A 96-well formatted method for exon and exon/intron boundary full sequencing of the CFTR gene

Analytical Biochemistry
Marco LucarelliRoberto Strom

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 ...Continue Reading

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Citations

Jun 17, 2011·Journal of Cystic Fibrosis : Official Journal of the European Cystic Fibrosis Society·C BombieriC Ferec
Aug 14, 2010·Genetics in Medicine : Official Journal of the American College of Medical Genetics·Marco LucarelliSerena Quattrucci
Jun 2, 2011·Clinical Chemistry and Laboratory Medicine : CCLM·Giampiero FerragutiMarco Lucarelli
Feb 1, 2008·Expert Opinion on Medical Diagnostics·Maria Rosaria D'ApiceFederica Sangiuolo
Dec 6, 2011·Molecular Genetics and Metabolism·S K CordovadoP W Mueller
Jun 20, 2012·American Journal of Medical Genetics. Part a·Samantha A SchrierMatthew A Deardorff
Sep 25, 2012·Journal of Cystic Fibrosis : Official Journal of the European Cystic Fibrosis Society·Sha TangPing Fang
May 6, 2008·Journal of Cystic Fibrosis : Official Journal of the European Cystic Fibrosis Society·C CastellaniJ S Elborn
Apr 26, 2011·Clinica Chimica Acta; International Journal of Clinical Chemistry·Marie C EarleyW Harry Hannon
May 10, 2016·The Journal of Molecular Diagnostics : JMD·Marco LucarelliSerena Quattrucci
Dec 28, 2018·Fetal and Pediatric Pathology·Pratiksha ChhedaSushant Vinarkar
Dec 24, 2018·BMC Pulmonary Medicine·Vito TerlizziFederica Zarrilli

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