Splicing biomarkers of disease severity in myotonic dystrophy

Abstract

Objective: To develop RNA splicing biomarkers of disease severity and therapeutic response in myotonic dystrophy type 1 (DM1) and type 2 (DM2).

Methods: In a discovery cohort, we used microarrays to perform global analysis of alternative splicing in DM1 and DM2. The newly identified splicing changes were combined with previous data to create a panel of 50 putative splicing defects. In a validation cohort of 50 DM1 subjects, we measured the strength of ankle dorsiflexion (ADF) and then obtained a needle biopsy of tibialis anterior (TA) to analyze splice events in muscle RNA. The specificity of DM-associated splicing defects was assessed in disease controls. The CTG expansion size in muscle tissue was determined by Southern blot. The reversibility of splicing defects was assessed in transgenic mice by using antisense oligonucleotides to reduce levels of toxic RNA.

Results: Forty-two splicing defects were confirmed in TA muscle in the validation cohort. Among these, 20 events showed graded changes that correlated with ADF weakness. Five other splice events were strongly affected in DM1 subjects with normal ADF strength. Comparison to disease controls and mouse models indicated that splicing changes were DM-specific, mainly attributable to MBNL1 sequestration, and reversible in mice by targeted knockdown of toxic RNA. Splicing defects and weakness were not correlated with CTG expansion size in muscle tissue.

Interpretation: Alternative splicing changes in skeletal muscle may serve as biomarkers of disease severity and therapeutic response in myotonic dystrophy.

Figure 1

Relationships between alternative splicing in TA muscle and strength of ankle dorsiflexion in DM1. For each splice event the fractional inclusion or exclusion of the indicated exon is shown for 45 DM1 subjects with full mutations (> 100 CTG repeats) and 8 healthy controls. Representative gel images of RT-PCR products are shown for each splice event. The exon inclusion splice product is the top band and the exon exclusion product is the lower band. ADF strength on the side of the TA biopsy was determined by manual testing and expressed as an MRC score. (A) Three examples of splice events that showed correlation of splicing outcome with muscle weakness. (B) Examples of “early transition” events that are strongly affected even in TA muscles that exhibit normal strength.

Figure 2

CTG expansion size is not associated with splicing misregulation or weakness in TA. (A) CTG expansion size in leukocytes is not correlated with ADF strength (left) or misregulated splicing of DTNA DB2 exons 11A and 12 (right). (B) CTG expansion size in TA muscle tissue is not correlated with ADF strength or DTNA splicing (right). (C) CTG expansion size in TA muscle tissue is not correlated with leukocytes (left). However, the difference of repeat size between muscle tissue and leukocytes (Δ repeat size) is correlated with age (right) (r = 0.68, P < 0.01)

Figure 3

Molecular features of the DM1 protomutation. (A) Tissue-specific somatic instability of the DM1 protomutation. (Top) Representative data from small-pool PCR + Southern blot analysis of CTG repeat length. DM1 expansions were amplified from genomic DNA at high dilution, so that each reaction contains one or several amplifiable alleles, then detected by Southern blot. * indicates normal DMPK allele. (Bottom) Histograms showing allelic distribution of CTG expansions in leukocytes or muscle from subjects with DM1 protomutations. CTG expansions are binned in groups spanning 10 repeats. More than 44 alleles were sized for each sample. M, molecular weight standard. (B) Fluorescence in situ hybridization and immunofluorescence of TA section showing colocalization of MBNL1 protein (green) with CUG^exp foci (red) in a myonucleus (blue) of a subject who carries a DM1 protomutation. Bar = 5 μm. (C) Alternative splicing of early transition events is misregulated in subjects with DM1 protomutations (Proto-DM1, CTG expansion size of 80 to 90 repeats), compared to healthy controls (Cont). * P<0.05, t test.