Direct Detection of Smn1 Copy Number by Capillary Electrophoresis for Preimplantation Genetic Testing in Challenging Spinal Muscular Atrophy Cases

Objective To evaluate whether capillary electrophoresis (CE)-based preimplantation genetic testing (PGT) can distinguish zero from non-zero survival motor neuron 1 (SMN1) copy number in embryos without probe development or linkage analysis, and demonstrate clinical utility in challenging spinal muscular atrophy (SMA) cases. Design Analytical validation study with retrospective clinical case review. Materials and Methods Fifty samples with known SMN1 status including Coriell cell lines and embryo specimens were analyzed. DNA was diluted to picogram concentrations, followed by whole genome amplification (WGA). Amplified DNA underwent CE to assess SMN1 copy presence. Results were confirmed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Concordance between CE, PCR-RFLP and known SMN1 status was calculated. Clinical application was assessed in two at-risk couples undergoing PGT for SMA. Results CE-based PGT demonstrated 99% concordance with known SMN1 status, with PCR-RFLP confirming all concordant results. In the first case, both partners were increased-risk SMA carriers, but conventional PGT-M was not feasible because phase determination could not be established, and no informative family members were available for linkage analysis. Two embryo biopsies were analyzed; both were euploid and demonstrated one or more copies of SMN1, reducing risk of SMA. In the second case, both partners were confirmed SMA carriers. Three embryo biopsies were analyzed: one euploid embryo demonstrated one or more copies of SMN1, one mosaic embryo demonstrated zero SMN1 copies consistent with affected status, and one triploid embryo demonstrated one or more copies of SMN1. Conclusions Direct assessment of SMN1 copy number using CE provides a reliable method for identification of SMA-affected embryos and expands access to PGT in cases where traditional linkage-based approaches are not feasible, including unphased or silent carrier scenarios.