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Repeat Interruptions Modify Age at Onset in Myotonic Dystrophy Type 1 by Stabilizing DMPK Expansions in Somatic Cells

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Repeat Interruptions Modify Age at Onset in Myotonic Dystrophy Type 1 by Stabilizing DMPK Expansions in Somatic Cells

Jovan Pešović et al. Front Genet.

Abstract

CTG expansions in DMPK gene, causing myotonic dystrophy type 1 (DM1), are characterized by pronounced somatic instability. A large proportion of variability of somatic instability is explained by expansion size and patient's age at sampling, while individual-specific differences are attributed to additional factors. The age at onset is extremely variable in DM1, and inversely correlates with the expansion size and individual-specific differences in somatic instability. Three to five percent of DM1 patients carry repeat interruptions and some appear with later age at onset than expected for corresponding expansion size. Herein, we characterized somatic instability of interrupted DMPK expansions and the effect on age at onset in our previously described patients. Repeat-primed PCR showed stable structures of different types and patterns of repeat interruptions in blood cells over time and buccal cells. Single-molecule small-pool PCR quantification of somatic instability and mathematical modeling showed that interrupted expansions were characterized by lower level of somatic instability accompanied by slower progression over time. Mathematical modeling demonstrated that individual-specific differences in somatic instability had greater influence on age at onset in patients with interrupted expansions. Therefore, repeat interruptions have clinical importance for disease course in DM1 patients due to stabilizing effect on DMPK expansions in somatic cells.

Keywords: CTG expansion; DMPK; age at onset; myotonic dystrophy 1; repeat interruptions; somatic instability.

Figures

FIGURE 1
FIGURE 1
Single-molecule small-pool PCR analysis of DMPK expanded alleles with repeat interruptions. Analysis of the blood sample for DF3-1 when she was 50 years old (the first time point, blood t1) and 52.5 years old (the second time point, blood t2), showing allele size heterogeneity. Each lane of agarose gel (24 cm in length) contained PCR products amplified from ∼30–60 pg of DNA (5–10 genomic equivalents). Arrows with number indicate the position and size of the mode, the 10th and the 90th percentile allele length in number of trinucleotide repeats, inferred from more than 200 alleles sized per sample. M, DNA Molecular Weight Marker X (Roche Life Science, Mannheim, Germany) shown in number of CTG repeats.
FIGURE 2
FIGURE 2
Tissue specific somatic instability of DMPK expansions. Density plots show the allele frequency distributions skewed toward larger expansions in blood (solid lines) and buccal cells (dashed lines), sampled at the same time point (t2). All allele frequency distributions were derived from sizing at least 200 alleles and their comparisons were performed by Wilcoxon–Mann–Whitney test (W and p-values are shown). DF1 family members (top graphs), carrying identical pattern of CGG interruptions, showed difference in the allele frequency distribution between examined tissues. DF5 family members (bottom graphs) showed no difference in the allele frequency distribution between examined tissues. DF5-2 has a single de novo CTC interruption, while her sister DF5-3 has an uninterrupted DMPK expansion.
FIGURE 3
FIGURE 3
DM1 patients with repeat interruptions show a lower level of somatic instability. The graphs represent the correlation of the level of somatic instability (SI), the 10th percentile (10thp) allele length (as an estimate of the progenitor allele length (Higham, 2013)), and age at sampling (AS) according to model 8. The analyzed group of DM1 patients included our patients with interrupted expansions, our patients with pure expansions (the control group), and 136 patients from the reference group (Morales et al., 2012). Observed level of SI in blood was estimated as the range between the 10th and the 90th percentile of the allele frequency distribution (Morales et al., 2012). Patients with interrupted expansions are shown in X and the control group is shown in O, at the first time point (blood t1) and the second time point (blood t2) (2.5–4 years later for patients with interrupted expansions and 4–11 years later for the control group). Patients with interrupted DMPK expansions showed a large residual variance, and the median of their standardized residuals of SI was significantly lower relative to the reference group. The control group showed no difference. AFD, allele frequency distribution.
FIGURE 4
FIGURE 4
Somatic instability of DMPK expansions in blood cells over time. Density plots show the allele frequency distributions from blood samples at the first time point (t1, dashed lines) and the second time point (t2, solid lines). All allele frequency distributions were derived from sizing at least 200 alleles and their comparisons were performed by Wilcoxon–Mann–Whitney test (W and p-values are shown). Among patients with repeat interruptions, DF1-1, DF2-1, and DF3-1 showed no difference in the allele frequency distribution during the examined time interval, while DF1-2, DF1-3, DF3-2, and DF5-2 showed statistical difference. As expected, patients with uninterrupted repeats (DF5-3 – sister of DF5-2, MD70, MD179 and MD180), showed statistical difference. Time interval in years (y) between sampling at t1 and t2 is indicated in parenthesis, beside patient’s ID.
FIGURE 5
FIGURE 5
Somatic stability of repeat interruptions at the 3′ end of DMPK expansions between tissues and over time. (A) Identical repeat-primed PCR profiles in blood sample at the first (t1) and the second time point (t2) and in buccal swab sample (only for patients DF1-1 and DF5-2) in patients with interrupted DMPK expansions. All profiles were obtained using repeat-binding primer P4CTG-for (Pešović et al., 2017). The only exception was DF2-1, whose profiles were obtained using P5CCG primer, binding to a large CCG block containing 36 repeats. DF1-2 and DF1-3 are not shown, as they had the identical repeat-primed PCR profiles from all analyzed samples as their mother DF1-1 due to identical wild-type allele of 5 CTG repeats and the same pattern of repeat interruptions. (B) Repeat-primed PCR profiles in blood sample at the first time point (t1) and the second time point (t2) and in buccal swab sample in patient DF5-3 with an uninterrupted DMPK expansion. The profiles were obtained using repeat-binding primer P4CTG-for. Time interval in years (y) between sampling at t1 and t2 is indicated in parenthesis, beside patient’s ID.
FIGURE 6
FIGURE 6
Repeat interruptions are positive genetic modifiers of age at onset in DM1 patients. The graphs represent the correlation of age at onset (AO) with the 10th percentile (10thp) allele length (as an estimate of the progenitor allele length, Higham, 2013) according to model 10(top graphs), and additionally with standardized residual of somatic instability (SI) according to model 11 (bottom graphs). The analyzed group of DM1 patients included our patients with interrupted expansions, our patients with pure expansions (the control group), and 121 symptomatic patients from the reference group (Morales et al., 2012). Observed values of AO (self-reported by patients) are shown in X in patients with interrupted expansions and in O in the control group, at the first time point (blood t1) and the second time point (blood t2) (2.5–4 years later for patients with interrupted expansions and 4–11 years later for the control group). Patients with interrupted expansions showed a large residual variance when AO was correlated with the 10th allele length (model 10) and median of their standardized residuals was statistically higher than in the reference group (top graphs). The inclusion of residual variance of SI as a variable (model 11) resulted in no statistical difference in medians of standardized residuals between patients with interrupted expansions and the reference group (bottom graphs). The control group showed no statistical difference according to both models. AFD, allele frequency distribution.

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