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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.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.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.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.

International audience; Pathogenic variants of the CFTR gene are responsible for a broad phenotypic spectrum characterized by malfunction of some exocrine tissues, with an autosomal recessive mode of inheritance. More than 2,000 variants, distributed throughout the CFTR gene, have been identified, with different effects on the gene and protein expression and function. Genotype-phenotype correlation studies have associated severe variants with a typical multi-organ form of cystic fibrosis, while mild variants are involved in monosymptomatic or adult-onset diseases, called CFTR-related disorders. However, the interpretation of rare variants remains challenging. This review presents an overview of the epidemiology of CFTR variants worldwide and in France and describes the functional classification. Finally, some frequent cystic fibrosis-causing and mild CFTR variants are used as example to depict the molecular pathology of the CFTR locus. Finally, we give the recommendations concerning nomenclature and classification that are useful for appropriate genetic counseling. © 2020 French Society of Pediatrics. Published by Elsevier Masson SAS.

International audience; Exome sequencing used for molecular diagnosis of Mendelian disorders considerably increases the number of missense variants of unclear significance, whose pathogenicity can be assessed by a variety of prediction tools. As the performance of algorithms may vary according to the datasets, complementary specific resources are needed to improve variant interpretation. As a model, we were interested in the cystic fibrosis transmembrane conductance regulator gene (CFTR) causing cystic fibrosis, in which at least 40% of missense variants are reported. Cystic fibrosis missense analysis (CYSMA) is a new web server designed for online estimation of the pathological relevance of CFTR missense variants. CYSMA generates a set of computationally derived data, ranging from evolutionary conservation to functional observations from three-dimensional structures, provides all available allelic frequencies, clinical observations, and references for functional studies. Compared to software classically used in analysis pipelines on a dataset of 141 well-characterized missense variants, CYSMA was the most efficient tool to discriminate benign missense variants, with a specificity of 85%, and very good sensitivity of 89%. These results suggest that such integrative tools could be adapted to numbers of genes involved in Mendelian disorders to improve the interpretation of missense variants identified in the context of diagnosis.

International audience; Most of the 2,000 variants identified in the CFTR (cystic fibrosis transmembrane regulator) gene are rare or private. Their interpretation is hampered by the lack of available data and resources, making patient care and genetic counseling challenging. We developed a patient-based database dedicated to the annotations of rare CFTR variants in the context of their cis- and trans-allelic combinations. Based on almost 30 years of experience of CFTR testing, CFTR-France (https://cftr.iurc.montp.inserm.fr/cftr) currently compiles 16,819 variant records from 4,615 individuals with cystic fibrosis (CF) or CFTR-RD (related disorders), fetuses with ultrasound bowel anomalies, newborns awaiting clinical diagnosis, and asymptomatic compound heterozygotes. For each of the 736 different variants reported in the database, patient characteristics and genetic information (other variations in cis or in trans) have been thoroughly checked by a dedicated curator. Combining updated clinical, epidemiological, in silico, or in vitro functional data helps to the interpretation of unclassified and the reassessment of misclassified variants. This comprehensive CFTR database is now an invaluable tool for diagnostic laboratories gathering information on rare variants, especially in the context of genetic counseling, prenatal and preimplantation genetic diagnosis. CFTR-France is thus highly complementary to the international database CFTR2 focused so far on the most common CF-causing alleles.

International audience; Several protocols have illustrated the possibility of deriving cells, such as fibroblasts, from different organs. These techniques generally concern organs sampled from living persons, but have already been described for cadavers, especially concerning the skin and tendons. We present, for the first time, an easy way to derive pulmonary fibroblasts from a lung tissue sampled from a cadaver and directly culture plated. The fibroblast output was checked daily. We obtained lung fibroblasts from 3 (60%) cadavers and 2 (100%) living persons. The fibroblast output took about 3 days for cells from living persons and took up to 39 days for those from cadavers. We did not clearly identify any parameters that could explain these differences. Nevertheless, these derived cells had the same features as the source cells, especially in terms of morphology and proliferation, and could potentially be used in different research domains such as forensic or regeneration medicine.

