P2864A novel mechanism of sinus node dysfunction: intergenic deletion between PITX2 and ANK2 disrupts chromatin structure in pacemaker cell differentiation

Abstract

Abstract Background Genetics of sinus node dysfunction (SND) remains poorly understood with few genes identified (HCN4, SCN5A, GJA5, KCNQ1, MYH6 and ANK2) and further genetic heterogeneity. Purpose We report two families with SND segregating with an intergenic deletion associated with long-range cis-regulatory elements (CREs) of PITX2. Methods We applied 30x whole genome sequencing (WGS) to two French families in which exome sequencing did not allow to detect the causing genes of SND and analyzed them bioinformatically. Results We first applied WGS to a 4-generation family presenting 16 patients with SND, atrial fibrillation and/or repolarization abnormalities and identified a 15-Kb deletion within a 16.5-cM linkage interval (Zmax = 5.9, θ = 0) on chromosome 4q25. We also applied WGS on a second family presenting 5 patients with SND and identified a 91-Kb deletion overlapping the initial deletion. This intergenic deletion, which was located between PITX2 and ANK2 and contained a binding motif of CCCTC-binding factor (CTCF), was segregating in all patients and was not found in 855 French control genomes. CTCF functions as a genome organizer mediating genomic interactions between genes and CREs. To clarify a possible regulatory function of the 1.5-Mb intergenic region containing the deletion, we interrogated 3 epigenetic databases. High-throughput chromosome conformation capture (Hi-C) (1) revealed that this intergenic region was placed in a topologically associating domain (TAD) containing PITX2. Chromatin interaction analysis by paired-end tag (ChIA-PET) of CTCF (2) suggested several possible functional chromatin loops in the TAD. Of them, a CTCF-mediated chromatin loop corresponding to a 300-Kb genomic region was clarified as a possible CRE using histone modification data from Roadmap. The region ranging between the intra-deletion CTCF motif and another distal motif was repressed in H3K27me3 profiles of fetal heart, while the region was activated in H3K27ac profiles of H1 ESC-derived mesendoderm. Conclusion This CRE may regulate expression of PITX2 and/or ANK2 in pacemaker cell differentiation. Ongoing experiments on patient's iPSC and transgenic mouse will further characterize the disease mechanism.