Identification of a novel TLE6 mutation linked to embryonic arrest and limited rescue by mRNA supplementation

Ben Yuan¹Tian Xu²Bingbing Luo¹Ziqin Lu³Junbiao Mao¹Xiumei Wang³Junling Wang^4*

• ¹Affiliated Hospital of Hubei Polytechnic University, Huangshi, China
• ²Hubei Academy of Agricultural Sciences, Wuhan, China
• ³Wuhan University of Science and Technology, Wuhan, China
• ⁴Huangshi Central Hospital, Huangshi, China

Background The subcortical maternal complex (SCMC) orchestrates early embryogenesis; TLE6, a core SCMC component, stabilizes the complex and regulates cytoskeletal dynamics during the oocyte-to-embryo transition. Pathogenic TLE6 variants are linked to pre-implantation arrest and infertility, but the mutational spectrum and rescue strategies remain incompletely defined. This study aims to describe a case of embryonic arrest associated with a novel homozygous TLE6 variant, elucidate its pathogenic mechanism, and evaluate an mRNA add-back rescue approach. Materials and Methods A case of primary infertility with repeated IVF failure underwent whole-exome sequencing with confirmatory Sanger sequencing in the proband and parents. Variant function was assessed by a minigene splicing assay, Mutalyzer prediction, and structural modeling. A CRISPR/Cas9 Tle6 mouse model evaluated developmental consequences. Wild-type Tle6 mRNA was microinjected into mutant zygotes to test rescue potential. Results A novel homozygous splice-region variant in TLE6 (NM_001143986.1: exon7: c.286-7G>A) was identified; both parents were heterozygous carriers. The minigene assay showed aberrant splicing with a 5-nucleotide insertion, producing a frameshift and premature truncation. Structural modeling predicted loss of the C-terminal domain essential for SCMC integrity. The Tle6 mutant mouse recapitulated cleavage-stage arrest with reduced blastocyst formation. Zygotic add-back of wild-type Tle6 mRNA yielded only limited improvement, with persistent developmental failure. Conclusions Findings expand the TLE6 variant landscape and support a loss-of-function mechanism causing embryonic arrest via SCMC disruption. Acute zygotic mRNA supplementation was insufficient to restore developmental competence, indicating a need for alternative or earlier-stage interventions and informing genetic counseling for affected families.