Preimplantation Genetic Testing of Spinocerebellar Ataxia Type 2—Robust Tools for Direct and Indirect Detection of the ATXN2 CAG Repeat Expansion

Spinocerebellar ataxia type 2 (SCA2) is an autosomal dominant neurodegenerative disorder caused by a pathogenic CAG trinucleotide repeat expansion in the ATXN2 gene. At-risk couples can embark on unaffected pregnancies through preimplantation genetic testing of monogenic disorders (PGT-M) of SCA2, which should involve accurate repeat expansion detection together with risk haplotype tracking using informative linked markers. Two couples underwent SCA2 PGT-M involving analysis of whole genome amplified embryonic trophectoderm cells by ATXN2 (CAG)n triplet-primed PCR (TP-PCR) and linkage-based risk allele genotyping using customized markers. To simplify and expedite the identification of informative markers for future PGT-M cases, putative microsatellite markers closely linked to ATXN2 were initially screened for polymorphism using a small set of anonymous DNA samples obtained from Coriell Cell Repository. Shortlisted markers with high polymorphism likelihood were then multiplexed in a single-tube reaction and genotyped on 190 anonymous DNA samples to determine their polymorphic information content. Across both SCA2 PGT-M clinical cases, the linked marker genotypes corroborated the TP-PCR results, allowing clear differentiation between unaffected and affected embryos. In both cases, transfer of an unaffected embryo led to a successful pregnancy and live birth of a healthy baby. In silico mining, filtering, and curation identified 287 microsatellites located within 1.65 Mb of either side of the ATXN2 CAG repeat. Of these, eight upstream and nine downstream polymorphic markers were successfully co-amplified in a single-tube assay and demonstrated high overall heterozygosity in both Chinese and Caucasian populations. Conclusion: To ensure high diagnostic accuracy for PGT-M of SCA2, we developed a heptadecaplex microsatellite marker panel for haplotype-based linkage analysis to complement TP-PCR-based direct detection of the ATXN2 CAG repeat. The panel can rapidly identify informative markers from virtually any couple, and it works equally well on MDA-amplified DNAs for embryonic haplotype analysis.