Extensive sequencing of the cystic fibrosis transmembrane regulator gene: assay validation and unexpected benefits of developing a comprehensive test.
OBJECTIVE
To develop a sequencing assay for the gene to identify mutations in patients with cystic fibrosis (CF).


METHODS
An automated assay format was developed to sequence all exons and splice junctional sequences, the promotor region, and parts of introns 11 and 19.


RESULTS
After validating the assay using 20 known samples, DNA of seven patients, four of whom were heterozygous for a known CF mutation, was sequenced. Known CF mutations were detected in seven of the eight chromosomes, and a novel missense mutation was detected in the eighth. In addition, this assay allowed 14 ambiguous results obtained using the Roche CF gold strips to be resolved. Three false-positive diagnoses were prevented; a different mutation at the same codon was identified in two patients and confirmation was provided in the remaining nine cases.


CONCLUSIONS
Sequencing of the gene provides important information for CF patients and is a valuable adjunct to a carrier screening program to resolve ambiguities in panel testing.
Strom
Charles M
CM
Molecular Genetics Laboratory, Quest Diagnostics Nichols Institute, San Juan Capistrano, California 92690, USA.
Huang
Donghui
D
Chen
Christina
C
Buller
Arlene
A
Peng
Mei
M
Quan
Franklin
F
Redman
Joy
J
Sun
Weimin
W
D003062:Codon / Q000235:genetics; D003550:Cystic Fibrosis / Q000175:diagnosis / Q000453:epidemiology / Q000235:genetics*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics*; D004247:DNA / Q000235:genetics; D004252:DNA Mutational Analysis / Q000379:methods*; D005091:Exons; D005820:Genetic Testing; D006801:Humans; D007438:Introns; D008969:Molecular Sequence Data; D009154:Mutation
C505032:CFTR protein, human; D003062:Codon; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D004247:DNA