Efficient and Highly Sensitive Screen for Myotonic Dystrophy Type 1 Using a One-Step Triplet-Primed PCR and Melting Curve Assay

Abstract

Instability and expansion of the DMPK CTG repeat cause myotonic dystrophy type 1 (DM1), the most common adult-onset neuromuscular disorder. Overlapping clinical features between DM1 and other myotonic disorders necessitate molecular confirmation for definitive diagnosis. Preconception screening could improve reproductive planning especially in DM1-affected women, who show diminished ovarian reserve and unfavorable in vitro fertilization-preimplantation genetic diagnosis outcome. We optimized triplet-primed PCR and melting curve analysis on 17 DNAs from DM1-affected/unaffected cell lines. A blinded test was performed on 60 genotype-known clinical samples. Plasmid constructs pDMPK(CTG)35 and pDMPK(CTG)48 were used to establish threshold temperatures separating DM1-affected from unaffected samples. Postscreen triplet-primed PCR amplicon sizing was achieved by short-cycle labeled-primer extension followed by capillary electrophoresis. Triplet-primed PCR melting curve analysis melt peak temperatures of unaffected and DM1-affected samples were lower and higher than the control plasmids' melt peak temperatures, respectively. Capillary electrophoresis of post-melting curve analysis amplicons was completely concordant with the screening results. Triplet-primed PCR melting curve analysis is a simple and cost-effective screening tool for rapid identification of DM1. The companion confirmation protocol allows quick determination of CTG repeat size when required. This strategy avoids the need to perform capillary electrophoresis sizing on all test samples, limiting capillary electrophoresis analysis to only a subset of cases that are screen-positive.

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.…”

“…An alternative TP-PCR/MCA method uses non-saturating conditions with SYBR Green I dye, where the limiting dye dissociates from melting lower T m molecules and re-intercalates to higher T m double-strand molecules during a slow temperature ramp, resulting in a unimodal derivative melt peak observed only at the highest melting temperature. 16, 25 While this elegant assay simplifies the melt curve interpretation, it requires precise matching of dye concentration with the final concentration of PCR amplicons that may be difficult with lower quality DNA samples.…”

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

“…The kit was developed based on previous studies by Lian et al . 10,22 . It utilizes a triplet-primed polymerase chain reaction, which includes a combination of primers targeting a flanking region as well as five CTG repeats.…”

Robust Preimplantation Genetic Testing Strategy for Myotonic Dystrophy Type 1 by Bidirectional Triplet-Primed Polymerase Chain Reaction Combined With Multi-microsatellite Haplotyping Following Whole-Genome Amplification

“…DM1 and non-DM1 cell lines were purchased from Coriell Cell Repositories (Coriell Institute for Medical Research, Camden, New Jersey, USA), and their genomic DNAs were extracted using the QIAsymphony DNA Midi Kit (Qiagen, Hilden, Germany) according to manufacturer’s instruction. The DM1 cell lines used were previously characterized DM1 reference materials which carry a mosaic small CTG repeat expansion (GM06075; 12, 56, 70 ± 0.9 repeats) or a large expansion (GM04648; 5, 1,008 ± 49 repeats, GM04567; 21, 637 ± 33 repeats, and GM03989; 13, 2000 repeats) (Kalman et al, 2013; Lian et al, 2015), whereas the non-DM1 cell lines used included GM16243, which carries GAA repeat expansions in the frataxin gene and was previously determined to carry 13 and 14 DMPK -CTG repeats (Lian et al, 2015), GM06890, GM06892, GM06852, GM09197, GM03813, GM03814, GM03815, AG17487, and GM00143, all of which are heterozygous for various normal-sized DMPK CTG repeat alleles. Isolation, lysis, and whole-genome amplification (WGA) of single cells from GM06075 ( n = 21), GM04648 ( n = 15), GM16243 ( n = 5), GM06890 ( n = 6), GM06892 ( n = 4), GM06852 ( n = 6), GM09197 ( n = 4), GM03813 ( n = 3), GM03814 ( n = 3), GM03815 ( n = 8), AG17487 ( n = 4), and GM00143 ( n = 6), as well as groups of six cells from GM06075 ( n = 3) and GM04648 ( n = 3), were performed as described (Lian et al, 2017).…”

“…TP-PCR reactions were performed in 50-μl volumes, each containing 2 U HotStarTaq DNA polymerase (Qiagen), 2× Q Solution (Qiagen), 1× supplied PCR buffer (Qiagen), 0.2 mmol/L each of deoxynucleotide triphosphates (Roche, Penzberg, Germany), and either 100 ng of genomic DNA or 2 μl of WGA product as template. The 5′ TP-PCR reaction included 0.6 μmol/L each of primers Fam -P2 and P3R (Warner et al, 1996), and 0.06 μmol/L of primer 5′ TPR (Lian et al, 2015), whereas the 3′ TP-PCR reaction utilized 0.6 μmol/L each of primers Fam -3′ R and 3′ Tail, and 0.06 μmol/L of primer 3′ TPF (Lian et al, 2015). Thermal cycling was performed on the GeneAmp PCR System 9700 (Applied Biosystems-Life Technologies, Carlsbad, CA, USA) and included an initial enzyme activation at 95°C for 15 min, followed by 30 cycles of 98°C for 45 s, 60°C for 1 min, and 72°C for 5 min, with a final extension at 72°C for 10 min.…”