F. Leturcq

Multitissular involvement in a family with LMNA and EMD mutations Role of digenic mechanism

Background: Mutations in the EMD and LMNA genes, encoding emerin and lamins A and C, are responsible for the X-linked and autosomal dominant and recessive forms of Emery–Dreifuss muscular dystrophy (EDMD). LMNA mutations can also lead to several other disorders, collectively termed laminopathies, involving heart, fat, nerve, bone, and skin tissues, and some premature ageing syndromes. Methods: Fourteen members of a single family underwent neurologic, electromyographic, and cardiologic assessment. Gene mutation and protein expression analyses were performed for lamins A/C and emerin. Results: Clinical investigations showed various phenotypes, including isolated cardiac disease (seven patients), axonal neuropathy (one patient), and a combination of EDMD with axonal neuropathy (two patients), whereas five subjects remained asymptomatic. Genetic analyses identified the coincidence of a previously described homozygous LMNA mutation (c.892C→T, p. R298C) and a new in-frame EMD deletion (c.110-112delAGA, p. delK37), which segregate independently. Analyses of the contribution of these mutations showed 1) the EMD codon deletion acts in X-linked dominant fashion and was sufficient to induce the cardiac disease, 2) the combination of both the hemizygous EMD and the homozygous LMNA mutations was necessary to induce the EDMD phenotype, 3) emerin was present in reduced amount in EMD-mutated cells, and 4) lamin A/C and emerin expression was most dramatically affected in the doubly mutated fibroblasts. Conclusions: This highlights the crucial role of lamin A/C–emerin interactions, with evidence for synergistic effects of these mutations that lead to Emery–Dreifuss muscular dystrophy as the worsened result of digenic mechanism in this family.

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.

G.P.75 Variable phenotype of del45–55 Becker patients correlated to nNOSμ mislocalization and RYR1 hypernitrosylation

Abstract Duchenne (DMD) and Becker (BMD) muscular dystrophies are muscle-wasting diseases caused by mutations in the DMD gene encoding dystrophin. Usually, out-of-frame deletions give rise to DMD whereas in-frame deletions result in BMD. BMD patients exhibit a less severe disease because an abnormal but functional dystrophin is produced. This is the rationale for attempts to correct the reading frame by using an exon-skipping strategy. In order to apply this approach to a larger number of patients, a strategy of exons 45–55 multi-skipping has been proposed, because it would correct the mRNA reading frame in almost 75% of DMD patients with a deletion. The resulting dystrophin lacks part of the binding site for the neuronal nitric oxide synthase (nNOSμ), which normally binds to spectrin-like repeats 16 and 17 of dystrophin. Knowing that these domains are encoded by exons 42–45, we investigated the nNOSμ status in muscle biopsies from twelve BMD patients with deletions of exons 45–55. We found a wide spectrum of nNOSμ expression and localization. The strictly cytosolic mislocalization of nNOSμμ was associated with the more severe phenotypes. The cytosolic NO production correlated with both hypernitrosylation of the sarcoplasmic reticulum calcium-release-channel RyR1 and release of calstabin-1, a central hub to Ca2+ signaling and contraction in muscle. This study shows that the terminal truncation of the nNOS binding domain in the “therapeutic” del45–55 dystrophin is not innocuous since, in some cases, it may perturb the nNOS dependent stability of the RyR1/calstabin-1 complex.