Pleiotropic Effects of an eQTL in the CELSR2/PSRC1/SORT1 Cluster That Associates With LDL-C and Resting Metabolic Rate

Abstract

Context: The locus CELSR2-PSRC1-SORT1, a primary genetic signal for lipids, has recently been implicated in different metabolic processes. Our investigation identified its association with energy metabolism.

Objective: This work aimed to determine biological mechanisms that govern diverse functions of this locus.

Methods: Genotypes for 491 265 variants in 7000 clinically characterized American Indians were previously determined using a custom-designed array specific for this longitudinally studied American Indian population. Among the genotyped individuals, 5205 had measures of fasting lipid levels and 509 had measures of resting metabolic rate (RMR) and substrate oxidation rate assessed through indirect calorimetry. A genome-wide association study (GWAS) for low-density lipoprotein cholesterol (LDL-C) levels identified a variant in CELSR2, and the molecular effect of this variant on gene expression was assessed in skeletal muscle biopsies from 207 participants, followed by functional validation in mouse myoblasts using a luciferase assay.

Results: A GWAS in American Indians identified rs12740374 in CELSR2 as the top signal for LDL-C levels (P = 1 × 10-22); further analysis of this variant identified an unexpected correlation with reduced RMR (effect = -44.3 kcal/day/minor-allele) and carbohydrate oxidation rate (effect = -5.21 mg/hour/kg-EMBS). Tagged variants showed a distinct linkage disequilibrium architecture in American Indians, highlighting a potential functional variant, rs6670347 (minor-allele frequency = 0.20). Positioned in the glucocorticoid receptor's core binding motif, rs6670347 is part of a skeletal muscle-specific enhancer. Human skeletal muscle transcriptome analysis showed CELSR2 as the most differentially expressed gene (P = 1.9 × 10-7), with the RMR-lowering minor allele elevating gene expression. Experiments in mouse myoblasts confirmed enhancer-based regulation of CELSR2 expression, dependent on glucocorticoids. Rs6670347 was also associated with increased oxidative phosphorylation gene expression; CELSR2, as a regulator of these genes, suggests a potential influence on energy metabolism through muscle oxidative capacity.

Conclusion: Variants in the CELSR2/PSRC1/SORT1 locus exhibit tissue-specific effects on metabolic traits, with an independent role in muscle metabolism through glucocorticoid signaling.

Keywords: American Indians; energy expenditure; glucocorticoid signaling; low-density lipoprotein cholesterol; skeletal muscle metabolism.

Figure 1.

Genome-wide association analysis and fine-mapping of the lead signal that associates with low-density lipoprotein cholesterol (LDL-C) levels in American Indians. A, Manhattan plot showing the results of genome-wide association studies with serum LDL-C levels in 5205 American Indians. Analysis was adjusted for age, sex, examination date, diabetes status, and first 5 principal genetic components, and identity by descent. Genes containing a variant(s) that exhibited genome-wide significance (P < 5 × 10⁻⁸ as represented by the horizontal red line) are labeled, with the CELSR2 variant rs12740374 showing the strongest association. B, Fine-mapping of the CELSR2-PSRC1-SORT1 signal using all imputed variants (minor allele frequency > 0.05) in this region. Rs12740374 is shown as a purple diamond and the variants in high linkage disequilibrium with rs12740374 at R² greater than or equal to 0.90 in American Indians are colored red.

Figure 2.

Association of CELSR2/PSRC1/SORT1 locus with energy metabolism in American Indians. A, Resting metabolic rate and B and C, rates of carbohydrate and lipid oxidation under basal and insulin-stimulated conditions, were examined based on genotypes of rs12740374. Data represent unadjusted trait measures plotted as mean + SE.

Figure 3.

Linkage disequilibrium (LD) structure in American Indians and Europeans. The R²-based LD plots were constructed using pairwise LD measures among 19 variants tagged by rs12740374 in American Indians (R² ≥ 0.90). LD information for the CELSR2/PSRC1/SORT1 locus in Europeans was obtained from the 1000Genomes EUR population and visualized using the LDlinkR package. R² value of 0 indicates alleles are independent, whereas an R² value of 1 indicates an allele of rs12740374 perfectly predicts an allele of another variant.

Figure 4.

Functional characterization of variant rs6670347. A, Orange highlights indicate genomic positions of the 19 variants that were in high linkage disequilibrium (LD) with rs12740374 in American Indians. CELSR2 intronic variant rs6670347 resides in the core binding site of the glucocorticoid receptor, NR3C1, within a credible enhancer element. Black arrowhead marks rs6670347. Comparison of various human tissues indicated that the enhancer is specific to skeletal muscle. Inspection of histone modification marks revealed substantial enrichment of the enhancer signature—H3K4Me1 in human skeletal muscle myoblast (HSMM) cells (data source: UCSC genome browser). B, NR3C1 binding sequence logo diagram retrieved from JASPAR 2024. The height of the letters and the corresponding numbers in the table represent the frequency at which a particular nucleotide is observed in that position. Black arrowhead represents rs6670347 within NR3C1 motif. C, The chromatin interaction data was obtained from the GeneHancer database within the UCSC genome browser, with the view set to double elite to ensure confident interactions derived from multiple significant sources. The enhancer element harboring rs6670347 showed a strong interaction with the promoters of CELSR2 and PSRC1.

Figure 5.

Global eQTL analysis of rs6670347 in muscle. Volcano plot illustrating the association between rs6670347 and global gene expression in skeletal tissue biopsies obtained from 207 American Indians. The effect is presented as SD in gene expression relative to the C-allele. Upregulated genes are visualized in red, whereas downregulated genes are represented in green. A nominal significance level of .05 for P values is indicated by a dotted line parallel to the X axis.

Figure 6.

Allele-specific effect of NR3C1 enhancer on CELSR2 expression. A, Reporter plasmids carrying the alternate alleles of rs6670347 were constructed by inserting the enhancer containing the binding site sequence for NR3C1 upstream of the promoter element of CELSR2 cloned upstream of the luciferase gene in a pGL4.10 vector. The constructs were cotransfected in mouse myoblast C2C12 cells with a human NR3C1 expression plasmid, and the cells were assayed for luciferase activity 48 hours post transfection. B, The enhancers did not elicit any change in luciferase activity, signifying no influence on the promoter activity of CELSR2. C, However, in the presence of 25µM synthetic glucocorticoid (dexamethasone), there is a notable increase (40%) in promoter activity when the enhancers were present. The luciferase activity of the enhancers is plotted as mean + SE relative to the activity of the promoter alone. The experiment was performed in triplicate and repeated on 3 independent days, with statistical significance determined through a t test. *P = .0002; **P = 4.1 × 10⁻⁵.