Chromatin Capture Identifies SCARB1-LAG3 Proinflammatory Cardiovascular Disease Gene Networks [RNA-Seq]

Purpose: Utilized Next-generation sequencing (NGS) to profile transcriptional differences between two populations, a selected carrier (C) alele of SNP (rs10846744) associated with cardiovascular disease (CVD) and a selected non-carrier (G) allele. Methods: RNA was isolated from three subjects homozygous for the rs10846744 reference (G) allele and three subjects homozygous for the rs10846744 risk (C) allele and then subjected to full transcriptome sequencing. Triplicates of one sample from each group were also sequences to overlay with chromatin data from each group. Knockdown of NR2F2 using lentiviral particles assessed differential expression between the treated and untreated risk replicates. The Illumina NextSeq 500 next gen sequencing platform (UCONN CGI, Storrs, CT) was used for RNA-Seq. HiSeq 2000 was used for biological replicates at Perkin Elmer. RNA targets of interest were validated by qPCR using TaqMan assays and proteins were blotted using standard western methodologies. Results: We normalized the raw read count by FPKM and using Cufflinks, identified differential transcripts. RNA-seq data confirmed differential expression of NR2F2 and LAG3, validated by qPCR. Conclusions: Our study represents the first detailed analysis of significant changes in gene expression by an altered chromatin interaction network from SCARB1 to NR2F2 and LAG3, contributing to CAD proinflammatory gene networks. Overall design: Transcriptional profiles from 3 carriers of the risk (C) allele and 3 non-carriers of the (G) reference allele, were generated by NGS, in triplicate, using Illumina platform technology. Replicates of one sample from each group and knockdown of NR2F2 in the risk replicate were also sequenced (n=3) to overlay with chromatin interaction data.