Interference of Small Sequence Variants with MLPA in CLCN1: Implications for Congenital Myotonia Diagnosis.

BACKGROUND/OBJECTIVES: Congenital myotonia (CM) is an inherited neuromuscular disorder caused by mutations in the Chloride Voltage-Gated Channel 1 (CLCN1) gene, encoding the Chloride Channel 1 (CIC-1) in skeletal muscle. These mutations can be inherited in either an autosomal dominant (Thomsen's disease) or recessive pattern (Becker's disease). Within the diagnostic workflow for this pathology, gene sequencing represents one of the main analytical approaches, while Multiplex Ligation-dependent Probe Amplification (MLPA) is used as a complementary method, particularly in patients with autosomal recessive inheritance, to detect deletions or duplications involving one or more exons of the CLCN1. This study aims to highlight a specific limitation of MLPA in CLCN1 analysis. METHODS: Patients carrying pathogenic CLCN1 variants, previously identified by sequencing, subsequently underwent MLPA analysis to investigate the presence of a second pathogenic allele. RESULTS: In the analysed patients, MLPA identified apparent exon deletions involving the same exons in which the previously identified variants were present. However, no actual variation in copy number was observed at the exon level. This discrepancy is linked to the efficiency of probe hybridization and ligation to target sequences. Small sequence variations can interfere with probe binding, preventing their correct ligation and resulting in a signal pattern consistent with apparent exon loss, thereby generating false positives. CONCLUSIONS: Our findings highlight the importance of integrating sequencing and MLPA data in CLCN1 analysis. Awareness of variant-induced MLPA artefacts is essential to avoid misinterpretation of apparent exon deletions and to ensure an accurate molecular diagnosis of CM.