Abstract
SCN4A codes for a skeletal muscle sodium channel (SCN) that is essential for muscle excitability and contraction. SCN4A mutations cause a number of neuromuscular phenotypes including myotonia and periodic paralysis. Infants with SCN4A mutations have common and/or severe respiratory compromise include life-threatening apnoeas, whilst adults do not. Recently we described SCN4A-linked sudden infant death. Surviving infants show a spontaneous reduction in respiratory episodes with age, even if untreated, suggesting a developmental contribution to the clinical phenotype. Lack of isoform-specific SCN monoclonal antibodies are a major bottleneck in functional and pathological studies of SCN gene mutations. To study the expression of SCNs in relation to the fibre type and developmental stage we have developed two multiplex assays for testing on whole transverse frozen sections from control human skeletal muscle biopsies (0-16 years) in whom a primary neuromuscular disorder is excluded. Samples are recruited from the MRC CNMD biobank in London. Assay 1 (ISH-IHC) includes fluorescent multiplex labelling of isoform-specific mRNA probes to SCN4A, SCN5A (ACDBio), and antibodies to pan-fast/slow myosin heavy chain and laminin alpha-2, latter used as mask for myofibre identification. Assay 2 is a fully automated multi-coloured chromogenic IHC protocol (Roche Diagnostics) allowing detection of myosin heavy chain isoforms I, IIA and IIX in a single skeletal muscle section. Further assay optimisation including creating an Assay-1-2 hybrid and developing a digital script for mRNA quantification are in progress. Age-stratified data on fibre-type composition and fibre-specific SCN4A/5A-mRNA expression will be presented. Knowledge of developmental changes in fibre-type proportions may help in understanding differences in phenotype of infants and adults with SCN4A mutations. mRNA expression data will be valuable in validating future isoform-specific SCN antibodies.
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