The renin-angiotensin-aldosterone system (RAAS) is the core regulator of systemic blood pressure. An essential component of this system is angiotensinogen (AGT), a serum renin glycoprotein substrate synthesized and released in the liver as the precursor of angiotensin, the hormone that constitutes part of the systemic blood pressure control system [1]. By virtue of the function of AGT in regulating blood pressure, changes in its gene sequence are likely to play an important role in the pathogenesis of cardiovascular risk traits, such as hypertension, as well as manifestation of coronary artery disease (CAD) [2, 3]. Several AGT variants, including the rs4762 (p.M207T, also formerly known as p.T174M) and rs699 (p.M268T, also referred to as p.M235T), have been associated with susceptibility to various cardiovascular risk traits, such as primary hypertension (HTN) [4–10] and type 2 diabetes (T2DM) [11, 12], that are linked to the development of CAD. These variants have similarly been implicated not only in the manifestation of CAD [13–23], but also in the severity of atherosclerosis [24, 25]. The fact that the AGT gene constitutes a risk for these disease traits as well as CAD strongly implicates gene-disease interactions in pathways that trigger atherosclerosis, a subject that has attracted considerable research interest recently. Indeed, a number of studies have indicated that such gene-trait or gene-gene interactions contribute to events leading to complex disorders, such as CAD [5, 26–31]. However, this subject has not been exhaustively addressed yet. Besides, the notion of some of the AGT variants or their interactions with such disease risk traits playing a role in CAD/myocardial infarction (MI) has been refuted by a number of studies in various ethnic groups [21, 22]. We recently described associations of several AGT variants with CAD in heterozygous familial hypercholesterolaemia through linkage and population-based association studies in a large, angiographed and ethnically homogeneous Saudi cohort [9]. In the present study, we specifically asked the question whether alterations in the AGT gene are a common cause for different cardiovascular risk factors, thereby posing an additional risk to acquiring CAD in individuals harbouring such risk traits. We employed the same study population previously engaged in the early onset CAD study.

In the present study, we tested the likelihood of AGT polymorphism interacting with important cardiovascular risk traits, such as HTN, T2DM and obesity, contributing to disease pathways leading to atherosclerosis, following our previous finding of its linkage to early onset of CAD in heterozygous familial hyperlipidaemia (unpublished results). Accordingly, we employed the eight SNPs investigated in the early onset CAD study in the same ethnically homogeneous, angiographed cohort of native Saudi individuals, in which each subset of the cardiovascular disease traits of interest was well-documented. Our observations point to the different AGT variants as invariably conferring risk for these traits in our study population, whereby the most significant relationships involved the HTN patient group. This disease was linked to six of the eight studied SNPs (rs2067853, rs7071, rs699, rs3789679, rs2148582 and rs5051) in a causative fashion, unequivocally demonstrating the impact of AGT polymorphism as a risk for acquiring HTN. Of these variants, the rs699 (p.268 T) exhibited by far the most significant relationship, in agreement with several other studies that have implicated it in this disease in other ethnic groups, including Caucasians[36], Chinese[8], Slovaks[37], Polish[17] and Tunisians[19], among others. Therefore, our study reaffirms its place as a risk variant for HTN globally. However, current literature does not seem to hold much data on the other SNPs implicated in HTN in the present study. Therefore, our observations with respect to their involvement in HTN probably point to the novelty of their role as risk factors for the disease, at least in native Saudis. On the other hand, the lack of association for the rs4762 (p.M207T) with HTN in the present study appears to be in conflict with those reports implicating it in the disease [8–10].

Interestingly, rather than being involved in HTN, our data indicates that the rs4762 is associated with obesity and MI in the Saudi general population. To our knowledge, apart from a report suggesting an association of the SNP with central obesity-related HTN in the Hong Kong Chinese [8], literature seems to be also lacking with respect to its potential role in metabolic disorders, in general. Besides, we also observed some sharing of causative properties for four of the variants by HTN and CAD/MI as well as one (rs3789679) by HTN and obesity, all of which similarly appear to be yet undocumented in the literature. Thus, put together, our study identified partly novel associations for various AGT variants concomitantly predisposing individuals to multiple cardiovascular disease traits, pointing to potential pleiotropic activity of the AGT polymorphism on the risk of acquiring atherosclerosis.

The primary question raised in this study was whether or not the interactions of the AGT gene with these risk traits might explain some of the disease pathways to atherosclerosis. To begin with our data suggested that although initially the studied SNPs appeared to be associated with CAD almost all association were abolished when adjusted for age. On the other hand, the relationships of the variants with the disease traits were retained in the CAD population, pointing to these relationships as being essentially independent of the presence or absence of CAD. Notably, however, T2DM, which exhibited no delineable relationship with any of the variants in the general population, was weakly linked to three of the variants, rs1926723, rs2148582 and rs5051, possibly suggestive of the potential requirement for CAD presence in their interactions with the disease. Furthermore, each disease subset seemed to display unique variants that were linked to CAD. However, some similarities between the T2DM and HTN groups were observed in the associations of the variants with CAD, despite the above-mentioned lack of association with the former in the general population. Moreover, different combinations of these traits also produced unique trends. Thereby, the rs699 and rs5051 appeared to be invariably implicated in the different traits. These results imply therefore that the impact of the changes in the AGT gene sequence on CAD may be partly dependent on the type of disease trait involved.

