P166 Deciphering the genetic cause of oculopharyngodistal myopathy in a French cohort using Cas9-targeted long-read sequencing

International audience ; Oculopharyngodistal myopathy (OPDM) is a rare neuromuscular disorder characterized by progressive ptosis, ophtalmoplegia, dysphagia and facial and distal limb muscle weakness. OPDM is caused by a CGG triplet expansion in the 5’-untranslated region (5’-UTR) in 4 different genes: LRP12, GIPC1, NOTCH2NLC and RILPL1 genes. Short tandem repeat (STR) such as CGG triplet expansion are repetitive DNA motifs, highly polymorphic and variable in length and their abnormal expansion have been associated with several diseases. STR characterization is time consuming and imprecise when using short-read sequencing, due to their inherent characteristics. In a cohort of 11 OPDM patients, we performed whole exome sequencing but failed to identify the causative gene. However, knowing the challenges to identify STR expansion using short-read sequencing, we cannot exclude the implication of one of the OPDM genes yet. A CRISPR Cas9-based approach combined to long-read sequencing (Oxford Nanopore Technologies) enables the sequencing of large and complex STR expansion. We established sequencing conditions for the 4 known OPDM genes in our cohort of 11 French OPDM patients using Cas9-targeted long read sequencing. Positive samples for each gene were used to set up the sequencing conditions. The first results allowed us to exclude LRP12, NOTCH2NLC and GIPC1 genes as causative in this cohort. Several other candidates, among which RILPL1 gene, are currently being investigated. Once the causative gene is identified, functional validations will be performed on patients’ muscle biopsies available at the lab. Several mechanisms will be investigated 1) RNA toxicity to highlight RNA foci, comprising CGG-expanded transcripts and RNA binding proteins, in the nucleus of patients, using RNA FISH, and 2) polyglycine protein toxicity targeting polyglycine motifs giving rise to toxic proteins by immunofluorescence.