Association Study of MIA3 rs17465637 Polymorphism with Cardiovascular Disease in Rheumatoid Arthritis Patients

Abstract

Rheumatoid arthritis (RA) is a complex polygenic inflammatory disease associated with accelerated atherosclerosis. Melanoma inhibitor protein 3 (MIA3) is required for the export of collagen VlI (COL7A1) from the endoplasmic reticulum and it appears to be a tumor suppressor of malignant melanoma. Genome-wide association studies have described an association between MIA3 rs17465637 A/C polymorphisms and coronary artery disease and myocardial infarction. Because of that, we assessed the MIA3 rs17465637 polymorphism in 1505 RA Spanish patients stratified according to the presence/absence of cardiovascular (CV) disease. Also, a subgroup of patients without CV events was assessed for the presence of subclinical atherosclerosis using carotid ultrasound to establish carotid intima-media wall thickness and carotid plaques and brachial ultrasonography to determine the presence of endothelial dysfunction by flow-mediated endothelium-dependent and independent vasodilatation. MIA3 rs17465637 allele A showed a trend for association with the presence of carotid plaques (odds ratio 1.56, 95% confidence interval [0.96–2.51]; p=0.07). However, apart from an association of the MIA3 rs17465637 A allele with the risk of CV events in RA patients with dyslipidemia (p=0.018), no other significant associations were found between the presence of MIA3 rs17465637 A allele and the risk of suffering CV events or other surrogate markers of atherosclerosis. In conclusion, our results suggest a potential association of the MIA3 rs17465637 with CV disease in dyslipidemic patients with RA. However, additional studies are required to better establish the role of the MIA3 gene in mechanisms leading to the accelerated atherogenesis observed in RA.

Introduction

R heumatoid arthritis (RA) is a complex autoimmune disease associated with progressive disability, systemic complications, and early death. Mortality is higher among RA patients than among healthy people, and cardiovascular (CV) complications remain a major challenge (McInnes and Schett, 2011). Atherosclerosis is the main cause of increased CV morbidity and mortality in RA patients (González-Gay et al., 2005). Besides traditional CV risk factors, chronic systemic inflammation plays a pivotal role in the development of accelerated atherosclerosis observed in RA (Nurmohamed, 2009).

Improvement of our understanding of the molecular basis of accelerated atherosclerosis occurring in RA is of main importance to establish predictive models of CV disease in RA.

Genetic factors may underlie susceptibility to CV disease in RA. In this regard, recent studies have disclosed the importance of genetic factors and the influence of several gene polymorphisms in the susceptibility to (Gregersen, 2010) and/or in the risk of accelerated atherosclerosis of patients with RA (Palomino-Morales et al., 2010; Rodriguez-Rodriguez et al., 2011a, 2011b; Teruel et al., 2011).

Recent genome-wide association studies (GWAS) have identified a number of genetic loci that confer risk of or protection from coronary artery disease and myocardial infarction, including rs17465637 on chromosome 1q41, rs2943634 on 2q36.3, rs6922269 on 6q25.1, rs599839 on 1p13.3, rs9818870 on 3q22.3, and rs501120 on 10q11.21 (Samani et al., 2007; Erdmann et al., 2009; Kathiresan et al., 2009; Samani et al., 2009), among others.

The rs17465637 single-nucleotide polymorphism (SNP) is located on chromosome 1q41, in an intronic region of the melanoma inhibitory activity family, member 3 (MIA3) gene (also known as ARNT or TANGO). MIA3 gene, coding for a 14 kDa protein of so far unknown function, was originally identified as a new member of MIA gene family. MIA3 is widely expressed in vivo in contrast to the highly restricted expression pattern for the other family members (Bosserhoff et al., 2004). MIA3 gene encodes the melanoma inhibitor protein 3 (MIA3) required for the export of collagen VlI (COL7A1) from the endoplasmic reticulum (Saito et al., 2009). This gene seems to be a suppressor of malignant melanoma and has also been implicated in the susceptibility to colon and hepatocellular carcinomas (Arndt and Bosserhoff, 2007). MIA3 may be involved in facilitating the migration of monocyte cells through human microvascular endothelial cells (Arndt et al., 2007), which may increase the risk of plaque formation, as monocytes differentiate into macrophages and macrophage foam cells and form the fatty streak, an early indicator of atherosclerosis in the arterial intima (Libby et al., 2009). Atherosclerotic plaques reflect the presence of large irregular arterial wall deposits, and constitute established measures of subclinical atherosclerotic disease (Bis et al., 2011), being a predictor of CV events (Nambi et al., 2010; Mathiesen et al., 2011).

