S. Laval

Limb-girdle muscular dystrophies - from genetics to molecular pathology

The limb‐girdle muscular dystrophies are a diverse group of muscle‐wasting disorders characteristically affecting the large muscles of the pelvic and shoulder girdles. Molecular genetic analyses have demonstrated causative mutations in the genes encoding a disparate collection of proteins involved in all aspects of muscle cell biology. Muscular dystrophy includes a spectrum of disorders caused by loss of the linkage between the extracellular matrix and the actin cytoskeleton. Within this are the forms of limb‐girdle muscular dystrophy caused by deficiencies of the sarcoglycan complex and by aberrant glycosylation of α‐dystroglycan caused by mutations in the fukutin‐related protein gene. However, other forms of this disease have distinct pathophysiological mechanisms. For example, deficiency of dysferlin disrupts sarcolemmal membrane repair, whilst loss of calpain‐3 may exert its pathological influence either by perturbation of the IκBα/NF‐κB pathway, or through calpain‐dependent cytoskeletal remodelling. Caveolin‐3 is implicated in numerous cell‐signalling pathways and involved in the biogenesis of the T‐tubule system. Alterations in the nuclear lamina caused by mutations in laminA/C, sarcomeric changes in titin, telethonin or myotilin at the Z‐disc, and subtle changes in the extracellular matrix proteins laminin‐α2 or collagen VI can all lead to a limb‐girdle muscular dystrophy phenotype, although the specific pathological mechanisms remain obscure. Differential diagnosis of these disorders requires the careful application of a broad range of disciplines: clinical assessment, immunohistochemistry and immunoblotting using a panel of antibodies and extensive molecular genetic analyses.

Automated genomic sequence analysis of the three collagen VI genes: applications to Ullrich congenital muscular dystrophy and Bethlem myopathy

Introduction: Mutations in the genes encoding collagen VI (COL6A1, COL6A2, and COL6A3) cause Bethlem myopathy (BM) and Ullrich congenital muscular dystrophy (UCMD). BM is a relatively mild dominantly inherited disorder with proximal weakness and distal joint contractures. UCMD is an autosomal recessive condition causing severe muscle weakness with proximal joint contractures and distal hyperlaxity. Methods: We developed a method for rapid direct sequence analysis of all 107 coding exons of the COL6 genes using single condition amplification/internal primer (SCAIP) sequencing. We have sequenced all three COL6 genes from genomic DNA in 79 patients with UCMD or BM. Results: We found putative mutations in one of the COL6 genes in 62% of patients. This more than doubles the number of identified COL6 mutations. Most of these changes are consistent with straightforward autosomal dominant or recessive inheritance. However, some patients showed changes in more than one of the COL6 genes, and our results suggest that some UCMD patients may have dominantly acting mutations rather than recessive disease. Discussion: Our findings may explain some or all of the cases of UCMD that are unlinked to the COL6 loci under a recessive model. The large number of single nucleotide polymorphisms which we generated in the course of this work may be of importance in determining the major phenotypic variability seen in this group of disorders.

Metazoan Scc4 homologs link sister chromatid cohesion to cell and axon migration guidance

Saccharomyces cerevisiae Scc2 binds Scc4 to form an essential complex that loads cohesin onto chromosomes. The prevalence of Scc2 orthologs in eukaryotes emphasizes a conserved role in regulating sister chromatid cohesion, but homologs of Scc4 have not hitherto been identified outside certain fungi. Some metazoan orthologs of Scc2 were initially identified as developmental gene regulators, such as Drosophila Nipped-B, a regulator of cut and Ultrabithorax, and delangin, a protein mutant in Cornelia de Lange syndrome. We show that delangin and Nipped-B bind previously unstudied human and fly orthologs of Caenorhabditis elegans MAU-2, a non-axis-specific guidance factor for migrating cells and axons. PSI-BLAST shows that Scc4 is evolutionarily related to metazoan MAU-2 sequences, with the greatest homology evident in a short N-terminal domain, and protein–protein interaction studies map the site of interaction between delangin and human MAU-2 to the N-terminal regions of both proteins. Short interfering RNA knockdown of human MAU-2 in HeLa cells resulted in precocious sister chromatid separation and in impaired loading of cohesin onto chromatin, indicating that it is functionally related to Scc4, and RNAi analyses show that MAU-2 regulates chromosome segregation in C. elegans embryos. Using antisense morpholino oligonucleotides to knock down Xenopus tropicalis delangin or MAU-2 in early embryos produced similar patterns of retarded growth and developmental defects. Our data show that sister chromatid cohesion in metazoans involves the formation of a complex similar to the Scc2-Scc4 interaction in the budding yeast. The very high degree of sequence conservation between Scc4 homologs in complex metazoans is consistent with increased selection pressure to conserve additional essential functions, such as regulation of cell and axon migration during development.

