Genetic determinants of disease severity in the myotonic dystrophy type 1 OPTIMISTIC cohort

Abstract

Objective: To evaluate the role of genetic variation at the DMPK locus on symptomatic diversity in 250 adult, ambulant patients with myotonic dystrophy type 1 (DM1) recruited to the Observational Prolonged Trial in Myotonic Dystrophy Type 1 to Improve Quality of Life-Standards, a Target Identification Collaboration (OPTIMISTIC) clinical trial.

Methods: We used small pool PCR to correct age at sampling biases and estimate the progenitor allele CTG repeat length and somatic mutational dynamics, and AciI digests and repeat primed PCR to test for the presence of variant repeats.

Results: We confirmed disease severity is driven by progenitor allele length, is further modified by age, and, in some cases, sex, and that patients in whom the CTG repeat expands more rapidly in the soma develop symptoms earlier than predicted. We revealed a key role for variant repeats in reducing disease severity and quantified their role in delaying age at onset by approximately 13.2 years (95% confidence interval 5.7-20.7, 2-tailed t test t = -3.7, p = 0.0019).

Conclusions: Careful characterization of the DMPK CTG repeat to define progenitor allele length and presence of variant repeats has increased utility in understanding clinical variability in a trial cohort and provides a genetic route for defining disease-specific outcome measures, and the basis of treatment response and stratification in DM1 trials.

Figure 1. Estimation of progenitor and modal CTG repeat length and detection of AciI-sensitive variant repeats

(A) Shown are representative small pool PCR (SP-PCR) analyses of repeat length variation at the DMPK CTG repeat in blood DNA from 5 individuals. For each participant, the analysis of 2 DNA samples is shown: historical sample taken at time of initial diagnosis (H) and at baseline (V2). The age at sampling for each sample is indicated in years. The approximate positions of the molecular weight standards converted into the number of CTG triplet repeats are shown on the left. The position of the estimated progenitor allele length for each individual is indicated with the lower dashed white line. The modal allele length at each time point is indicated by the upper dashed white line. For each sample, 4 replicate PCRs were performed with ∼180–300 pg DNA. (B) Shown are representative SP-PCR and AciI digestions of the DMPK CTG repeat in blood DNA from 7 individuals. For each participant, 3 replicate PCRs were performed with ∼500 pg DNA, digested (+) or undigested (−) with AciI, resolved by agarose gel electrophoresis and Southern blot hybridized with a repeat unit probe. The approximate positions of the molecular weight standard converted into the number of CTG triplet repeats of undigested products is shown on the left. For each participant the presence (blue) or absence (red) of AciI-sensitive variant repeats within the expanded allele is indicated by filled pedigree symbols. Note OP177 contains AciI-sensitive variant repeats within the non-disease-associated allele as indicated by the blue triangle. (C) Detection of expanded alleles using repeat-primed PCR (RP-PCR). Shown are 5′- and 3′-RP-PCR assays for 2 participants (OP251 and OP121) in which we were not able to amplify the expanded allele using flanking primers. For both participants, a ladder of products extending beyond 50 repeats was observed using RP-PCR, confirming the presence of a disease-causing expansion. Notably, the ladders were discontinuous, consistent with the presence of variant repeat blocking amplification from the (CAG)₅ or (CTG)₅ repeat primer at some positions within the array. The approximate positions of the molecular weight standard converted into the number of triplet repeats are shown.

Figure 2. Age at onset and somatic instability correlations

(A) Age at onset is highly correlated with estimated progenitor allele length (ePAL). The scatterplots show the relationship between ePAL and age at onset. The relevant line of best fit under a logarithmic model for female individuals (circles, orange line), male individuals (squares, lavender line), and sex-averaged for all individuals (black line, model 1a) (table 1) are shown. Individuals with (blue) and without (red) AciI-sensitive variant repeats are also depicted, along with the logarithmic regression line for AciI-sensitive variant repeat carriers (blue line). (B) Effect of variant repeats on age at onset. Shown are boxplots for the difference in observed age at onset minus predicted age at onset for Observational Prolonged Trial in Myotonic Dystrophy Type 1 to Improve Quality of Life—Standards, a Target Identification Collaboration (OPTIMISTIC) participants with (yes, blue) and without (no, red) AciI-sensitive variant repeats in their expanded DMPK allele. Predicted age at onset was derived using model 1a (Age_o = 80.3 + [−23.4 × log(ePAL)]) (table 1). The mean difference in age at onset for participants carrying AciI-sensitive variant repeats was 13.2 years (95% confidence interval [CI] 5.7 to 20.7, 2-tailed t test t = −3.7, p = 0.0019). The bottom and top of the box are the lower and upper quartiles, respectively. The band near the middle of the box is the median and the notches approximate to the 95% CI for the medians. The whiskers represent the full range of observations bounded by an upper limit equal to the upper quartile plus 1.5× the interquartile range, and a lower limit equal to the lower quartile minus 1.5× the interquartile range. Any points outside these bounds are displayed individually as small circles. (C) Modal allele length, ePAL, and presence of AciI variant repeats. The scatterplots show the relationship between ePAL and modal allele length at the V2 time point. Individuals with (blue) and without (red) AciI-sensitive variant repeats are indicated. (D) Sampling bias in the OPTIMISTIC cohort. The scatterplots show the relationship between ePAL and age at onset for the OPTIMISTIC cohort (open diamonds, black line) and a family-based DM1 population characterized by Morales et al. (green triangles and line). The relevant line of best fit under a logarithmic model (model 1a, table 1) is shown for each population (for the OPTIMISTIC cohort n = 222, r = 0.178, p = 3.1 × 10⁻¹¹ and for Morales et al., n = 137, r² = 0.640, p < 2.2 × 10⁻¹⁶). Note that the Morales et al. cohort has more very mildly affected participants with small expansions, and more severely affected patients with large expansions. (E) ePAL is more informative than the diagnostic measure in predicting age at onset. The scatterplots show the relationship between age at onset and ePAL (ePAL, open diamonds and black line) and the diagnostic allele length (diagnostic CTG, maroon diamonds and line) for the OPTIMISTIC cohort. The relevant line of best fit under a logarithmic model (model 1a, table 1) is shown for each CTG measure (for ePAL n = 222, r² = 0.178, p = 3.1 × 10⁻¹¹ and for diagnostic CTG n = 105, r² = 0.123, p = 0.00014).

Figure 3. Estimated progenitor allele length (ePAL) and variant repeat correlations with age-dependent phenotypes

The scatterplots show the relationship between ePAL and a number of age-dependent phenotypes. The relevant line of best fit under a logarithmic model (dependent variable = β₀ + β₁Log[ePAL]) for female individuals (circles, orange line), male individuals (squares, lavender line), and sex-averaged for all individuals (black line) are shown. Individuals with (blue) and without (red) AciI-sensitive variant repeats are also depicted, along with the logarithmic regression line for AciI-sensitive variant repeat carriers (blue line).