Polymorphisms within the C-reactive protein (CRP) promoter region are associated with plasma CRP levels

Abstract

Elevated plasma levels of C-reactive protein (CRP), an inflammation-sensitive marker, have emerged as an important predictor of future cardiovascular disease and metabolic abnormalities in apparently healthy men and women. Here, we performed a systematic survey of common nucleotide variation across the genomic region encompassing the CRP gene locus. Of the common single-nucleotide polymorphisms (SNPs) identified, several in the CRP promoter region are strongly associated with CRP levels in a large cohort study of cardiovascular risk in European American and African American young adults. We also demonstrate the functional importance of these SNPs in vitro.

Figure 1

A GeneSNPs image of CRP, showing the region scanned for polymorphism. Coding regions of exons are shown in blue, and the UTR is shown in green. Polymorphic positions are indicated with ticks below the axis, and the length of the tick represents MAF in the combined population. A highly repetitive region of the intron (shown in pink) could not be screened for polymorphism by resequencing. Nonsyn = nonsynonymous; Synon = synonymous; CDS - coding sequence.

Figure 2

Visual genotype diagram of patterns of common variation at the CRP locus. Each row corresponds to an individual DNA sample in the polymorphism-discovery panel; E001–E023 are EAs, and D001–D040 are AAs. Each column corresponds to a polymorphic site. Sites are ordered by LD, not position along the chromosome, with sites showing similar patterns of genotype shown adjacent to one another. Site numbers are relative to GenBank accession number AF449713. Selected tagSNPs are indicated with arrows.

Figure 3

Phylogeny of common CRP haplotypes. Haplotypes were inferred for all 18 common SNPs (MAF >10% in either AA or EA sequenced samples) by use of PHASE v 2.0. A neighbor-joining phylogenetic tree of the major CRP haplotypes resolved using the seven selected tagSNPs is shown, with branch lengths representing evolutionary distance as the proportion of alleles shared at all 18 common SNPs. The tagSNP alleles associated with each resolved haplotype are shown.

Figure 4

Relative effects of tagSNP haplotypes on ln(CRP). Regression coefficients from haplo.glm analysis were estimated for each haplotype, with phenotypic variables included as covariables. Coefficients reflect difference in mean ln(CRP) per copy relative to H2, the most frequent haplotype. H1 was significantly lower than H2, and H5–H8 were significantly higher (see P values in tables 7 and 8).

Figure 5

CRP promoter and predicted USF1 sites. Upper panel, A vista plot (Mayor et al. 2000) of mouse homology across the human CRP locus. The CRP transcript and repeat elements are annotated across the top of the plot (see key at left), and strong evolutionary conservation of the coding region in the second exon is apparent as the shaded blue peak. A putative promoter region (75% conserved) is visible as a pink peak stretching from 1444 to 1650; the two putatively functionally polymorphic USF1 sites (1421 and 1440) are on the edge of this region. Lower panel, Haplotypes of the polymorphisms at 1421 and 1440, with the putative USF1-binding sites indicated in uppercase letters. Alleles predicted to disrupt USF1 binding are shown in red, and alleles predicted to retain USF1 binding are shown in green. H1–H3 represents the ancestral haplotype, as inferred from the chimpanzee alleles at these SNPs. LINE = long interspersed nuclear element; LTR = long terminal repeat; SINE = short interspersed nuclear element.

Figure 6

Native promoter construct activity. Short (−703 to +1, relative to TSS) and extended (−1186 to +1) versions of the CRP promoter region were cloned into a luciferase-reporter–gene plasmid. Transient transfection data show the increase in transcription levels for each construct over that of the plasmid-lacking promoter in both uninduced and IL6-induced environments. Shaded circles identify minor alleles at a given SNP. Unshaded circles identify one of the triallelic SNP variants at bp 1440. Error bars reflect SEM in 20 replicate experiments. The short construct group of H1–H4 is divided into two categories of long clones on the basis of the unique 1009 SNP in H4 that is not included in the short-clone constructs. Within the IL6-induced extended constructs, t tests show that H4 activity is significantly reduced, whereas H7–H8 activity is significantly increased (P<.05), consistent with the induced short constructs in which t tests show that H1–H4 activity is significantly reduced, whereas H7–H8 activity is significantly increased (P<.05). Uninduced activities showed similar trends, but the signal was relatively weak.

Figure 7

SV40-replacement promoter construct activity. To increase the level of uninduced expression, an IL6-responsive negative regulatory element in the proximal segment of the CRP promoter (−107 to +1) was replaced with the SV40 minimal promoter. Transient transfection data shows the increase in transcription levels for each construct over that of the SV40 minimal promoter alone. Shaded circles identify minor alleles at a given SNP. Unshaded circles identify one of the triallelic SNP variants at bp 1440. Error bars reflect SE measurements. Within each combination of construct length and induction condition, t tests show that all differences between haplotypes’ behavior are significant (P<.05), except for H1–H3 versus H7–H8 in IL6-induced extended constructs.