Dysferlin associates with the developing T‐tubule system in rodent and human skeletal muscle

Mutations in the dysferlin gene cause limb‐girdle muscular dystrophy type 2B, Miyoshi myopathy, and distal anterior compartment myopathy. Dysferlin mainly localizes to the sarcolemma in mature skeletal muscle where it is implicated in membrane fusion and repair. In different forms of muscular dystrophy, a predominantly cytoplasmic localization of dysferlin can be observed in regenerating myofibers, but the subcellular compartment responsible for this labeling pattern is not yet known. We have previously demonstrated an association of dysferlin with the developing T‐tubule system in vitro. To investigate the role of dysferlin in adult skeletal muscle regeneration, we studied dysferlin localization at high resolution in a rat model of regeneration and found that the subcellular labeling of dysferlin colocalizes with the developing T‐tubule system. Furthermore, ultrastructural analysis of dysferlin‐deficient muscle revealed primary T‐tubule anomalies similar to those seen in caveolin‐3–deficient muscle. These findings indicate that dysferlin is necessary for correct T‐tubule formation, and dysferlin‐deficient skeletal muscle is characterized by abnormally configured T‐tubules. Muscle Nerve : 166–173, 2010

From T-tubule to sarcolemma: damage-induced dysferlin translocation in early myogenesis

The dysferlin gene is mutated in limb‐girdle muscular dystrophy type 2B, Miyoshi myopathy, and distal anterior compartment myopathy. In mature skeletal muscle, dysferlin is located predominantly at the sarcolemma, where it plays a role in membrane fusion and repair. To investigate the role of dysferlin during early muscle differentiation, its localization was studied at high resolution in a muscle cell line. This demonstrated that dysferlin is not expressed at the plasmalemma of myotubes but mostly localizes to the T‐tubule network. However, dysferlin translocated to the site of injury and toward the plasma membrane in a Ca2+‐dependent fashion in response to a newly designed in vitro wounding assay. This reaction was specific to the full‐length protein, as heterologously expressed deletion mutants of distinct C2 domains of dysferlin did not show this response. These results shed light on the dynamics of muscle membrane repair and are highly indicative of a specific role of dysferlin in this process in early myogenesis.—Klinge, L., Laval, S., Keers, S., Haldane, F., Straub, V., Barresi, R., Bushby, K. From T‐tubule to sarcolemma: damage‐induced dys‐ferlin translocation in early myogenesis. FASEB J. 21, 1768–1776 (2007)

Exon skipping mutations in collagen VI are common and are predictive for severity and inheritance

Mutations in the genes encoding collagen VI (COL6A1, COL6A2, and COL6A3) cause Bethlem myopathy (BM) and Ullrich congenital muscular dystrophy (UCMD), two related conditions of differing severity. BM is a relatively mild dominantly inherited disorder characterized by proximal weakness and distal joint contractures. UCMD was originally regarded as an exclusively autosomal recessive condition causing severe muscle weakness with proximal joint contractures and distal hyperlaxity. We and others have subsequently modified this model when we described UCMD patients with heterozygous in‐frame deletions acting in a dominant‐negative way. Here we report 10 unrelated patients with a UCMD clinical phenotype and de novo dominant negative heterozygous splice mutations in COL6A1, COL6A2, and COL6A3 and contrast our findings with four UCMD patients with recessively acting splice mutations and two BM patients with heterozygous splice mutations. We find that the location of the skipped exon relative to the molecular structure of the collagen chain strongly correlates with the clinical phenotype. Analysis by immunohistochemical staining of muscle biopsies and dermal fibroblast cultures, as well as immunoprecipitation to study protein biosynthesis and assembly, suggests different mechanisms each for exon skipping mutations underlying dominant UCMD, dominant BM, and recessive UCMD. We provide further evidence that de novo dominant mutations in severe UCMD occur relatively frequently in all three collagen VI chains and offer biochemical insight into genotype–phenotype correlations within the collagen VI–related disorders by showing that severity of the phenotype depends on the ability of mutant chains to be incorporated in the multimeric structure of collagen VI. Hum Mutat 29(6), 809–822, 2008.

