Molecular Diagnosis of DM1

Accurate sizing of expanded triplet repeat sequences is essential in establishing diagnosis of DM1. The Genetic Testing Registry at NCBI/NIH currently lists 50 facilities that provide molecular genetics tests for DM1. Current commercially available testing includes the Athena Diagnostics’ Myotonic Syndrome Advanced Evaluation (detection of abnormal splicing of ATP2A1, CAV3, CLCN1, HSPG2, and SCN4A and repeat expansions in DMPK and CNBP), GeneDx’s Myotonic Dystrophy, Type 1 (detection of DMPK repeat expansion), and MNG Laboratories’ Myotonic Dystrophy 1 (DMPK) Genetic Testing (detection of DMPK repeat expansion).

Commercial tests offer considerable value for rapid diagnosis, but all have limited research value due to inability to accurately size repeat sizes above 150. More accurate repeat sizing is essential for understanding of genotype/phenotype relationships and likely needed for interventional clinical trials. Continued efforts to develop and validate robust and broadly available molecular diagnostics for DM carry considerable importance.

Assessing a New, Commercially Available Test for DM1

Drs. Lonneke Haer-Wigman (Radboud University), Ming Guan (The BioFactory Pte Ltd, Singapore), and colleagues have published work in Science Reports evaluating the performance of a new commercially available triplet-repeat primed PCR (TP-PCR) methodology for sizing expanded CTG repeats, the FastDM1^TM DMPK sizing kit (Leferink et al., 2019). The new kit sought to overcome limitations in existing TP-PCR diagnostics, including inabilities to distinguish between intermediate (150>x >51) and large (x>150) pathogenic alleles and to identify and size samples from patients with interrupted repeats.

In an assessment of sizing accuracy, 19 reference samples of established expanded repeat length (consensus from three independent labs for 53% of samples and from one independent lab for the remainder) were sized with the FastDM1^TM kit—all determinations were within 3 repeats of the reference samples. To evaluate assay sensitivity, testing of a 1-200 ng input DNA range showed that sample sizes from 25-200 ng retained sizing accuracy. Mosaic samples (artificially created by mixing reference samples) could accurately detect up to 2.5% mosaicism, but that repeat size could be underestimated at under 20% mosaicism.

The specificity of the FastDM1^TM kit for DMPK repeat expansions was demonstrated by the failure to detect and size CTG expansions in TCF4 in DNA samples containing either only the TCF4 repeat or samples containing expanded repeat TCF4 mixed with reference samples with DMPK expanded repeats. The research team also established intra-assay consistency and intra- and inter-kit batch (including kits from separate batches produced over a two-year period) reproducibility in use of the FastDM1^TM kit.

Use of the FastDM1^TM DMPK Sizing Kit with Patient Samples

Performance of the FastDM1^TM kit was evaluated using 225 postnatal and 10 prenatal clinical samples. The research team reported 100% clinical sensitivity and specificity in classifying samples tested into four genotypic groups (normal: 5-35 repeats, non-pathogenic pre-expansion: 36-50, unstable intermediate sized: 51-150, and unstable pathogenic: >150) recognized by the European Molecular Genetics Quality Network. In contrast to prior technologies, the kit showed the ability to distinguish between unstable intermediate and unstable pathogenic alleles (over 150 repeats)—a distinction of considerable diagnostic importance. Finally, the research team evaluated eight samples with known interrupted repeats and found that the kit had the ability to detect such repeats, albeit with less accuracy in sizing. These findings suggest that the kit may be an important new tool for the DM1 field.

Reference:

Robust and accurate detection and sizing of repeats within the DMPK gene using a novel TP-PCR test.
Leferink M, Wong DPW, Cai S, Yeo M, Ho J, Lian M, Kamsteeg EJ, Chong SS, Haer-Wigman L, Guan M.
Sci Rep. 2019 Jun 4;9(1):8280. doi: 10.1038/s41598-019-44588-3.