Myotonic Dystrophy: Accurately Scoring the Boundaries that Define Regions of Triplet Repeat Expansion Mutations

Abstract

Aims: Myotonic Dystrophy type 1 (DM1) is an autosomal dominant neuromuscular multi-systemic disorder caused by a CTG triplet repeat expansion mutation in the DMPK gene. The clinical decision points defining the CTG repeat boundaries between normal, premutation and mild disease ranges are poorly characterised with a lack of commercially available sequenced controls. There are no US Food and Drug Administration (FDA) approved tests for DM1 so testing protocols are developed and managed by individual laboratories. Study Design: This paper presents a cross-laboratory exchange scheme between Auckland City Hospital and Concord Hospital, which took place between October 2013 and January 2014, in order to validate the scoring of CTG repeats within the DMPK gene and to build comprehensive allelic libraries. Methodology: Seven samples ranging from 30-59 repeats, spanning the critical clinical decision points, were sequenced to confirm the “true” repeat sizes, and 19 samples were tested by both laboratories using standard and triplet repeat-primed PCR methods. Results: The results showed a very strong correlation between the sequencing results and the standard PCR results for the 7 selected samples with a Pearson correlation coefficient of 0.999 and P = 1.20x10-7. The results from the inter-laboratory comparison also showed a very strong correlation between the diagnostic tests of the two labs with a Pearson correlation coefficient of 0.999 and P = 1x10-29. A paired t-test showed no significant difference between the two laboratories data with a Mean (SD) = 0.263 (0.828), P = .058. Conclusion: This study provides two critical outcomes. The first is that the extrapolations that were used by each of the participating laboratories in determining the number of CTG repeats in the absence of well-characterised controls in the 35-51 repeat range were within their reported margins-of-error. The second outcome is that small regional laboratories can gain confidence in the accuracy of their reported allele calls, specifically around clinically critical decision points, with inter-laboratory exchange studies and in-house sequencing of relevant control samples.