Robust and accurate detection and sizing of repeats within the DMPK gene using a novel TP-PCR test

Abstract

Myotonic dystrophy type 1 is a multisystem disorder caused by the expansion of a trinucleotide repeat in the DMPK gene. In this study we evaluated the performance of the FastDM1 TM DMPK sizing kit in myotonic dystrophy type 1 testing. This commercially available triplet repeat-primed PCR based kit was validated using reference and clinical samples. Based on testing with 19 reference samples, the assay yielded repeat sizes within three repeats from the consensus reference length, demonstrating an accuracy of 100%. Additionally, the assay generated consistent repeat size information with a concentration range of template-DNA, and upon repetition and reproduction (CV 0.36% to 0.41%). Clinical performance was established with 235 archived prenatal and postnatal clinical samples, yielding results of 100% sensitivity (95% CI, 97.29% to 100%) and 100% specificity (95% CI, 96.19% to 100%) in classifying the samples into the respective genotype groups of 5–35 (normal), 36–50 (non-pathogenic pre-expansion), 51–150 (unstable intermediate-sized pathogenic) or >150 (unstable pathogenic) CTG repeats, respectively. Furthermore, the assay identified interrupted repeat expansions in all samples known to have interruptions, and also identified interruptions in a subset of the clinical samples.

High Throughput Screening for Expanded CTG repeats in Myotonic Dystrophy Type 1 Using Melt Curve Analysis

“…Past recommendations to include both 5’ and 3’ TP-PCR stem from the possibility that expanded samples may be missed in TP-PCR reactions due to interruptions of the CTG repeat. 11, 16, 29, 30 However, bidirectional assays are likely unnecessary, particularly in the setting of a screening test as we have proposed here. With traditional TP-PCR careful examination of the electropherograms from published reports of DM1 cases with interrupted CTG repeats show that the TP-PCR reaction does not fail, but proceeds with interruptions to the typical sawtooth pattern seen from uninterrupted CTG repeats.…”

“…10 There are a number of challenges with this technique, including the requirement for a large amount of DNA and the amount of time required to perform the method. More recently, fluorescently labeled triplet-primed PCR (TP-PCR) and fragment sizing by capillary electrophoresis has been proposed as an alternative for diagnosis of DM1 11, 12 and for other triplet repeat expansion disorders such as Fragile X syndrome and Huntington disease 13, 14 . The TP-PCR methodology uses a (CTG) n or (CAG) n repeat primer to generate a nested set of fragments from the expanded allele, and the observed ‘ladder’ of amplicons is detected by capillary electrophoresis.…”

The Need for Establishing a Universal CTG Sizing Method in Myotonic Dystrophy Type 1

“…It would be interesting to determine how novel, recently described technologies for CTG sizing [11,12] compare to the methods we assessed here. The sizing kit used by Leferink et al, was based on tripled repeat primed PCR, which is a robust and accurate technique to determine the presence of a CTG expanded allele [11]. However, the sizing of the repeat was limited in their study, set at 180 CTG repeats.…”

“…Indeed, the number of CTG repeats can be inversely and directly related with age of disease onset and clinical severity, respectively [9,10]. Although different approaches have been described to assess CTG expansion size in patients with DM1 [5,[11][12][13][14][15], some methodological issues remain to be solved, mainly related to the inherent repeat instability and technical difficulties when amplifying long CTG fragments.…”

DM1 Phenotype Variability and Triplet Repeat Instability: Challenges in the Development of New Therapies

“…Polymerase chain reaction (PCR) amplifications are difficult for a high number of CTG repeats, and Southern blotting and triplet-primed (TP)-PCR do not allow accurate CTG number measurements. Although optimized protocols have been developed more recently, precise determination of repeat numbers is limited to~1000 CTG repeats [16,17]. In addition, age at the time of diagnosis and somatic instability in the blood are confounding factors that can bias correlation studies [18].…”