Several studies indicate the presence of an additive genetic model for susceptibility to CV disease (Lluís-Ganella et al., 2010). In this model an influence of the MIA3 rs17465637 gene variant with coronary artery disease has been proposed. Although there is no information showing that MIA3 locus may confer higher risk of CV disease than other gene polymorphisms associated with coronary artery disease and no association of the MIA3 rs17465637 gene variant with autoimmune inflammatory diseases has been reported, the similarities between atherosclerosis and RA, a disease associated with a process of accelerated atherogenesis, make plausible the search for a potential association of MIA3 rsl7465637 gene variant with CV disease in RA. Therefore, we aimed to determine the potential role of rs17465637 located in the MIA3 gene in a large cohort of RA Spanish patients, stratified by the presence or absence of CV events.

Patients and Methods

Patients and study protocol

A cohort of 1505 RA Spanish patients was included in the present study. Blood samples were obtained from patients recruited from Hospital Xeral-Calde (Lugo), Hospital Universitario Marqués de Valdecilla (Santander), Hospital Universitario Bellvitge (Barcelona), and Hospital Universitario La Paz, Hospital de La Princesa, and Hospital Clínico San Carlos (Madrid). The study was approved by the ethics committee of the corresponding hospitals and a subject's written consent was obtained in all the cases. All the patients fulfilled the 1987 American College of Rheumatology criteria for the classification of RA (Arnett et al., 1988).

Information on the main demographical data, clinical characteristics of the patients enrolled in the study, CV risk factors, and CV events of patients is shown in Table 1. Two hundred and seventy-four (17.74%) of these 1505 RA patients had experienced clinically evident CV events (ischemic heart disease, heart failure, cerebrovascular accident, or peripheral arteriopathy). Classic CV risk factors were established as previously described (González-Gay et al., 2007; González-Juanatey et al., 2009). With respect to this, patients were considered to have dyslipidemia if they had hypercholesterolemia and/or hypertriglyceridemia (defined as diagnosis of hypercholesterolemia or hypertriglyceridemia by the patients' family physicians prior to the diagnosis of RA, or total cholesterol and/or triglyceride levels in fasting plasma greater than 240 and 160 mg/dL, respectively, at the time of disease diagnosis or over the extended follow-up) (González-Gay et al., 2007).

Table 1.

Demographic Characteristics of the Rheumatoid Arthritis Patients Included in the Study

Variables	n=1505
Women	1118 (74.29)
Age of patients at the time of disease diagnosis (years), median (IQR)	54 (43–64)
Time of follow-up (years), median (IQR)	10.8 (5.5–17)
Anti-CCP positive (n=1217)	712 (58.50)
Rheumatoid factor positive (n=1472)	1027 (69.77)
Shared epitope presence (n=1153)	726 (62.97)
CV events	267 (17.74)
Ischemic heart disease	144 (9.60)
Cerebrovascular accidents	74 (4.95)
Heart failure	79 (5.29)
Peripheral arteriopathy	29 (1.95)
Hypertension (n=1498)	590 (39.39)
Diabetes mellitus (n=1492)	190 (12.73)
Dyslipidemia (n=1478)	562 (38.02)
Obesity (n=1410)	276 (19.57)
Smoking habit (n=1431)	358 (25.02)

Except where indicated otherwise, values are n (%).

IQR, interquartile range; Anti-CCP, anti-cyclic citrullinated peptide antibodies; CV, cardiovascular.

To determine the potential association between the MIA3 rs17465637 polymorphism and subclinical atherosclerosis, between March 2007 and September 2010 a randomly selected subgroup of patients from the Lugo and Santander cohorts with no previous history of CV events was selected. Presence of endothelial dysfunction was assessed by a brachial artery reactivity study in 135 Lugo patients. [Flow-mediated endothelium-dependent (FMD) (post-ischemia) and endothelium-independent (NTG) (post-nitroglycerin) vasodilatation were measured by brachial ultrasonography as previously reported (González-Juanatey et al., 2003; González-Gay et al., 2008).] Also, carotid ultrasonography studies were performed in 270 patients to determine the carotid artery intima-media (IMT) wall thickness and the presence of carotid plaques as previously reported (González-Juanatey et al., 2006; González-Gay et al., 2008).

