Search

International audience; 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.

International audience; In 80% of infertile men with obstructive azoospermia caused by a congenital bilateral absence of the vas deferens (CBAVD), mutations are identified in the cystic fibrosis transmembrane conductance regulator gene (CFTR). For the remaining 20%, the origin of the CBAVD is unknown. A large cohort of azoospermic men with CBAVD was retrospectively reassessed with more stringent selection criteria based on consistent clinical data, complete description of semen and reproductive excurrent ducts, extensive CFTR testing, and kidney ultrasound examination. To maximize the phenotypic prioritization, men with CBAVD and with unilateral renal agenesis were considered ineligible for the present study. We performed whole-exome sequencing on 12 CFTR-negative men with CBAVD and targeted sequencing on 14 additional individuals. We identified three protein-truncating hemizygous mutations, c.1545dupT (p.Glu516Ter), c.2845delT (p.Cys949AlafsTer81), and c.2002_2006delinsAGA (p.Leu668ArgfsTer21), in ADGRG2, encoding the epididymal- and efferent-ducts-specific adhesion G protein-coupled receptor G2, in four subjects, including two related individuals with X-linked transmission of their infertility. Previous studies have demonstrated that Adgrg2-knockout male mice develop obstructive infertility. Our study confirms the crucial role of ADGRG2 in human male fertility and brings new insight into congenital obstructive azoospermia pathogenesis. In men with CBAVD who are CFTR-negative, ADGRG2 testing could allow for appropriate genetic counseling with regard to the X-linked transmission of the molecular defect.

Kartagener syndrome (KS) is a trilogy of symptoms (nasal polyps, bronchiectasis, and situs inversus totalis) that is associated with ultrastructural anomalies of cilia of epithelial cells covering the upper and lower respiratory tracts and spermatozoa flagellae. The axonemal dynein intermediate-chain gene 1 (DNAI1), which has been demonstrated to be responsible for a case of primary ciliary dyskinesia (PCD) without situs inversus, was screened for mutation in a series of 34 patients with KS. We identified compound heterozygous DNAI1 gene defects in three independent patients and in two of their siblings who presented with PCD and situs solitus (i.e., normal position of inner organs). Strikingly, these five patients share one mutant allele (splice defect), which is identical to one of the mutant DNAI1 alleles found in the patient with PCD, reported elsewhere. Finally, this study demonstrates a link between ciliary function and situs determination, since compound mutation heterozygosity in DNAI1 results in PCD with situs solitus or situs inversus (KS).

We have collated the results of cystic fibrosis (CF) mutation analysis conducted in 19 laboratories in France. We have analyzed 7, 420 CF alleles, demonstrating a total of 310 different mutations including 24 not reported previously, accounting for 93.56% of CF genes. The most common were F508del (67.18%; range 61-80), G542X (2.86%; range 1-6.7%), N1303K (2.10%; range 0.75-4.6%), and 1717-1G>A (1.31%; range 0-2.8%). Only 11 mutations had relative frequencies >0. 4%, 140 mutations were found on a small number of CF alleles (from 29 to two), and 154 were unique. These data show a clear geographical and/or ethnic variation in the distribution of the most common CF mutations. This spectrum of CF mutations, the largest ever reported in one country, has generated 481 different genotypes. We also investigated a cohort of 800 French men with congenital bilateral absence of the vas deferens (CBAVD) and identified a total of 137 different CFTR mutations. Screening for the most common CF defects in addition to assessment for IVS8-5T allowed us to detect two mutations in 47.63% and one in 24.63% of CBAVD patients. In a subset of 327 CBAVD men who were more extensively investigated through the scanning of coding/flanking sequences, 516 of 654 (78. 90%) alleles were identified, with 15.90% and 70.95% of patients carrying one or two mutations, respectively, and only 13.15% without any detectable CFTR abnormality. The distribution of genotypes, classified according to the expected effect of their mutations on CFTR protein, clearly differed between both populations. CF patients had two severe mutations (87.77%) or one severe and one mild/variable mutation (11.33%), whereas CBAVD men had either a severe and a mild/variable (87.89%) or two mild/variable (11.57%) mutations.

