Large-Scale Functional Assessment of Genes Involved in Rare Diseases with Intellectual Disabilities Unravels Unique Developmental and Behaviour Profiles in Mouse Models.

Major progress has been made over the last decade in identifying novel genes involved in neurodevelopmental disorders, although the task of elucidating their corresponding molecular and pathophysiological mechanisms, which are an essential prerequisite for developing therapies, has fallen far behind. We selected 45 genes for intellectual disabilities to generate and characterize mouse models. Thirty-nine of them were based on the frequency of pathogenic variants in patients and literature reports, with several corresponding to de novo variants, and six other candidate genes. We used an extensive screen covering the development and adult stages, focusing specifically on behaviour and cognition to assess a wide range of functions and their pathologies, ranging from basic neurological reflexes to cognitive abilities. A heatmap of behaviour phenotypes was established, together with the results of selected mutants. Overall, three main classes of mutant lines were identified based on activity phenotypes, with which other motor or cognitive deficits were associated. These data showed the heterogeneity of phenotypes between mutation types, recapitulating several human features, and emphasizing the importance of such systematic approaches for both deciphering genetic etiological causes of ID and autism spectrum disorders, and for building appropriate therapeutic strategies.

Assessment of a Targeted Gene Panel for Identification of Genes Associated With Movement Disorders.

Importance: Movement disorders are characterized by a marked genotypic and phenotypic heterogeneity, complicating diagnostic work in clinical practice and molecular diagnosis. Objective: To develop and evaluate a targeted sequencing approach using a customized panel of genes involved in movement disorders. Design, Setting and Participants: We selected 127 genes associated with movement disorders to create a customized enrichment in solution capture array. Targeted high-coverage sequencing was applied to DNA samples taken from 378 eligible patients at 1 Luxembourgian, 1 Algerian, and 25 French tertiary movement disorder centers between September 2014 and July 2016. Patients were suspected of having inherited movement disorders because of early onset, family history, and/or complex phenotypes. They were divided in 5 main movement disorder groups: parkinsonism, dystonia, chorea, paroxysmal movement disorder, and myoclonus. To compare approaches, 23 additional patients suspected of having inherited cerebellar ataxia were included, on whom whole-exome sequencing (WES) was done. Data analysis occurred from November 2015 to October 2016. Main Outcomes and Measures: Percentages of individuals with positive diagnosis, variants of unknown significance, and negative cases; mutational frequencies and clinical phenotyping of genes associated with movement disorders. Results: Of the 378 patients (of whom 208 were male [55.0%]), and with a median (range) age at disease onset of 31 (0-84) years, probable pathogenic variants were identified in 83 cases (22.0%): 46 patients with parkinsonism (55% of 83 patients), 21 patients (25.3%) with dystonia, 7 patients (8.4%) with chorea, 7 patients (8.4%) with paroxysmal movement disorders, and 2 patients (2.4%) with myoclonus as the predominant phenotype. Some genes were mutated in several cases in the cohort. Patients with pathogenic variants were significantly younger (median age, 27 years; interquartile range [IQR], 5-36 years]) than the patients without diagnosis (median age, 35 years; IQR, 15-46 years; P = .04). Diagnostic yield was significantly lower in patients with dystonia (21 of 135; 15.6%; P = .03) than in the overall cohort. Unexpected genotype-phenotype correlations in patients with pathogenic variants deviating from the classic phenotype were highlighted, and 49 novel probable pathogenic variants were identified. The WES analysis of the cohort of 23 patients with cerebellar ataxia led to an overall diagnostic yield of 26%, similar to panel analysis but at a cost 6 to 7 times greater. Conclusions and Relevance: High-coverage sequencing panel for the delineation of genes associated with movement disorders was efficient and provided a cost-effective diagnostic alternative to whole-exome and whole-genome sequencing.

Detection of TRIM32 deletions in LGMD patients analyzed by a combined strategy of CGH array and massively parallel sequencing.

