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• Exonic variants may have various molecular mechanisms of pathogenicity. • The impact on splicing of exonic sequence variations should be systemically assessed. • Splicing default of exonic variants may hamper the efficiency of targeted pharmacotherapy. • Functional in vitro experiments are key tools to classify CFTR rare variants in order to offer personalized therapies. 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. [Display omitted] [ABSTRACT FROM AUTHOR]

Purpose: 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., Methods: 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., Results: 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., Conclusions: 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.

Purpose: 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)., Methods: 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., Results: 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., Discussion: 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.

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.