Genomic and transcriptomic analyses of aortic stenosis enhance therapeutic target discovery and disease prediction

Figures

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  Fig. 1. Circos plot of multi-ancestry AS GWAS. Circular Manhattan plot of our multi-ancestry AS GWAS results in both autosomes and the X chromosome. Genome-wide significance (P = 5 × 10−8), indicated by the inner red circle. Prioritized genes are highlighted at chromosomal position at outer circle, new loci are red and replicated loci are black.

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  Fig. 2. Trumpet plot of lead variants in multi-ancestry as well as ancestry-stratified and sex-stratified AS GWASs. Trumpet plot representing effect size and effect allele frequencies for lead variants. Lead variants are colored green if identified in multi-ancestry, gray if a prior AS risk locus, red if specific to African ancestry GWAS, blue if specific to European ancestry and yellow if specific to male sex GWAS. Variants with high effect sizes are labeled by the nearest gene.

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  Fig. 3. Manhattan plot of TWAS using human AV eQTLs. Manhattan plot of TWAS using European AS GWAS summary statistics and expression eQTLs data from human AVs. Z score represents magnitude and direction of association between genetically determined AV gene expression and AS risk. Genes in red are new TWAS associations. Only the most associated gene is labeled when multiple genes were identified at the same locus.

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  Fig. 4. Overview of causal gene prioritization. Schematic depicting variants with a gene prioritized by at least three methods. Green represents multi-ancestry GWAS; blue represents European genetic ancestry. A total of 154 multi-ancestry unique lead variants, 8 European genetic unique ancestry lead variants, 5 male sex unique lead variants, 1 African genetic ancestry unique lead variant and 3 X chromosome unique lead variants with less than three supportive methods are not shown.

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  Fig. 5. Performance of AS PRS in European genetic ancestry individuals from the UK Biobank, TIMI trials and UCLA ATLAS. a, Forest plot of AS PRS validation in the UK Biobank, TIMI clinical trials and UCLA ATLAS. b, Forest plot comparing the HR for categories of genetic risk and individual risk factors in the UKB. Forest plot in a demonstrating HRs/ORs and 95% CI per 1 s.d. PRS for the new AS PRS compared to a previously published PRS in the UK Biobank (n = 446,895), TIMI trials (n = 59,866) and the UCLA ATLAS (n = 34,024), as well as a meta-analysis (Meta) of the UK Biobank and TIMI trial results. All analyses are adjusted for age, sex, five principal components and clinical risk factors including T2D, HTN, CAD, HLD, elevated BMI, current smoking and renal failure. Forest plot in b demonstrating HRs for genetic risk categories and individual risk factors with AS in the UKB (n = 446,895). eGFR, estimated glomerular filtration rate; CI, confidence interval; BMI, body mass index.

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  Fig. 6. Suppression of calcification in human VICs cultured in OM by silencing of LTBP4 and CMKLR1, and their colocalization with calcification in human AV. a, Alizarin red staining identifies calcification in human VICs (example with two of eight donors). Silencing of LTBP4 and CMKLR1 significantly suppressed calcification in human VICs cultured in OM. Total n = 11 VIC donors. b, Validation of silencing of LTBP4 and CMKLR1 by siRNA transfection; qPCR. Silencing of LTBP4 and CMKLR1 significantly suppressed their gene expression levels in human VICs (mean ± s.d., n = 8; P < 0.05, two-tailed paired t test). Box plots show the distribution from minimum to maximum values. The box represents the interquartile range (25th to 75th percentiles), with the line inside the box indicating the median. Whiskers extend to the minimum and maximum data points. c, Immunofluorescence staining of LTBP4 and CMKLR1 showed colocalization with calcific nodules and calcifying cells (OsteoSense680; yellow arrows) in human AV (n = 3 donors). LTBP4 expression in smooth muscle-like cells (orange arrow). CMKLR1 is colocalized with microvessels in human AV (green arrow, inset). Scale bars, 100 µm.