Showing papers by "Francesco Salvatore published in 1999"

Efficiency of Two Different Nine-Loci Short Tandem Repeat Systems for DNA Typing Purposes

Abstract: Background: Genotyping based on short tandem repeat (STR) regions is widely used in human identification and parentage testing, in gene mapping studies, and as an approach to studies on the etiopathogenesis and diagnosis of hereditary diseases. We wished to study a new analytical approach that uses capillary electrophoresis and multicolor fluorescence in place of slab gel electrophoresis. Methods: We evaluated the efficiency for parentage and forensic purposes of the AmpF L STR Profiler PlusTM typing kit that is used with the ABI Prism 310 Genetic Analyzer (System-2 STR), and that of a widely used panel of nine STRs analyzed with conventional slab-gel electrophoresis followed by radioactive detection (System-1 STR). System-2 STR, based on automated capillary electrophoresis and automated sizing of the alleles by Genotyper 2.0 software, was used to determine the allele frequency of the nine loci in 157 Caucasian subjects from southern Italy. On the basis of the data obtained, we submitted 40 trios to parentage testing. Results: A higher median probability of paternity attribution and power of exclusion were obtained with System-2 STR vs System-1 STR: respectively, 99.99% and 99.95% ( P <0.05) for attribution; and five and four excluding loci ( P <0.05) for exclusion. The most informative and highly discriminating loci were D18S51 , D21S11 , and FGA . The combined probability of matching-by-chance for all nine STRs was 1.36 × 10−12 for System-2 compared with 1.11 × 10−7 obtained with the other system. The internal standard and allelic ladder of the System-2 STR facilitated accurate and precise genotyping; furthermore, System-2 STR and was faster than the conventional System-1 STR. Conclusions: The System-2 STR allows rapid testing with higher probabilities of attribution and a higher power of exclusion than with the comparison method with slab-gel electrophoresis.

Detection of five rare cystic fibrosis mutations peculiar to Southern Italy: implications in screening for the disease and phenotype characterization for patients with homozygote mutations.

Abstract: Background: The search for the eight most frequent mutations (i.e., ΔF508, G542X, W1282X, N1303K, 1717-1G→A, R553X, 2183AA→G, and I148T) by allele-specific oligonucleotide dot-blot analysis revealed 78% of 396 cystic fibrosis alleles in Southern Italy. The observation of frequent haplotypes on the unidentified cystic fibrosis alleles suggested that a few mutations could account for a large number of unidentified alleles. Methods: We screened most of the coding sequence of the cystic fibrosis transmembrane regulator gene by denaturing gradient gel electrophoresis to determine the spectrum of these mutations in 68 unrelated cystic fibrosis patients bearing one or both unidentified mutations. Results: The screening revealed five mutations, R1158X, 711+1G→T, 4016insT, L1065P, and G1244E, each of which had a frequency of 1.3–1.8% (7% collectively). The 7% increase in the detection rate (85% vs 78%) reduces by >50% the residual risk of being cystic fibrosis carriers for couples who had tested negative by molecular analysis. We therefore designed a second allele-specific oligonucleotide set to analyze the five mutations. Among the patients analyzed, one patient homozygous for the L1065P mutation expressed a mild pulmonary and intestinal form of the disease with pancreatic insufficiency. Two other patients, homozygous for mutations R1158X and 4016insT, both expressed a severe cystic fibrosis phenotype. Conclusions: Five cystic fibrosis mutations are peculiar to patients from Southern Italy. The method described for their analysis is efficient, inexpensive, and can be semi-automated by use of a robotic workstation. The results obtained in patients from Southern Italy may have an impact on laboratories in other countries, given the large migrations of populations from Southern Italy to other countries in the last two centuries.

Novel six-nucleotide deletion in the hepatic fructose-1,6-bisphosphate aldolase gene in a patient with hereditary fructose intolerance and enzyme structure-function implications.

Abstract: Hereditary fructose intolerance (HFI) is an autosomal recessive human disease that results from the deficiency of the hepatic aldolase isoenzyme. Affected individuals will succumb to the disease unless it is readily diagnosed and fructose eliminated from the diet. Simple and non-invasive diagnosis is now possible by direct DNA analysis that scans for known and unknown mutations. Using a combination of several PCR-based methods (restriction enzyme digestion, allele specific oligonucleotide hybridisation, single strand conformation analysis and direct sequencing) we identified a novel six-nucleotide deletion in exon 6 of the aldolase B gene (delta 6ex6) that leads to the elimination of two amino acid residues (Leu182 and Val183) leaving the message inframe. The three-dimensional structural alterations induced in the enzyme by delta 6ex6 have been elucidated by molecular graphics analysis using the crystal structure of the rabbit muscle aldolase as reference model. These studies showed that the elimination of Leu182 and Val183 perturbs the correct orientation of adjacent catalytic residues such as Lys146 and Glu187.

A case of discordance between genotype and phenotype in a malignant hyperthermia family.

Abstract: Malignant hyperthermia (MH) is an inherited autosomal dominant pharmacogenetic disorder and is the major cause of anaesthesia-induced death. Malignant hyperthermia susceptibility is usually diagnosed by the in vitro contracture test (IVCT) performed on fresh muscle biopsies exposed to caffeine and halothane, respectively. Around 50% of affected families are linked to the ryanodine receptor (RYR1) gene. The human RYR1 gene maps to chromosome 19q13.1 and encodes a protein that associates as a homotetramer and acts as a calcium-release channel from the sarcoplasmic reticulum. To date, 17 mutations have been identified in the coding region of the RYR1 gene and appear to be associated to the MH-susceptible phenotype. Here we describe a rare case of discordance between genotype (characterised by the presence of the Arg614Cys mutation in the RYR1 gene) and MH-normal typed phenotype. Although the IVCT remains a very reliable procedure for the assessment of MH status, genetic data can provide in some cases an additional aid to clinical diagnosis.

Serum γ-Glutamyltransferase Isoform Complexed to LDL in the Diagnosis of Small Hepatocellular Carcinoma

Abstract: Up to one-third of cirrhoses evolve into hepatocellular carcinoma (HCC), with ∼90% of these cases developing on a preexisting cirrhosis (1). However, the detection of early-stage neoplastic transformation remains a challenge. In a recent series of studies, we reported that multivariate discriminant functions based on the serum concentrations of various biochemical indexes efficiently discriminate between chronic hepatobiliary diseases at different stages of their natural history (2)(3)(4). The multivariate analysis also allowed us to detect the neoplastic evolution in five of six cirrhotic patients ∼6 months earlier than instrumental approaches (2). This result prompted us to evaluate the efficiency of serum biochemical indexes in detecting small HCC, i.e., <3 cm (5), developed …