Identification and characterization of three novel mutations in theCASQ1gene in four patients with tubular aggregate myopathy

Abstract

Here, we report the identification of three novel missense mutations in the calsequestrin-1 (CASQ1) gene in four patients with tubular aggregate myopathy. These CASQ1 mutations affect conserved amino acids in position 44 (p.(Asp44Asn)), 103 (p.(Gly103Asp)), and 385 (p.(Ile385Thr)). Functional studies, based on turbidity and dynamic light scattering measurements at increasing Ca concentrations, showed a reduced Ca -dependent aggregation for the CASQ1 protein containing p.Asp44Asn and p.Gly103Asp mutations and a slight increase in Ca -dependent aggregation for the p.Ile385Thr. Accordingly, limited trypsin proteolysis assay showed that p.Asp44Asn and p.Gly103Asp were more susceptible to trypsin cleavage in the presence of Ca in comparison with WT and p.Ile385Thr. Analysis of single muscle fibers of a patient carrying the p.Gly103Asp mutation showed a significant reduction in response to caffeine stimulation, compared with normal control fibers. Expression of CASQ1 mutations in eukaryotic cells revealed a reduced ability of all these CASQ1 mutants to store Ca and a reduced inhibitory effect of p.Ile385Thr and p.Asp44Asn on store operated Ca entry. These results widen the spectrum of skeletal muscle diseases associated with CASQ1 and indicate that these mutations affect properties critical for correct Ca handling in skeletal muscle fibers.

Tubular aggregate myopathy and Stormorken syndrome: Mutation spectrum and genotype/phenotype correlation

“…It is however conceivable that amino acid substitutions in the SAM domain, encoded by exons 4 and 5, or within the cytosolic coiled‐coil domain 3, encoded by exons 9 and 10 and forming the inhibitory clamp with CC1, can have a similar pathogenic effect as the known STIM1 GOF mutations and cause TAM/STRMK. If no mutation is detected, all other exons including the muscle‐specific long exon 11 should be analyzed, followed by the other TAM/STRMK gene ORAI1 and finally CASQ1 , associated with a moderate TAM phenotype restricted to skeletal muscle (Barone et al, ; Böhm et al, ).…”

Molecular determinants of homo- and heteromeric interactions of Junctophilin-1 at triads in adult skeletal muscle fibers

“…In all immunostaining experiments, primary antibodies were prepared in 2% BSA and incubated overnight at 4°C. Slides were mounted with Mowiol (Mowiol 4-88, Sigma-Aldrich, 20% diluted in PBS) and analyzed with a LSM-510 META confocal microscope (ZEISS, Jena, Germany) as previously described (55). Fibers expressing low levels of GFP fluorescence were selected to avoid potential problems due to overexpression.…”

Abnormal Excitation-Contraction Coupling and Calcium Homeostasis in Myopathies and Cardiomyopathies

“…Calsequestrin also actively participates in the Ca 2+ release process by providing a large Ca 2+ pool in close proximity to the RyR release channel [77]. Heterozygous CASQ1 missense mutations were found in vacuolar myopathy (OMIM #616231) [78] and in tubular aggregate myopathy (TAM) [79,80], both involving mild muscle weakness, but differing at the histopathological level. Vacuolar myopathy biopsies show vacuoles containing diverse SR proteins and enlargement of the terminal SR cisternae [78], while TAM muscle fibers typically accumulate densely packed membrane tubules [81].…”

“…Vacuolar myopathy biopsies show vacuoles containing diverse SR proteins and enlargement of the terminal SR cisternae [78], while TAM muscle fibers typically accumulate densely packed membrane tubules [81]. Both disorders arise from adverse mutational effects: the TAM-related CASQ1 mutations were shown to significantly impair calsequestrin polymerization, resulting in reduced Ca 2+ storage abilities of the SR, while the vacuolar myopathy mutation promoted the formation of high molecular mass polymers [78][79][80]. Noteworthy, Casq1 null mice exhibit a susceptibility to heatand anesthetics-induced sudden death resembling malignant hyperthermia [82].…”