Mitochondrial disorders are the most common inherited conditions, characterized by defects in oxidative phosphorylation and caused by mutations in nuclear or mitochondrial genes. Due to its high energy request, skeletal muscle is typically involved. According to the International Workshop of Experts in Mitochondrial Diseases held in Rome in 2016, the term Primary Mitochondrial Myopathy (PMM) should refer to those mitochondrial disorders affecting principally, but not exclusively, the skeletal muscle. The clinical presentation may include general isolated myopathy with muscle weakness, exercise intolerance, chronic ophthalmoplegia/ophthalmoparesis (cPEO) and eyelids ptosis, or multisystem conditions where there is a coexistence with extramuscular signs and symptoms. In recent years, new therapeutic targets have been identified leading to the launch of some promising clinical trials that have mainly focused on treating muscle symptoms and that require populations with defined genotype. Advantages in next-generation sequencing techniques have substantially improved diagnosis. So far, an increasing number of mutations have been identified as responsible for mitochondrial disorders. In this review, we focused on the principal molecular genetic alterations in PMM. Accordingly, we carried out a comprehensive review of the literature and briefly discussed the possible approaches which could guide the clinician to a genetic diagnosis.

SCN4A encodes the skeletal-muscle voltage-gated sodium channel NaV1.4. Pathogenic variation in this gene produces fundamentally different disease mechanisms, including dominant alpha-pore gain of function, dominant S4 gating-pore currents, and reduced channel availability, with severe biallelic loss of function causing congenital myopathy and fetal hypokinesia. This heterogeneity makes variant interpretation difficult because formal variant classification, the direction of channel dysfunction, and patient-level disease attribution are closely related but distinguishable conclusions. In this review, we synthesize the clinical, genetic, electrophysiological, structural, and functional evidence relevant to SCN4A variant interpretation, with particular attention to missense variants of uncertain significance. We examine how inheritance, channel topology, phenotype, population data, segregation, RNA evidence, regional and residue-level context, and mechanism-matched functional assays can be integrated within the ACMG/AMP framework. Published pathogenic-enriched regions, gnomAD regional missense constraint, and same-residue observations may contribute to variant interpretation when the requirements of an applicable ACMG/AMP criterion are met; when they have not been specifically validated or calibrated for SCN4A, they are best used as contextual information to prioritize additional evidence generation. Based on this literature, we organize these evidence domains into a practical phenotype-first, mechanism-informed workflow for SCN4A variant interpretation. This workflow is not intended as an alternative to or extension of ACMG/AMP but as an SCN4A-specific application of established variant-interpretation principles in a gene associated with multiple inheritance patterns and directionally distinct disease mechanisms. After technical confirmation of a candidate variant, as well as phenotype and inheritance context guide selection of the relevant disease model, appropriately validated functional evidence may contribute directly to ACMG/AMP classification, while functional mechanism and patient-level disease attribution are documented as related but distinguishable interpretative outputs. The same approach applies when a variant is identified through genotype-first sequencing: interpretation should return to deliberate phenotyping, inheritance assessment, electrophysiological characterization, and consideration of the differential diagnosis before disease causality is inferred.