Mate-Pair Sequencing Enables Identification and Delineation of Balanced and Unbalanced Structural Variants in Prenatal Cytogenomic Diagnostics.

Background: Mate-pair sequencing detects both balanced and unbalanced structural variants (SVs) and simultaneously informs in relation to both genomic location and orientation of SVs for enhanced variant classification and clinical interpretation, while chromosomal microarray analysis (CMA) only reports deletion/duplication. Herein, we evaluated its diagnostic utility in a prospective back-to-back prenatal comparative study with CMA.

Methods: From October 2021 to September 2023, 426 fetuses with ultrasound anomalies were prospectively recruited for mate-pair sequencing and CMA in parallel for prenatal genetic diagnosis. Balanced/unbalanced SVs and regions with absence of heterozygosity (AOH) were detected and classified independently, and comparisons were made between mate-pair sequencing and CMA to assess concordance. In addition, novel SVs were investigated for potential RNA perturbations using cultured cells, whenever available.

Results: Mate-pair sequencing and CMA successfully yielded results for all 426 fetuses without the need for cell culturing. In addition, mate-pair sequencing identified 19 cases with aneuploidies, 16 cases with pathogenic simple deletions/duplications, and 5 cases with pathogenic translocations/insertions, providing a 25% incremental diagnostic yield compared to CMA (9.4%, 40/426 vs 7.6%, 32/426). Furthermore, by identifying the location and orientation of SVs, mate-pair sequencing improved the variant interpretation and/or follow-up approach for 40.0% (12) of the 30 cases with likely clinically significant deletions/duplications reported by CMA. Lastly, both platforms reported 3 cases (3/426) with multiple regions of AOH likely attributable to parental consanguinity.

Conclusions: Mate-pair sequencing detects additional balanced/unbalanced SVs and improves variant interpretation in comparison to CMA, indicating its potential to serve as a comprehensive prenatal cytogenomic diagnostic method.