International audience; New technologies, which constantly become available for mutation detection and gene analysis, have contributed to an exponential rate of discovery of disease genes and variation in the human genome. The task of collecting and documenting this enormous amount of data in genetic databases represents a major challenge for the future of biological and medical science. The Locus Specific Databases (LSDBs) are so far the most efficient mutation databases. This review presents the main types of databases available for the analysis of mutations responsible for genetic disorders, as well as open perspectives for new therapeutic research or challenges for future medicine. Accurate and exhaustive collection of variations in human genomes will be crucial for research and personalized delivery of healthcare.

DNA microarray technology enables investigators to measure the expression of several 1000 mRNA species simultaneously in a biological specimen. However, the reliability of the microarray technology to detect transcriptional differences representative of the original samples is affected by the quality of the extracted RNA. Thus, it is of critical importance to standardize sample-handling protocols and to perform a quality assessment of RNA preparations. In this report, 59 human tissue samples were used to evaluate the relationships between RNA quality and gene expression. From Affymetrix GeneChip array data analysis of these samples, we compared the performance of the 28S/18S ratio, two computer methods (RIN and degradometer) and our in-house RNA quality scale (RQS) in assessing RNA quality. The optimal RNA reliability threshold was determined for each method using statistical discrimination measures. We showed that RQS, RIN and degradometer have a similar capacity to detect reliable RNA samples whereas the 28S/18S ratio leads to a misleading categorization. Furthermore, we developed a new approach, based on clustering analyses of full chip expression, to control RNA quality after hybridization experiments. The combination of these methods, allowing monitoring of RNA quality prior to and after the hybridization experiments, ensured reliable and reproducible microarray data.

Analysis of circulating cell-free fetal DNA (cffDNA) in maternal plasma is very promising for early diagnosis of monogenic diseases. However, this approach is not yet available for routine use and remains technically challenging because of the low concentration of cffDNA, which is swamped by the overwhelming maternal DNA.To make clinical applications more readily accessible, we propose a new approach based on mutant enrichment with 3′-modified oligonucleotides (MEMO) PCR along with real-time PCR to selectively amplify from the maternal blood the paternally inherited fetal allele that is not present in the maternal genome.The first proof of concept of this strategy was displayed for cystic fibrosis by the accuracy of our detection of the p.Gly542* mutation used as the initial developmental model. Subsequently, a retrospective study of plasmas originating from two pregnant women carrying a fetus with private mutation confirmed the effectiveness of our method. We confirmed the presence of cffDNA in the studied samples by the identification of a tri-allelic DNA profile using a miniSTR kit.This new non-invasive prenatal diagnosis test offers numerous advantages over current methods: it is simple, cost effective, time efficient and does not require complex equipment or bioinformatics settings. Moreover, our assays for different private mutations demonstrate the viability of this approach in clinical settings for monogenic disorders.

Retinitis pigmentosa (RP) is a clinically and genetically heterogeneous group of retinal diseases leading to blindness. By performing full genome linkage analysis in a consanguineous French family affected with severe autosomal recessive RP, we have excluded linkage to known loci involved in RP and mapped a novel locus to chromosome 16q13-q21 (Zmax=2.83 at theta=0 at the D16S3089 locus). Two candidate genes KIFC3 and CNGB1 mapping to this critical interval have been screened for mutations. The CNGB1 gene, which encodes the beta-subunit of the rod cGMP-gated channel, is mutated in the family presented in this study.

Mutations in the gene encoding rhodopsin, the visual pigment in rod photoreceptors, were shown to be the most common cause of autosomal retinitis pigmentosa (RP). In order to determine the prevalence of rhodopsin alterations in southern French populations, we examined 52 unrelated patients/families with autosomal dominant RP (adRP=29), RP simplex (6), or unclassified RP (17).The full coding and flanking sequences of the rhodopsin (RHO) gene were scanned using an improved DGGE (denaturing gradient gel electrophoresis) assay, followed by sequencing of abnormal fragments.This study revealed three RHO mutations in patients with adRP (G106R, R135W, and c.998999ins4) and a number of frequent or rare polymorphisms. No disease-causing sequence variation was found in simplex and unclassified RP pedigrees. Mutation c.998999ins4 has not been previously reported, and appears as the first duplication identified so far in the RHO gene. This frameshift mutation, which is associated with a severe RP, alters the carboxy terminus and predicts a 353-amino acid mutant rhodopsin instead of 348.Our study demonstrates that rhodopsin mutations are responsible for only 10.3% of adRP in French populations living in the Mediterranean area in contrast to the 25-35% reported in other populations.

