Journal: Nature Genetics

Article Title: Discovery and systematic characterization of risk variants and genes for coronary artery disease in over a million participants

doi: 10.1038/s41588-022-01233-6

Figure Lengend Snippet: New loci for CAD from primary meta-analysis

Article Snippet: Gene expression was assessed by quantitative PCR with Taqman probes (Invitrogen) for genes of interest ( MYO9B : Hs00994622_m1; HAUS8 : Hs00928622_m1 ; OCEL1 : Hs00928613_m1; USE1 : Hs00218426_m1; NR2F6 : Hs00172870_m1; GAPDH : Hs03929097_g1).

Techniques: Variant Assay

Journal: Nature Genetics

Article Title: Discovery and systematic characterization of risk variants and genes for coronary artery disease in over a million participants

doi: 10.1038/s41588-022-01233-6

Figure Lengend Snippet: a , Regional association plot from the primary CAD meta-analysis for the new gene-dense region around MYO9B . Colored dots represent the position ( x axis) in GRCh37 coordinates and –log 10 (meta-analysis P value) ( y axis) of each variant in the region. Dots are shaded to represent the r 2 with the lead CAD variant ( rs7246865 ), estimated using a random sample of 5,000 European ancestry participants from the UK Biobank. Recombination peaks are plotted in blue based on estimates of recombination from 1000 Genomes European ancestry individuals. b , Identification of a noncoding enhancer in the region around the CAD association signal. The plot shows an inset of a 5-kb window surrounding the lead CAD variant ( rs7246865 ). The top three tracks (blue) show H3K27Ac ChIP-seq of human CA, aorta and tibial artery, identifying a vascular tissue enhancer element overlying rs7246865 . The bottom three tracks (purple) show ATAC-seq of human monocytes, immortalized human aortic ECs and CA-VSMCs, identifying a region of open chromatin in all three cell types around rs7246865 . The plot also shows the location of the sgRNAs used for deletion of the noncoding enhancer. c , Efficiency of CRISPR editing in primary human cells. The Cas9-sgRNA ribonucleoprotein nucleofection method resulted in noncoding enhancer deletion efficiency ( x -axis) of greater than 0.5 by densitometry and was comparable across monocytes, ECs and CA-VSMCs. Points indicate enhancer deletion efficiency for each of the 12 replicates. Horizontal bars indicate mean enhancer deletion efficiency, and whiskers indicate 95% CIs. d , Relative expression of nearby genes after enhancer deletion in ECs. The y axis shows mean expression of five local genes expressed in ECs compared to expression levels of a control gene ( GAPDH ). Blue bars indicate gene expression with Cas9–control sgRNA. Red bars indicate expression with tandem enhancer-deleting guides as identified in b . Points indicate gene expression levels for each of the six replicates. Vertical bars indicate mean expression levels and whiskers indicate 95% CIs. Gene expression was quantified by qPCR. Expression levels were compared using an unpaired two-way Student’s t test. Reduced expression of MYO9B and HAUS8 was identified after 131-bp enhancer deletion as in b . ** P = 0.0020; *** P < 0.0001. e , Relative expression of nearby genes after enhancer deletion in CA-VSMCs. The y -axis shows mean expression of five local genes expressed in CA-VSMCs compared to expression levels of a control gene ( GAPDH ). Blue bars indicate gene expression with Cas9–control sgRNA. Red bars indicate expression with tandem enhancer-deleting guides as identified in b . Points indicate gene expression levels for each of the six biological replicates. Vertical bars indicate mean expression levels and whiskers indicate 95% CIs. Gene expression was quantified by qPCR. Expression levels were compared using an unpaired two-way Student’s t test. Reduced expression of MYO9B was identified after 131-bp enhancer deletion as in b . ** P = 0.0044. f , In vitro endothelial wound healing with enhancer and gene deletions. The y -axis indicates fluorescence intensity, a read-out for endothelial wound healing and a composite of migration and proliferation. ECs with CRISPR–Cas9 genome editing for enhancer deletion (red) or single-gene knock-outs exhibited diminished wound healing relative to nontargeting control with no deletions (blue). Dots indicate endothelial wound healing for each of the six replicates. Vertical bars indicate mean wound-healing levels and whiskers indicate 95% CIs. Levels of wound healing were compared by one-way ANOVA. * P = 0.0464; ** P = 0.0013; *** P = 0.0003; **** P < 0.0001; NS, not significant.

Article Snippet: Gene expression was assessed by quantitative PCR with Taqman probes (Invitrogen) for genes of interest ( MYO9B : Hs00994622_m1; HAUS8 : Hs00928622_m1 ; OCEL1 : Hs00928613_m1; USE1 : Hs00218426_m1; NR2F6 : Hs00172870_m1; GAPDH : Hs03929097_g1).

Techniques: Variant Assay, ChIP-sequencing, CRISPR, Expressing, Control, Gene Expression, In Vitro, Fluorescence, Migration