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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; 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.

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.

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.

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.