V. Allamand group: Collagen type VI-related myopathies
Mutations in the genes encoding collagen type VI (ColVI), a heterotrimeric molecule linking the basement membrane and interstitial matrix, are responsible for a group of neuromuscular disorders, clinically and genetically heterogeneous (Lampe and Bushby, 2008
; Allamand et al., in press
). ColVI-related myopathies have long been under-recognized and under-estimated; indeed, Ullrich congenital muscular dystrophy (UCMD), which represents the most severe end of the clinical spectrum, is most likely the most common form of “merosin-positive” CMD (i.e. without involvement of the alpha2 chain of laminin-211).
Over the last 5 or 6 years, we set up a diagnostic strategy which consists first, of the analysis of ColVI expression and secretion in patient-derived skin fibroblasts in culture, followed by sequencing of the coding regions of the genes encoding ColVI (COL6A1, COL6A2, COL6A3). This second step is now performed in the Cardio & Myogenetics Fonctional Unit (P Richard, Service de Biochimie Métabolique, GH Pitié-Salpêtrière). Following this strategy, we identified 56 disease-causing mutations in the COL6A genes a cohort of 49 patients who were extensively characterized clinically, cellularly and genetically. Importantly, 61% of the mutations were de novo with a dominant-negative effect (Briñas/Richard et al, en révision). The frequency of such de novo mutations bears important consequences for diagnosis and genetic counselling. In addition, we demonstrated the usefulness of quantifying the transcript levels of each chain (using RT-qPCR) in order to point to the mutation-bearing gene. Finally, we were able to establish genotype-phenotype correlations in this cohort, which are interesting for the follow up of patients and to determine relevant outcome measures for future clinical trials (Briñas/Richard et al, in rev; Allamand et al., in press
In parallel to these genetic analyses, and in the context of the European Network TREAT-NMD, we have developed Locus Specific databases for each of the three COL6A gene which contain mutation data (from the litterature and our work), and will also contain clinical data in the future (Sarkozi et al., 2009
; Allamand et al., in press
). The development and maintenance of these databases is carried out in close collaboration R. Ben Yaou et K. Chikhaoui (U974, Gisèle Bonne’s group), and C Béroud who created the UMD tool (Montpellier) (Béroud et al., 2005
Nevertheless, no disease-causing mutation could be identified in about 10% of the patients that were referred to us for diagnosis, although they do present an abnormal secretion of ColVI in the fibroblast cultures. Indeed, other genes may be involved and we set out to identify and investigate candidate genes for these “ColVI-like myopathies”.
- Functional analyses of the mutations in the COL6A1, A2, A3 genes
Although the pathogenicity of mutations causing a deficiency in ColVI is clear, the underlying leading to the muscle phenotype remain elusive.
We are particularly interested in a sub-group of patients harbouring autosomal recessive mutations leading to a complete absence of ColVI secretion in cultured fibroblasts (a widely recognized observation), but also in the muscle biopsies, a very unusual finding. We are pursuing this retention of ColVI in the cytosol, at the cellular level, and its consequences. Using electronic microscopy, we demonstrated that ColVI is specifically retained in the endoplasmic reticulum (ER) in skin fibroblasts in culture, thereby preventing its secretion. Preliminary data indicate that this accumulation in ER does not appear to trigger the ER-associated surveillance mechanisms (Allamand et al., in press
To date, the most advanced pathophysiological hypothesis is the mitochondrial dysfunction of ColVI-deficient muscle fibers leading to their cellular death by apoptosis with was first described in a mouse model of ColVI-related myopathy (Irwin et al., 2003
) and in patients’ cells thereafter (Angelin et al., 2007
; Merlini et al., 2008
). Indeed, these studies open the way to interesting therapeutic approaches which remain nevertheless somewhat open to discussion due to its apparent lack of specificity toward ColVI (Hicks et al., 2009
). We have undertaken to detect and quantify the occurrence of apoptosis in skeletal muscle from patients.
Update: April 2010