IDENTIFICATION OF EXON 12 MUTATIONS IN THE CFTR GENE USING A COST-EFFECTIVE CAPILLARY ELECTROPHORESIS (CE) ASSAY

Abstract

Cystic Fibrosis (CF) is an autosomal recessive genetic disorder caused by mutations in the CFTR gene. The F508del mutation in exon 11 of the CFTR gene is prevalent worldwide, affecting approximately 70% of CF patients, but it is less common in the local Pakistani population. Exon 12 mutations, such as S549N and S549R, have been observed in CF patients with Pakistani ancestry. This research provides accessible and affordable mutation detection using reproducible and cost-effective capillary-electrophoresis (CE) methods. Therefore, this study investigated whether local Pakistani CF patients lacking exon 11 mutations, such as F508del, harbor any common or rare CF-causing mutations in exon 12 of the CFTR gene using a cost-effective Sanger sequencing assay. To achieve this, a new set of primers was meticulously designed and optimized for the amplification of exon 12 through PCR. Additionally, Sanger sequencing-based CE assay was fine-tuned for the sequencing of exon 12 amplicons. The Sanger sequencing results revealed no mutations in exon 12 among the 17 local CF patients who participated in the study. This absence of mutations in both exons 11 and 12 suggests that CF-causing mutations may be located in these patients' other regions of the CFTR gene. Furthermore, it indicates that exon 12 mutations are less prevalent among local Pakistani CF patients. The optimized   Sanger sequencing-based CE assay is at least five times more cost-effective and can be employed for the identification of mutations in any sequence with a length of up to 500 bases.