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Title: Correlation of clinical and molecular features in myotonic dystrophy type 1 (DM1)
Author: Sedehizadeh, Saam
ISNI:       0000 0004 7965 8082
Awarding Body: University of Nottingham
Current Institution: University of Nottingham
Date of Award: 2019
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Objectives: (1) To correlate RNA splicing misregulation in human DM1 vastus lateralis (VL) muscle of two DM1 splice events identified from the literature and the myotonic dystrophy deep sequencing data repository (nuclear factor I/X (NFIX) and insulin receptor (INSR)), with primary clinical outcome measures. (2) Determine if the clinical outcome measures and these splicing defects changed after 18 months follow-up. (3) Compare differential gene expression in DM1 VL skeletal muscle from patients with a severe phenotype to controls and DM1 patients with a mild phenotype. (4) Establish preliminary proteomic profile of DM1 VL skeletal muscle using mass spectrometry. Methods: I performed a cross-sectional evaluation of 45 Southern blot genotyped DM1 patients who underwent VL needle muscle biopsy (for RT-PCR splicing analysis in NFIX and INSR), bilateral grip dynamometry, quantitative muscle testing (QMT) of ankle dorsiflexion strength and the six-minute walk test. In addition, I established prospective 18 month interval data of these parameters and used a targeted custom annotated qPCR array for the differential gene expression analysis in a subset of DM1 patients and healthy control skeletal muscle. In addition, with a collaborator (Dr Marzena Wojciechowska) mass spectrometry was used to establish a proteomic profile of DM1 VL skeletal muscle comparing four DM1 samples with four control samples. Results: Splicing misregulation in INSR and NFIX differentiated DM1 patients with severe phenotype from healthy controls and DM1 patients with a mild phenotype (one way ANOVA p < 0.05). In addition, linear regression of alternative splicing in these genes correlated with all clinical outcome measures (p < 0.05, r values 0.3-0.5). DM1 patients who underwent a second review visit after 18 months showed significant decline in various clinical outcome measures assessed but not in the degree of alternative splicing of INSR or NFIX in VL skeletal muscle. The Taqman® low density microarray analysis to profile the expression of 188 target transcripts, annotated based on functional categories and relevance to DM1 molecular pathophysiology, identified 39 transcripts significantly differentially expressed between DM1 patients with a severe phenotype (n=16) and healthy control VL muscle (n=16). Four out of these 38 transcripts were significantly differentially expressed between severely affected DM1 patients (n=16) and mildly affected DM1 patients (n=8). Mass spectrometry proteomic screening identified 115 targets (majority up-regulated) that were differentially expressed between DM1 VL skeletal muscle (n=4) and control skeletal muscle (n=4). Conclusions: A quantifiable clinical protocol of neuromuscular decline in our population over the 18 month interval has been established; however sampling of more distal muscles for alternative splicing events (e.g. tibialis anterior) may yield improved sensitivity in a longitudinal study. Candidate differentially expressed genes and proteins have been identified that warrant further mechanistic studies in DM1 models.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: RC Internal medicine