Preimplantation Genetic Testing for Cornelia de Lange Syndrome with Low-Level Maternal Gonadal Mosaicism using nanopore sequencing and digital PCR

Background Presently, to address the limited resolution at the single-cell level within the preimplantation genetic testing for aneuploidy (PGT-A) framework, our institution implemented a preimplantation genetic testing for monogenic disease (PGT-M) strategy based on haplotype linkage analysis for families with copy number variants (CNVs) < 1 Mb. Objective This study aims to deliver an accurate diagnosis for a Chinese family affected by Cornelia de Lange syndrome 5 (CDLS5) resulting from a microdeletion del(X)(q13.1q13.2) in the HDAC8 gene, characterized by notably low-level gonadal mosaicism. Furthermore, we execute preimplantation genetic testing for aneuploidy and monogenic disorders leveraging the diagnostic outcomes. Methods A de novo CNV was identified through chromosomal microarray analysis (CMA) and Whole Exome Sequencing (WES) in a family experiencing two unsuccessful pregnancies, indicating the existence of germline mosaicism. Validation of this CNV was performed via real-time quantitative polymerase chain reaction (PCR). Whole-genome low-coverage mate-pair sequencing (WGL-MPS) was conducted on female peripheral blood to exclude cryptic chromosomal abnormalities or mosaic states. Long-PCR was utilized to amplify the deleted fragment in insufficient miscarriage samples, with primers designed at breakpoints identified through WES and CMA results. After purifying the Long-PCR products, Oxford Nanopore Technology (ONT) third-generation sequencing was employed to pinpoint specific breakpoint positions. Designed primers and probes for droplet-digital polymerase chain reaction (ddPCR) were utilized to confirm the presence and proportion of germline mosaicism in ovarian samples obtained during in vitro fertilization procedures, such as granulosa cells and follicular fluid. Results The disease-causing microdeletion at Xq13.1q13.2 disrupting the HDAC8 Gene in the two male miscarriage tissues was not detected in the parents' peripheral blood cells by CMA, ES, quantitative PCR, and WGL-MPS. The maternal gonadal tissues were assumed to be the source of inheritance as Cornelia de Lange syndrome 5 (CDLS5) is an X-linked dominant disease. Specific breakpoint positions (chrX:g.71666527–71838853, 172 kb) were identified through third-generation sequencing of Long-PCR products. ddPCR quantitatively revealed approximately 1% mosaic state for the deletions in ovarian granulosa cells and none in peripheral blood cells, confirming the presence of CNV-induced gonadal mosaicism, a novel finding in maternal ovarian tissues. PGT investigations indicated 16.7% (1/6) of embryos with the deletion, demonstrating a low-level gonadal mosaicism. Conclusion Our findings underscore the efficacy of PGT-M utilizing haplotype linkage analysis for CNVs < 1 Mb, even in cases of gonadal mosaicism, emphasizing the significance of parental testing in CDLS5 families and the reproductive utility of in vitro fertilization (IVF) with PGT for families affected by low-level parental gonadal mosaicism. By employing a spectrum of methodologies, including NGS-based sequencing, microarray-based comparative genomic hybridization, and ddPCR for precise breakpoint determination, we showcase approaches to address and resolve uncommon genetic mechanisms underlying microdeletions in cases of gonadal mosaicism. Our results advocate for the expanded application of PGT-M based on haplotype linkage analysis for families with minor pathogenic CNVs.