Diagnosis of cystic fibrosis by new generation sequencing

The mutations within the CFTR gene affect the lungs, the digestive tract, the sweat glands or the pancreas and the reproductive system in males. Over 1800 CFTR mutations have been listed. Extensive genetic and clinical heterogeneity of the disease exists, depending on the type of CFTR gene mutation and the ethnic origin of the parents.

CFTR gene mutations can be screened for using several different methods. Commercial diagnostic kits can detect the most frequently encountered mutations (between 20 and 36 mutations depending on the kit used), which cover approximately 80% of the mutations responsible for cystic fibrosis. Sanger sequencing enables the entire coding sequence of the CFTR gene to be screened and offers better detection for the mutations for this gene. However, the method remains cumbersome and costly.

New generation sequencing techniques (NGS: MiSeq, Illumina) enable high sensitivity analysis that is rapid and less costly of the entire CFTR gene when compared to other reference method such as Sanger sequencing.

In Biomnis, a pilot study has been carried out on 70 samples from patients and 7 external quality control samples. This study, which used the Nextera XT® (Illumina) kit and multiplex PCR of 32 amplicons, simultaneously analysed the 5′ and 3′ UTRs, 27 CFTR exons and the intronic regions implied in the alternative splicing of the CFTR gene (flanking intronic junctions of the 27 exons and intron 19 (3849+10kb C>T) and 11 (1811+1.6kb A>G) mutations).

The total number of mutations currently identified in the CFTR gene is 1818. The total number of mutations included in the gene screening by the OLA® (Abbott) kit is 32. The number of mutations included in the NGS method for the CFTR gene is 1707.