Integrative analysis reveals regulatory effects of tandem repeat expansions in tetralogy of Fallot

Congenital heart disease (CHD) affects approximately 1% of live births, yet a substantial proportion of cases remain genetically unexplained. Tetralogy of Fallot (TOF) is among the most common cyanotic CHDs. Tandem repeat expansions (TREs) can alter gene regulation and have been implicated in complex cardiac disease, but their contribution to structural CHD remains poorly understood.

We performed genome-wide analysis of rare TREs using short-read genome sequencing in 835 individuals with TOF and 386 controls, complemented by PacBio HiFi long-read sequencing, DNA methylation profiling, myocardial RNA sequencing, and fetal human heart single-cell RNA-seq. We further compared TRE-associated genes and biological processes between TOF and a previously characterized cardiomyopathy cohort.

We identified 1,043 rare TREs in individuals with TOF, corresponding to 1.25 rare TREs per individual, compared with 0.85 per individual in controls. TOF-associated rare TREs were enriched for GC-rich motifs, more often found in the 5′ untranslated regions (UTR; OR = 7.0, p = 2.1 × 10 ⁻² ) and splicing regions (OR = 3.0, p = 9 × 10 ⁻⁴ ), and located closer to transcription start sites and splice junctions, consistent with a regulatory role. We identified 91 recurrent genic TRE loci, including recurrent 5′UTR TREs absent from controls and TREs in the CHD-associated genes PACS1 and TRIP4 . Long-read analysis identified 36 loci at which repeat size was significantly associated with DNA methylation, revealing both expansion-associated hypermethylation and hypomethylation. TRE-associated genes were preferentially expressed in smooth muscle cells, pericytes, and cardiomyocytes in the developing human heart. TRE-associated biological pathways were largely distinct between TOF and cardiomyopathy. Notably, rare TRE burden was higher among individuals without positive clinical genetic testing results. We estimated an excess burden of rare genic TREs of approximately 4.4% in the TOF cohort.

Rare TREs represent an underrecognized class of genetic variation contributing to TOF. Their enrichment in regulatory regions, association with locus-specific DNA methylation and transcriptional alterations, and increased burden among genetically unexplained cases support a role for TRE-mediated regulatory dysfunction in CHD pathogenesis and suggest that assessment of tandem repeat variation may improve genetic characterization of TOF.