NIH Research Festival
Using GRASP cohort, we examined the hypothesis that common noncoding variants are identifying gene regions that are enriched for rare, functional variants that can increase disease burden in African Americans (AAs) with scleroderma (SSc). Exome sequencing and gene-based testing was performed in 969 SSc and 771 controls of African ancestry for 32 genes reported with SSc in GWAS catalog. In both discovery and replication series only functional variants in NOTCH4 remained statistically significant. Majority of NOTCH4 variants were African ancestry-predominant or novel. On analyzing SSc-subsets, diffuse cutaneous SSc, anti-fibrillarin antibody, and severe Raynauds‚Äô remained significant. We theorized that the NOTCH4 variants transcriptionally regulate NOTCH4 signaling pathway either by increasing NOTCH4 or downstream signaling molecules- HEY2 and HES1. eQTL analysis using GTEx data showed that NOTCH4 promoter (c.-117G>A) variant increased NOTCH4 transcripts and missense (p.Gly942Arg) variant increased HEY2 in LCLs. RT-PCR and ELISA revealed higher NOTCH4 in LCLs with c.-117A allele. Stimulation of NOTCH4 in LCLs carrying missense variant showed higher HEY2 and HES1 expression than wild type LCLs. SSc patients carrying c.-117A allele had higher NOTCH4 and the missense variant was associated with increased downstream NOTCH4 signaling in skin than WT alleles. Single cell analysis identified NOTCH4 principally being expressed in endothelial cells (ECs). SSc ECs with c.-117A allele had increased NOTCH4, downstream signaling, and decreased tube formation. Inhibiting the NOTCH4 pathway using a NOTCH4-specific blocking antibody, rescued normal tube formation. To summarize, NOTCH4 variants in AA SSc patients increase NOTCH4 expression and signaling that can cause impaired angiogenesis.
Scientific Focus Area: Health Disparities
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