Pathogenic CGG expansions in oculopharyngodistal myopathy exhibit distinct characteristics of each causative gene on the flanking sequences as well as methylation status

Background: Oculopharyngodistal myopathy (OPDM) is a hereditary muscle
disease caused by CGG/CCG repeat expansions in six genes. Although the
clinical features are often similar, such as ptosis, dysphagia, and distal
muscle weakness, the age at onset vary widely, and the mechanisms
underlying this variation remain unclear. In particular, the contributions
of repeat size, flanking sequence variation, and DNA methylation to
phenotype have not been systematically explored using single-molecule
resolution. Methods: We applied CRISPR/Cas9-targeted nanopore sequencing
(nCATS) to genomic DNA from 91 individuals carrying expanded CGG repeats
in three OPDM-related genes (LRP12, GIPC1, and NOTCH2NLC). This approach
enabled the simultaneous analysis of CGG repeat length, flanking sequence
architecture, single-nucleotide variant haplotypes, structural variation,
and CpG methylation profiles. Genotype–phenotype correlations were
evaluated by integrating molecular and clinical data. Results: Expanded
LRP12 and GIPC1 alleles in the patients showed respective single
nucleotide variant patterns around repeat regions, suggesting founder
haplotypes. Repeat regions essentially comprised pure CGG expansions, but
exhibited size variability, even within patients. Additionally,
LRP12-expanded repeats lacked flanking nucleotide sequences present in
non-expanded repeats, whereas GIPC1 expanded repeats contained specific
discontinued CGG patterns in their 5'-regions. Structural variations
were also identified in some patients. A significant inverse correlation
was observed between repeat length and age at onset in patients with GIPC1
or NOTCH2NLC expansions, while this was disturbed by higher methylation of
upstream regions in patients with LRP12 expansions, leading to delayed
onset. Conclusions: This study highlights gene-specific differences in CGG
repeat architecture and epigenetic regulation in OPDM. Founder haplotypes,
expanded allele-specific flanking sequences, and the combined effects of
repeat size and methylation contribute to patient regional frequency,
repeat stability, and clinical variability, respectively, offering insight
into disease pathomechanism and potential therapeutic targets.