The pro-inflammatory cytokine LTA (lymphotoxin-α) has multiple functions in regulating the immune system and may contribute to inflammatory processes leading to CHD (coronary heart disease). The aim of the present study was to investigate whether the common C804A (resulting in a Thr26→Asp amino acid substitution) and A252G polymorphisms of the LTA gene and the C3279T polymorphism of the galectin-2 (LGALS2) gene, which affects LTA secretion, are associated with inflammatory parameters and cell adhesion molecules, and whether these polymorphisms are related to CHD in American women and men. We conducted a prospective nested case-control study within the Nurses' Health Study and Health Professionals Follow-Up Study. Among participants free of cardiovascular disease at baseline, 249 women and 266 men developed CHD during 8 and 6 years of follow-up respectively, and we matched controls 2:1 based on age and smoking. The LGALS2 gene variant was significantly associated with a decreased risk of CHD in women [odds ratio (95% confidence interval), 0.70 (0.50–0.97); P=0.03]. In addition, the LGALS2 polymorphism was directly associated with CRP (C-reactive protein) levels in cases from both studies (P<0.05). The LTA gene polymorphisms were directly associated with levels of sTNFRs (soluble tumour necrosis factor receptors) and VCAM-1 (vascular cell adhesion molecule-1) in both women and men with CHD (P<0.05). However, no overall effect was demonstrated between LTA gene polymorphisms and risk of CHD.
- coronary artery disease
- cellular adhesion molecule
- gene polymorphism
- lymphotoxin-α (LTA)
- myocardial infarction
Inflammation plays an important role in the development and progression of atherosclerosis and is likely to play a critical role in the pathogenesis of plaque rupture which precedes a myocardial infarction . The pro-inflammatory cytokine LTA [lymphotoxin-α; or TNF (tumour necrosis factor)-β] is found in atherosclerotic lesions  and may contribute to these processes. Furthermore, LTA may also induce adhesion molecules and cytokines from vascular endothelial and smooth muscle cells . A large-scale association study from the Japanese Osaka Acute Coronary Insufficiency Study group identified functional SNPs (single nucleotide polymorphisms) within the LTA gene that were associated with a risk of myocardial infarction [LTA C804A (resulting in a Thr26→Asp amino acid substitution) and LTA A252G] . Among these SNPs, the LTA C804A polymorphism induced an almost 2-fold higher expression of E-selectin and VCAM-1 (vascular cell adhesion molecule-1) in cultured human coronary artery smooth muscle cells, and the presence of the LTA A252G gene polymorphism was associated with a 1.5-fold greater transcriptional activity of LTA . The LTA and TNF (encoding TNF-α) genes are in significant linkage disequilibrium and are situated close to each other within the HLA (human leucocyte antigen) class III cluster on the short arm of chromosome 6.
Several other studies have examined the association between LTA gene polymorphisms and CHD (coronary heart disease), but these studies were based on identification of prevalent cases and the results were inconsistent [5–10]. In addition, genotype distribution in the original study by Ozaki et al.  deviated from Hardy–Weinberg equilibrium in the control group. A prospective longitudinal study is needed to investigate the association between LTA gene polymorphisms and CHD. Furthermore, the relationship between LTA gene polymorphisms and plasma levels of inflammatory markers and cell adhesion molecules are unknown.
Recently, the Japanese Osaka Acute Coronary Insufficiency Study group identified the galectin-2 protein as a regulator of LTA protein secretion and, therefore, also potentially important in modifying the degree of inflammation . Both LTA and galectin-2 are expressed in smooth muscle cells and macrophages in the intima of atherosclerotic lesions of the coronary artery. Furthermore, the functional SNP (C3279T) in the galectin-2 (LGALS2) gene may be inversely associated with a risk of myocardial infarction . No study has yet replicated these results in a prospective design.
We hypothesize that the common C804A and A252G polymorphisms of the LTA gene and the C3279T polymorphism of the LGALS2 gene are associated with circulating inflammatory markers and cell adhesion molecules [i.e. CRP (C-reactive protein), IL-6 (interleukin-6), sTNF-R1 (soluble TNF receptor 1), sTNF-R2, VCAM-1 and E-selectin] and we aim to investigate whether these polymorphisms are related to CHD in a large nested case-control study among American women and men.
