title: Complete gene scanning by temperature gradient capillary electrophoresis using the cystic fibrosis transmembrane conductance regulator gene as a model.
abstract: Many inherited diseases involve large genes with many different mutations. Identifying a wide spectrum of mutations requires an efficient gene-scanning method. By differentiating thermodynamic stability and mobility of heteroduplexes from heterozygous samples, temperature gradient capillary electrophoresis (TGCE) was used to scan the entire coding region of the cystic fibrosis transmembrane conductance regulator gene. An initial panel (29 different mutations) showed 100% agreement between TGCE scanning and previously genotyped results for heterozygous samples. Different peak patterns were observed for single base substitutions and base insertions/deletions. Subsequently, 12 deidentified clinical samples genotyped as wild type for 32 mutations were scanned for the entire 27 exons. Results were 100% concordance with the bidirectional sequence analysis. Ten samples had nucleotide variations including a reported base insertion in intron 14b (2789 + 2insA) resulting in a possible mRNA splicing defect, and an unreported missense mutation in exon 20 (3991 G/A) with unknown clinical significance. This methodology does not require labeled primers or probes for detection and separation through a temperature gradient eliminates laborious temperature optimization required for other technologies. TGCE automation and high-throughput capability can be implemented in a clinical environment for mutation scanning with high sensitivity, thus reducing sequencing cost and effort.
authors.affiliation: Institute for Clinical and Experimental Pathology, Associated Regional University Pathologists Laboratories, 500 Chipeta Way, Salt Lake City, UT 84108-1221, USA. choulb@aruplab.com
authors.forename: Lan-Szu
authors.initials: LS
authors.lastname: Chou
authors.forename: Friederike
authors.initials: F
authors.lastname: Gedge
authors.forename: Elaine
authors.initials: E
authors.lastname: Lyon
mesh_terms: D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000379:methods*; D019075:Electrophoresis, Capillary / Q000379:methods*; D005820:Genetic Testing / Q000379:methods*; D015894:Genome, Human; D006801:Humans; D007438:Introns / Q000235:genetics; D008957:Models, Genetic; D009154:Mutation / Q000235:genetics
chemical_list: C505032:CFTR protein, human; D019005:Cystic Fibrosis Transmembrane Conductance Regulator