title: A 96-well formatted method for exon and exon/intron boundary full sequencing of the CFTR gene.
abstract: Full genotypic characterization of subjects affected by cystic fibrosis (CF) is essential for the definition of the genotype-phenotype correlation as well as for the enhancement of the diagnostic and prognostic value of the genetic investigation. High-sensitivity diagnostic methods, capable of full scanning of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, are needed to enhance the significance of these genetic assays. A method for extensive sequencing of the CFTR gene was optimized. This method was applied to subjects clinically positive for CF and to controls from the general population of central Italy as well as to a single subject heterozygous for a mild mutation and with an uncertain diagnosis. Some points that are crucial for the optimization of the method emerged: a 96-well format, primer project and purification, and amplicon purification. The optimized method displayed a high degree of diagnostic sensitivity; we identified a subset of 13 CFTR mutations that greatly enhanced the diagnostic sensitivity of common methods of mutational analysis. A novel G1244R disease causing mutation, leading to a CF phenotype with pancreatic sufficiency but early onset of pulmonary involvement, was detected in the subject with an uncertain diagnosis. Some discrepancies between our results and previously published CFTR sequence were found.
authors.affiliation: Department of Cellular Biotechnologies and Hematology, University of Rome La Sapienza, 00161 Rome, Italy. lucarelli@bce.uniroma1.it
authors.forename: Marco
authors.initials: M
authors.lastname: Lucarelli
authors.forename: Lorena
authors.initials: L
authors.lastname: Narzi
authors.forename: Rita
authors.initials: R
authors.lastname: Piergentili
authors.forename: Giampiero
authors.initials: G
authors.lastname: Ferraguti
authors.forename: Francesco
authors.initials: F
authors.lastname: Grandoni
authors.forename: Serena
authors.initials: S
authors.lastname: Quattrucci
authors.forename: Roberto
authors.initials: R
authors.lastname: Strom
mesh_terms: D001483:Base Sequence; D003550:Cystic Fibrosis / Q000175:diagnosis* / Q000235:genetics; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000379:methods*; D005091:Exons; D006801:Humans; D007438:Introns; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D011897:Random Allocation
chemical_list: D019005:Cystic Fibrosis Transmembrane Conductance Regulator