R497K polymorphism in epidermal growth factor receptor gene is associated with the risk of acute coronary syndrome

Acute coronary syndrome (ACS) is a clinical manifestation of preceding atherosclerosis, inflammation and thrombosis [1]. It constitutes an important cause of mortality and morbidity in many countries including China. Although the exact mechanism of ACS pathogenesis is not fully known, many factors such as age, gender, smoking, hypertension, diabetes mellitus, hypercholesterolemia, obesity and sedentary lifestyle are involved in the disease [2‐4]. In addition, several studies have shown that genetic factors such as single nucleotide polymorphisms (SNPs) of angiotensin converting enzyme (ACE), platelet glycoprotein IIb HPA-3, and matrix metalloproteinase-3 (MMP-3) also play a crucial role in the development of ACS [5‐7]. Despite many gene SNPs [5‐14] have been reported to be associated with ACS, it is necessary to discover novel gene variants in the risk of ACS. Firstly, ACS is a disease related to multiple genes. Secondly, the molecular mechanism of ACS is unclear, and it may be benefit to elucidate the pathogenesis of ACS by finding novel gene variants.

The epidermal growth factor receptor (EGFR) is a transmembrane glycoprotein of 170 kDa (1186 amino acids) encoded by a gene located in the short arm of chromosome 7 (chromosome 7p12.1–12.3) [15]. And it contains 26 exons [16]. A polymorphic variant of the EGFR arising from a single nucleotide change (G → A) leading to an arginine (Arg) →Lysine (Lys) substitution in codon 497 (HER-1 R497K) in the extracellular domain within subdomain IV of the EGFR gene has been identified [17]. It is one of the key polymorphisms within the EGFR signaling pathway. An in vitro study has shown that the variant HER-1 497K has attenuated functions in ligand binding, growth stimulation, tyrosine kinase activation, and induction of protooncogenes myc, fos, and jun compared with the "wild-type" HER-1 497R [18]. Another polymorphic variant of the EGFR is a highly polymorphic region in intron 1 of the EGFR gene which is associated with transcription levels of EGFR in vitro and in vivo [19, 20]. The length of this (CA)_n dinucleotide polymorphism correlates inversely with the transcriptional activity of the gene. In vitro studies have shown that the transcriptional activity in cell lines containing a prolonged polymorphic region (>20 CA repeats) was markedly reduced compared with cells containing a shorter allele (16 repeats). Recently, several studies reported that genetic polymorphisms of the EGFR gene had been implicated in the risk of a range of tumors [21‐26]. In addition to its prominent role in the development of several cancers [27], EGFR has been currently implicated in vascular pathophysiological processes associated with excessive remodeling and atherosclerosis [28, 29]. Moreover, EGFR has been identified on intimal smooth muscle cells within human atherosclerotic plaque and cultured rat aortic smooth muscle cells, which mediates cell proliferation and DNA synthesis [30‐32]. Atherosclerosis, regarded as a chronic inflammatory process [33], plays a fundamental role in the pathogenesis of ACS.

Therefore, we hypothesized that HER-1 R497K and EGFR intron 1 (CA)_n repeat polymorphisms may modulate the susceptibility to ACS. To test this hypothesis, we conducted a hospital-based case-control study to evaluate the potential association between HER-1 R497K and intron 1 (CA)_n repeat polymorphisms in the EGFR gene and the risk of ACS in a Chinese population.

In this retrospective, hospital-based case-control study, we found that the frequency of Lys allele in the ACS group was significantly higher than that in control. Additionally, multivariable logistic regression analysis showed that R497K was an independent risk factor for ACS besides some classical risk factors, such as smoking status, total cholesterol, LDL-cholesterol, and triglycerides. It indicates that Lys allele may be a risk factor for ACS in a Chinese population. This finding is in consistent with our hypothesis and suggests that the R497K polymorphism may be used as a genetic susceptibility marker of the ACS.

For the (CA)n repeat in intron 1 of EGFR polymorphism, alleles 20 (42.9%) and 16 (15.7%) were the two most common in a Chinese population in our current study, which was similar to that reported by Liu et al. [34] in a U.S. Asian population (62.9% for allele 20 and 17.4% for allele 16). However, the allelic distribution was reversed in Caucasians and African-Americans, with allele 16 the most common (43.2% and 41.7%, respectively), followed by allele 20 at a much lower frequency (21.0% and 14.3%, respectively). It suggests that the allelic frequencies of the EGFR intron 1 polymorphism might vary among different ethnic groups.

