Abstract
Huntington disease (HD) is a lethal neurodegenerative disorder caused by expansion of a CAG repeat within the huntingtin (HTT) gene. Disease prevention can be facilitated by preimplantation genetic testing for this monogenic disorder (PGT-M). We developed a strategy for HD PGT-M, involving whole genome amplification (WGA) followed by combined triplet-primed PCR (TP-PCR) for HTT CAG repeat expansion detection and multi-microsatellite marker genotyping for disease haplotype phasing. The strategy was validated and tested pre-clinically in a simulated PGT-M case before clinical application in five cycles of a PGT-M case. The assay reliably and correctly diagnosed all embryos, even where allele dropout (ADO) occurred at the HTT CAG repeat locus or at one or more linked markers. Ten of the 27 embryos analyzed were diagnosed as unaffected. Four embryo transfers were performed, two of which involved fresh cycle double embryo transfers and two were frozen-thawed single embryo transfers. Pregnancies were achieved from each of the frozen-thawed single embryo transfers and confirmed to be unaffected by amniocentesis, culminating in live births at term. This strategy enhances diagnostic confidence for PGT-M of HD and can also be employed in situations where disease haplotype phase cannot be established prior to the start of PGT-M.
Highlights
preimplantation genetic testing for this monogenic disorder (PGT-M) for Huntington disease (HD) began in 1998, employing PCR amplification across the CAG repeat and subsequent capillary electrophoresis for direct expansion mutation detection[14,15,16,17,18,19,20]
We report the first clinical application of this strategy for IVF PGT-M of HD in an at-risk couple for which family members of the affected spouse were unavailable for development of a standalone linkage-based PGT-M
triplet primed PCR (TP-PCR) has been shown to provide reliable amplification and detection of repeat expansions regardless of size[25,26,27,28,30,34], and the American College of Medical Genetics and Genomics has indicated that it is the preferred method for genetic testing of HD35
Summary
PGT-M for HD began in 1998, employing PCR amplification across the CAG repeat and subsequent capillary electrophoresis for direct expansion mutation detection[14,15,16,17,18,19,20]. Current HD PGT-M assays based on standard PCR rely on observing two normal alleles of different size to unequivocally identify unaffected embryos. This strategy, requires that couples must be informative for their normal alleles (i.e. carry normal alleles of different size), and cannot be applied to couples with uninformative normal alleles. Compared to standard PCR, the triplet primed PCR (TP-PCR) method, which was first described by Warner et al.[22], consistently detects the presence of the expanded allele regardless of its repeat length[27,28,29,30]. We report the first clinical application of this strategy for IVF PGT-M of HD in an at-risk couple for which family members of the affected spouse were unavailable for development of a standalone linkage-based PGT-M
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