenetic molecular lesion. Reverse transcriptase-polymerase chain reaction (RT-PCR) performed on lymphocyte mRNA showed that the mutant transcript is normally present and stable. No aberrantly spliced mRNAs were detected. Interestingly, the mutant PKD1 chromosome in this family also bears two missense mutations downstream (A12341G and C12384T), not found in the other ADPKD families studied.

Adolescent↗
PubMed1996

A common polymorphism in exon 46 of the human autosomal dominant polycystic kidney disease 1 gene (PKD1).

Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common single gene diseases in humans. We have identified a synonymous T to C transition polymorphism in exon 46 of the PKD1 gene (12838T-->C, Pro4209Pro). The polymorphism was present with similar frequencies in ADPKD patients and unaffected individuals. The heterozygosity, determined in 89 Italian individuals, was 0.347. The frequency of the rarer allele was 0.222. This polymorphism is easy to determine as it abolishes a naturally occurring Ddel restriction site. The availability of an additional intragenic marker in the PKD1 gene will improve the accuracy of linkage studies in ADPKD families.

Chromosome Mapping↗
PubMed1996

Detection of two different nonsense mutations in exon 44 of the PKD1 gene in two unrelated Italian families with severe autosomal dominant polycystic kidney disease.

Sixty-seven Italian patients with autosomal dominant polycystic kidney disease (ADPKD) were screened for mutations in the PKD1 gene. We used PCR, heteroduplex and single-strand conformation polymorphism DNA analysis, and automated DNA sequencing for exons 35, 36, 38, 44 and 45. We detected abnormal heteroduplexes in affected individuals from two unrelated families with clinically severe ADPKD phenotype. These changes were absent in other, unaffected members, as well as in the probands of the other families studied. DNA sequencing revealed in both cases different C to T transitions in exon 44, which created premature stop codons. Both mutations altered restriction sites, and the abnormal patterns were observed in all the affected family members. RT-PCR performed on lymphocyte mRNA showed that both the mutant and the normal transcript are represented. To our knowledge these are the first nonsense mutations described in the PKD1 gene.

Exons↗
PubMed1996

X-linked Alport syndrome: an SSCP-based mutation survey over all 51 exons of the COL4A5 gene.

The COL4A5 gene encodes the alpha5 (type IV) collagen chain and is defective in X-linked Alport syndrome (AS). Here, we report the first systematic analysis of all 51 exons of COL4A5 gene in a series of 201 Italian AS patients. We have previously reported nine major rearrangements, as well as 18 small mutations identified in the same patient series by SSCP analysis of several exons. After systematic analysis of all 51 exons of COL4A5, we have now identified 30 different mutations: 10 glycine substitutions in the triple helical domain of the protein, 9 frameshift mutations, 4 in-frame deletions, 1 start codon, 1 nonsense, and 5 splice-site mutations. These mutations were either unique or found in two unrelated families, thus excluding the presence of a common mutation in the coding part of the gene. Overall, mutations were detected in only 45% of individuals with a certain or likely diagnosis of X-linked AS. This finding suggests that mutations in noncoding segments of COL4A5 account for a high number of X-linked AS cases. An alternative hypothesis is the presence of locus heterogeneity, even within the X-linked form of the disease. A genotype/phenotype comparison enabled us to better substantiate a significant correlation between the degree of predicted disruption of the alpha5 chain and the severity of phenotype in affected male individuals. Our study has significant implications in the diagnosis and follow-up of AS patients.

Adult↗
PubMed1995

Comparison of heteroduplex and single-strand conformation analyses, followed by ethidium fluorescence visualization, for the detection of mutations in four human genes.

