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

## TL;DR

A new TLE6 mutation linked to infertility and embryonic arrest was identified, and attempts to rescue it with mRNA were only partially successful.

## Contribution

A novel TLE6 splice-region variant is described, with functional and structural analysis and an evaluation of mRNA-based rescue.

## Key findings

- The TLE6 variant causes aberrant splicing, leading to a frameshift and loss of the C-terminal domain.
- Tle6 mutant mice showed cleavage-stage arrest and reduced blastocyst formation.
- Wild-type Tle6 mRNA microinjection provided limited developmental rescue in mutant zygotes.

## Abstract

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 associated with 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.

A case of primary infertility with repeated IVF failure underwent whole-exome sequencing, followed by confirmatory Sanger sequencing performed in the proband and parents. The variant function was assessed using a minigene splicing assay, Mutalyzer prediction, and structural modeling. A CRISPR/Cas9 Tle6 mouse model was evaluated for developmental consequences. Wild-type Tle6 mRNA was microinjected into mutant zygotes to test its rescue potential.

A novel homozygous splice-region variant in TLE6 (NM_001143986.1: exon7: c.286-7G > A) was identified, with both parents confirmed as heterozygous carriers. The minigene assay showed aberrant splicing with a 5-nucleotide insertion, producing a frameshift and premature truncation. Structural modeling predicted the 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.

These findings expand the TLE6 variant spectrum and support a loss-of-function mechanism causing embryonic arrest through SCMC disruption. Acute zygotic mRNA supplementation was insufficient to restore developmental competence, indicating the need for alternative or early-stage interventions and informing genetic counseling for affected families.