We conducted a prospective nested case-control study within the NHS (Nurses' Health Study) and HPFS (Health Professionals Follow-Up Study). Among participants free of cardiovascular disease at baseline, 249 women and 266 men developed non-fatal myocardial infarction or fatal CHD during 8 and 6 years of follow-up respectively. As a secondary end point, we additionally identified 564 men who had CABG (coronary artery bypass graft surgery) or PTCA (percutaneous transluminal coronary angioplasty) during follow-up. Myocardial infarction was confirmed using World Health Organization criteria. Deaths were identified from State vital records and the National Death Index, or were reported by subjects' next of kin or the postal system. Fatal CHD was confirmed by hospital records or on autopsy, or if CHD was listed as the cause of death on the death certificate, if it was the underlying and most plausible cause, and if evidence of previous CHD was available. Confirmation of CABG/PTCA was based on self-reporting only. The study protocol was approved by the Institutional Review Board of the Brigham and Women's Hospital and the Harvard School of Public Health Human Subjects Committee Review Board; all participants provided informed consent.
Controls were selected 2:1 matched for age, smoking and month of blood draw. In addition, female controls were matched for fasting status. Biomarkers were measured for non-fatal myocardial infarction and fatal CHD cases and their controls only (the set of CABG/PTCA cases and controls did not have available plasma biomarkers).
CRP concentrations were determined using an immunoturbidimetric high-sensitivity assay (Denka Seiken) with day-to-day assay variability between 1 and 2%. Levels of IL-6, sTNF-R1, sTNF-R2, VCAM-1 and E-selectin were measured by ELISA (R&D Systems) , which have a day-to-day variability of 3.5–9.0%. HDL (high-density lipoprotein)-cholesterol and directly obtained LDL (low-density lipoprotein)-cholesterol were measured using standard methods with reagents from Roche Diagnostics and Genzyme.
Genotyping of polymorphisms
DNA was extracted from the buffy coat fraction of centrifuged blood using the QIAmp Blood Kit (Qiagen). We studied two SNPs in the LTA gene on chromosome 6p21 (HLA cluster): the LTA C804A (rs1041981) in exon 3 (resulting in the amino acid substitution Thr26→Asn) and LTA A252G (rs909253) in intron 1. In addition, we genotyped the C3279T (rs7291467) polymorphism in intron 1 of the LGALS2 gene on chromosome 22q using Taqman SNP allelic discrimination by means of an ABI 7900HT (Applied Biosystems). Primer and probe sequences are available on request.
Continuous data are reported as means±S.E.M. or medians (interquartile range) if the data were skewed. Categorical data are presented as per group percentages. Differences between subgroups were evaluated by Student's t test for the normally distributed continuous variables or by the Mann–Whitney test if data were skewed. Differences in genotype frequencies and other categorical data between cases and controls were compared with the χ2 test or Fisher's exact test. Consistency of genotype frequencies with the Hardy–Weinberg equilibrium was tested using a χ2 goodness-of-fit test on a contingency table of observed compared with expected genotype frequencies in cases and controls. Genotype–phenotype associations were examined with additive, dominant and recessive models using multivariate logistic regression analyses. Odds ratios for the occurrence of CHD and their 95% CIs (confidence intervals) were calculated after adjustment for matching factors. Linear mixed models were used to investigate the age-adjusted association between genotypes and inflammatory markers. In addition, linear mixed models were used to investigate the gene–environment interaction between BMI (body mass index) and LTA and LGALS2 gene polymorphisms on inflammatory markers. All results were considered statistically significant if the two-sided P value for the test statistic was less than or equal to the set type I error rate (α) of 0.05. No adjustment for multiple comparisons was performed, because there were few statistical tests and there is good biological evidence that each of the biochemical systems being studied is functionally involved in regulating inflammatory status either directly or indirectly, suggesting the universal null hypothesis that is assumed for a Bonferroni-type correction does not apply to these data . Analyses were performed using SAS version 9.1 (SAS Institute).
