Influence of coronary artery disease and subclinical atherosclerosis related polymorphisms on the risk of atherosclerosis in rheumatoid arthritis

A genetic component influences the development of atherosclerosis in the general population and also in rheumatoid arthritis (RA). However, genetic polymorphisms associated with atherosclerosis in the general population are not always involved in the development of cardiovascular disease (CVD) in RA. Accordingly, a study in North-American RA patients did not show the association reported in the general population of coronary artery disease with a series of relevant polymorphisms (TCF21, LPA, HHIPL1, RASD1-PEMT, MRPS6, CYP17A1-CNNM2-NT5C2, SMG6-SRR, PHACTR1, WDR12 and COL4A1-COL4A2). In the present study, we assessed the potential association of these polymorphisms with CVD in Southern European RA patients. We also assessed if polymorphisms implicated in the increased risk of subclinical atherosclerosis in non-rheumatic Caucasians (ZHX2, PINX1, SLC17A4, LRIG1 and LDLR) may influence the risk for CVD in RA. 2,609 Spanish patients were genotyped by TaqMan assays. Subclinical atherosclerosis was determined in 1,258 of them by carotid ultrasonography (assessment of carotid intima media thickness and presence/absence of carotid plaques). No statistically significant differences were found when each polymorphism was assessed according to the presence/absence of cardiovascular events and subclinical atherosclerosis, after adjustment for potential confounder factors. Our results do not show an association between these 15 polymorphisms and atherosclerosis in RA.

A genetic component influences the development of atherosclerosis in the general population and also in patients with rheumatoid arthritis (RA) 1,2 . Several pieces of evidence support the hypothesis that both pathologies share many similarities and exhibit analogous pathophysiological mechanisms 3 . However, genetic polymorphisms associated with atherosclerosis in the general population are not always involved in the development of cardiovascular disease (CVD) in RA 2 . In this respect, a recent study performed in patients with RA from North-America did not disclose that a series of gene polymorphisms related to coronary artery disease in the general population 4 were involved in the development of atherosclerotic disease in RA 5  In the present study, we assessed the potential association of these polymorphisms with CVD in Southern European individuals with RA. Moreover, we also assessed if other gene polymorphisms implicated in the increased risk of subclinical atherosclerosis in non-rheumatic Caucasian individuals 6 may influence the risk for CVD in RA. These polymorphisms associated with subclinical atherosclerosis in the general population were the following: Epidemiological and clinical characteristics of patients enrolled in the study are shown in Table 1. Definitions of cardiovascular (CV) events and for traditional CV risk factors were established as previously described 8 . Genotyping. DNA from patients was obtained from peripheral blood using standard methods.
For the selection of the gene polymorphisms studied in the present report, we carried out a search of genes associated with CVD or subclinical atherosclerosis in the general population. Based on this analy- and LDLR rs6511720 were assessed in patients with RA. For this purpose, a TaqMan predesigned single-nucleotide polymorphism genotyping assays in a 7900 HT Real-Time polymerase chain reaction (PCR) system, according to the conditions recommended by the manufacturer (Applied Biosystems, Foster City, CA, USA), was performed.
Negative controls and duplicate samples were included to check the accuracy of genotyping.

Carotid ultrasonography (US) examination. cIMT values and presence/absence of carotid plaques
were evaluated in 1,258 cases. Patients from Santander, Granada, Tenerife, Valencia, Ciudad Real and Madrid were assessed using a commercially available scanner, Mylab 70, Esaote (Genoa, Italy) 9 . Patients from Lugo were assessed using high-resolution B-mode ultrasound, Hewlett Packard SONOS 5500 8 . cIMT was measured at the far wall of the right and left common carotid arteries, 10 mm from the carotid bifurcation, over the proximal 15 mm-long segment. cIMT value was determined as the average of three measurements in each common carotid artery. The final cIMT was the largest average cIMT (left or right) 9,10 . The plaque criteria in the accessible extracranial carotid tree were focal protrusion in the lumen at least cIMT > 1.5 mm, protrusion at least 50% greater than the surrounding cIMT, or arterial lumen encroaching > 0.5 mm 10,11 . Agreement between these two US methods was previously reported 12 . Experts with a high reproducibility, excellent inter-observer reliability and close collaboration in the assessment of subclinical atherosclerosis in RA performed the studies. The relationship between allelic frequencies and the presence/absence of CV events was tested using logistic regression adjusting for sex, age at RA diagnosis, follow-up time and traditional CV risk factors as potential confounder factors. Results were expressed as odds ratios (OR) with 95% confidence intervals (CI).
Association between allelic frequencies and cIMT values was tested using unpaired t test. Results were adjusted for sex, age at the time of US study, follow-up time and traditional CV risk factors as potential confounder factors using analysis of covariance (ANCOVA).
Differences in the allelic frequencies according to the presence/absence of carotid plaques were calculated by χ 2 or Fisher tests. Strength of associations was estimated using OR and 95% CI. Results were adjusted for sex, age at the time of US study, follow-up time and traditional CV risk factors as potential confounder factors by logistic regression.

Discussion
Results from our study show that a large number of gene polymorphisms associated with CV disease or subclinical atherosclerosis in the general population are not implicated in the increased risk or CV disease found in Caucasian individuals with RA, which is the prototype of chronic inflammatory rheumatic disease. Since a recent study that included a smaller series of North-American patients with RA found no association with CV disease of most of the gene polymorphisms assessed in our study, our results are also confirmatory and they further enhance the fact that the genetic predisposition for CV disease in RA may not be the same as that of the general population.
Similarities between atherosclerosis and chronic inflammatory diseases have been reported 3 . This is especially true for RA 3,13 . In this respect, recruitment of blood mononuclear cells, up-regulation of adhesion molecules and production of pro-inflammatory cytokines and matrix-degrading enzymes were described as potential common mechanisms involved in the initiation and perpetuation of both pathologies 3 . However, as pointed out before, a recent study performed in North-American patients with RA failed to demonstrate the implication of a series of pro-atherogenic genes, which were associated with CV disease in the general population, in the development of CVD in RA 5 .
According to our data, and in contrast to the general population 4 Table 3. Association between ZHX2, LRIG1, PINX1, SLC17A4 and LDLR polymorphisms and CV events or subclinical atherosclerosis in patients with RA. CV: cardiovascular; RA: rheumatoid arthritis; cIMT: carotid intima-media thickness; OR: odds ratio; CI: confidence interval. * Adjusted for sex, age at RA diagnosis, follow-up time and traditional CV risk factors using logistic regression. † Adjusted for sex, age at the time of ultrasonography study, follow-up time and traditional CV risk factors using analysis of covariance (ANCOVA). ‡ Adjusted for sex, age at the time of ultrasonography study, follow-up time and traditional CV risk factors by logistic regression.
In our study, we also aimed to determine if other gene polymorphisms implicated in the increased risk of subclinical atherosclerosis in non-rheumatic Caucasians individuals 6 could also influence the risk for CVD in RA. Unfortunately, unlike non-rheumatic Caucasians 6 , we did not disclose a relationship between ZHX2, PINX1, SLC17A4 2, LRIG1 and LDLR variants and CVD in patients with RA.
Taken together, our results suggest that RA itself should be considered an independent CV risk factor. Noteworthy, a former report from our group already highlighted genetic differences between the atherosclerosis disease in the general population and that associated to RA 14 . Consequently, our findings further support that the genetic component implicated in the development of atherosclerosis in RA may be different from that associated to "idiopathic" atherosclerosis. In this line, factors that are intrinsic to RA may independently contribute to the development of CVD.