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.

CFTR gene variants, epidemiology and molecular pathology.

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. All rights reserved.

Recommendations for the classification of diseases as CFTR-related disorders.

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.

[Genetic mutation databases: stakes and perspectives for orphan genetic diseases]. / Banques de données de mutations: enjeux et perspectives pour les maladies génétiques orphelines.

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.

Nonprogressive autosomal recessive ataxia maps to chromosome 9q34-9qter in a large consanguineous Lebanese family.

Congenital ataxias are a heterogeneous group of predominantly nonprogressive disorders characterized by hypotonia, developmental delay followed by the appearance of ataxia, and often associated with dysarthria, mental retardation, and atrophy of the cerebellum. We performed a genome-wide screen on a large inbred Lebanese family presenting a nonprogressive autosomal recessive congenital cerebellar ataxia associated with short stature (MIM 213200), already described by Mégarbané and colleagues. The disease locus was assigned to a 12.1 cM interval on chromosome 9q34-9qter between D9S67 and D9S312. Differential diagnosis with other hereditary ataxias linked to the same region is discussed.

Segregation of a mutation in CNGB1 encoding the beta-subunit of the rod cGMP-gated channel in a family with autosomal recessive retinitis pigmentosa.

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.

A mutation in periaxin is responsible for CMT4F, an autosomal recessive form of Charcot-Marie-Tooth disease.

Charcot-Marie-Tooth (CMT) disease is a heterogeneous group of inherited peripheral motor and sensory neuropathies characterized by chronic distal weakness with progressive muscular atrophy and sensory loss in the distal extremities. Inheritance can be autosomal dominant, X-linked or autosomal recessive (ARCMT). Recently, a locus responsible for a demyelinating form of ARCMT disease, named CMT4F, has been mapped on 19q13 in a large consanguineous Lebanese family. L- and S-periaxin are proteins of myelinating Schwann cells and homozygous periaxin-null mice display extensive demyelination of myelinated fibers in the peripheral nervous system, which suggests that the periaxin gene is a good candidate gene for an ARCMT disease. The human gene encoding the periaxins (PRX) was mapped to 19q13, in the CMT4F candidate interval. After characterizing the human PRX gene, we identified a nonsense R196X mutation in the Lebanese family which cosegregated with CMT. Histopathological and immunohistochemical analysis of a sural nerve biopsy of one patient revealed common features with the mouse mutant and the absence of L-periaxin from the myelin sheath. These data confirm the importance of the periaxin proteins to normal Schwann cell function and substantiate the utility of the periaxin-null mouse as a model of ARCMT disease.

Mapping of a new locus for autosomal recessive demyelinating Charcot-Marie-Tooth disease to 19q13.1-13.3 in a large consanguineous Lebanese family: exclusion of MAG as a candidate gene.

Autosomal recessive Charcot-Marie-Tooth disease (CMT) type 4 (CMT4) is a complex group of demyelinating hereditary motor and sensory neuropathies presenting genetic heterogeneity. Five different subtypes that correspond to six different chromosomal locations have been described. We hereby report a large inbred Lebanese family affected with autosomal recessive CMT4, in whom we have excluded linkage to the already-known loci. The results of a genomewide search demonstrated linkage to a locus on chromosome 19q13.1-13.3, over an 8.5-cM interval between markers D19S220 and D19S412. A maximum pairwise LOD score of 5.37 for marker D19S420, at recombination fraction [theta].00, and a multipoint LOD score of 10.3 for marker D19S881, at straight theta = .00, strongly supported linkage to this locus. Clinical features and the results of histopathologic studies confirm that the disease affecting this family constitutes a previously unknown demyelinating autosomal recessive CMT subtype known as "CMT4F." The myelin-associated glycoprotein (MAG) gene, located on 19q13.1 and specifically expressed in the CNS and the peripheral nervous system, was ruled out as being the gene responsible for this form of CMT.

[Molecular genetics of pigmentary retinopathies: identification of mutations in CHM, RDS, RHO, RPE65, USH2A and XLRS1 genes]. / Génétique moléculaire des rétinopathies pigmentaires: identification de mutations des gènes CHM, RDS, RHO, RPE65, USH2A et XLRS1.

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.

Molecular analysis of the rhodopsin gene in southern France: identification of the first duplication responsible for retinitis pigmentosa, c.998999ins4.

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.

Autosomal recessive retinal dystrophy associated with two novel mutations in the RPE65 gene.

Retinal dystrophies are a complex set of hereditary diseases of the retina that result in the degeneration of photoreceptors. Recent studies have shown that mutations in RPE65, a gene that codes for a retinal pigment epithelium (RPE)-specific protein thought to be involved in the 11-cis-retinoid metabolism, a key process in vision, cause severe, early onset retinal dystrophy. We describe two novel missense RPE65 mutations, L22P and H68Y, in a compound heterozygote with autosomal recessive retinal dystrophy. The relatively mild phenotype associated with these mutations suggests a possible link between the severity of the disease and the type of mutations in the RPE65 gene.

[Clinical features and genetic analysis in a family with X-linked incomplete congenital stationary night blindness (CSNBi)]. / Particularités cliniques et analyse génétique dans une famille atteinte de cécité nocturne non progressive congénitale héréditaire liée au chromosome X dans sa forme incomplète (CSNBi).

PURPOSE: We describe particular clinical features in a three-generation family with X-linked CSNBi and present the genetic analysis. METHOD: 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. RESULTS: 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. CONCLUSION: 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.

Mutation analysis of the dystrophin gene in Southern French DMD or BMD families: from Southern blot to protein truncation test.

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.

A complex allele (1064delTC and IVS2 + 22ins7) in the peripherin/RDS gene in retinitis pigmentosa with macular dystrophy.

Because mutations in the peripherin/RDS gene have been found in retinal dystrophies involving the macula, we examined various types of macular dystrophies from southern France to characterize sequence variations that may be associated with these conditions. DNA sequence analysis of the full coding and flanking regions of the peripherin/RDS gene was performed in fifteen unrelated patients with different types of macular dystrophy, including nine with retinitis pigmentosa (RP). Of the 15 probands with macular disease, two (13.3%) were found to carry a mutation in the peripherin/RDS gene. The recurrent mutation P216S was identified in a pedigree with autosomal dominant RP. A previously unreported complex allele (1064delTC associated with IVS2 + 22ins7) that is predicted to result in the premature termination of peripherin/RDS synthesis was identified in a sporadic case of macular atrophy with RP. We also report eight novel neutral sequence variations in the peripherin/RDS gene, most of them found in the 3' untranslated part of the gene.

Stability of AML1 (core) site enhancer mutations in T lymphomas induced by attenuated SL3-3 murine leukemia virus mutants.

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.

An exonic polymorphism (381A/G) in the choroideremia gene.

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.

[Rapid genetic diagnosis of females carriers related to patients with choroideremia]. / Diagnostic génétique rapide des femmes conductrices apparentées à des sujets atteints de choroïdérémie.

PURPOSE: 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. METHODS: 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. RESULTS: We could follow the segregation of the mutation in the pedigree, and unambiguously determine the genetic status of the females. CONCLUSION: 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.

The protein truncation test (PTT) as a method of detection for choroideremia mutations.

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.