Diagnostic anoctamin-5 protein defect in patients with ANO5-mutated muscular dystrophy.

AIMS: Previously, detection of ANO5 protein has been complicated by unspecific antibodies, most of which have not identified the correct protein. The aims of the study were to specify ANO5 protein expression in human skeletal muscle, and to investigate if the ANO5 protein levels are affected by different ANO5 mutations in anoctaminopathy patients. METHODS: Four different antibodies were tested for ANO5 specificity. A sample preparation method compatible with membrane proteins, combined with tissue fractionation was used to determine ANO5 expression in cell cultures expressing ANO5, in normal muscles and eight patient biopsies with six different ANO5 mutations in homozygous or compound heterozygous states, and in other dystrophies. RESULTS: Only one specific monoclonal N-terminal ANO5 antibody was efficient in detecting the protein, showing that ANO5 is expressed as a single 107 kD polypeptide in human skeletal muscle. The truncating mutations c.191dupA and c.1261C>T were found to abolish ANO5 expression, whereas the studied point mutations had variable effects; however, all the ANO5 mutations resulted in clearly reduced ANO5 expression in the patient muscle membrane fraction. Attempts to detect ANO5 using immunohistochemistry were not yet successful. CONCLUSIONS: The data presented here indicate that the ANO5 protein expression is decreased in ANO5-mutated muscular dystrophy and that most of the non-truncating pathogenic ANO5 mutations likely destabilize the protein and cause its degradation. The method described here allows direct analysis of human ANO5 protein, which can be used in diagnostics, for evaluating the pathogenicity of the potentially harmful ANO5 variants of uncertain significance.

Altered expression and splicing of Ca(2+) metabolism genes in myotonic dystrophies DM1 and DM2.

AIMS: Myotonic dystrophy types 1 and 2 (DM1 and DM2) are multisystem disorders caused by similar repeat expansion mutations, with similar yet distinct clinical features. Aberrant splicing of multiple effector genes, as well as dysregulation of transcription and translation, has been suggested to underlie different aspects of the complex phenotypes in DM1 and DM2. Ca(2+) plays a central role in both muscle contraction and control of gene expression, and recent expression profiling studies have indicated major perturbations of the Ca(2+) signalling pathways in DM. Here we have further investigated the expression of genes and proteins involved in Ca(2+) metabolism in DM patients, including Ca(2+) channels and Ca(2+) binding proteins. METHODS: We used patient muscle biopsies to analyse mRNA expression and splicing of genes by microarray expression profiling and RT-PCR. We studied protein expression by immunohistochemistry and immunoblotting. RESULTS: Most of the genes studied showed mRNA up-regulation in expression profiling. When analysed by immunohistochemistry the Ca(2+) release channel ryanodine receptor was reduced in DM1 and DM2, as was calsequestrin 2, a sarcoplasmic reticulum lumen Ca(2+) storage protein. Abnormal splicing of ATP2A1 was more pronounced in DM2 than DM1. CONCLUSIONS: We observed abnormal mRNA and protein expression in DM affecting several proteins involved in Ca(2+) metabolism, with some differences between DM1 and DM2. Our protein expression studies are suggestive of a post-transcriptional defect(s) in the myotonic dystrophies.

Zaspopathy in a large classic late-onset distal myopathy family.

Distal myopathies have been associated with mutations in titin, dysferlin, GNE, desmin and myosin. Of these, only titin mutations were previously known to cause dominant late-onset distal myopathy. Recent findings, however, have indicated that patients affected with myofibrillar myopathy have a more distal than proximal muscle phenotype and a proportion of these may have mutations in myotilin, ZASP or filamin C, besides previously known desmin and alphaB-crystallin. Here we report that the disorder in one of the well-characterized autosomal dominant distal myopathy families, the Markesbery et al. family, first reported in 1974, is caused by ZASP mutation A165V. Previous linkage to the titin locus 2q31 proved incorrect. ZASP expression by immunoblotting shows normal presence of the main 32 and 78 kDa bands and immunohistochemistry in patients reveals normal Z-disc localization except for moderate accumulations together with myotilin, desmin alphaB-crystallin and alpha-actinin. Muscle imaging reveals involvement in both the posterior and anterior compartments of the lower leg and considerable affection of proximal leg muscles at later stages. Haplotype studies in this family and in five other unrelated families with European ancestry carrying the identical A165V mutation share common markers at the locus suggesting the existence of a founder mutation.

Titinopathies and extension of the M-line mutation phenotype beyond distal myopathy and LGMD2J.

