View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Journal of the American College of Cardiology Vol. 50, No. 12, 2007 © 2007 by the American College of Cardiology Foundation ISSN 0735-1097/07/$32.00 Published by Elsevier Inc. doi:10.1016/j.jacc.2007.06.012 STATE-OF-THE-ART PAPER C-Reactive Protein Gene Polymorphisms, C-Reactive Protein Blood Levels, and Cardiovascular Disease Risk Fadi G. Hage, MD, Alexander J. Szalai, PHD Birmingham, Alabama C-reactive protein (CRP), a blood marker of inflammation and a hallmark of the acute-phase response, has been shown to be a powerful and specific predictor of cardiovascular event risk in populations of otherwise healthy persons. Here we review what is known about CRP gene polymorphisms, discuss how these might affect the epidemiology of CRP and our understanding of CRP’s contribution to cardiovascular disease, and examine their potential clinical usefulness. Evidence shows that certain subtle variations in the CRP gene sequence, mostly single nucleotide polymorphisms, predictably and strongly influence the blood level of CRP. Some of these varia- tions are associated with clinical correlates of cardiovascular disease. If future studies can establish with cer- tainty that CRP influences cardiovascular biology, then CRP gene profiling could have clinical utility. (J Am Coll Cardiol 2007;50:1115–22) © 2007 by the American College of Cardiology Foundation C-reactive protein (CRP) was discovered in 1930 by Tillet “high-sensitivity CRP” assays, technologies that allow cli- and Francis during their studies of patients with acute nicians to detect the low levels of blood CRP in ostensibly pneumonia (1). They found that when serum from febrile healthy people, has resulted in accumulation of vast patients was mixed with a cell-wall component of pneumo- amounts of data linking blood CRP to various kinds of cocci that they called “Fraction C,” a precipitate formed. cardiovascular diseases (5). At the same time, new evidence This property was subsequently found to be due to reactivity from animal models indicates that CRP might actually have of CRP, present at high levels in “acute-phase” sera, with a a pathogenic role in vascular disease (6–9). polysaccharide in the pneumococcal cell wall (C- It has long been recognized that environmental variables polysaccharide). C-reactive protein was the first of many and patient behaviors and traits such as smoking, infections, acute-phase reactants subsequently discovered (2), but today age, gender, lipid levels, and blood pressure can contribute elevation of CRP is still considered the hallmark of the to variation in baseline CRP level1 (10). Indeed, obesity is a acute-phase response. major determinant of CRP levels in humans, and elevated C-reactive protein is a member of the phylogentically levels of CRP predict the development of type 2 diabetes ancient and conserved pentraxin family of proteins. Human mellitus and the metabolic syndrome (11,12). Elevated CRP (Fig. 1) consists of 5 noncovalently bound subunits of CRP levels in these conditions are related to insulin resis- 206 amino acids, each arranged symmetrically around a tance (13), and weight loss can reduce CRP levels (14). central pore. The molecule has a ligand recognition face that Newer evidence indicates an additional and substantial ϩ contains a Ca2 -dependent binding site, as well as an genetic component (10,15,16). The realization that CRP effector molecule binding face that is able to initiate fluid- genetic polymorphisms do exist and that certain of these phase pathways of host defense (by activating the comple- directly and predictably influence steady-state blood CRP ment system) and cell-mediated pathways (by activating level could be of substantial clinical importance, because complement and by binding to the Fc receptors of immu- genetic predisposition to high baseline CRP might account noglobulin G) (3)(Fig. 1). The rise in blood CRP after for a significant proportion of people with a higher than tissue insult or injury is rapid and robust, with levels average risk of heart disease (17). Herein we review the increasing by as much as 1,000-fold above baseline within available evidence showing that a variety of CRP gene 24 h. This behavior makes blood CRP an ideal clinical polymorphisms are statistically associated with blood CRP marker of a patient’s general health status, and it has thus level, that certain of these are biologically functional in that been used for decades (4). The recent introduction of they directly alter CRP blood levels, and that some CRP variants are linked to a risk of heart and blood-vessel disease. From the Department of Medicine, University of Alabama at Birmingham, Birming- ham, Alabama. 1Because of the highly skewed distribution of human CRP blood levels, the Manuscript received March 14, 2007, revised manuscript received May 7, 2007, population statistic most often reported in the epidemiologic literature is the median. accepted June 5, 2007. Mean CRP levels and transformed CRP levels are also reported. 