A simple, fast and inexpensive method for mutation scanning of CFTR gene

• Published in: BMC medical genetics Vol. 18; no. 1; p. 58
• Main Authors: Figueredo Lago, Juan Emilio, Armas Cayarga, Anny, González González, Yaimé Josefina, Collazo Mesa, Teresa
• Format: Journal Article
• Language: English
• Published: England BioMed Central 25-05-2017; BMC
• Subjects: Allele-specific multiplex real-time PCR; Alleles; Benign; Biopsy; CF-causing mutations; Conductance; Cost-Benefit Analysis; Cystic fibrosis; Cystic Fibrosis - diagnosis; Cystic Fibrosis - genetics; Cystic fibrosis transmembrane conductance regulator; Cystic Fibrosis Transmembrane Conductance Regulator - genetics; Deoxyribonucleic acid; DNA; DNA Mutational Analysis - methods; Genetic disorders; Genetic screening; Genotype; Genotyping Techniques; Humans; Infant, Newborn; Latin America; Methods; Mutation; Mutation scanning; Neonates; Point mutation; Real-Time Polymerase Chain Reaction; Reproducibility of Results; Scanning; Technical Advance; Zygosity; Cuba; Mutation scanning; Allele-specific multiplex real-time PCR; CF-causing mutations
• ISSN: 1471-2350; 1471-2350
• DOI: 10.1186/s12881-017-0420-9

Mutation scanning methods in Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene may not distinguish between a Cystic Fibrosis (CF) causing mutation and a benign variant. We have developed a simple and fast method for scanning 14 selected CF-causing mutations which have high frequency in Latin America.
In a group of 35 samples coming from CF patients previously characterized and using two allele-specific real-time multiplex PCRs targeting wild-type and mutant alleles respectively, we detect the presence of mutations by analyzing the Ct variation. Twenty-five samples without mutations considered non-carrier samples, were also included in this study. High Resolution Melting Analysis (HRMA) was performed to confirm the result of the scanning method and in most cases allowed the genotype determination.
The results validate this method for CF diagnosis. A least one CFTR gene mutation was detected in the samples of CF patients, as predicted by their ΔCt values. The ΔCt value also indicated the zygosity of the sample according to the distribution of CFTR gene mutations. In most cases, HRMA allowed the identification of the mutation(s), thereby confirming the efficiency of this scanning strategy.
This strategy simplifies the detection of CF, reducing the analysis of 14 CF-causing mutations to two parallel reactions and making the procedure compatible with the analysis of a large number of samples. As the method is fast, inexpensive and highly reliable, it is advisable for scanning CFTR gene mutations in newborns, patients with a clinical suspicion of CF as well as in the preconception carrier screening.