A novel PADI6 splice-site variant induces non-canonical GC-AG splicing and embryonic arrest in humans

Early embryonic arrest (EEA) represents the predominant cause of assisted reproductive technology (ART) failure. PADI6 was a disease-associated gene identified for EEA. To date, 42 likely causal PADI6 variants have been documented, predominantly located in exonic regions with clustering outside the N-terminal domain. Whole-exome sequencing identified candidate variants, with pedigree validation via Sanger sequencing. PCR amplification and sequencing of PADI6 transcripts from arrested embryos characterized splicing alterations. Single-embryo RNA sequencing assessed transcriptomic perturbations in PADI6-variant carrier. We report a homozygous PADI6 splicing variant (c.104_116 + 10del, p.Leu35_Gly39delinsTrpGluLeuCysGlnArgTrpGlnAlaAspArg) inducing non-canonical "GC-AG" splicing, causing a 13-bp exon 1 deletion and 31-bp intron 1 retention. This aberrant splicing altered the N-terminal domain, replacing five wild-type residues with eleven novel amino acids. Transcriptome analysis revealed dysregulation enriched in RNA metabolism pathways (down-regulated genes) and Rho GTPase signaling (up-regulated genes), with ribosomal dysfunction implicated as a potential pathogenic mechanism. This study expands the PADI6 mutational landscape and provides the first transcriptome profiling of human embryos harboring PADI6 variant. Our findings establish a framework for genetic counseling in female infertility characterized by embryonic arrest.