4 Valuable collaboration between a molecular CFTR database and a national CF registry: the French experience C. Bareil, L. Lemonnier, C. Dehillotte, V. Colomb-Jung, C. Theze, M.-P. Audrezet, C. Ferec, T. Bienvenu, E. Girodon, P. Fanen, C. Mekki, E. Bieth, V. Gaston, P. Fergelot, M.-P. Reboul, A. Kitzis, G. Lalau, A. Pagin, M.-C. Malinge, C. Raynal, M. Claustres. CHRU de Montpellier, Laboratoire de Genetique Moleculaire, Montpellier, France; Association Vaincre La Mucoviscidose, Paris, France; CHRU Brest, Laboratoire de Genetique Moleculaire, Brest, France; Hopital Cochin, APHP, Service de Genetique et Biologie Moleculaires, Paris, France; GH Henri Mondor, Departement de Genetique, Creteil, France; CHRU de Toulouse, Service de Genetique Medicale, Toulouse, France; Hopital Pellegrin – CHU de Bordeaux, Laboratoire de Genetique Moleculaire, Bordeaux, France; CHU de Poitiers, Service de Genetique, Poitiers, France; CHRU de Lille, Service de Toxicologie et Genopathies, Lille, France; CHU Angers, UF de Genetique Moleculaire – Departement de Biochimie Genetique, Angers, France In 2013 the French CF Registry and CFTR-France database managers started collaborating to address the limitations of each base, due to the lack of genetics and clinical data cross comparison. The aim was to check and update clinical data in CFTR-France and genetic data in the Registry. Records from the two databases were compared using name, date of birth, gender, sex and mutations. Matched and unmatched records were identified. Discrepancies were checked and confirmed by the laboratories and then sent to the CF centres. Comparison of 8807 patients in the Registry and 4617 in CFTR-France identified 1924 full match and 321 partially matched records. Molecular laboratories confirmations added 216 full matches. The remaining 105 patients still need confirmation by laboratories and CF centres. Among the 321 patients, personal data and/or genotype discrepancies were identified for respectively 70 and 129 patients. This database crossing allowed 110 genotypes to be corrected/completed in the Registry, which is of major interest. Indeed, if those incorrect data are also present in the CF centre medical files, they can impact familial genetic counselling and impair patients’ access to optimal mutation targeted therapies. Clinical data provided by the Registry are currently being analysed to complete the CFTR-France phenotypic status of patients. In-depth analysis of these data allows patients to be re-classified in different phenotypic groups (CF, CBAVD, isolated bronchiectasis or pancreatitis) and genotype/phenotype relationships to be refined, in order to better manage rare variants interpretation, medical care strategy and genetic counselling to families.

Background: Cystic fibrosis (CF) is caused by compound heterozygosity or homozygosity of CF transmembrane conductance regulator gene (CFTR) mutations. Phenotypic variability associated with certain mutations makes genetic counselling difficult, notably for R117H, whose disease phenotype varies from asymptomatic to classical CF. The high frequency of R117H observed in CF newborn screening has also introduced diagnostic dilemmas. The aim of this study was to evaluate the disease penetrance for R117H in order to improve clinical practice. Methods: The phenotypes in all individuals identified in France as compound heterozygous for R117H and F508del, the most frequent CF mutation, were described. The allelic prevalences of R117H (p R117H), on either intron 8 T5 or T7 background, and F508del (p F508del) were determined in the French population, to permit an evaluation of the penetrance of CF for the [R117H]+[F508del] genotype. Results: Clinical details were documented for 184 [R117H]+[F508del] individuals, including 72 newborns. The disease phenotype was predominantly mild; one child had classical CF, and three adults' severe pulmonary symptoms. In 5245 healthy adults, p F508del was 1.06%, p R117H;T7 0.27% and p R117H;T5 ,0.01%. The theoretical number of [R117H;T7]+[F508del] individuals in the French population was estimated at 3650, whereas only 112 were known with CF related symptoms (3.1%). The penetrance of classical CF for [R117H;T7]+[F508del] was estimated at 0.03% and that of severe CF in adulthood at 0.06%. Conclusions: These results suggest that R117H should be withdrawn from CF mutation panels used for screening programmes. The real impact of so-called disease mutations should be assessed before including them in newborn or preconceptional carrier screening programmes .