The general characteristics of both the NHS as well as the HPFS, divided on the basis of cases and controls, are shown in Table 1. Cases were more likely to have diabetes, hypertension and a family history of myocardial infarction than matched controls. In addition, cases from both studies had significantly higher levels of LDL-cholesterol and lower levels of HDL-cholesterol. Women with CHD had a higher BMI and higher levels of inflammatory markers, including CRP, IL-6, sTNF-R1, sTNF-R2 and E-selectin, than the matched control group. Men with CHD had significantly higher levels of CRP, IL-6 and VCAM-1, but mean sTNF-R1 and sTNF-R2 levels were not different between the groups. The characteristics did not change substantially when including men and matched controls who needed cardiac revascularization, the secondary end point (Men II). The distributions of genotypes were in Hardy–Weinberg equilibrium both in cases as well as controls (P>0.10). The genotype frequencies are shown in Table 2. Only the distribution of the LGALS2 gene polymorphism was significantly different between female cases compared with matched event-free controls. The pairwise linkage disequilibrium (D′) and the correlation coefficient between LTA C804A and LTA A252G were 0.99. No correlation was present between the LTA gene polymorphisms and LGALS2 gene polymorphism.
Association between LTA and LGALS2 gene polymorphisms and markers of inflammation and cell adhesion molecules
Tables 3–5 show the age-adjusted levels of the inflammatory markers and cell adhesion molecules (i.e. CRP, IL-6, sTNF-R1, sTNF-R2, VCAM-1 and E-selectin) among the different genotypes. The LTA C804A polymorphism was associated with plasma levels of sTNF-R2 and VCAM-1 in both female and male cases. In addition, the LTA C804A gene polymorphism was significantly associated with IL-6 in men without CHD (Table 3). Similar results were found for the LTA A252G gene polymorphism, which was also associated with sTNF-R1 levels in both women as well as men with CHD (Table 4).The LGALS2 gene polymorphism was associated with CRP levels in both male and female cases (Table 5). Furthermore, no significant interaction was present between BMI and the gene polymorphisms on the inflammatory markers in cases or controls from both the female and male cohorts.
Association between LTA and LGALS2 gene polymorphisms and risk of CHD
Table 6 shows the results from unconditional multivariate logistic regression analyses for CHD. The LGALS2 gene variant was inversely associated with a risk of CHD in women [odds ratio (95% CI), 0.70 (0.50–0.97); P=0.03]. This effect was independent of cardiovascular risk factors predictive of cardiovascular disease (diabetes, history of hypertension, BMI, family history of myocardial infarction, HDL-cholesterol, LDL-cholesterol, CRP, IL-6, sTNF-R1, sTNF-R2, VCAM-1 and E-selectin). The odds ratio (95% CI) for CHD in women after adjustment for all these factors was 0.36 (0.22–0.59) (P<0.001). This association was not present in men. After pooling the data from both women and men, we found a significant gender interaction between the LGALS2 gene polymorphism and risk of CHD (P=0.01 for interaction).
In the present large prospective nested case-control study among American women and men, we investigated the relationship between LTA and LGALS2 gene polymorphisms and levels of inflammatory markers, cell adhesion molecules and risk of CHD. This study showed significant associations between the polymorphisms in the LTA and LGALS2 genes and markers of inflammation and cell adhesion molecules, but no association was found between LTA gene polymorphisms and risk of CHD in women and men. For the LGALS2 gene polymorphism, we found evidence of a significant gender interaction, with a significant association for women, but not men, with the risk of CHD.
Previous case-control and cross-sectional studies have examined the association between LTA gene polymorphisms and cardiovascular disease, but the results are inconsistent. The first study by Ozaki et al.  described significant associations between LTA gene polymorphisms and myocardial infarction; however, the authors did not adjust for relevant covariates, including gender and age, and the genotype distributions among the control subjects were not in Hardy–Weinberg equilibrium. The association between the LTA gene polymorphisms and CHD was confirmed in another Japanese population  and in the family-based European PROCARDIS (precocious coronary artery disease) study . Furthermore, a significant association was found between the LTA C804A genotype and the extent of coronary atherosclerosis in Caucasian patients with angiographically confirmed coronary atherosclerosis . In concordance with the present results, several other studies did not detect an association between LTA gene polymorphisms and myocardial infarction [8–10], and our findings are in agreement with a recent meta-analysis performed by Clarke et al. , which showed no relationship between LTA gene polymorphisms and CHD. In contrast with the previous reports included in this meta-analysis, we used unrelated controls selected from the same population as the cases.