Confirmatory Evidence for Linkage of Relative Hand Skill to 2p12-q11

To the Editor: We previously reported in the Journal the first genomewide linkage screen for a measure related to handedness in humans (Francks et al. 2002), in which we found evidence for a quantitative trait locus (QTL) influencing relative hand skill on chromosome 2p12-q11 (P=.00007). The screen was performed using 195 reading-disabled (RD) sibling pairs (Fisher et al. 2002), although reading ability was apparently unrelated to handedness in this sample. The 2p12-q11 linkage was the most significant in the screen by 1.5 orders of magnitude and approached the threshold for genomewide significance proposed by Lander and Kruglyak (1995) (threshold P=.00002). However, we failed to replicate the QTL in a second sample of a similar composition (143 sibling pairs). Therefore, the possibility remained that this was a false positive result, brought about by multiple testing of markers across the entire genome. Now, we have found further evidence for the 2p12-q11 QTL in a new sample of 105 pairs of adult brothers drawn from a sample of 168 unrelated male sibships (338 brothers) that was originally collected for investigating X-linked effects on handedness (described by Laval et al. [1998]). As before, we assessed relative hand skill using the test of Annett (1985), which involves measuring the time taken to move, with each hand, a row of pegs from one set of slots on a board to another. A relative hand skill quotient, PegQ, was derived for each subject as (L-R)/[(L+R)/2]; that is, the difference between left and right hand times, adjusted for overall hand skill (fig. 1a). Figure 1 a, PegQ distribution in 222 siblings with RD who appeared normal for this measure and were previously analyzed genomewide for linkage (black) and in 338 left-writing-handed brothers from whom the current study sample was drawn (gray). Positive scores ... The recruitment criterion that all brothers in each sibship should write with their left hands constituted a form of imperfect phenotypic selection for PegQ. This resulted in curtailed PegQ variance in the 338 brothers (fig. 1a) and suggested that quantitative linkage analysis of the whole sample might be underpowered. We therefore selected sibships on the basis of their suitability for linkage analysis with basic DeFries-Fulker regression (Fulker et al. 1991), which can derive power from extreme phenotypic selection. Extreme left handed “probands” were designated as scoring below −1.5 SD (fig. 1a), relative to the sample of reading-disabled siblings (who scored as an unselected population for relative hand skill). This yielded 101 probands in 88 independent sibships. The threshold of −1.5 SD was chosen to balance increased power from increasing severity of selection against diminishing power because of reduced sample size. No other threshold scores for designating probands were used. We genotyped the 88 sibships at seven microsatellite markers spanning 2p16-q14 and obtained multipoint identity-by-descent (IBD) sharing information across this interval, using the software Genehunter 2.1 (Pratt et al. 2000). Allele frequencies were calculated using data from all parents plus one random sibling in each family, and the genetic marker map was the same as used by Francks et al. (2002) (see fig. 1b). We then assessed the regression of PegQ in brothers of extreme left-handers toward the population mean, as a function of proband scores and IBD sharing with probands, using basic DeFries-Fulker regression as implemented in SAS macros by Lessem and Cherny (2001). A double entry procedure was used when a sibship contained more than one proband, as recommended (Fulker et al. 1991). This yielded a total of 91 independent proband-cosib pairs and 105 total proband-cosib pairs. Unbiased pointwise empirical significance levels for multipoint linkage results were obtained by performing 100,000 genotype simulations while fixing the family structures and phenotypes of the real sample (as described by Francks et al. [2002] and Fisher et al. [2002]) and then analyzing these replicates for linkage. The peak linkage t score was −3.51 (fig. 1b), asymptotic pointwise P=.00035, empirical pointwise P=.00090, thus greatly exceeding significance guidelines for confirmation of linkage (guideline P=.01; Lander and Kruglyak [1995]). The new linkage curve was strikingly similar to that found in the genomewide screen, and this concordance provides confirmatory evidence for the QTL over and above the significance level of the linkage (fig. 1b). This linkage evidence confirms that, although handedness variation may be etiologically complex, there is at least one polymorphic genetic influence that is located on 2p12-q11. Epidemiological studies of twins have provided ambiguous data that point either to weak or else to nonsignificant genetic effects on handedness (Bishop 2001), but no large-scale twin studies have used the greater potential power inherent in a continuous description of the trait, whereas PegQ has shown familialities of up to 35% in our samples (Francks et al. 2002). Linkage analysis of handedness as a dichotomous trait is, therefore, likely to be underpowered, but only one study has so far attempted this approach, and for only six genomic regions (not including 2p12-q11), without identifying suggestive or significant linkage (Van Agtmael et al. 2002). Sex-dependent effects on cerebral lateralization and on the inheritance of handedness have pointed to the involvement of an X-linked genetic effect on handedness (Corballis et al. 1996; McKeever 2000), and suggestive or weak evidence for linkage of relative hand skill to a locus on Xq21 has been identified in both our RD siblings and the left-handed brothers (Laval et al. 1998; Francks et al. 2002), although Crow (2002) has suggested that any X-linked effect may be mediated by an epigenetic mechanism. Roughly 90% of individuals perform complex manual tasks preferentially with their right hands, whereas slightly <10% are left-handed, and a small proportion are ambidextrous (McManus and Bryden 1992). No other primates show a population-level bias in handedness, and individual differences in human handedness are correlated with cerebral hemispheric asymmetries that underlie much complex human cognition, including language (McGrew and Marchant 1997; Geschwind et al. 2002), as well as with asymmetries of the motor cortex (Amunts et al. 1996). We predict that genes containing variants that influence handedness have an important role in the development of cerebral lateralization and may have been involved in the evolution of complex human cognition.