Genotyping

DNA from patients was obtained from peripheral blood using standard methods. DNA from the patients was tested for rs17465637 polymorphism located in the MIA3 gene.

The rs17465637 gene variant was genotyped by TaqMan SNP genotyping assays in a 7900 HT real-time polymerase chain reaction system, according to the recommended conditions by the manufacturer (Applied Biosystem, Foster City, CA). Negative controls and duplicate samples were included to check the accuracy of genotyping.

Statistical analysis

All genotype data were checked for deviation from Hardy–Weinberg equilibrium (HWE) using http://ihg.gsf.de/cgi-bin/hw/hwa1.pl. Both allelic and genotypic frequencies were calculated and compared by χ² or Fisher's test using the StatsDirect software V2.6.6 (StatsDirect www.statsdirect.com, England: StatsDirect 2008). Strength of associations between CV events and genotypes or alleles was estimated using odds ratios (ORs) and 95% confidence intervals (CIs), via multiple logistic regression; estimates were further adjusted for sex, age at RA diagnosis, time of follow-up, and classic CV risk factors (hypertension, diabetes mellitus, dyslipidemia, obesity, and smoking habit).

The association between genotypes of the MIA3 rs17465637 polymorphism and surrogate markers of subclinical atherosclerosis—carotid IMT, FMD-endothelium–dependent, or NTG-endothelium-independent vasodilatation—was tested using unpaired t-test to compare between two groups, and one-way analysis of variance to compare among more than two groups. Moreover, we also tested the association between these parameters and alleles using analysis of covariance (ANCOVA) adjusting for sex, age, and duration of the disease at the time of the ultrasonographic study and traditional CV risk factors. The association between the MIA3 rs17465637 polymorphism and the presence of carotid plaques was assessed by logistic regression model; estimates were further adjusted for sex, age at RA diagnosis, time of follow-up, and classic CV risk factors (hypertension, diabetes mellitus, dyslipidemia, obesity, and smoking habit) and anti-cyclic citrullinated peptide (CCP) antibody status.

Statistical significance was defined as p<0.05. All analyses were performed with STATA statistical software 9.1 (Stata Corp., College Station, TX).

Statistical power for the study was calculated using “CaTS—Power Calculator for Two Stage Association Studies” (www.sph.umich.edu/csg/abecasis/CaTS).

Results

Frequencies of the MIA3 rs17465637 variant and CV events in RA patients

No deviation from HWE was detected for MIA3 rs17465637 polymorphism, and the genotyping success rate in our study was 93.75%.

The study had 70% power to detect modest OR ≥1.3 (Wang et al., 2011), at the stated significance level (α=0.05), and a prevalence of RA disease in Spanish population was 0.5% (Carmona et al., 2002).

As shown in Table 2, no significant differences in the genotype and allele frequencies were found when patients who had experienced CV disease were compared with those who had not suffered any kind of CV events.

Table 2.

Differences in Genotype and Allele Frequencies of MIA3 rs17465637 Variant Between Rheumatoid Arthritis Patients With CV Events or Without CV Events

rs17465637	With CV events	Without CV events	p	OR [95% CI]
Genotype
CC	127 (49.80)	599 (51.82)	–	Ref.
CA	104 (40.78)	472 (40.83)	0.79	1.04 [0.77–1.40]
AA	24 (9.41)	85 (7.35)	0.25	1.37 [0.79–2.23]
Alleles
C	358 (70.19)	1670 (72.23)
A	152 (29.80)	642 (27.77)	0.35	1.10 [0.89–1.37]

Values in CV event columns express number of patients (%).

MIA3, melanoma inhibitor protein 3; OR [95% CI]: odds ratio with 95% confidence interval.

