Investigation of genomic and transcriptomic risk factors in clopidogrel response in African Americans

Figures

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  Figure 1:. Overview of Study Infographic showing the multiple stages of this study including discovery cohort analysis, functional in vitro follow up and validation cohorts for both the genomic (blue) and transcriptomic (green) findings.

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  Figure 2.. Miami and QQ plots from GWAS Miami plot representing the association between single nucleotide polymorphisms (SNPs) and High on Treatment Platelet Reactivity (HTPR) (Top) and the association between SNPs and the change of PRU value (Bottom). The x-axis shows the position of each SNP on the chromosome, while the y-axis displays the association (−log10 P-value) between the SNP and the trait of interest. The red line represents the threshold for genome-wide significance (p = 5*10−8), and the blue line represents the suggestive level of significance (p = 1*10−6). The QQ plot displays the observed p-values of the genome-wide association study (GWAS) summary data on the y-axis, compared to the expected p-values under the null hypothesis of no association on the x-axis. Deviations from the diagonal line indicate departures from the null hypothesis, where points above the line suggest more significant associations than expected, while points below the line suggest fewer significant associations than expected. (A) The GWAS identified one locus (THSD7A on chromosome 7) to be associated with higher risk of HTPR at genome-wide significance level (p < 5* 10−8). (B) QQ plot for GWAS. The…

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  Figure 3.. Regional plot of top SNP The colors of the circles refer to linkage disequilibrium (LD) (r2) between top SNP rs7807369 (purple diamond) and nearby SNPs (based on pairwise r2 values from the 1000 Genomes Project reference panel). The blue line and right y-axis show the estimated recombination rate. The x-axis represents the genomic position in chromosome 7 and the left y-axis represents the −log10 p of association with HTPR in discovery cohort. Region plot showed THSD7A variant rs7792678 is high LD with top SNP rs7807369. Both are located at the intronic region of THSD7A gene. SNPs rs7792678 and rs7811849 located at the potential enhancer/promoter region based on ENCODE annotation displayed in the UCSC Genome Browser dataset.

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  Figure 4.. THSD7A expression associated with higher risk of HTPR (A) The violin box plot of the expression of THSD7A gene in HTPR (PRU >230) and non-HTPR groups from the GEO dataset GSE32226. The THSD7A gene expression is significant higher in HTPR group compared with non-HTPR group (p = 0.004). (B) Fold-change in expression of THSD7A gene in carriers of the effect alleles compared with carriers wild-type alleles in HUVEC cell cultures. Bar heights indicate the mean expression of THSD7A gene in groups of white wild-type, AA wild-type, and AA effect alleles carriers. Error bars indicate 95% confidence interval estimates of the mean expressions. (*) = p<0.05, (**) = p<0.01, (***) = p<0.001, (****) = p<0.0001, ns = non-significant. (C) Quantified difference in protein expression from the THSD7A ELISA show in the bar chart for the same groups.

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  Figure 5.. Volcano plot of the differential gene expression analysis The volcano plot illustrates the statistical significance and fold change for each gene in the RNA-seq dataset from whole blood. Each point represents a gene, with the x-axis representing the log2 fold change and the y-axis representing the negative logarithm of the p-value. The red color point represents the gene with significant p-value (< 4*10−6) and log2 fold change.

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  Figure 6.. RT-qPCR Fold-change expression of target genes LAIR1 (A), SRC (B), SYK (C), and AKT (D), between MEG-01 cells after LAIR1 knockdown and wild-type cells. Bar heights indicate the mean expression of relative genes. Error bars indicate 95% confidence interval estimates of the mean expressions. (*) = p<0.05, (**) = p<0.01, (***) = p<0.001, (****) = p<0.0001, ns = non-significant. Lack of LAIR1 expression increased the expression of SYK and AKT significantly with no change in SRC gene expression.

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  Figure 7.. LAIR1 knockdown cells increases AKT pathway. (A) Whole-cell lysates of MEG-01 from wild-type and LAIR1 knockdown groups were Western blotted with anti-LAIR1, anti-SRC, anti-p-SRC, anti-Syk, anti-p-Syk, anti-Akt, anti-p-Akt, and GAPDH antibodies. Representative images are shown. (B) Quantification of western blotting images. Bar heights indicate the mean value of relative proteins. Error bars indicate 95% confidence interval estimates of the mean values. (*) = p<0.05, (**) = p<0.01, (***) = p<0.001, (****) = p<0.0001, ns = non-significant. The LAIR1 knockdown MEG-01 cells show significant lower LAIR1 protein level compared with wild-type MEG-01 cells. The LAIR1 knockdown cells increased the phosphorylation levels of Syk and Akt proteins.