CFTR gene variant detection in moroccan individuals via nanopore long-read sequencing

Cystic fibrosis (CF) is an autosomal recessive disease resulting from pathogenic CF transmembrane conductance regulator (CFTR) pathogenic gene variants. While CF's frequency varies among ethnicities, its epidemiology, clinical manifestations, and mutational profiles in Africa still must be explored due to the absence of a comprehensive public health strategy there. This study postulates that complete sequencing of CFTR using Oxford Nanopore Technology (ONT)-based long-read sequencing enhances the diagnostic yield.

To amplify ∼25-kb fragments covering the whole CFTR gene (NM_000492.4), we designed 11 primer pairs, and barcoded libraries were prepared and sequenced on ONT flow cells (R10.4.1) using an Mk1C device. Variant pathogenicity was assessed by expressing the variant channel in HEK293 cells and examining expression through immuno-blotting. With sequencing data obtained from 9 Moroccan individuals (6 probands with suspected CF diagnoses and 3 parents), we identified the following variants: c.680T>G p.Leu227Arg, c.1521_1523del p.Phe508del, c.3484C>T p.Arg1162*, c.1090T>C p.Ser364Pro, c.3233T>C p.Phe1078Ser and c.2991G>C p.Leu997Phe. The analytical pipeline we developed allowed the phasing of the variants. Sanger sequencing confirmed all these results. The previously uncharacterised CFTR variants p.Ser364Pro and p.Phe1078Ser exhibit diminished expression in HEK293 cells, substantiating their pathogenic nature, with p.Phe1078Ser responding positively to the in vitro treatment with CFTR-modulator molecules.

This study demonstrates the potential of long-read sequencing using ONT as an efficient means to detect CF-causing variants in African populations. Given the significant genetic heterogeneity in Africa, this technique can serve as an affordable molecular screening tool for CF, especially in areas with constrained access to genetic screening.