The predominance of truncative mutations responsible for choroideremia (CHM) led us to investigate the use of the protein truncation test (PTT) applied to lymphocyte RNA derived from affected males as a scanning method. The entire CHM coding region was reversed-transcribed in three overlapping cDNA segments (RT-PCR) which were amplified and further analysed by PTT after in vitro transcription/translation. This strategy enabled us to detect the CHM-causative alteration in each of the four unrelated patients from southern France who were investigated. We describe three novel mutations (E177X, 323delT, 1313delTC), and report one recurrent mutation (R267X) in CHM. We believe this to be the first attempt at applying RT-PCR-PTT to CHM mutation detection.

Udgivelsesdato: 1997-Jul Murine retrovirus SL3-3 is highly T lymphomagenic. Its pathogenic properties are determined by the transcriptional enhancer of the U3 repeat region which shows preferential activity in T cells. Within the U3 repeats, the major determinant of T-cell specificity has been mapped to binding sites for the AML1 transcription factor family (also known as the core binding factor [CBF], polyomavirus enhancer binding protein 2 [PEBP2], and SL3-3 enhancer factor 1 [SEF-1]). SL3-3 viruses with AML1 site mutations have lost a major determinant of T-cell-specific enhancer function but have been found to retain a lymphomagenic potential, although disease induction is slower than for the SL3-3 wild type. To compare the specificities and mechanisms of disease induction of wild-type and mutant viruses, we have examined lymphomas induced by mutant viruses harboring transversions of three consecutive base pairs critical to AML1 site function (B. Hallberg, J. Schmidt, A. Luz, F. S. Pedersen, and T. Grundström. J. Virol. 65:4177-4181, 1991). Our results show that the mutated AML1 sites are genetically stable during lymphomagenesis and that ecotropic provirus numbers in DNA of tumors induced by wild-type and mutant viruses fall within the same range. Moreover, proviruses were found to be integrated at the c-myc locus in similar proportions of wild-type and mutant SL3-3-induced tumors, and the mutated AML1 sites of proviruses at c-myc are unaltered. In some cases, however, including one c-myc-integrated provirus, a single-base pair change was detected in a second, weaker AML1 binding site. By DNA rearrangement analysis of the T-cell receptor beta-locus, tumors induced by the AML1 site mutants are found to be of the T-cell type. Thus, although the AML1 site mutants have weakened T-cell-specific enhancers they are T-lymphomagenic, and wild-type- and mutant-virus-induced tumor DNAs are similar with respect to the number of overall ecotropic and c-myc-integrated clonal proviruses. The SL3-3 wild-type and AML1 site mutant viruses may therefore induce disease by similar mechanisms.

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.

International audience; Several diseases have been clinically or genetically related to cystic fibrosis (CF), but a consensus definition is lacking. Here, we present a proposal for consensus guidelines on cystic fibrosis transmembrane conductance regulator (CFTR)-related disorders (CFTR-RDs), reached after expert discussion and two dedicated workshops. A CFTR-RD may be defined as "a clinical entity associated with CFTR dysfunction that does not fulfil diagnostic criteria for CF". The utility of sweat testing, mutation analysis, nasal potential difference, and/or intestinal current measurement for the differential diagnosis of CF and CFTR-RD is discussed. Algorithms which use genetic and functional diagnostic tests to distinguish CF and CFTR-RDs are presented. According to present knowledge, congenital bilateral absence of vas deferens (CBAVD), acute recurrent or chronic pancreatitis and disseminated bronchiectasis, all with CFTR dysfunction, are CFTR-RDs.