OBJECTIVE: To determine the phenotype variability associated with the specific C-terminal M-line titin mutation known to cause autosomal dominant distal myopathy, tibial muscular dystrophy (TMD; MIM 600334), and limb girdle muscular dystrophy 2J (LGMD2J). METHODS: Three hundred eighty-six individuals were genotyped for the Finnish founder mutation in titin (FINmaj) causing TMD/LGMD2J. RESULTS: Two hundred seven patients were heterozygous for the mutation. Among these patients, 189 (91%) had a more common phenotype compatible with the classic description of TMD. However, 18 (9%) had unusual phenotypes such as proximal leg or posterior lower leg muscle weakness and atrophy even at onset. Four patients were confirmed homozygotes representing the LGMD2J phenotype. These homozygotes were half of the eight LGMD patients previously described in the original large consanguineous kindred. CONCLUSIONS: Large variability of phenotypic expression caused by just one mutation, the Finnish FINmaj, suggests that no certain phenotype of myopathy/dystrophy can be excluded from being caused by mutated titin. Yet unknown homozygous or compound heterozygous titin mutations without phenotype in the heterozygote carriers may be responsible for undetermined recessive MD and LGMD.

New methods for molecular diagnosis and demonstration of the (CCTG)n mutation in myotonic dystrophy type 2 (DM2).

Myotonic dystrophy types 1 and 2 are autosomal dominant, multisystemic disorders with many similarities in their clinical manifestations. Myotonic dystrophy type 1 is caused by a (CTG)n expansion in the 3' untranslated region of the DMPK gene in 19q13.3 and myotonic dystrophy type 2 by a (CCTG)n expansion in intron 1 of ZNF9 in 3q21.3. However, the clinical diagnosis of myotonic dystrophy type 2 is more complex than that of myotonic dystrophy type 1, and conventional molecular genetic methods used for diagnosing myotonic dystrophy type 1 are insufficient for myotonic dystrophy type 2. Herein we describe two in situ hybridization protocols for the myotonic dystrophy type 2 mutation detection. Chromogenic in situ hybridization was used to detect both the genomic expansion and the mutant transcripts in muscle biopsy sections. Chromogenic in situ hybridization can be used in routine myotonic dystrophy type 2 diagnostics. Fluorescence in situ hybridization on extended DNA fibers was used to directly visualize the myotonic dystrophy type 2 mutation and to estimate the repeat expansion sizes.

Histopathological differences of myotonic dystrophy type 1 (DM1) and PROMM/DM2.

Muscle biopsy findings in DM2 have been reported to be similar to those in DM1. The authors used myosin heavy chain immunohistochemistry and enzyme histochemistry for fiber type differentiation on muscle biopsies. Their results show that DM2 patients display a subpopulation of type 2 nuclear clump and other very small fibers and, hence, preferential type 2 fiber atrophy in contrast to type 1 fiber atrophy in DM1 patients.

Secondary calpain3 deficiency in 2q-linked muscular dystrophy: titin is the candidate gene.

BACKGROUND: Tibial muscular dystrophy (TMD), a late-onset dominant distal myopathy, is caused by yet unknown mutations on chromosome 2q, whereas MD with myositis (MDM) is a muscular dystrophy of the mouse, also progressing with age and linked to mouse chromosome 2. For both disorders, linkage studies have implicated titin as a potential candidate gene. METHODS: The authors analyzed major candidate regions in the titin gene by sequencing and Southern blot hybridization, and performed titin immunohistochemistry on TMD patient material to identify the underlying mutation. Western blot studies were performed on the known titin ligands in muscle samples of both disorders and controls, and analysis of apoptosis was also performed. RESULTS: The authors identified almost complete loss of calpain3, a ligand of titin, in the patient with limb-girdle MD (LGMD) with a homozygous state of TMD haplotype when primary calpain3 gene defect was excluded. Apoptotic myonuclei with altered distribution of transcription factor NF-kB and its inhibitor IkBalpha were encountered in muscle samples of patients with either heterozygous or homozygous TMD haplotype. Similar findings were confirmed in the MDM mouse. CONCLUSIONS: These results imply that titin mutations may be responsible for TMD, and that the pathophysiologic pathway following calpain3 deficiency may overlap with LGMD2A. The loss of calpain3 could be a downstream effect of the deficient TMD gene product. The significance of the secondary calpain3 defect for the pathogenesis of TMD was emphasized by similar calpain3 deficiency in the MDM mouse, which is suggested to be a mouse model for TMD. Homozygous mutation at the 2q locus may thus be capable of producing yet another LGMD.