CARMALIs a Long Non-coding RNA Locus That RegulatesMFGE8Expression

Genome-wide association studies have identified several genetic loci linked to coronary artery disease (CAD) most of them located in non-protein coding regions of the genome. One such locus is the CAD Associated Region betweenMFGE8andABHD2(CARMA), a similar to 18 kb haplotype that was recently shown to regulate vicinal protein coding genes. Here, we further investigate the region by examining a long non-coding RNA gene locus (CARMAL/RP11-326A19.4/AC013565) abutting the CARMA region. Expression-genotype correlation analyses of public databases indicate thatCARMALlevels are influenced by CAD associated variants suggesting that it might have cardioprotective functions. We foundCARMALto be stably expressed at relatively low levels and enriched in the cytosol.CARMALfunction was investigated by several gene targeting approaches in HEK293T: inactive CRISPR fusion proteins, antisense, overexpression and inactivation by CRISPR-mediated knock-out. Modest increases inCARMAL(3-4x) obtained via CRISPRa using distinct single-guided RNAs did not result in consistent transcriptome effects. By contrast,CARMALdeletion or reducedCARMALexpression via CRISPRi increasedMFGE8levels, suggesting thatCARMALis contributing to reduceMFGE8expression under basal conditions. While future investigations are required to clarify the mechanism(s) by whichCARMALacts onMFGE8, integrative bioinformatic analyses of the transcriptome ofCARMALdeleted cells suggest that this locus may also be involved in leucine metabolism, splicing, transcriptional regulation and Shwachman-Bodian-Diamond syndrome protein function.