Our present study has shown a significant association between the LGALS2 gene polymorphism and reduced risk for CHD in women; however, this association could not be replicated in our male population. This statistical gender interaction might be a true biological interaction or may reflect differences in cardiovascular risk factors in the male and female study populations. The present study is in concordance with the findings of Ozaki et al. , who reported an association between the LGALS2 gene polymorphism and myocardial infarction. However, their study did not provide any information about gender differences. Other functional studies published so far do not report differences in LTA secretion between genders, but, as shown in Table 1, levels of inflammatory markers differ between genders and, therefore, it is possible that LTA has a sex-specific range too. Future studies are needed to investigate whether LTA secretion differs between genders.
Surprisingly, no relationship was found between LTA gene polymorphisms and CRP. LTA is a pro-inflammatory cytokine acting through activation of NF-κB (nuclear factor κB), and previous reports have demonstrated a weak, but significant, association between an LTA gene polymorphism and CRP levels [10,13]. Galectin-2 has been shown  to affect LTA expression levels and might therefore influence CRP levels as well; however, the relationship between LGALS2 genotype and CRP in the present study was opposite to that expected. We cannot exclude the role of chance or some counter-regulatory action which we did not capture with the genetic variation in LGALS2. Clearly, further study is needed to confirm or reject the present findings.
Interestingly, we found a significant association between LTA gene polymorphisms and the level of sTNF-R2 in both women and men. LTA is a pro-inflammatory cytokine that may contribute to atherosclerosis by activation of growth factors and cytokines, and by affecting the synthesis and stimulation of adhesion molecules . LTA, like TNF-α, interacts with sTNF-R1 and sTNF-R2. sTNF-R concentrations are increased in patients with infectious diseases and may be useful as an indicator of LTA-induced inflammation . On the other hand, the observed association between LTA gene polymorphisms and inflammatory markers might also represent an effect of the TNF gene or other genetic products of the HLA cluster, because the LTA gene is in significant linkage disequilibrium with the TNF gene located on chromosome 6 and the HLA cluster .
Furthermore, we detected a weak association between VCAM-1 and LTA gene polymorphisms; however, the direction of the associations between VCAM-1 and LTA gene polymorphisms found in the present study were not consistent among the cohorts. In contrast with the males, the variant genotype was associated with lower VCAM-1 levels in the female cases. This might be due to chance considering the borderline significance levels or indicate a true gender difference. Ozaki et al.  demonstrated previously that variant protein LTA 26A induced an increase in VCAM-1 and E-selectin in human coronary artery vascular smooth muscle cells. Elevated expression of adhesion molecules, such as VCAM-1 and E-selectin, might contribute to the pathogenesis of myocardial infarction, but despite the association between LTA gene polymorphisms and VCAM-1 no relationship between LTA gene polymorphisms and CHD could be demonstrated in the present study.
In conclusion, the present study has demonstrated an association between LTA and LGALS2 gene polymorphisms and markers of inflammation and cell adhesion molecules, but did not detect a significant association between LTA gene polymorphisms and CHD in American women and men. Future studies are needed to replicate the observed association between the LGALS2 gene polymorphism and reduced risk of CHD in women.
This study has been funded by the Jan Kornelis de Cock foundation (06-05), Groningen, The Netherlands, and by the National Institutes of Health (HL35464, CA55075 and HL34594). We gratefully acknowledge Patrice Soule and Hardeep Ranu of the Harvard Scholl of Public Health Molecular Epidemiology Core Facility for genotyping. We thank Alan Paciorek, Helena Ellis and Jeanne Sparrow for coordinating sample collection and laboratory management, and Lydia Liu for programming review. F.W.A. is a research fellow of the Netherlands Heart Foundation (2003T010) and the Interuniversity Cardiology Institute of The Netherlands.
Abbreviations: BMI, body mass index; CABG, coronary artery bypass graft surgery; CHD, coronary heart disease; CI, confidence interval; CRP, C-reactive protein; HDL, high-density lipoprotein; HLA, human leucocyte antigen; HPFS, Health Professionals Follow-Up Study; IL-6, interleukin-6; LDL, low-density lipoprotein; LTA, lymphotoxin-α; NHS, Nurses' Health Study; PTCA, percutaneous transluminal coronary angioplasty; SNP, single nucleotide polymorphism; TNF, tumour necrosis factor; sTNF-R, soluble TNF receptor; VCAM-1, vascular cell adhesion molecule-1
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