In a further step, we constructed a logistic regression model to explain the presence of CV disease according to the MIA3 rs17465637 allele distribution, which was adjusted for sex, age at the time of RA diagnosis, follow-up time from the disease diagnosis, and traditional CV risk factors as potential confounders. However, no association was found in the crude or in adjusted model (OR: 1.10, 95% CI [0.89–1.36], p=0.36 and OR: 1.10, 95% CI [0.86–1.43], p=0.44, respectively). Also, no association was detected for the risk of developing each one of the CV disease complications described before—heart failure, ischemic heart disease, cerebrovascular accidents, or peripheral vascular events (data not shown).

Also, when data were stratified according to shared epitope status or anti-CCP antibody positivity, the adjusted logistic regression model did not show any change in patient's CV risk (results not shown). However, when we stratified RA patients according to the presence of dyslipidemia—since lipid alterations appear to predate the diagnosis of RA and may influence the increased risk of accelerated atherosclerosis observed in patients with RA, and a moderate association of MIA3 rs17465637 variant with HDL-cholesterol was recently reported (Xie et al., 2011)—we observed that A allele conferred a higher risk of CV events in the subgroup of patients with dyslipidemia (OR=1.66, 95% CI [1.09–2.52], p=0.018).

MIA3 rs17465637 gene polymorphism and subclinical atherosclerosis markers

The results of the comparison between the different genotype frequencies of MIA3 rs17465637 polymorphism according to well-established surrogate markers of subclinical atherosclerosis, such as FMD%, NTG%, and carotid IMT, were as follows:

Carotid IMT—mean (SD): CC (n=132) 0.73 mm (0.17), CA (n=120) 0.73 mm (0.18), AA (n=18) 0.75 (0.19); p=0.95.

FMD%—mean (SD): CC (n=67) 4.95 (4.05), CA (n=61) 5.09 (4.92), AA (n=7) 7.81 (4.65); p=0.28.

NTG%—mean (SD): CC (n=67) 15.80 (6.80), CA (n=61) 15.09 (7.16), AA (n=7) 14.72 (11.07); p=0.83.

No significant association was observed when allele frequencies of MIA3 rs17465637 polymorphism were analyzed according to the carotid IMT or FMD% and NTG%. It was also the case when association between MIA3 rs17465637 polymorphism and results from FMD%, NTG%, and carotid IMT was adjusted for potential confounders (sex, age at RA diagnosis, follow-up time from the disease diagnosis, and classic CV risk factors—hypertension, diabetes mellitus, dyslipidemia, obesity, and smoking habit) in the ANCOVA model (MIA3 rs17465637 carotid IMT: p=0.22; FMD%: p=0.18; NTG%: p=0.85). Nevertheless, an adjusted logistic regression model disclosed that presence of the minor allele A yielded a trend of association with an increased risk of carotid plaques in this series of patients (Table 3).

Table 3.

Logistic Regression Model to Explain the Presence of Carotid Plaques in Patients with Rheumatoid Arthritis According to MIA3 rs17465637 Allele Distribution

	p	OR [95% CI]	p^a	OR [95% CI] ^a
rs17465637 A vs. C	0.19	1.28 [0.88–1.88]	0.07	1.56 [0.96–2.51]

Analyses adjusted for sex, age at rheumatoid arthritis diagnosis, follow-up time from the disease diagnosis, anti-CCP status, and classic CV risk factors—hypertension, diabetes mellitus, dyslipidemia, obesity, and smoking habit.

Discussion

Lately, GWAS have become a powerful approach to identify rapidly genetic variants that influence susceptibility to common diseases. In this regard, GWAS have described several novel putative loci that seem to increase risk or protect against coronary artery disease and myocardial infarction (Helgadottir et al., 2007; Morgan et al., 2007; Samani et al., 2007).

SNP rs17465637 in MIA3 gene was identified as a potential genetic locus for coronary artery disease after combining the Wellcome Trust Case Control Consortium (2007) and the German Myocardial Infarction family studies (more than 80% probability of a true association) (Samani et al., 2007). A follow-up study by the Myocardial Infarction Genetics Consortium also supported the potential association between rs17465637 and myocardial infarction (Kathiresan et al., 2009). However, a study by the Coronary Artery Disease Consortium (Samani et al., 2009) and another that included African-American and North-American Caucasian individuals (Bressler et al., 2010) failed to confirm this association. Contradictory results were obtained from a replication study (Wang et al., 2011) and a meta-analysis (Conde et al., 2011). However, a recent study suggested a potential association between MIA3 haplotypes and the risk of myocardial infarction (Koch et al., 2011).