International audience; Molecular pathophysiology of facioscapulohumeral muscular dystrophy (FSHD) involves the heterozygous contraction of the number of tandemly repeated D4Z4 units at chromosome 4q35.2. FSHD is associated with a range of 1-10 D4Z4 units instead of 11-150 in normal controls. Several factors complicate FSHD molecular diagnosis, especially the cis-segregation of D4Z4 contraction with a 4qA allele, whereas D4Z4 shortening is silent both on alleles 4qB and 10q. Discrimination of pathogenic 4q-D4Z4 alleles from highly homologous 10q-D4Z4 arrays requires the use of the conventional Southern blot, which is not suitable at the single-cell level. Preimplantation genetic diagnosis (PGD) is a frequent request from FSHD families with several affected relatives. We aimed to develop a rapid and sensitive PCR-based multiplex approach on single cells to perform an indirect familial segregation study of pathogenic alleles. Among several available polymorphic markers at 4q35.2, the four most proximal (D4S2390, D4S1652, D4S2930 and D4S1523

Purpose In patients with advanced colorectal cancer, leucovorin, fluorouracil, and irinotecan (FOLFIRI) is considered as one of the reference first-line treatments. However, only about half of treated patients respond to this regimen, and there is no clinically useful marker that predicts response. A major clinical challenge is to identify the subset of patients who could benefit from this chemotherapy. We aimed to identify a gene expression profile in primary colon cancer tissue that could predict chemotherapy response. Patients and Methods Tumor colon samples from 21 patients with advanced colorectal cancer were analyzed for gene expression profiling using Human Genome GeneChip arrays U133. At the end of the first-line treatment, the best observed response, according to WHO criteria, was used to define the responders and nonresponders. Discriminatory genes were first selected by the significance analysis of microarrays algorithm and the area under the receiver operating characteristic curve. A predictor classifier was then constructed using support vector machines. Finally, leave-one-out cross validation was used to estimate the performance and the accuracy of the output class prediction rule. Results We determined a set of 14 predictor genes of response to FOLFIRI. Nine of nine responders (100% specificity) and 11 of 12 nonresponders (92% sensitivity) were classified correctly, for an overall accuracy of 95%. Conclusion After validation in an independent cohort of patients, our gene signature could be used as a decision tool to assist oncologists in selecting colorectal cancer patients who could benefit from FOLFIRI chemotherapy, both in the adjuvant and the first-line metastatic setting.

To evaluate the occurrence and inheritance of various types of pigmentary retinopathy in patients followed at the outpatient clinic in the university hospital, Montpellier, France. To characterize genes and mutations causing these conditions.Ophthalmic examination and various visual tests were performed. Mutations were sought from genomic DNA by PCR amplification of exons associated with single-strand conformation analysis and/or direct sequencing.Among 315 patients over an 8-year period, cases of retinitis pigmentosa (63.2%), Usher's syndrome (10.2%), Stargardt's disease (5.4%), choroideremia (3.2%), Leber's congenital amaurosis (3.2%), congenital stationary night blindness (2.9%), cone dystrophy (2.5%), dominant optic atrophy (1.9%), X-linked juvenile retinoschisis (1.6%), Best's disease (1.6%), and others (4.3%) were diagnosed. In retinitis pigmentosa, inheritance could be determined in 54.2% of the cases including dominant autosomic (26.6%), recessive autosomic (22.6%), and X-linked cases (5%) while it could not be confirmed in 45.7% of the cases (simplex cases in the majority). For the 6 examined genes, mutations were found in 22 out of 182 propositus (12.1%). Analysis of phenotype-genotype correlations indicates that in retinitis pigmentosa, RDS is more frequently associated with macular involvement and retinal flecks, RHO with regional disease, and RPE65 with the great severity of the disease with some cases of Leber's congenital amaurosis.Identification of genes may help in diagnosis and in genetic counseling, especially in simplex cases with retinitis pigmentosa. In this latter condition, molecular diagnosis will be necessary to rationalize future treatments.

We describe particular clinical features in a three-generation family with X-linked CSNBi and present the genetic analysis.The diagnosis of CSNBi was established on clinical and electrophysiological criteria. Polymorphic DNA markers of the Xp region were analyzed by fluorescent polymerase chain reaction.Clinical findings evidenced an atypical association of both myopia and hyperopia in the same brotherhood. The most interesting feature in this family was the observation of major worsening of the clinical shape between the first and the third generation of affected individuals. DNA analysis did not show significant linkage between the disease and markers of the Xp11-p21 region. Southern analysis did not show expansion of trinucleotide repeat CAG/CTG and CCG/CGG over the three generation.Haplotypic analysis together with clinical observations allow to exclude the existence of a myopia gene closely linked to the CSNB2 locus. The clinical anticipation observed in this family does not seem to be linked with trinucleotide repeat expansion CAG/CTG or CCG/CGG.

