Atheromatous plaques start to progress from childhood and may eventually become prone to plaque rupture in adulthood leading to clinical events, such as acute myocardial infarction, unstable angina or stroke. In this large prospective cohort study, both a Mendelian randomization approach, in which confounding is controlled for by using genetic variants as instruments for the unconfounded association, and standard multivariable regression analyses (adjusting for a range of potential confounding factors) were consistent in showing no independent association of CRP with CIMT. These findings could be explained if CRP does not itself contribute to the development of atherosclerosis but rather marks pro-atherogenic exposures, the presence of atheroma, or a combination of the two.
We used haplotypes in the CRP
gene that were constructed on the basis of tag SNPs rs1205, rs1130864 and rs3093077 that capture comprehensively the common variability at the CRP
locus in subjects of European descent 
. A recent large-scale meta-analysis of genetic association studies of 8 CRP
polymorphisms and CRP concentration used a novel Bayesian approach that allows integration of informative data from a wide range of studies, irrespective of the specific CRP
polymorphism typed 
. All the three SNPs we studied were found to mark haplotypes likely to harbour functional variants in the vicinity of the CRP
gene that could regulate its level. In the present study, these SNPs were consistently associated with serum CRP levels across two time points 12 years apart suggesting that the haplotypes defined groups with long-term differences in circulating CRP. However, there was no strong statistical evidence that these haplotypes influencing serum CRP levels were related to CIMT after taking into account the magnitude of their association with CRP. This null finding is assumed to represent a non-confounded and unbiased estimate of the association between CRP and CIMT because the existence of early stages of atherosclerosis cannot alter inherited haplotypes 
, and the potential confounders of the CRP-atherosclerosis association (e.g., obesity, smoking, physical inactivity or socioeconomic adversity) were distributed evenly among the different CRP haplotypes.
Our findings are consistent with the null findings in two smaller studies on CRP
genotypes and CIMT, one related to young adults aged 24 to 39 years 
and the other to an older cohort than ours 
. In combination, these and other genetic studies related to less direct correlates of atherosclerosis, such as blood pressure 
and metabolic syndrome 
provide evidence against the status of CRP as a causal factor for atherosclerosis. Lange et al. 
suggest that CRP may affect plaque rupture rather than atherosclerosis in a study reporting an association of CRP
genotype with incident CHD in a subgroup but no association with CIMT in the same subgroup or in the study population as a whole. However, the association between CRP
genotype and CHD has not been confirmed by other studies or meta-analyses 
. A companion for this study is the largest meta-analysis on this issue to date, based on Whitehall II and four other general population cohorts. That study showed no association between a single CRP
polymorphism and incident or prevalent CHD in a total of 18,637 participants (4,610 cases) 
. However, a very large sample size (around 20,000 cases and controls), with comprehensive tag SNP typing, such as that being assembled by the CRP-CHD genetics collaboration (CCGC) 
, will be necessary to confirm or refute a causal association of CRP with risk of CHD events.
Several issues may compromise the value of the Mendelian randomisation approach in determining causality 
. First, such an approach requires the existence of genetic variants that have been shown to be robustly (replicated in several independent studies) associated with the non-genetic modifiable exposure of interest. For the haplotypes that we have used here, such a robust association has been established in multiple independent studies 
, and was confirmed in our dataset. Furthermore, the association of the haplotype (instrumental variable) was strong enough for the instrumental variables analysis to be consistent as the F-statistic was above the value of 10 suggested as a threshold to distinguish weak vs. strong instruments 
Second, population stratification, resulting from factors such as ancestral patterns of geographical migration and differences in mating practices and reproductive behaviors between populations, may confound genotype-phenotype associations and is often speculated to be the reason for non-replication of genetic associations 
. There is some evidence of such confounding in relation to ethnic groups, i.e., relationships between genotype and phenotype that were found in multiethnic populations disappeared when analysed separately in each ethnic group 
. Population stratification may not only potentially lead to such false positive genotype-phenotype associations but can also, in principle, mask associations. To increase protection against bias from population stratification we restricted our analyses on white Europeans only. We also confirmed that there was no stratification in CRP
haplotypes between socioeconomic groups. Furthermore, the null finding of CRP
haplotype and CIMT is replicable as consistent findings have been obtained from this UK study and studies in a US and Finnish population 
. For these reasons, it seems unlikely that population stratification would have masked a causal association between CRP and CIMT.
Third, the Mendelian randomisation approach may be compromised if genetic variants used as instruments have multiple effects on phenotype (pleiotropy) or if the variants are in linkage disequilibrium with another genetic variant, that influences the pathway of interest in the opposite direction. We think pleiotropy is unlikely for the variants that we used to generate the CRP
haplotypes as they are in very close linkage disequilibrium with variation within a putative transcription factor binding site located 5′ of the CRP
gene that has been associated with circulating concentrations of CRP and thought to be functional 
. The variants also lie in a block of allelic association that does not contain any other gene with a role in CRP regulation 
Fourth, developmental compensation (or canalization) in early life whereby genetically-determined alterations in CRP might be buffered by compensatory changes in other systems may compromise the validity of the Mendelian randomisation approach 
. However, most recognised examples of developmental compensation relate to dramatic genetic or environmental insults 
and it is unclear whether the generally smaller phenotypic differences induced by common functional polymorphisms, as used in our study, will be sufficient to induce compensatory responses.
Fifth, the most important limitation is that the instrumental variables analysis provided wide confidence intervals for the effects of CRP on CIMT suggesting that larger samples are needed to obtain more precise estimation. Moreover, CIMT, although a valid non-invasive index of carotid atherosclerosis 
, may not comprehensively capture the general atherosclerotic process.
Nevertheless, despite these limitations, standard multivariable regression analyses of CRP levels and CIMT produced converging support for the conclusions from Mendelian randomization analyses. The association between serum CRP and CIMT attenuated towards the null in adjustments for obesity and other risk factors and this is consistent with several previous studies 
. It has been argued that systemic inflammation (of which CRP is a marker) might cause increases in blood pressure, BMI and changes in lipid profiles that might mediate an increase in CIMT and CHD risk 
. If so, adjustment for these variables in a multivariable model might actually be controlling for factors in the causal pathway. However, adjustment for BMI (which had the most potent attenuating effect) is unlikely to represent an overadjustment, since weight gain is associated with an increase in CRP, and weight loss with a CRP reduction 
. Furthermore, CRP
genotypes that are associated with higher CRP were not associated with BMI, nor with a range of other established or novel risk factors for CHD 
In conclusion, the consistency of evidence from both the Mendelian randomisation approach and the multivariable regression analysis approach (each of which has distinct, but differing potential limitations) implies that the association of CRP with CIMT may be better explained by CRP marking the presence of atheroma, or other risk factors rather than having a direct causal role itself, as has been suggested 
. However, much larger analyses using the genetic approach we and others have described, as well as intervention studies involving a new, specific CRP-inhibitor 
are needed to more definitively assess the potential causal role for CRP in atherosclerosis and CHD.