LAUSR

Myotonic dystrophy type 1 (DM1), an autosomal dominant inherited disease, is the most common form of adult-onset muscular dystrophy with a prevalence of 1 in 8000 worldwide and no disease-modifying treatment is currently available [1]. The molecular defect is an expanded (CTG)n repeat within the noncoding 3’ untranslated region of the myotonic dystrophy protein kinase (DMPK) gene on chromosome 19q35 [2]. Mutant RNA transcripts containing expanded, non-translated repeats accumulate in nuclear foci, causing abnormal regulation of alternative RNA splicing which is one of the molecular hallmarks of the disease [3]. Agents that promote mutant DMPK transcript degradation are the topic of intense pre-clinical research [4], therefore, development of primary and secondary outcomes to determine therapeutic response in human trials are necessary to facilitate clinical trial readiness. RNA splicing is a regulated post-transcriptional process that occurs prior to the translation of the mRNA. In this process, the coding regions or exons are either retained or excluded to generate different sets of mRNAs, which further result in several protein isoforms from a single gene [5]. DM1-specific abnormal alternative splicing or mis-splicing in tibialis anterior (TA) muscle has been shown to correlate with functional impairment, with various splicing events showing graded changes with ankle dorsiflexion (ADF) weakness [6]. We have examined whether splicing patterns in vastus lateralis (VL) muscle (a) correlate with clinical phenotype and (b) change over time after 18 months followup to further evaluate their utility as biomarkers. Fifteen mis-spliced genes in DM1 that have been identified in the literature to demonstrate good test-re-test reliability in 32 pairs of human TA DM1 biopsy samples that were taken 2 to 3 months[7] apart were interrogated on the international collaborative DMseq (available www. dmseq. org) deep sequencing data repository isoform expression browser that contains data on both DM1 and healthy control human VL and TA skeletal muscles. Four transcripts were identified to be mis-spliced in VL muscle compared to control VL muscle. These were Insulin receptor (INSR), nuclear factor I/X (NFIX) and muscleblind-like 1 and 2 (MBNL1 and MBNL2) splice events. We decided to validate INSR and NFIX DM1-splice events in VL muscle in our cohort of patients. Forty-five DM1 patients, aged 23–73 years, were recruited. The diagnosis of DM1 was confirmed by molecular genetic testing in all patients. All patients were assessed by one of us (SS) both at the first visit and after 18 months according to standardised hand-held dynamometry (Microfet2®), grip dynamometry (Microfet4®), 6 min walk test (6MWT) protocols described previously [8–11] and needle VL muscle biopsy under local anaesthetic. Reasons for nonattendance at follow up included excessive travelling distance involved (n = 5), mortality (n = 1), could not tolerate study protocol (n = 2) and lost to follow-up (n = 3). The clinical characteristics of the patient cohort are shown in Table 1. RT-PCR (Reverse transcriptase-polymerase chain reaction) products of VL total cellular RNA evaluating alternative splicing of INSR exon 11 and NFIX exon 7 were analyzed. Using linear regression, aberrant splicing of INSR and NFIX correlated with all clinical outcome measures (p = < 0.01, r values 0.4–0.5, Fig. 1a–f) in a cross-sectional analysis. In addition, aberrant splicing of INSR and NFIX * Saam Sedehizadeh [email protected]