Three complex alleles of CFTR gene identified in Lebanese, Egyptian and French population and their potential impact on splicing

Abstract

Cystic Fibrosis (CF) in Arab Mediterranean countries has a different CFTR mutational profile if compared either to Caucasians or in the Arabian Peninsula. The c.3909C>G (N1303K, p.Asn1303Lys) mutation of the Cystic Fibrosis Transmembrane Conductance Regulator gene (CFTR). This mutation represents a higher frequency in the Mediterranean countries in association with different polymorphisms or mutations in cis position constituting various complex alleles. N1303K mutation induces many phenotypes, especially pancreatic insufficiency from mild to severe and it is associated in cis with other polymorphisms. The aim of this investigation is therefore to screen complex alleles carrying N1303K mutation among Lebanese, Egyptian and French patients. All exons of the CFTR and their flanking regions were performed by PCR amplification, followed by automated direct DNA sequencing. Two complex alleles are more frequent corresponding to Wild Type and mutated haplotype. Besides that two other very rare complex alleles have been detected, one in Egyptian and French samples, and then another one in Lebanon samples. We have studied their impact on the CFTR mRNA splicing using a minigene strategy. Constructs containing wild-type and mutant CFTR cloned into the pTBNdeI hybride minigene have been expressed in HeLa, HT29 and HEK293 cells. RT-PCR analysis of mRNA using β-globin-specific primers revealed that N1303K and the polymorphisms associated with cis induce weak abnormal splicing and a modification of the quality and the quantity of CFTR protein. These different associations of identified polymorphisms with N1303K in cis could have an impact on the severity of the disease.

Beyond Kaftrio : mechanistic insights to maximize N1303K-CFTR rescue

“…The level of improvement was similar to that observed for F508del patients. The variability of the response was not explained by lung severity at baseline nor additional mutations in cis of N1303K (34). Interestingly, we identified S/Z alpha1 antitrypsin genotype and gain of function SLC26A4 and SCNN1A (35,36) variants in non or transient responders, suggesting that additional genes must be investigated in pwCF displaying an atypical response to ETI.…”

“…Investigation of potential other genetic factors accounting for the variability in response to ETI was performed in non or transient responders from the French cohort (#6, #8, #9, #15, and #16) and in 2 patients from the Italian cohort (patient ME084 and ME087) respectively weak responder and best responder (Supplemental Manuscript). Only the frequent haplotype described in cis with N1303K (c.[744-33GATT [6];869+11C>T]) was observed (34) and no other CFTR variant was identified as a complex allele. Three of the five French patients carried variants in other genes: patient #6 was a compound heterozygote for SERPINA1 Z and S variants, a genotype responsible for alpha1-antitrypsin deficiency ; Patient #15 carried a pathogenic variant in the SLC26A4 gene, R185T, involved in Pendred syndrome (35), patient #16 carried a gain-of-function variant in the SCNN1A gene, R181W (36).…”

“…Twenty-eight French patients entered a compassionate program and were evaluated at baseline and at 1 to 2 months of ETI for Forced Expiratory Volume, expressed in percentage predicted (ppFEV1) and sweat chloride concentration (mmol/L) completed by β-adrenergic secretion in selected patients (Institutional Review Board of the Société de Pneumologie de Langue Française (#2020-003) (14,18,33). Extensive genetic screening was performed in nonresponder patients (e.g ; less than 5% improvement in ppFEV1) or patients who experienced a respiratory degradation after initial improvement, including next generation sequencing to search for CFTR complex alleles and variants of genes that were shown to modify airway surface liquid homeostasis (TMEM16A, SLC26A4, ATP12A, and SCNN1A/B/G), or respiratory status such as SERPINA1 gene (34).…”