Protein studies in dysferlinopathy patients using llama-derived antibody fragments selected by phage display

Mutations in dysferlin, a member of the fer1-like protein family that plays a role in membrane integrity and repair, can give rise to a spectrum of neuromuscular disorders with phenotypic variability including limb-girdle muscular dystrophy 2B, Myoshi myopathy and distal anterior compartment myopathy. To improve the tools available for understanding the pathogenesis of the dysferlinopathies, we have established a large source of highly specific antibody reagents against dysferlin by selection of heavy-chain antibody fragments originating from a nonimmune llama-derived phage-display library. By utilizing different truncated forms of recombinant dysferlin for selection and diverse selection methodologies, antibody fragments with specificity for two different dysferlin domains could be identified. The selected llama antibody fragments are functional in Western blotting, immunofluorescence microscopy and immunoprecipitation applications. Using these antibody fragments, we found that calpain 3, which shows a secondary reduction in the dysferlinopathies, interacts with dysferlin.

ANO5 gene analysis in a large cohort of patients with anoctaminopathy: confirmation of male prevalence and high occurrence of the common exon 5 gene mutation

Limb girdle muscular dystrophy type 2L or anoctaminopathy is a condition mainly characterized by adult onset proximal lower limb muscular weakness and raised CK values, due to recessive ANO5 gene mutations. An exon 5 founder mutation (c.191dupA) has been identified in most of the British and German LGMD2L patients so far reported. We aimed to further investigate the prevalence and spectrum of ANO5 gene mutations and related clinical phenotypes, by screening 205 undiagnosed patients referred to our molecular service with a clinical suspicion of anoctaminopathy. A total of 42 unrelated patients had two ANO5 mutations (21%), whereas 14 carried a single change. We identified 34 pathogenic changes, 15 of which are novel. The c.191dupA mutation represents 61% of mutated alleles and appears to be less prevalent in non‐Northern European populations. Retrospective clinical analysis corroborates the prevalently proximal lower limb phenotype, the male predominance and absence of major cardiac or respiratory involvement. Identification of cases with isolated hyperCKaemia and very late symptomatic male and female subjects confirms the extension of the phenotypic spectrum of the disease. Anoctaminopathy appears to be one of the most common adult muscular dystrophies in Northern Europe, with a prevalence of about 20%–25% in unselected undiagnosed cases.

Cyclosporine A treatment for Ullrich congenital muscular dystrophy: a cellular study of mitochondrial dysfunction and its rescue

Mutations in COL6A1, COL6A2 and COL6A3, the genes which encode the extra-cellular matrix component collagen VI, lead to Bethlem myopathy and Ullrich congenital muscular dystrophy (UCMD). Although the Col6a1(-/-) null mouse has an extremely mild neuromuscular phenotype, a mitochondrial defect has been demonstrated, linked to dysregulation of the mitochondrial permeability transition pore (PTP) opening. This finding has been replicated in UCMD muscle cells in culture, providing justification for a clinical trial using cyclosporine A, an inhibitor of PTP opening. We investigated whether PTP dysregulation could be detected in UCMD fibroblasts (the predominant source of muscle collagen VI), in myoblast cells from patients with other diseases and its response to rescue agents other than collagen VI. Although we confirm the presence of PTP dysregulation in muscle-derived cultures from two UCMD patients, fibroblasts from the same patients and the majority of fibroblasts from other well-characterized UCMD patients behave normally. PTP dysregulation is found in limb girdle muscular dystrophy (LGMD) type 2B myoblasts but not in myoblasts from patients with Bethlem myopathy, merosin-deficient congenital muscular dystrophy, LGMD2A, Duchenne muscular dystrophy and Leigh syndrome. In addition to rescue by cyclosporine A and collagen VI, this cellular phenotype was also rescued by other extra-cellular matrix constituents (laminin and collagen I). As the muscle derived cultures demonstrating PTP dysregulation shared poor growth in culture and lack of desmin labelling, we believe that PTP dysregulation may be a particular characteristic of the state of these cells in culture and is not specific to the collagen VI defect, and can in any case be rescued by a range of extra-cellular matrix components. Further work is needed on the relationship of PTP dysregulation with UCMD pathology.