Integrated targeted whole-genome and RNA-sequencing analysis of an intronic GNE variant in GNE myopathy

GNE myopathy is a rare autosomal recessive myopathy caused by biallelic pathogenic variants in GNE, which encodes an essential enzyme for sialic acid biosynthesis. Most variants are located in exonic regions, whereas significance of intronic variants remains unclear, leaving many suspected cases genetically unresolved. We aimed to assess the pathogenicity of intronic GNE variants using targeted whole-genome and RNA sequencing. We performed combined long amplicon sequencing in two siblings with GNE myopathy. DNA and RNA were extracted from muscle biopsy specimens and peripheral blood mononuclear cells. Variants were validated using Sanger sequencing. Splicing effects were predicted using SpliceAI and SpliceRover and validated by RNA sequencing. Muscle sialylation was assessed using peanut agglutinin lectin staining. An uncharacterized intronic variant, NC_000009.12(NM_001128227): c.1163+5G>T, was identified in trans with a known missense variant (NM_001128227): c.1807C>G, p.V603L. RNA sequencing demonstrated aberrant splicing caused by intronic mutation, leading to a non-productive transcript in muscle and blood cells. Reduced sialic acid biosynthesis further supported the pathogenicity of the intronic mutation. These findings demonstrate that combined long amplicon sequencing can elucidate the genetic basis of GNE myopathy undetectable by exome sequencing. This approach provides a clinically applicable, less invasive diagnostic strategy and expands the spectrum of pathogenic GNE variants.