Data from 6 years of experience in molecular diagnosis of Duchenne (DMD) and Becker (BMD) muscular dystrophy in Southern France are reported. DMD and BMD patients have been extensively analyzed for deletions and for point mutations in the dystrophin gene. By scanning the whole coding sequence as reverse-transcribed from lymphocytes or muscular RNA by the protein truncation test, we have reached a minimum of an 86% detection rate for point mutations responsible for DMD; these mutations consist of nonsense, frameshifting, and splicing mutations. Four of 12 small alterations identified in our sample are novel and described in this study. We also present an improved protocol for the automated detection of fluorescently labeled duplex polymerase chain reactions of six known intragenic microsatellites (Dys II, TG 15, STRs 44, 45, 49, and 50). Accurate sizing of the alleles at each locus was performed, and we elucidated the sequence of several repeat units. Allele frequencies at each of the six microsatellite loci and at one restriction fragment length polymorphism site (intron 16/TaqI) were defined in a sample of normal, DMD, and BMD X chromosomes from Southern France. The determination of the grandparental origin of either deletions or point mutations revealed differences depending on the type of the mutation, with most of the deletions occurring in oogenesis and most of the point mutations occurring in spermatogenesis.

By using the single strand conformational analysis (SSCA) to search for point mutations in the choroideremia gene, we have identified a previously undescribed polymorphism within exon 5a (381A/G). We have studied the frequency of this polymorphism in a population from Southern France. The sequence variation creates a new restriction site for HhaI, allowing a convenient DNA-based genetic counseling in families in which the causal disease mutation is unknown.

By using the single strand conformation analysis to search for point mutations in the choroideremia gene, we had previously identified the first truncative mutation responsible for CHM in France. The aim of the present study was to perform a simple and nonisotopic routine test to identify carriers and non carriers in the relevant family.We used a PCR-based restriction analysis to detect the presence or absence of the mutation in the family members, as the mutation creates a restriction site in the coding sequence of the CHM gene.We could follow the segregation of the mutation in the pedigree, and unambiguously determine the genetic status of the females.When a mutation responsible for choroideremia modifies a restriction site, the PCR-restriction provides an efficient and unexpensive one-day test to detect heterozygosity in the family.

La detection de mutations dans l'ADN represente une etape essentielle de la biologie moleculaire, pour la recherche fondamentale comme pour les applications medicales. Les strategies actuellement disponibles pour deceler les alleles mutes sont fondees, soit sur l'utilisation de methodes rapides mais limitees a l'identification d'un nombre restreint de mutants determines, soit sur l'utilisation de techniques couteuses et laborieuses mais capables de deceler la moindre alteration de sequence dans les portions essentielles des genes. La detections de mutations est une demarche complexe et aucune des methodes disponibles n'est applicable seule a toutes les situations, leur choix dependant de nombreux criteres (nature des mutations, taille et structures du gene, acces a l'ARNm, degres d'efficacite et de sensibilite recherches). Nous presentons les avantages relatifs d'une strategie de recherche specifique des mutations qui introduisent un signal d'arret premature de la synthese proteique, fondee sur le test de troncation des proteines (PTT), par rapport aux techniques classiques de balayage d'un fragment d'ADN ou d'ARN telles que l'electrophores en gel de gradient denaturant (DGGE), l'analyse d'heteroduplex (HA), l'analyse de conformation de l'ADN en simple brin (SSCA) ou le clivage chimique ou enzymatique de mesappariements (CCM/EMC). Le PTT, fonde sur l'analyse de fragments d'ARN ou d'ADN transcrits et traduits in vitro, parait etre, pour certaines maladies genetiques, la facon la plus efficace et la moins couteuse de deceler les mutations de l'ADN responsables de troncation proteique.