Functional validation at RALGPS2. (A) Coordinated peaks at RALGPS2 with driver caPeak21014 (purple arrow). Read depth–normalized ATAC signal tracks averaged by genotype for rs17361251, which is the lead variant in the 1 kb caQTL analysis and is in very strong LD (r2 = 0.998, TOPMed Europeans) with the 1 Mb lead (rs6701606). (B) Colocalized GWAS (GGT), eQTL (RALGPS2), and caQTL (peak21014) associations. The caQTL lead variant (rs17361251) is indicated in each plot by a purple diamond, and colors represent LD r2 values from 1000G Europeans. (C) Study design showing the location of the three variants within caPeak21014 that were tested in a haplotype for luciferase activity (for results, see D) and positions of gRNAs for CRISPRi (blue) relative to the peak (for results, see E). (D) Transcriptional activity in HepG2 cells of a 323 bp DNA element spanning caPeak21014 and containing rs17361251, rs17276513, and rs17276527. The DNA element was tested in both orientations relative to the genome. (EV) Empty vector. Symbols represent the average of two transfected wells for each of eight independent clones for each haplotype; bars indicate mean and standard deviation; P-values are from t-tests of haplotype differences. (E) Gene expression measured after CRISPRi of the caPeak21014 region. Compared with a pool of nontargeting control gRNAs, a pool of gRNAs targeted to the enhancer led to lower expression of RALGPS2. Each point represents the mean of three qPCR replicates for an independently transfected well. Lines indicate the mean of the 10–11 wells; P-value is from a t-test. (F) Predicted mechanism at the RALGPS2 locus. The AAA haplotype of variants rs17361251, rs17276513, and rs17276527 showed higher transcriptional activity and is associated with higher liver chromatin accessibility, higher liver expression of RALGPS2, and higher plasma levels of gamma-glutamyltransferase.