The EGFR, also known as HER-1 or erbB-1, is a member of the human epithelial receptor tyrosine kinase family. The EGFR gene is located on chromosome 7 [15] and contains 26 exons [16]. Exons 1 to 14 code for the extracellular domain. Exon 15 codes for the transmembrane domain, and exons 16 to 26 code for the intracellular domain [35]. Transcription of the EGFR gene is regulated by two enhancer elements. The first is located upstream near the transcription initiation site and the second downstream in intron 1 [36]. The latter contains a characteristic simple sequence repeat (SSR) of CA dinucleotides, the length of which exhibits interethnic differences [34]. In addition, the length of this polymorphism has been observed to correlate inversely with EGFR transcription in both in vitro experiments and breast cancer specimens. The longer allele with 21 repeats was shown an 80% reduction of gene expression compared with the shorter allele with 16 repeats [19, 20]. In this present study, no significant difference was observed in EGFR intron 1 (CA)_n repeat polymorphism between cases and controls. When taking HER-1 R497K and EGFR intron 1 (CA)_n repeat polymorphisms into together, there was no statistically significant difference between patients with ACS and controls as well.

Currently, EGFR is considered as a convergence point in the complex signaling network and regulation of cellular functions, such as cell growth, differentiation, motility, survival and death [37‐39]. Thereby, it plays a crucial role in various pathophysiological processes, including atherosclerosis. EGFR has been demonstrated on both intimal smooth muscle cells within human atherosclerotic plaque and cultured rat aortic smooth muscle cells [30‐32]. Furthermore, it has previously reported that EGFR is present on rabbit peripheral blood leucocytes, and co-localises with macrophages within lesions of atherosclerosis in the cholesterol-fed rabbit [40], which mediates both chemotactic responses towards monocytes and mitogenic responses towards monocyte-derived macrophages in vitro. Transactivation of the EGFR mediates endothelin-1 (a powerful vasoconstrictor, involving in vasospastic diseases such as coronary artery disease) signaling in vascular smooth muscle cells and isolated arteries [41]. This identification heightens the potential importance of EGFR in atherogenesis and other inflammatory disease. A few studies have shown that polymorphism of EGFR was associated with susceptibility of some inflammatory diseases [42, 43]. For example, Wang et al. [42] reported that asthmatic patients had a significantly higher incidence of having shorter alleles (≤ 16 CA repeats) compared with control subjects (P < 0.05). Martin et al. [43] reported that the frequency of the WT (Arg) allele at position R497K in the EGFR gene was significantly higher in ulcerative colitis than in controls (P = 0.04).

However, no previous study has shown the relationship between HER-1 R497K and EGFR intron 1 (CA)_n repeat polymorphisms and patients with ACS. To our knowledge, the present pilot study is the first to investigate the association between them. We found that R497K but not (CA)_n repeat polymorphism might be used as a genetic susceptibility marker of the ACS. The exact mechanism by which the Lys allele may be associated with the risk of ACS is unclear. In 1993, a variant EGFR of an arginine to lysine substitution at codon 497 was identified [17]. Wild-type EGFR (Arg allele) differs from mutant EGFR (Lys allele) with respect to epithelial proliferation following the administration of EGF and TGF-α in rodents [18]. Thus, it is possible that mutations which influence the function or expression of EGFR (such as attenuating its ligand binding as well as subsequent activation of its downstream effectors) might predispose to the development of ACS.

To conclude, in the present retrospective study, we found that HER-1 R497K polymorphism was significantly associated with the risk of ACS in a Chinese population. These results suggested that genetic polymorphism of EGFR (arginine →Lysine mutation) might be clinically important in the development and progression of ACS. Nevertheless, there are some limitations in our study, and several questions still should be addressed. For instance, the molecular mechanism by which the EGFR gene polymorphisms are associated with ACS is unknown. It should also be emphasized that other polymorphisms in linkage disequilibrium with the R497K and (CA)_n repeat may also contribute to variability in EGFR expression. In addition, the inadequate study design such as non-random sampling and a limited sample size should also be considered. Therefore, One needs to explore further, whether EGFR polymorphisms are an independent risk factor or an indirect marker of different genetic and environmental factors. Further studies are required to evaluate haplotype, relationship among genotypes, and the molecular mechanisms by which EGFR is involved in susceptibility to ACS in diverse ethnic populations and larger number of patients.