L26/O-334 A reference-free, 3D genome-based method and clinical application for haplotype phasing and carrier status determination of structural rearrangements in preimplantation genetic testing

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Abstract Study question Preimplantation screening for structural variations (SVs) has limitations, requiring specific embryos as references and being applicable only to certain types of variations. Summary answer We developed a carrier status determination method based on 3D genome that requires no reference samples and is applicable to any type of SV. What is known already Preimplantation screening of in vitro fertilized embryos to select those without SVs helps reduce the risk of birth defects and prevent reproductive issues in the next generation. Through Mapping Allele with Resolved Carrier Status (MaReCs) techniques (Xu et al. PNAS 2017 114:E8695-E8702), both chromosomal ploidy and carrier status screening for reciprocal translocations and Robertsonian translocations can be simultaneously performed during preimplantation genetic testing. However, about 18% of cases failed to be determinated due to the absence of reference embryos or family samples, particularly in Robertsonian translocations. It is also unavailable for inversions, insertions, or complex SVs. Study design, size, duration We analyzed 3,178 embryos from 672 families, covering variations including reciprocal translocations, Robertsonian translocations, insertions, inversions, and complex SVs. Among these, 181 families carried reciprocal translocations or Robertsonian translocations and had available reference embryos, enabling carrier status analysis using standard MaReCs methods. After excluding the reference embryos, our improved method performed independent embryo predictions on the same pedigree data. The results of the two methods were subsequently compared. Participants/materials, setting, methods We designed and developed a novel method, MaReCs_MoKa, based on 3D genome method. By leveraging paired sequence features within the 3D genome, it enables haplotype phasing using only a single sample from a variant carrier. This approach distinguishes the pathogenic haplotype from the normal haplotype in heterozygous SNPs without requiring reference embryos or family samples, and is applicable to all variant types. We subsequently validated the haplotype results in embryos. Main results and the role of chance MaReCs_MoKa achieved a 100% detection rate for SV breakpoints, whereas normal MaReCs detected only 81.03% of breakpoints through embryonic CNVs. The remaining 18.97% of breakpoints could not be detected due to the absence of reference embryos. 97.19% of the breakpoints detected by both methods were consistent, while MaReCs_MoKa yielded more accurate results for the remaining samples. For MaReCs breakpoints obtained using karyotyping, there were 6 false negatives, 3 false positives, and 2 errors in primary banding or mutation type detection. This indicates that MaReCs_MoKa significantly reduces both false negatives and false positives. The two methods yielded consistent embryo status assessments in 98.96% of cases. Inconsistencies arose from the absence of reference embryos, imprecise chromosomal breakpoint detection, and misclassifications due to recombination events. Additionally, we identified several families carrying insertions or complex SVs that are not applicable to the MaReCs approach. The diploid embryos from these families underwent MaReCs_MoKa testing, with results validated through amniotic fluid karyotyping and live birth cases, confirming consistency in outcomes. This demonstrates the high reliability and broad applicability of our methodology. Limitations, reasons for caution 3D genome-based sequencing lacks sufficient coverage of genomic repetitive regions, limiting MaReCs_MoKa’s ability to effectively distinguish haplotypes for SVs in inversion polymorphisms or heterochromatic regions. Additionally, background noise in the 3D genome increases the error rate of SNP sites, necessitating sufficient data volume to statistically exclude interference. Wider implications of the findings The MaReCs_MoKa method offers improvements in the following three key aspects. First, it integrates the detection of SV types, precise breakpoint, and haplotype into a single assay. Second, it significantly expanding its applicability and reducing the likelihood of test failure. Third, it demonstrates enhanced capability in resolving complex SVs. Trial registration number No

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