Non-isotopic DNA single-strand conformation analysis and heteroduplex analysis by ethidium bromide fluorescence visualization (SSCAE and HAE, respectively) were compared for the detection of 15 different naturally occurring mutations in 15 different DNA samples. The mutations included single nucleotide transitions, transversions and deletions, in CFTR (cystic fibrosis transmembrane conductance regulator), COL4A5 (collagen type IV alpha 5 chain), HEXB (hexosaminidase B), and COL1A2 (collagen type 1 alpha 2 chain) genes, responsible for diseases of medical interest. Genomic DNA from peripheral blood leukocytes or cDNA from reverse-transcribed fibroblast mRNA were amplified by polymerase chain reaction (PCR), and then analysed by two SSCAE and one HAE protocol. Fourteen out of 15 mutations (93%) were detected with one or the other method. HAE was more sensitive than SSCAE for the larger products (257-426 bp). The only undetected mutation was then identified with the use of a different primer, located farther from the mutation was then identified with the use of a different primer, located farther from the mutation site, thus increasing the combined efficiency of the two methods to 100%. We believe that combined use of SSCAE and HAE is a good, cheap and safe approach for mutation screening in a human gene.

Base Sequence↗
PubMed1995

A novel nonsense mutation in the PKD1 gene (C3817T) is associated with autosomal dominant polycystic kidney disease (ADPKD) in a large three-generation Italian family.

We have looked for disease-causing mutations in the PKD1 gene in 20 unrelated ADPKD probands from northern Italy, all members of families in which our previous studies had indicated linkage to PKD1. Using PCR with primer pairs located in the 3' unique region of the gene and heteroduplex DNA analysis, we have detected novel aberrant bands in five affected individuals from the same family, which were absent in 13 other unaffected family members. Cloning and automated DNA sequencing revealed a C to T transition at nucleotide position 3817 of the published cDNA sequence, which created a premature stop codon. The mutation destroyed a MspA1I restriction site, and the abnormal restriction pattern was observed on genomic DNA from all the affected family members. RT-PCR and restriction analysis performed on peripheral white blood cell mRNA showed that in the affected members, both the mutant and the normal transcript are represented. This mutation was not found in the probands of the other families studied. To our knowledge, this is the first nonsense mutation described in the PKD1 gene.

Adult↗
PubMed1995

A novel missense mutation in exon 3 of the COL4A5 gene associated with late-onset Alport syndrome.

We have identified a novel missense transition (362G-->A) in exon 3 of the COL4A5 gene in a male patient with late-onset Alport syndrome. We used non-isotopic single strand conformation polymorphism, heteroduplex analysis, and automated DNA sequencing. The mutation changes a conserved glycine at codon 54 for an aspartic acid (Gly54Asp), which abolishes a BstNI site. Using restriction analysis, we identified the heterozygous carrier status in the two daughters of the proband. Our findings are in keeping with the hypothesis that slower progressive forms of Alport syndrome are more often associated with missense mutations rather than large deletions or frameshifts. This is the first mutation described in the N-terminus triple helical 7S domain of the COL4A5 gene in an Alport syndrome patient.

Adolescent↗
PubMed1995

Gene linkage analysis and DNA based detection of autosomal dominant polycystic kidney disease (ADPKD) in a newborn infant. Case report.

Bilateral polycystic kidneys were detected by ultrasound at 23 weeks gestation in a male fetus. Bilateral renal cysts were subsequently also found in the asymptomatic propositus' mother and grandmother, suggesting the diagnosis of autosomal dominant polycystic kidney disease (ADPKD). The renal ultrasonograms showed cortical cysts with normal or decreased-sized kidneys. Renal function was normal. Seven available members of the family were genotyped for flanking DNA markers tightly linked to the PKD1 gene on chromosome 16p, and for a polymorphism close to a second putative disease gene (PKD2) on chromosome 2. The genetic linkage approach allowed us to detect with a high degree of accuracy the ADPKD1 at risk chromosome in the three patients, as well as in a 28-year-old unaffected female. This report illustrates the feasibility and the usefulness of recent molecular genetic strategies for diagnostic purposes in ADPKD, especially when clinical and radiological data are atypical. Furthermore, it also confirms that early or very early onset forms of the disease are not uncommon, and should be considered in the differential diagnosis of childhood cystic disease.

Blotting, Southern↗