This study highlighted the association between TREML4 mRNA expression and polymorphisms as a potential biomarker for coronary lesion extent. Chagas et al. studied 337 patients who were undergoing coronary angiography for suspected CAD and observed that anthropometric measures were not correlated with the extent of coronary lesions12. The authors found only a few associations between classic risk factors and atherosclerotic burden, similar to our findings shown in Table 1. Together, these observations strengthen the hypothesis that classic risk factors for CVD are not sufficiently sensitive to evaluate the extent of coronary lesions in patients with CAD, highlighting the importance of studies to discover novel non-invasive, specific, and sensitive biomarkers for early assessment of atherosclerotic lesions.

Although the 13 genes evaluated in this study reportedly are associated with ACS8, in this study, only two genes, TREML4 and ECHDC3, were associated with coronary artery lesion extent. Hypotheses may explain these results are that ALOX15, AREG, BCL2A1, BCL2L1, CA1, COX7B, IL18R1, IRS2, KCNE1, MMP9, and MYL4 are involved in atherosclerosis destabilization7. Importantly, the patients in the present study did not have destabilized atherosclerotic plaques. Therefore, we evaluated the physiological processes that precede ACS. Among the 13 genes associated with ACS, some reportedly are involved in atherothrombotic processes, e.g. IRS2 and MMP9 are involved in plaque disruption and acute myocardial infarction in humans13,14,15 and KCNE1 is involved in ischemic events, such as acute myocardial infarction, in a rat model17. TREML4 also has an important association with the extent of coronary lesion, but few studies have evaluated its relationship with the process of atherosclerosis. We previously observed increased TREML4 expression in patients with ACS8. This result reinforces our findings, as ACS is a complex pathological process that predisposes for cardiac injury and a common risk factor in patients with CAD.

TREM and TREML are receptors composed of a single extracellular variable-type immunoglobulin-like domain and are structurally similar, with a transmembrane domain and a short cytoplasmic tail lacking any known signalling motif18. They are expressed predominantly on myeloid cells19. Studies have demonstrated that TREM clusters are involved in the development of atherosclerosis. Most studies on TREM clusters focused on two members, TREM-1 and TREM-2, both of which are involved in the innate immunity inflammatory process induced by Toll-like receptors. Considering that inflammation is an important mechanism in the atherosclerotic process, the association of TREM-1 and TREM-2 expression with atherosclerotic plaque formation20,21 and polymorphisms in TREM-1 have been reported to be associated with CAD20.

In our study population, 22.1% of patients with CAD were carriers of the rs2803495 AG + GG genotypes and the minor-allele frequency of the G allele was 11.8% (Table 2). These results are similar to those in a European population in phase 3 of the 1000 Genomes project (http://www.internationalgenome.org/), in which 21.2% of subjects were carriers of the AG + GG genotypes and the G allele frequency was 13.0%. The SNP rs2803496 showed similar frequencies for CT + CC genotypes (23.1%) and C allele (11.9%), which are comparable to those found in the European population (16.2% and 11.0%, respectively). TREML4 mRNA expression was higher in patients with CAD carrying the C allele (genotypes CT + CC) for rs2803496 in leukocytes than in non-C allele carriers (p = 0.030). In contrast, the G allele (genotypes AG + GG) for rs2803495 did not influence mRNA expression levels (p = 0.409). Similarly, Sen et al. reported that the presence of the C allele was strongly associated with increased TREML4 expression in patients with CAC in the USA9. This is the first study to evaluate these polymorphisms and TREML4 mRNA expression in CAD patients in an admixture Brazilian population.

Both rs2803495 and rs2803496 variants are in the 5′-untranslated region (UTR) of TREML4. The 5′-UTR contains regulatory regions, such as selenocysteine insertion sequence elements, AU-rich elements, riboswitches, and microRNA-binding sites that affect translational efficiency, subcellular localization, and mRNA stability22. Therefore, the 5′-UTR plays an important role in controlling gene expression23, and polymorphisms in this region may influence mRNA stability and, consequently, gene expression levels.

We previously reported a significant increase in TREML4 mRNA expression in leukocytes of patients with ACS compared to in controls8. However, the role of this gene in the presence and progression of CVD remained unclear, and only two studies have evaluated TREML4 in CVD. Studies on mRNA expression in the peripheral blood have revealed vascular inflammatory markers and other indicators of tissue injury reflecting cellular damage anywhere in the body24. In this study, TREML4 mRNA expression was correlated with the extent of coronary lesions. Patients with major lesions showed higher TREML4 expression than other groups (Fig. 1). Sen et al.9 evaluated leukocyte subpopulations of patients with CAC, and also detected higher TREML4 expression levels by qRT-PCR in human leukocytes. CAC is well known to be closely related to atherosclerosis25. These results suggest that TREML4 expression is involved in the progression and extent of coronary lesions, and its consequences, such as CAC. Therefore, TREML4 expression is a potential marker of atherosclerosis severity and may be useful for monitoring patients with CAD.

A high frequency of diabetes mellitus (OR 3.7, CI 1.1–11.8, p = 0.029) as well as antidiabetic use (OR 13.0, CI 2.2–33.9, p = 0.002) were observed in patients who had high TREML4 mRNA levels, and these patients also presented more extensive atherosclerotic lesions (Table 3). Hyperglycaemia, a diabetes mellitus symptom, is a well-established, important risk factor for CAD. Increased use of antidiabetics in the diabetes mellitus group was expected; however, these medications may be related to the progression of atherosclerotic lesions, as previous studies examining the use of some antidiabetics showed controversial results, in which mortality was not reduced26,27.

Our results suggest that TREML4 mRNA expression in leukocytes is increased in patients with CAD with more extensive atherosclerotic lesions in the coronary arteries. Moreover, it is likely that carriers of rare alleles of TREML4 polymorphisms are more prone to having more extensive coronary lesions. Both findings support that TREML4 is involved in the formation, progression, and severity of atherosclerotic lesions. However, considering that the polymorphisms in this study are mutations in the 5′-UTR of mRNA, an important regulatory region for protein synthesis, further studies on the involvement miRNA, proteins, and TREML4 in other CAD stages are necessary to confirm this hypothesis.

In conclusion, this study evaluated the relationship between gene expression and TREML4 polymorphisms in patients undergoing coronary angiography. The results suggest that TREML4 mRNA expression in leukocytes is influenced by the extent of coronary artery lesions and gene polymorphisms in patients with CAD. Therefore, the TREML4 mRNA level may be an important biomarker for evaluating the progression and severity of CAD.

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