Sylvain Baulande

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.

G.P.281

Defects in TRIM32 were reported in limb-girdle muscular dystrophy type 2H (LGMD2H). Few cases have been described to date, but this gene is not systematically analysed due to the absence of specific signs and difficulties in protein analysis. By using high-throughput mutation screening techniques, we identified mutations in TRIM32 in two patients presenting nonspecific progressive 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 since 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 ASTN2 inactivation could be associated with cognitive impairment.

D.P.2 Next generation sequencing after selected DNA capture as a tool for molecular diagnosis of neuromuscular disorders

Abstract Currently in most laboratories, molecular investigations of neuromuscular disorders (NMDs) are based on a differential molecular diagnosis by a complex and time-consuming gene by gene approach guided by a clinical approach. As a consequence, it is estimated that 35–50% of patients remain devoid of a precise molecular diagnosis. On one hand, many causal genes for NMDs are still to be identified but, on the other hand, it becomes evident that the current approaches are not sufficiently powerful and accurate to perform exhaustive screenings even in known genes, and that around 50% of patients without a definite molecular diagnosis carry unidentified mutations in known genes. In the recent years, technological tools have been developed towards performing massive molecular analyses using DNA sequence capture (SC) and next generation sequencing (NGS). In the context of the European project “NMD-Chip” (FP7 Health call), we have taken advantage of these technologies to develop specific custom SC DNA arrays to explore via NGS, known and candidate genes for NMDs. A custom in-solution DNA SC library has been designed. It includes 820 genes: 50 are known to be involved in common muscular dystrophies or myopathies (Duchenne/Becker, limb girdle, congenital), the others are candidate genes for those disorders, selected from experimental and published data. To validate this DNA chip, we have selected 20 patient DNAs: five with one known mutation, including point mutations and large rearrangements (1 COL6A2 , 1 COL6A1 , 1 DOK7 , 1 DES , 1 TNXB ) and 15 with no molecular diagnosis. We also explored the DNA of three of these patients via Exome sequencing to further compare and validate our custom SC design. The analysis of our data will help to demonstrate the proof of concept of NGS as a robust and accurate approach towards exploring multiple genes in NMD patients.