The Elusive Quantification of Repeat Length

In a disease that exhibits somatic mosaicism, somatic cell instability, and the consequent tissue-to-tissue variability in expanded repeat length, assigning an “actual progenitor repeat length” value to individual DM1 patients has been problematic. The connotations here are obvious—how do we use repeat length for essential drug development functions from molecular biomarkers to genotype-phenotype analyses to stratification of patients in interventional clinical trials, if the parameter is hard to pin down? A recent paper from a multi-site team (Universidad de Costa Rica, University of Texas MD Anderson Cancer Center, and University of Glasgow) attempts to determine the optimal body fluid/tissue to sample and thereby yield insight into the best path forward for clinical studies and interventional clinical trials (Corrales et al., 2019).

Saliva as an Accessible and Reliable Source for DM1 Mutation Testing

Dr. Fernando Morales and colleagues sought to build on their prior findings (Morales et al., 2012 & 2016) that used small pool-PCR (SP-PCR) to control for somatic instability in estimations of progenitor allele length measured in blood. The goal was to improve upon allele length correlations with age of DM1 onset. In their latest work, the research team reports out on comparison of saliva vs. blood as the analyte source for progenitor allele length determinations.

This report was based upon analysis of progenitor allele length in saliva and blood from 40 DM1 patients that had been characterized for age of onset; screening also assessed for presence of variant repeats and methylation of CTCF binding sites adjacent to the DMPK locus, as these may be modifiers of somatic instability. Modal allele length was slightly larger in saliva (529 repeats) than concurrently collected blood samples (486 repeats). Progenitor allele length then was estimated as the lower boundary of allele distribution from SP-PCR—values from the two sample sources were highly correlated and, again, were higher in saliva than blood (414 vs. 310).

Analyses showed that progenitor allele length estimated from blood samples explained 75% of the variation in DM1 age of onset, while that from saliva explained 66% of the variation. The authors suggest that the “true progenitor allele length” needed for genotype-phenotype studies and other preclinical and clinical development purposes is more likely reflected by the values obtained from blood samples.

Additional single molecule SP-PCR studies, excluding two CDM cases, revealed greater somatic instability in blood than in saliva. The research team also showed that the lower boundary of allele distribution was slightly higher in saliva than in blood, while the overall degree of somatic variation was typically lower in saliva than in blood. Finally, analyses of repeat variants and methylation levels as putative modifiers of somatic instability showed that neither were significant factors.

Blood or Saliva?

The authors of this paper summarize the compelling literature case against use of skeletal muscle samples (essentially the confounding effect of tissue-specific rate of somatic expansion) to estimate progenitor allele size, bringing the choice down to blood or saliva. These data show that somatic mosaicism is comparable in blood and saliva DNA from DM1 patients, while saliva is obtained by considerably less invasive means—a feature that is potentially vital for interventional clinical trials in CDM or repeated sampling to assess efficacy of a candidate therapeutic in either CDM or DM1.

References:

Analysis of mutational dynamics at the DMPK (CTG)n locus identifies saliva as a suitable DNA sample source for genetic analysis in myotonic dystrophy type 1.
Corrales E, Vásquez M, Zhang B, Santamaría-Ulloa C, Cuenca P, Krahe R, Monckton DG, Morales F.
PLoS One. 2019 May 2;14(5):e0216407. doi: 10.1371/journal.pone.0216407. eCollection 2019.

Somatic instability of the expanded CTG triplet repeat in myotonic dystrophy type 1 is a heritable quantitative trait and modifier of disease severity.
Morales F, Couto JM, Higham CF, Hogg G, Cuenca P, Braida C, Wilson RH, Adam B, del Valle G, Brian R, Sittenfeld M, Ashizawa T, Wilcox A, Wilcox DE, Monckton DG.
Hum Mol Genet. 2012 Aug 15;21(16):3558-67. doi: 10.1093/hmg/dds185. Epub 2012 May 16.

A polymorphism in the MSH3 mismatch repair gene is associated with the levels of somatic instability of the expanded CTG repeat in the blood DNA of myotonic dystrophy type 1 patients.
Morales F, Vásquez M, Santamaría C, Cuenca P, Corrales E, Monckton DG.
DNA Repair (Amst). 2016 Apr;40:57-66. doi: 10.1016/j.dnarep.2016.01.001. Epub 2016 Mar 8.