Whole genome sequencing and UMI-4C provide novel insights into the genetic architecture and mechanisms underlying North Carolina macular dystrophy, a cis-regulatory disease

Abstract

Purpose : North Carolina macular dystrophy (NCMD) is a rare autosomal dominant disease, characterized by loss of central vision. With the identification of noncoding single nucleotide variants (SNVs) and duplications overlapping with a DNase I hypersensitive site (DHS) near PRDM13 or IRX1 as underlying genetic cause, NCMD is hypothesized to be a cis-regulatory disease. Here we investigate the genetic architecture of and mechanisms underlying NCMD, to further solve its missing heritability, and to provide mechanistic insight into its molecular pathogenesis.

Methods : Twenty-five unrelated NCMD families underwent targeted testing of PRDM13 and IRX1 regions followed by whole genome sequencing (WGS) in a selection of unsolved cases (n=11). Chromosome conformation capture, in particular UMI-4C sequencing, was applied on retinas from human donor eyes to fine-map interactions of cis-regulatory elements with the PRDM13 and IRX1 promoter. This data, together with other human retinal (epigen)omics data, were integrated in a UCSC browser session to advance the interpretation of the WGS data. Luciferase assays in ARPE-19 cells were performed to evaluate the functional effect of 6 previously reported (V1-V3, V10-V12) and 2 novel candidate NCMD-associated SNVs.

Results : A known noncoding SNV upstream of PRDM13 (V1) was found in a first family, while a novel SNV at the same position was identified in a second family. WGS revealed a novel tandem duplication, encompassing the known DHS upstream of PRDM13, in a third family. An additional novel noncoding SNV upstream of PRDM13 was found in a fourth family. Interestingly, UMI-4C showed an interaction of this region with the PRDM13 promoter. This region is active at a specific developmental stage (D103) that is compatible with the timepoint when retinal progenitor cells of the central retina exit mitosis. Using luciferase assays we demonstrated that the noncoding SNVs, located in two mutational hotspots, displayed up- and downregulation of expression, respectively.

Conclusions : Overall, the genetic architecture of NCMD was expanded with novel noncoding variants with a likely effect on PRDM13 regulation. The retinal interaction profiles of PRDM13 and IRX1 advance the interpretation of novel noncoding variants identified using WGS and provide insight into the cis-regulatory mechanisms underlying NCMD.

This is a 2021 ARVO Annual Meeting abstract.