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 <i>ATXN2</i> 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 <i>ATXN2</i> (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 <i>ATXN2</i> 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 <i>ATXN2</i> 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 <i>ATXN2</i> 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.