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

Figures

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  Figure 1. Schematic illustration of the DM1 PGT-M strategy. (A) Bidirectional 5′ and 3′ TP-PCR at the DMPK CTG repeat locus, depicting annealing positions of primers (arrows), as well as the expected electropherogram patterns from unaffected and DM1-affected embryos after capillary electrophoresis. (B) Location of 12 microsatellite markers relative to the DMPK CTG repeat. (C) Expected electropherogram results after multiplex PCR amplification of the dodecaplex marker panel and capillary electrophoresis, showing the electrophoretic peak color and approximate size range of each marker. Adapted from Lian et al. (2016, 2017).

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  Figure 2. Electropherograms of whole-genome amplified single lymphoblasts from three cell lines after bidirectional 5′ and 3′ DMPK TP-PCR (A), and dodecaplex marker panel PCR (B) amplifications. A non-DM1 cell line (top row), a DM1-affected cell line carrying a modest expansion (rows 2–4), and a DM1-affected cell line carrying a large expansion (row 5) were used. The 50-repeat cutoff for full penetrance DMPK allele is represented by a vertical dotted red line. Numbers in the TP-PCR and dodecaplex marker panel PCR electropherograms indicate CTG repeat number and product fragment size in base pairs, respectively. RFU, relative fluorescence units; ADO, allele dropout; AF, amplification failure.

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  Figure 3. Electropherograms of cell line-extracted genomic DNA after bidirectional 5′ and 3′ DMPK TP-PCR (A) and dodecaplex marker panel PCR (B) amplifications. RFU, relative fluorescence units.

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  Figure 4. Bidirectional 5′ and 3′ DMPK TP-PCR electropherograms of GM06075 (A) and GM04648 (B) genomic DNAs (row 1; also shown in Figure 3), whole-genome amplified single lymphoblasts showing allele dropout of the normal allele (row 2), allele dropout of the expanded allele (row 3), and whole-genome amplified multi-cell samples with no allele dropout (row 4). RFU, relative fluorescence units; ADO, allele dropout.

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  Figure 5. Comparison of the modified TP-PCR and linked marker assay of Kakourou et al. (2010) (A) and our combined bidirectional TP-PCR and linked multi-marker assay (B) on two DM1-affected samples. The modified TP-PCR consisted of 5′ TP-PCR amplification of DMPK locus using P2/P4CAG/P3R primers (top lane), standard PCR amplification of DMPK locus using P2/DMPK2 primers (middle lane), and PCR of linked marker D19S112 (bottom lane). Black arrow indicates bleed-through signal from VIC channel that was initially misidentified as a second D19S112 allele in GM03989.

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  Figure 6. Bidirectional DMPK TP-PCR (A) and dodecaplex marker panel PCR electropherogram (B) profiles from a UKNEQAS simulated PGT-M case, which consisted of a parent-child trio and three simulated trophectoderm samples. ADO, allele dropout.

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  Figure 7. Haplotypes generated from analysis of 12 linked markers in the simulated PGT-M case. Haplotype pairs of the DM1-affected father, unaffected mother, affected daughter, and three simulated “Embryo” samples are shown. Markers are ordered from the telomere (top) to centromere (bottom) of the q-arm. Dark blue- and light blue-shaded columns denote haplotypes linked to the paternal mutant/expanded and normal alleles, respectively. Light purple- and orange-shaded columns denote haplotypes linked to both of the maternal normal alleles. ^Allele size determined from simplex PCR.