Clinical, molecular and histopathological analysis of myotonia congenita in a Malaysian family with a rare c.1667T>A (p.Ile556Asn) variant in CLCN1

Myotonia congenita (MC) is a rare neuromuscular disease resulting from genetic variants in the CLCN1 gene, which encodes skeletal muscle chloride channels. It has a prevalence of approximately 1 in 100,000 individuals worldwide. It is characterised by muscle stiffness which causes by delayed muscle relaxation during contraction. This study was aimed to determine the clinical, molecular aspects and histopathological changes of MC. The study reports a 49-year-old female proband who presented with muscle stiffness started in early childhood. Clinical diagnosis was established using electrophysiological and blood analysis, whole exome sequencing (WES), in silico and Sanger sequencing, molecular gene expression, and epigenetic studies. The functional effects of the variant were characterised through protein expression and histopathological analysis. The proband was diagnosed with MC after myotonic discharge on electromyography, clinical examination with transient muscle weakness, warm-up phenomenon, and muscular hypertrophy. No additional family member had MC except her brother who had similar symptoms. Serum creatine kinase levels were elevated in both proband and her brother with 447 U/L and 228 U/L respectively. Furthermore, WES revealed a previously reported pathogenic CLCN1 heterozygous variant NM_000083.3(CLCN1):c.1667T>A (p.Ile556Asn), in the proband, her brother, and observed in all their asymptomatic children, showing a dominant with an incomplete penetrance inheritance. Characterisation of this variant using multiple in silico prediction tools showed a damaging and deleterious effect on the ClC-1 protein, and with low allele frequency, it indicates that the variant is a rare and uncommon. Thus, the variant was classified as pathogenic according to ACMG guidelines. Additionally, most allele counts were found in South Asian ancestry, highlighting the need to monitor asymptomatic carriers in the Southeast Asian population. The multiplex ligation-dependent probe amplification analysis revealed neither exon deletion nor duplication found in CLCN1, which rules out the recessive form of MC. Meanwhile, epigenetics analysis through DNA methylation demonstrated a hypermethylated CpG site in the proband located in the CLCN1 enhancer region. This hypermethylation is implicated in the reduction of gene expression and subsequently, protein expression, which suggests that epigenetics might play a role in the genotype-phenotype correlation. Histopathological analysis showed an increase in the internal nuclei by 33%, indicating hindrance of muscle contraction. Additionally, the diameter of muscle fibres varied, displaying hypertrophic muscle fibres and the occurrence of type 2 fast-twitch fibres predominance, reflecting a hypertrophic condition and rapid relaxation of muscle stiffness in the patient, respectively. Ultrastructural analysis using transmission electron microscopy showed a normal Z- line with an irregular arrangement of fibres showing narrowing and splitting of myofibrils and reduction of mitochondria. Thus, these histopathological findings correlate with the mild phenotype in the MC patient. In conclusion, this study describes the first case of MC in Malaysia caused by a known pathogenic CLCN1 variant. Collectively, the c.1667T>A (p.Ile556Asn) variant is a rare variant that displayed incomplete penetrance causing clinical, molecular, and mild histopathological changes. The findings of this study have elucidated the c.1667T>A (p.Ile556Asn) variant in MC and provide an understanding of its impact will lead to personalised medicine thereby improving diagnosis, genetic counselling, and treatment.

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