The multi-faceted nature of 15 CFTR exonic variations: Impact on their functional classification and perspectives for therapy.

BACKGROUND: The majority of variants of unknown clinical significance (VUCS) in the CFTR gene are missense variants. While change on the CFTR protein structure or function is often suspected, impact on splicing may be neglected. Such undetected splicing default of variants may complicate the interpretation of genetic analyses and the use of an appropriate pharmacotherapy. METHODS: We selected 15 variants suspected to impact CFTR splicing after in silico predictions on 319 missense variants (214 VUCS), reported in the CFTR-France database. Six specialized laboratories assessed the impact of nucleotide substitutions on splicing (minigenes), mRNA expression levels (quantitative PCR), synthesis and maturation (western blot), cellular localization (immunofluorescence) and channel function (patch clamp) of the CFTR protein. We also studied maturation and function of the truncated protein, consecutive to in-frame aberrant splicing, on additional plasmid constructs. RESULTS: Six of the 15 variants had a major impact on CFTR splicing by in-frame (n = 3) or out-of-frame (n = 3) exon skipping. We reclassified variants into: splicing variants; variants causing a splicing defect and the impairment of CFTR folding and/or function related to the amino acid substitution; deleterious missense variants that impair CFTR folding and/or function; and variants with no consequence on the different processes tested. CONCLUSION: The 15 variants have been reclassified by our comprehensive approach of in vitro experiments that should be used to properly interpret very rare exonic variants of the CFTR gene. Targeted therapies may thus be adapted to the molecular defects regarding the results of laboratory experiments.

Genetic diagnosis in practice: From cystic fibrosis to CFTR-related disorders.

Cystic fibrosis (CF) is a channelopathy caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR) protein. Diagnosis of CF has long relied on a combination of clinical (including gastrointestinal and/or respiratory) symptoms and elevated sweat chloride concentration. After cloning of the CFTR gene in 1989, genetic analysis progressively became an important aspect of diagnosis. Although combination of sweat test and genetic analysis have simplified the diagnosis of CF in most cases, difficult situations remain, especially in cases that do not fulfill all diagnostic criteria. Such situations are most frequently encountered in patients presenting with a single-organ disease (e.g., congenital absence of the vas deferens, pancreatitis, bronchiectasis) leading to a diagnosis of CFTR-related disorder, or when the presence/ absence of CF is not resolved after newborn screening. This article reviews the diagnostic criteria of CF, with special emphasis on genetic testing. © 2020 French Society of Pediatrics. Published by Elsevier Masson SAS. All rights reserved.

Functional characterization and phenotypic spectrum of three recurrent disease-causing deep intronic variants of the CFTR gene.

BACKGROUND: The CFTR genotype remains incomplete in 1% of Cystic Fibrosis (CF) cases, because only one or no disease-causing variants is detected after extended analysis. This fraction is probably higher in CFTR-Related Disorders (CFTR-RD). Deep-intronic CFTR variants are putative candidates to fill this gap. However, the recurrence, phenotypic spectrum and full molecular characterization of newly reported variants are unknown. METHODS: Minigenes and analysis of CFTR transcripts in nasal epithelial cells were used to determine the impact on CFTR splicing of intronic variants that we previously identified by next generation sequencing of the whole CFTR locus. Phenotypic data were collected in 19 patients with CF and CFTR-RD, in whom one of the deep intronic variants has been detected. RESULTS: Three deep-intronic variants promoted the inclusion of pseudo-exons (PE) in the CFTR transcript, hindering the synthesis of a functional protein. The c.2989-313A > T variant, detected in four patients with CF or CFTR-RD from three different families, led to the inclusion of a 118 bp PE. The c.3469-1304C > G variant promoted the inclusion of a 214 bp-PE and was identified in five patients with CF from four families. Haplotype analysis confirmed that this variant was associated with one CF chromosome of African origin. The most represented variant in our cohort was the c.3874-4522A > G, detected in 10 patients with various phenotypes, from male infertility to CF with pancreatic insufficiency. CONCLUSION: These three deep intronic CFTR variants are associated with a large phenotypic spectrum, including typical CF. They should be included in CF diagnostic testing and carrier screening strategies.

Congenital bilateral absence of the vas deferens: the impact of spermatogenesis quality on intracytoplasmic sperm injection outcomes in 108 men.

In azoospermic men with congenital bilateral absence of the vas deferens (CBAVD), it is not known whether the outcomes of intracytoplasmic sperm injection (ICSI) depend on the quality of testicular spermatogenesis (as determined histopathologically). We retrospectively studied the impact of spermatogenesis quality on ICSI outcomes in 108 azoospermic men with CBAVD consulting in a university hospital's department of andrology and reproductive biology. As part of an ICSI program, sperm samples were obtained from the epididymis [by microsurgical epididymal sperm aspiration (MESA); n = 47] or the testis [by testicular sperm extraction (TESE); n = 14] or both (MESA + TESE, n = 47). In the TESE group (i.e., TESE-only and MESA + TESE), spermatogenesis was normal in 21 of the 108 men (19.4%) and hypospermatogenesis occurred in 33 (30.5%). The fertilization rate was significantly lower in the hypospermatogenic group than in the normospermatogenesis group (65.6 and 72.9%, respectively; p = 0.02); this was also true for the embryo cleavage rate (88.6 and 92.1%, respectively; p = 0.007), and the proportion of embryos with fewer than 30% of enucleate fragments (79.5 and 86.9%, respectively; p = 0.02). Our study results showed that impaired spermatogenesis had a negative impact on certain early-stage biological outcomes of ICSI. In CBAVD, male factors are likely to exert a harmful effect on the early stages of embryo development.

Evaluation of a new panel of six mononucleotide repeat markers for the detection of DNA mismatch repair-deficient tumours.

BACKGROUND: Microsatellite instability (MSI) is a molecular phenotype due to defective DNA mismatch repair (MMR) system. It is used to predict outcome of colorectal tumours and to screen tumours for Lynch syndrome (LS). A pentaplex panel composed of five mononucleotide markers has been largely recommended for determination of the MSI status. However, its sensitivity may be taken in default in occasional situations. The aim of the study was to optimise this panel for the detection of MSI. METHODS: We developed an assay allowing co-amplification of six mononucleotide repeat markers (BAT25, BAT26, BAT40, NR21, NR22, NR27) and one polymorphic dinucleotide marker (D3S1260) in a single reaction. Performances of the new panel were evaluated on a cohort of patients suspected of LS. RESULTS: We demonstrate that our assay is technically as easy to use as the pentaplex assay. The hexaplex panel shows similar performances for the identification of colorectal and non-MSH6-deficient tumours. On the other hand, the hexaplex panel has higher sensitivity for the identification of MSH6-deficient tumours (94.7% vs 84.2%) and MMR-deficient tumours other than colorectal cancer (92.9% vs 85.7%). CONCLUSION: The hexaplex panel could thus be an attractive alternative to the pentaplex panel for the identification of patients with LS.