Next-Generation Sequencing for Screening Analysis of Cystic Fibrosis: Spectrum and Novel Variants in a South-Central Italian Cohort

Abstract

The incidence of cystic fibrosis (CF) and the spectrum of cystic fibrosis transmembrane conductance regulator (CFTR) gene variants differ among geographic regions. Differences in CF carrier distribution are also reported among Italian regions. We described the spectrum of the CFTR variants observed in a large group of subjects belonging from central-southern Italy. We also provide a predictive evaluation of the novel variants identified. CFTR screening was performed in a south-central Italian cohort of 770 subjects. We adopted a next-generation sequencing (NGS) approach using the Devyser CFTR NGS kit on the Illumina MiSeq System coupled with Amplicon Suite data analysis. Bioinformatics evaluation of the impact of novel variants was described. Overall, the presence of at least one alternative allele in the CFTR gene was recorded for 23% of the subjects, with a carrier frequency of CF pathogenic variants of 1:12. The largest sub-group corresponded to the heterozygous carriers of a variant with a conflicting interpretation of pathogenicity. The common CFTR p.(Phe508del) pathogenic variants were identified in 37% of mutated subjects. Bioinformatics prediction supported a potential damaging effect for the three novel CFTR variants identified: p.(Leu1187Phe), p.(Pro22Thr), and c.744-3C > G. NGS applied to CF screening had the benefit of: effectively identifying asymptomatic carriers. It lies in a wide overview of CFTR variants and gives a comprehensive picture of the carrier prevalence. The identification of a high number of unclassified variants may represent a challenge whilst at the same time being of interest and relevance for clinicians.

Keywords: CFTR; cystic fibrosis; cystic fibrosis carriers; cystic fibrosis transmembrane conductance regulator gene; next-generation sequencing.

Figure 1

Classification of CFTR variants. The pie chart shows the different groups of CFTR alterations identified in the cohort of the study, sliced by color. Annotations were in accordance with ClinVar database (accessed on 15 June 2023). CIP: conflicting interpretation of pathogenicity; VUS: variants of uncertain significance; SNVs: single-nucleotide variants.

Figure 2

Distribution of CFTR identified variants in the context of protein structure. The figure shows the linear map of the CFTR gene (NM_000492) and the exon/intron location of the genetic variants. Pathogenic and likely pathogenic variants are reported above (purple). Variants of uncertain significance (VUS, red), variants with conflicting interpretation of pathogenicity (CIP, orange), and novel single-nucleotide variants (blue, SNVs) are reported below. Protein domains are represented by different colored areas (https://proteinpaint.stjude.org/ (accessed on 15 June 2023)).

Figure 3

Pathogenic and likely pathogenic CFTR sequence variants (n = 23) distribution among the 62 carriers identified in our cohort. * One carrier of the complex allele: p.(Phe508del)/p.(Asn1303Lys); ** one carrier of the complex allele: p.(Leu1077Pro)/p.(Asp192Gly).

Figure 4

Details of the ClinVar interpretations for each variant with conflicting interpretation of pathogenicity identified in the study (accessed on 15 June 2023).

Figure 5

(A): Tridimensional structure of the CFTR protein as of the 5AUK structure in PDB data repository. Protein moieties are colored according to the vibrational entropy change upon mutation 22 Pro → Thr. Blue shades are representative of a rigidification of the structure while red shades indicate a gain in flexibility. Interatomic interactions of the wild-type Pro22 (B) and mutant Thr22 (C) protein. Wild-type and mutant residues are colored in light green and are represented along with the surrounding residues that are involved in any type of interaction.