Third-generation sequencing enables accurate preimplantation genetic testing of monogenic defects for de novo...

Can preimplantation genetic testing for monogenic defects (PGT-M) be achieved by performing third-generation sequencing (TGS) only on the proband for families with de novo variants or incomplete pedigrees?

Whole-genome TGS facilitates a simplified PGT-M workflow by establishing reliable haplotypes solely from proband sequencing involving de novo variants or incomplete pedigrees.

PGT-M enables the accurate exclusion of embryos carrying pathogenic variants. However, its application to de novo variants or incomplete pedigrees is hindered by haplotype phasing. Moreover, direct variant detection suffers from detection failure and erroneous genotyping due to uneven coverage and allele dropout caused by whole-genome amplification. Current solutions, such as gamete or embryo analysis and targeted TGS, remain constrained by procedural complexity and lack of universality across different genes and mutation types.

This prospective study enrolled 16 families requiring PGT-M with de novo variants or incomplete pedigrees at the Reproductive Medicine Center of Peking University Third Hospital from July 2023 to August 2025.

This study included 9 families with incomplete pedigrees and 7 families with de novo variants, covering 10 distinct disease-causing genes or regions. To assess the capability of TGS for haplotype phasing, we evaluated its performance regarding genomic coverage and the retrieval of informative single-nucleotide polymorphisms (SNPs). Haplotypes were constructed using proband TGS data, and linkage analysis was performed by integrating linked heterozygous SNPs with next-generation sequencing data from the couple and embryos to determine pathogenic status. Subsequently, we developed a simplified strategy that inferred inheritance by comparing heterozygous SNPs from the proband's haplotype directly against corresponding homozygous sites in the embryos. The diagnostic outcomes of this simplified workflow were statistically evaluated and compared with those of the standard TGS strategy to assess concordance.

Phase blocks generated by TGS achieved >75% coverage for the vast majority of OMIM genes, most of which contained more than 100 heterozygous informative SNPs located in the gene body and their 1 Mb flanking regions, indicating a wide range of applicability in a variety of gene variants. Haplotypes were successfully constructed for all 16 enrolled families using TGS data, with 14 families having completed embryo testing, while the 2 families withdrew due to personal reasons. To date, prenatal diagnosis via amniocentesis in three families has confirmed the fetuses to be free of pathogenic variants. A simplified strategy was further applied to 14 families that completed the embryo testing process. This approach achieved applicability rates of 91.9% and 80.0% in embryos from non-D4Z4 and D4Z4 families, respectively. While diagnosis was precluded in a subset of embryos due to aneuploidy or insufficient SNP retrieval, the diagnostic outcomes for all remaining embryos were fully concordant with those of the standard TGS strategy.

The applicability of this approach is primarily contingent upon embryo chromosomal euploidy and sufficient retrieval of informative SNPs. Additionally, the relatively high cost of whole-genome TGS remains a barrier to widespread adoption. Given the limited cohort size (n = 16) of this study, the applicability of this method necessitates further validation in larger clinical populations.

Direct haplotype construction via proband whole-genome TGS provides an effective clinical strategy to expand the applicability of PGT-M, particularly for families with de novo variants or incomplete pedigrees. Furthermore, the simplified TGS workflow demonstrates the potential to improve clinical efficiency and reduce costs relative to the standard TGS protocol within its applicable scope.

This work was supported by the National Natural Science Foundation of China (82125013, 82288102, 825B2046). The authors declare no competing interests.

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