The MIA3 protein directly binds to the leukocyte-specific β2-integrin CD11c/CD18, which is involved in leukocyte adhesive interactions with vascular endothelium (Imhof and Aurrand-Lions, 2004; Arndt et al., 2007). Experimental evidence suggests that MIA3 protein reduces attachment and promotes migration of monocytes across the endothelium by modulating CD11c/CD18 activity (Arndt et al., 2007). Upon transmigration, monocytes differentiate into macrophages and macrophage foam cells and form the fatty streak, an early indicator of atherosclerosis in the arterial intima (Libby et al., 2009). Thus, a functional link appears to exist between the MIA3 gene and the formation of atherosclerotic plaques, which may become unstable and give rise to thrombotic complications, such as myocardial infarction.

Our study constitutes the first attempt to determine the potential role of the MIA3 rs17465637 gene polymorphism in the susceptibility to CV disease in patients with RA. Our results suggest a potential association of the MIA3 rs17465637 with CV disease in dyslipidemic patients with RA. However, although a moderate association of MIA3 rs17465637 variant with HDL-cholesterol has recently been described (Xie et al., 2011), the exact relationship between this SNP and dyslipidemia in patients with RA is still unknown. In this regard, there is a large body of literature that states that there is a “lipid paradox” in RA, where these patients actually have reasonable lipid profile. Therefore, the effect of this SNP in patients with RA may be different from that observed in healthy individuals without a state of chronic inflammation.

Our data also suggest that the MIA3 rs17465637 polymorphism may be associated with increased risk of developing atherosclerotic plaques, which reflect the presence of large irregular arterial wall deposits and constitute an established measure of subclinical atherosclerotic disease (Bis et al., 2011), and a marker of CV disease that predicts the risk of CV events (Johnsen et al., 2007; Nambi et al., 2010; Mathiesen et al., 2011). The underlying pathobiology of myocardial infarction is a buildup of atherosclerotic plaques on the walls of major vessels, with plaque rupture and subsequent thrombosis (Schoenhagen et al., 2002). Collagen is the main constituent of the protein of the atherosclerotic plaque and the collagen-rich fibrous cap covering the lipid-dense core of the atherosclerotic plaque is the major factor in its stability (Adiguzel et al., 2009). An imbalance between collagen synthesis and degradation by macrophage- and smooth muscle cell–associated proteinases may lead to cap thinning, degradation of the collagen, and plaque instability. In keeping with that, recent studies regarding the role of MIA3 protein in the collagen transport involving knock-out mice lead to the conclusion that a reduction in MIA3 expression may tilt the balance toward a thinner cap and unstable plaque (Wilson et al., 2011).

As in many other human genetic studies, potential limitations may exist in our study. In this regard, the phenotypes involved in the atherosclerosis disease, which is considered an inflammatory autoimmune disease (Sherer and Shoenfeld, 2006), are complex and involve multiple small-to-modest effects of multiple genes and environmental factors. Genetic effects of MIA3 were reported to be modest (Samani et al., 2007; Kathiresan et al., 2009; Schunkert et al., 2011) and, because of that, it is possible that the size of our cohort may not be large enough to detect association with CV disease, as prevalence of RA in Spanish population seems to be lower than in other Caucasian populations. Therefore, additional studies are required to better establish the role of the MIA3 gene in the complex mechanisms leading to the accelerated atherogenesis observed in RA.

In summary, our results suggest a potential association of the MIA3 rs17465637 with CV disease in dyslipidemic patients with RA. However, additional studies are required to confirm the influence of the MIA3 gene in the mechanisms leading to the accelerated atherogenesis observed in RA.

Footnotes

Acknowledgments

The authors are thankful to all the patients who participate in this study.

The authors want to thank especially Rodrigo Ochoa, Gema Robledo, Sonia García, and Sofía Vargas for their technical assistance.

This work was supported by grants from Fondo de Investigaciones Sanitarias PI06-0024 and PS09/00748 (Spain), and by the RETICS Program RD08/0075 (RIER) from Instituto de Salud Carlos III (ISCIII), within the VI PN de I+D+i 2008–2011 (FEDER).

M.G.B. is a beneficiary of a grant from Fundación Española de Reumatología (FER).

Disclosure Statement

The authors declare that no competing financial interests exist.

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