title: Comprehensive and rapid genotyping of mutations and haplotypes in congenital bilateral absence of the vas deferens and other cystic fibrosis transmembrane conductance regulator-related disorders.
abstract: Available commercial kits only screen for the most common cystic fibrosis transmembrane conductance regulator (CFTR) mutations causing classic cystic fibrosis and for the Tn variant in IVS8. However, full scanning of CFTR is needed for the diagnosis of patients with cystic fibrosis or CFTR-related disorders (including congenital bilateral absence of the vas deferens) bearing rare mutations. Standard strategies for detecting point mutations rely on extensive scanning of the gene by denaturing gradient gel electrophoresis or denaturing high performance liquid chromatography, which are time-consuming. Moreover, the haplotyping of IVS8-(TG)m and Tn tracts is still challenging despite several recent improvements. We have optimized both the detection of mutations and the haplotyping of IVS8 polyvariants in developing two methods: i) a rapid and robust direct sequence analysis of all exons/flanking introns of the CFTR gene based on single condition touchdown amplification/sequencing in 96-well plates, and ii) a fluorescent assay that allows haplotyping of IVS8-(TG)mTn even without family linkage study. Combined with search for rare large rearrangements, this strategy detected 87.9% of CFTR defects in congenital bilateral absence of the vas deferens patients, a proportion considerably higher than those usually reported. These highly efficient tests, scanning each sample in a few days, greatly improve the genotyping of patients with CFTR-related symptoms and may be particularly important in emergency situations such as fetus with hyperechogenic bowel suggestive of cystic fibrosis.
authors.affiliation: Laboratoire de Génétique Moléculaire, Institut Universitaire de Recherche Clinique, 641 av du doyen Gaston Giraud, Montpellier cedex 5, France.
authors.forename: Corinne
authors.initials: C
authors.lastname: Bareil
authors.forename: Caroline
authors.initials: C
authors.lastname: Guittard
authors.forename: Jean-Pierre
authors.initials: JP
authors.lastname: Altieri
authors.forename: Carine
authors.initials: C
authors.lastname: Templin
authors.forename: Mireille
authors.initials: M
authors.lastname: Claustres
authors.forename: Marie
authors.initials: M
authors.lastname: des Georges
mesh_terms: D003550:Cystic Fibrosis / Q000235:genetics*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics*; D004252:DNA Mutational Analysis; D006239:Haplotypes; D006801:Humans; D008297:Male; D009154:Mutation / Q000235:genetics*; D011110:Polymorphism, Genetic; D015203:Reproducibility of Results; D014649:Vas Deferens / Q000002:abnormalities*
chemical_list: D019005:Cystic Fibrosis Transmembrane Conductance Regulator