VP.66 CRISPR/Cas9-targeted single molecule long-read sequencing reveals allelic microheterogeneity of triplet repeat expansion in oculopharyngodistal myopathy

Abstract

Oculopharyngodistal myopathy (OPDM) is a hereditary muscle disease characterized by progressive ptosis, ophthalmoplegia, dysphagia and predominantly distal muscle weakness. To date, CGG repeat expansion in 5'UTR of <i>LRP12, GIPC1, NOTCH2NLC</i> are known to be causative. Its pathogenesis has been suggested to be toxicities of the expanded RNAs or repeat-associated translated proteins, although it remains controversial. To elucidate the pathomechanism in OPDM, it is important to know precise sequences of expanded repeats. In this study, we sequenced the expanded repeats from 35 OPDM patients using CRISPR/Cas9-targeted long-read sequencing at single molecule level. A pair of guideRNAs was designed to cut both sides of CGG expansion in <i>LRP12, GIPC1, NOTCH2NLC</i>. Genomic DNAs from 5 patients were analyzed at single run. We successfully got certain number of reads regardless of expansion (20±17 reads/sample). The number of expanded reads in affected genes was about a half of total reads (47±11%), which was compatible with heterozygous inheritance. The observed length of repeats tended to be shorter than the estimated ones detected in Southern blotting. Interestingly, the expanded reads also frequently had several insertions and/or deletions in flanking regions as much as within expanded repeats, indicating that reading frame was not maintained in such reads. Also, the various nucleotide changes were detected. These suggest three possibilities; <i>1)</i> Instability of CGG expansion on genome replication, <i>2)</i> The transcripts from the alleles with abnormal expansion may be heterogeneous, and <i>3)</i> Frameshift within expanded repeats in the transcripts may be a rebuttal to the theory of repeat associated non-AUG repeat-associated translation. In conclusion, we have established a diagnostic tool for OPDM by use of long read sequencing, and it is useful for not only diagnosis but also the understanding of pathomechanism in OPDM.