Characterization of variants associated with Cerebral Small Vessel Disease identifies a functional SNV in Versican

Single nucleotide variants in the non-coding genome can significantly alter disease risk, but identifying the function of SNVs is a challenge. Increasing numbers of variants of unknown significance have been associated with the risk of Stroke, Cerebral Small Vessel Disease and burden of White Matter Hyperintensities, but without biological validation, the significance of these findings is uncertain. We use a Multiplexed Parallel Reporter Assay (MPRA) to assay the function of 44 SNVs associated with Cerebral Small Vessel Disease and stroke in EFEMP1, FOXF2, HAAO, KCNK3, NMT1, OPA1, RASL12, STAT3 and VCAN. A functional SNV in versican was further probed using transcriptional reporter and Chromatin Immunoprecipitation assays. We identify 26 allele-specific enhancers where a SNV increases or decreases transcriptional activity. We validate rs13176921 as an SNV that significantly modulates an intronic enhancer in the matrisome protein Versican and show it affects mRNA levels of the gene. We identify that the transcription factor NKX3.1 binds to the region of this SNV. Thus, using MRPA we were able to gain mechanistic insight into which GWAS-identified SNVs modulate transcriptional activity, and validate a functionally important SNV in Versican and provide a mechanism by which it controls its transcription. Author SummaryGenome wide association studies identify many single nucleotide variant changes associated with diseases such as stroke and Cerebral Small Vessel disease, but not all the variants lead to disease. In this work we screen for variants that control transcription and therefore expression of adjacent genes. We find that a human variant associated with CSVD in the young and elderly decreases the expression of the extracellular matrix protein Versican. We use molecular biology experiments to determine the mechanism by which the variant changes expression and determine that the variant changes a transcription factor binding site.