Summary by Jantima Tanboon, MD, PhD

Scheme by Nobuyuki Eura, MD, PhD

Short title: Flanking sequences and methylation statuses underlie clinical heterogeneity in OPDM

Publication

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

Eura N, Noguchi S, Ogawa M, Sonehara K, Yamanaka A, Kurashige T, Hayashi S, Okada Y, Sugie K, Nishino I.

Genome Med. 2026 Mar 27;18(1):33.

doi: 10.1186/s13073-026-01617-x. PMID: 41888971; PMCID: PMC13023157.

https://link.springer.com/article/10.1186/s13073-026-01617-x#citeas

Oculopharyngodistal myopathy (OPDM) is an autosomal dominant myopathy caused by CGG/CCG repeat expansions in six genes LRP12, GIPC1, NOTCH2NLC, RIPL1, LOC642361/NUTM2B-AS1, and ABCD3. This study, based on the implication of previous studies (Mohren L, et al. Nat Commun. 2024 and Pellerin D, et al. Nat Gen. 2024), that sequence variation flanking the repeats might influence repeat stability causing difference in age of the onset and clinical severity among patients. The authors used CRISPR/Cas9-targeted Nanopore sequencing (nCATS) to assess repeat size, sequencing variation, and methylation status of genomic DNAs from 91 individuals carrying CGG repeat expansions in OPDM (86 symptomatic OPDM_LRP12, OPDM_GIPC1 and OPDM_NOTCH2NLC patients and five family members) with genotype-phenotype correlation. Intrafamilial repeat analysis was performed on 12 individuals from four OPDM_LRP12 families and three individuals from one OPDM_GIPC1 family. Whole genome sequencing data from 1,162 Japanese individuals was used as reference population.

The results show intra-patient repeat size variability, especially in OPDM_LRP12, indicating somatic instability of the repeats, which may contribute to intrafamilial clinical heterogeneity. Three OPDM_GIPC1 and 3 OPDM_NOTCH2NLC showed additional low proportion of repeat expansion (<5%) in LRP12 gene, suggesting de novo expansion with negligible effect. Ten and seven single-nucleotide variants (SNVs) were present in 82.9% and 100% of expanded alleles in OPDM_LRP12 and OPDM_GIPC1, respectively. The corresponding SNVs were present in 2.9% of non-expanded alleles in OPDM_LRP12 and 7% of OPDM_GIPC1 and presence in a low proportion in the Japanese reference population implying a founder effect. No common SNVs pattern in OPDM_NOTCH2NLC.

The presence of flanking sequences in the non-expanded alleles of OPDM_LRP12, their absence in the expanded alleles and the distinct repeat sequence in the 5' region of the OPDM_GIPC1 expanded alleles suggesting a role in repeat length during meiosis and haplotype-specific expansion supported by free minimum energy single-strand RNA structural prediction. Loss of flanking sequences may represent a common molecular feature across LRP12-related CGG expansion as the expanded alleles in two cases of amyotrophic lateral sclerosis with LRP12-related CGG expansion showed matched haplotypes, lacked flanking sequences in expanded alleles but retained the flanking sequences observed in OPDM_LRP12. Notably, no distinct flanking sequences are present in OPDM_GIPC1 and OPDM_NOTCH2NLC.

The authors also demonstrated structural variations outside the repeat expansion regions resulting in complex repeat-associated non-AUG (RAN) translation products. The variations included a deletion in upstream region of two OPDM_LRP12 predicting frameshifts with different translated products; a duplication containing the repeat expansion and its upstream region in one OPDM_GIPC1 predicting two separate amino acid polymers; and a deletion of flanking region downstream to the repeat expansions in one OPDM_NOTCH2NLC predicting frameshift with fusion protein containing polyglycine and NOTCH2NLC. These results suggested the RAN-translational theory was not merely an alternative universal mechanism to the polyglycine aggregation hypothesis in CGG repeat disorders. On genotype-phenotype correlation, the authors demonstrated two asymptomatic fathers of two OPDM_LRP12 families with hypermethylated ultra-long reads (> 300 repeats); OPDM_LRP12 individuals with pathogenic reads (< 300 repeats) showing a bimodal methylation pattern; OPDM_GIPC1; and OPDM_NOTCH2NLC with pathogenic reads (< 300 and < 600 reads, respectively) showing a low methylation pattern, which suggested differences in epigenetic regulation in different genes. Interestingly, a symptomatic father of one OPDM_LRP12 family had hypermethylated ultra-long reads (599), implying partial methylation as a modulating mechanism in patients with ultra-long reads. Inverted correlation between repeat length and age of onset was observed across all three genes but the correlation coefficient was lower in patients with OPDM_LRP12. This was likely due to different methylation susceptibility in these genes: variable CpG methylated expand reads in OPDM_LRP12 vs. consistently unmethylated expand reads in OPDM_GIPC1 or OPDM_NOTCH2NLC.

In summary, this study used nCATS as a novel approach to analyse OPDM repeat expansions, providing new insights on genotype–phenotype relation­ships, founder effects, and potential mechanisms under­lying repeat instability, including flanking sequence architectures, haplotypes, structural variations and CpG methylation shaping clinical heterogeneity.

About the author

Noboyuki Eura, MD, PhD, is a clinical neurologist and Lecturer in the Department of Neurology at Nara Medical University, Japan. He graduated from Nara Medical University School of Medicine in 2008 and completed his neurology residency at Nara Medical University Hospital, where he trained in muscle pathology and neuroembryology. He received his PhD in 2020 for his work on brainstem organoid generation. From 2021 to 2023, he was a postdoctoral fellow in Ichizo Nishino’s laboratory at the National Center of Neurology and Psychiatry, where he studied the diagnosis and molecular pathomechanisms of oculopharyngodistal myopathy. His research focuses on neuromuscular disorders, including inherited myopathies and inflammatory myopathies, with particular emphasis on muscle pathology, skeletal muscle imaging, and genetics.

About the reviewer

Dr Jantima Tanboon is an assistant professor of anatomical pathology at Siriraj Hospital, Mahidol University, Thailand. Dr.Tanboon was a research student and a postdoctoral research fellow at the Department of Neuromuscular Research at the National Center of Neurology and Psychiatry (NCNP), Japan under the supervision of Dr.Satoru Noguchi and Dr.Ichizo Nishino where she grew in depth passion in neuromuscular research. Dr.Tanboon is one of a few specialists in her country responsible for muscle biopsy diagnosis.