Functional characterization of a non-coding RUNX1T1 variant in a kindred with esophageal atresia

Esophageal atresia (EA) is a congenital foregut malformation with substantial genetic heterogeneity. While coding variants explain some syndromic cases, the molecular basis of non-syndromic EA remains largely unknown. We investigated an Israeli-Bedouin kindred with a high incidence of non-syndromic EA.

Three of ten affected individuals underwent whole-exome/genome sequencing. Following variant filtering and prioritization using population databases and in silico tools, the candidate non-coding variant underwent Sanger sequencing in all five available affected individuals. Functional effects were assessed by minigene splicing assays, quantitative PCR, dual-luciferase reporter assays, and a fly overexpression model.

Sequencing data variant analysis of 3 affected individuals identified a shared heterozygous deep-intronic RUNX1T1 variant (chr8:91994544G > A, hg38), subsequently verified in the five participating affected individuals. Although incorporated into a rare RUNX1T1 transcript, the variant did not affect splicing. Instead, it increased expression of canonical RUNX1T1 isoforms and enhanced reporter activity, consistent with a regulatory gain-of-function effect. RUNX1T1 ortholog (nvy) global overexpression in Drosophila resulted in morphological alterations of the feeding apparatus, including reduced rigidity and elongation of the labral sense organ.

In a kindred with apparently partially penetrant dominant non-syndromic EA, we identified a novel non-coding RUNX1T1 variant with functional evidence of a regulatory gain-of-function effect. These findings implicate RUNX1T1 dysregulation as a contributor to EA pathogenesis and highlight the importance of non-coding regulatory variation in congenital foregut malformations.