Trio Exome Sequencing Reveal Paternal Transmission Bias and Novel Gene Loci in Recurrent Spontaneous Abortion

To systematically evaluate the contributions of parental and fetal genetic factors in recurrent spontaneous abortion (RSA) through trio-based exome sequencing and transmission disequilibrium test (TDT). We conducted whole-exome sequencing on 31 trios (mother, father, and miscarried fetus) affected by RSA, collected from Nanjing Drum Tower Hospital between March 2021 and December 2023. Using TDT, we analyzed common genetic variants to identify associations with RSA and performed parent-of-origin analysis to assess the independent contributions of paternal and maternal alleles. Rare variant TDT analysis was also conducted to identify associations at the gene level. Significant findings underwent computational validation (population genetics, functional prediction, gene constraint and mouse phenotypes). We identified one common variant (rs2034910825 in the DNA repair gene LIG1) and 15 suggestive SNPs associated with RSA. Computational validation showed 14/17 top SNPs enriched in East Asians. Functional predictions indicated potential deleterious effects or regulatory impacts for several variants, and mouse knockout models supported embryonic developmental roles for key genes (e.g., Lig1, Lrp2, and Flywch1). Additionally, our analysis revealed a paternal transmission bias at two loci (PRAMEF4 and SLC24A4), where alleles from fathers were preferentially transmitted to affected pregnancies. Gene-level rare variant analysis further implicated eight genes (PSG1, D2HGDH, OAS2, etc.) in placental development, angiogenesis, and DNA repair. This study reveals paternal genetic contributions and fetal-placental dysfunction pathways in RSA. Trio exome sequencing coupled with TDT provides a robust framework for unmasking familial transmission patterns, offering actionable markers for early risk prediction and personalized counseling.