1116 Hage and Szalai JACC Vol. 50, No. 12, 2007 CRP Gene Polymorphisms September 18, 2007:1115–22 Abbreviations Localization and and Acronyms Structure of the CRP Gene coronary artery On the basis of the amino acid ؍ CABG bypass graft sequence of the protein, White- C-reactive protein head et al. (18) synthesized ؍ CRP interleukin CRP-specific oligonucleotides ؍ IL systemic lupus and used these to screen a human ؍ SLE erythematosus liver c-deoxyribonucleic acid li- single nucleotide brary. The clone pCRP1 was ؍ SNP polymorphism thus isolated, sequenced, and used as a probe to localize the human CRP gene to chromosome 1. This launched the Figure 2 The CRP Gene Is Located on Chromosome 1 era of CRP genetics. C-reactive protein was subsequently mapped to the proximal long arm of chromosome 1 in the The gene per se is comprised of 2 exons (red boxes) separated by a single intron encoding a dinucleotide (gt) repeat. Exon 1 encodes an 18 amino acid 1q23.2 region (19,20)(Fig. 2). The CRP gene sequence leader peptide (green box) and the first 2 amino acids of the mature protein. was simultaneously determined in 1985 by 2 different The cross-hatched box demarcates the position of a reported alternative tran- groups (21,22), both reporting that it is composed of 1 script. CRP ϭ C-reactive protein; UTR ϭ untranslated regions (yellow boxes). intron separating 2 exons (Fig. 2). The first exon encodes a signal peptide and the first 2 amino acids of the mature protein. This is followed by a 278-nucleotide-long intron cleic acid production. Several reviews of that subject are that includes a GT repeat sequence. The second exon available (3,24,25). Fundamentally, regulation of CRP encodes the remaining 204 amino acids, followed by a expression occurs mostly at the transcriptional level, with stop codon. Goldman et al. (23) were the first to show interleukin (IL)-6 being the major inducer and IL-1 that the GT stretch in the intron is polymorphic in acting synergistically to enhance the effect (26). Accord- length. ingly, certain polymorphisms of the IL-1 (27,28) and Once the CRP gene was identified and its sequence IL-6 (29) genes associate with differences in blood CRP reported, several groups investigated its proximal pro- level. Both IL-1 and IL-6 polymorphisms have been linked moter region and how this regulates messenger ribonu- to myocardial infarctions and stroke (30,31) and to mortal- ity after acute coronary syndromes (32). Studies using mice expressing human CRP from the human CRP promoter have shown that although IL-6 is necessary for the induc- tion of the CRP gene, it is not by itself sufficient (33,34). In humans, CRP blood levels are generally higher in females than in males (35), whereas in mice, expression of the human CRP transgene exhibits the opposite pattern (33). It is important to note that in vitro studies of regulation of CRP gene expression have focused solely on primary hepa- tocytes, hepatocyte cell lines, or a variety of transfected cell lines (3,24,25). With the recent realization that CRP can be locally produced by the vasculature (7,36), however, this area needs to be reinvestigated to determine whether the same regulatory mechanisms are operating in cells of non- hepatic origin. In this review we will not discuss further IL-6, IL-1, gender hormones, or any other of the multitude of trans- acting factors and their genes that might themselves be polymorphic and might participate in CRP gene regulation. Rather, we will focus on the CRP gene per se and systematically discuss its many polymorphic cis-acting ele- Figure 1 Blood CRP Circulates as a Pentamer ments. We will in turn examine evidence that the CRP gene One face of the pentamer supports multipoint attachment to ligands and coding and promoter region is polymorphic, that this ligand-decorated surfaces; the other face binds C1q and Fc␥R. Only some of sequence variation affects how much blood CRP is made, the many C-reactive protein (CRP) ligands are listed. HDL ϭ high-density lipoprotein; oxLDL ϭ oxidized low-density lipoprotein. and that this in turn affects CRP’s association to cardiovas- cular disease. JACC Vol. 50, No. 12, 2007 Hage and Szalai 1117 September 18, 2007:1115–22 CRP Gene Polymorphisms SNP in the CRP Gene Table 1 SNP in the CRP Gene Nucleotide Associated SNP Position Commonly Relative Allele With Higher Gene Region Reported in the Literature NCBI refSNP ID Frequency* CRP Blood Level Association Studies† Promoter Ϫ757 rs3093059 TϾCT (47,58,60,72) Ϫ717 rs2794521 TϾCT(46,49,52,57,58,66,67) Ϫ409 rs3093062 GϾAG (51) Ϫ390 rs3091244 CϾTϾAT(16,51,52,55,67,68,70,71) Intron ϩ29 rs1417938 AϾTA(49,50,60,67,72) Exon 2 ϩ1059 rs1800947 GϾCG(50,52,60–63,65,67,71,72) 3= untranslated ϩ219 rs3093066 CϾAC (49) ϩ1444 rs1130864 CϾTT(46–48,52,67,69,71) 3= flanking ϩ1846 rs1205 CϾTC (65–67,69,72) ϩ2911 rs3093068 CϾGG (69) *Alleles are reported in order from most frequent to least frequent as listed on the National Center for Biotechnology Information (NCBI) website.