Defects in TRIM32 were reported in limb-girdle muscular dystrophy type 2H (LGMD2H), sarcotubular myopathies (STM) and in Bardet-Biedl syndrome. Few cases have been described to date in LGMD2H/STM, but this gene is not systematically analysed because of the absence of specific signs and difficulties in protein analysis. By using high-throughput variants screening techniques, we identified variants in TRIM32 in two patients presenting nonspecific LGMD. We report the first case of total inactivation by homozygous deletion of the entire TRIM32 gene. Of interest, the deletion removes part of the ASTN2 gene, a large gene in which TRIM32 is nested. Despite the total TRIM32 gene inactivation, the patient does not present a more severe phenotype. However, he developed a mild progressive cognitive impairment that may be related to the loss of function of ASTN2 because association between ASTN2 heterozygous deletions and neurobehavioral disorders was previously reported. Regarding genomic characteristics at breakpoint of the deleted regions of TRIM32, we found a high density of repeated elements, suggesting a possible hotspot. These observations illustrate the importance of high-throughput technologies for identifying molecular defects in LGMD, confirm that total loss of function of TRIM32 is not associated with a specific phenotype and that TRIM32/ASTN2 inactivation could be associated with cognitive impairment.

Assessment of the structural and functional impact of in-frame mutations of the DMD gene, using the tools included in the eDystrophin online database.

BACKGROUND: Dystrophin is a large essential protein of skeletal and heart muscle. It is a filamentous scaffolding protein with numerous binding domains. Mutations in the DMD gene, which encodes dystrophin, mostly result in the deletion of one or several exons and cause Duchenne (DMD) and Becker (BMD) muscular dystrophies. The most common DMD mutations are frameshift mutations resulting in an absence of dystrophin from tissues. In-frame DMD mutations are less frequent and result in a protein with partial wild-type dystrophin function. The aim of this study was to highlight structural and functional modifications of dystrophin caused by in-frame mutations. METHODS AND RESULTS: We developed a dedicated database for dystrophin, the eDystrophin database. It contains 209 different non frame-shifting mutations found in 945 patients from a French cohort and previous studies. Bioinformatics tools provide models of the three-dimensional structure of the protein at deletion sites, making it possible to determine whether the mutated protein retains the typical filamentous structure of dystrophin. An analysis of the structure of mutated dystrophin molecules showed that hybrid repeats were reconstituted at the deletion site in some cases. These hybrid repeats harbored the typical triple coiled-coil structure of native repeats, which may be correlated with better function in muscle cells. CONCLUSION: This new database focuses on the dystrophin protein and its modification due to in-frame deletions in BMD patients. The observation of hybrid repeat reconstitution in some cases provides insight into phenotype-genotype correlations in dystrophin diseases and possible strategies for gene therapy. The eDystrophin database is freely available: http://edystrophin.genouest.org/.

TM4SF2 gene involvement reconsidered in an XLMR family after neuropsychological assessment.

The TM4SF2 gene (localized at Xp11.4 between the loci DXS564 and DXS556) has been found to be mutated in one MRX family. In order to define the corresponding behavioral phenotype, global IQ and specific cognitive skills were assessed in seven males and three females of this family, independent of subject status. Mental retardation (MR) was mild in three patients and moderate in three others. Despite the broad variability of severity of MR, a cognitive profile specific to the MR in this family was documented. It was characterized by language disorder that was more marked in the articulatory component and spatial/verbal short-term memory dissociation with larger mnemonic span for spatial than for verbal cues. Linkage analysis was then performed on the basis of the cognitively determined status. Recombinations were observed with the loci DXS556 at Xp11.4 and DXS441 at Xq13.2 (maximum LOD score = 2.23 at theta = 0 for ALAS2). This localization region does not include the TM4SF2 gene that has been found mutated in both patients with MR and in one non-MR male subject of this family. The present results suggest two main hypotheses. First, TM4SF2 gene mutation could be involved in MR in this family, therefore representing accentuated intra familial phenotypic variability. Second, the structural particularity detected in the TM4SF2 gene might reflect a rare polymorphism rather than a pathogenic mutation, with the gene responsible for MR in this family being therefore more likely to be searched for in the pericentromeric region of the X chromosome.