The role of AGT polymorphism, particularly the rs699 and rs4762, in CAD has also been a subject of intense research interest recently. In the present setting, while the rs699 showed a strong link, the rs4762 exhibited only borderline association with CAD. As is the case with HTN, our findings concur with the reports of the rs699 predisposing individuals to developing CAD/MI in a diversity of ethnic groups, including the Egyptian [13], Japanese [16] and Spanish [21] populations. However, the finding of a weak relationship for the rs4762 is not in complete agreement with those linking it to CAD [8, 18, 19, 31]. Besides, both the p.207 T and p268T have not only been implicated in CAD, but also in the severity of atherosclerosis [24, 25], requirement for coronary artery bypass surgery [38], and even as predictors of restenosis recurrence after angioplasty [39, 40], suggesting a multi-faceted role for these two variants in CAD, as a whole. Our data also provides some support for an association of the AGT polymorphism with the severity of atherosclerosis, independently of other cardiovascular risk traits, possibly strengthening the hypothesis of its involvement in the extent of the disease reported elsewhere [24, 25]. Apart from these two, we also established the sharing of four other variants by HTN and CAD. Since alterations in blood pressure constitute a complex risk trait for CAD, this sharing of causative variants by CAD and HTN, its important predisposing disease, in this and other ethnic populations seems to strongly implicate gene-disease interactions in pathways leading to CAD. A number of studies have produced evidence pointing to the importance of gene-environment and gene-disease interactions in complex diseases such as CAD and its predisposing traits^43-50. Thus, our findings indeed furnish strong support for the notion of such AGT interactions with the cardiovascular risk traits as a triggering factor for CAD.

Nonetheless, it should be noted that various other studies involving different ethnic populations, including the Chinese [41, 42], Indians [28] and Germans [43], failed to establish any contribution of these variants to the risk of CAD or HTN, possibly pointing to ethnic variability in the practical relevance of these findings globally. Several factors might be responsible for these discrepancies. One of the most likely explanations is the probability of inter-ethnical variation in the minor allele frequencies among the various ethnic groups. Thus for example, the frequency of the 268 T has been found to vary significantly between black and white subjects in some studies, leading to the suggestion of the 268TT genotype as an independent risk for coronary events selectively in black post-myocardial infarction patients [30]. Besides, some studies have also implicated gender and age in the association of these genetic changes with disease. In the present study, gender appeared not to impact the overall relationships for the different traits. There was also no delineable confounding effect of family history or smoking on the interactions of the AGT gene with disease in our population.

The partial lack of consistency in some of our findings on the individual variants with other previous observations raised our curiosity as to whether haplotyping might provide better insight into potential gene-disease interactions that may contribute to CAD pathways. To begin with, as observed with the individual variants, by far the greater majority of the haplotype constructs were associated with HTN, in a much more significant fashion than did the constituent variants themselves. In particular, it was noteworthy that the most common 8-mer GGTGGGGT itself was implicated in HTN. Besides, these associations culminated in the 5-mer or 4-mer derivatives encompassing the three variants rs699G > A, rs4762G > A and rs3789679G > A exhibiting the most conspicuous relationships. Similar trends were also observed for obesity, T2DM and MI, whereby each of the traits was associated with a couple of the 8-mer haplotypes and their shorter derivatives. Most importantly, T2DM shared several causative haplotypes with CAD/MI. Moreover, many other haplotypes were also common to two or more of the disorders, similarly pointing to the presence of the rs699 and rs3789679 as the core loci for these traits. Not only were the haplotypes more significantly involved, but they also included causative sequences common to the risk traits among themselves and partly shared with CAD. Hence, while the associations of the individual variants with the disease traits do not unequivocally delineate the indicators for their interactive involvement in atherosclerosis, haplotyping seems to reveal several characteristics which may help decipher such relationships. These observations indeed implicate haplotypes as more reliable indicators for the involvement of the AGT gene in the different diseases. Notably, the involvement of haplotypes in the gene-environmental interactions in CAD has also been highlighted recently, with some studies describing events such a female sex-related segregation with hypertension [32]. Therefore, our findings support the notion of complex interactions of such traits offering some basis for a mechanistic explanation for the events leading to CAD. The putative mechanisms involved in these interactions need however to be addressed further to shed light on this complex subject.

In this regard, it is noteworthy that three of the variants investigated herein, which are also integral constituents of the reported causative haplotypes, are non-coding SNPs, residing either upstream in the promoter region or downstream in the untranslated portion of the gene. This raises the crucial question as to the nature of their role on disease pathways. Some other AGT variants, in particular those residing in the promoter region of the gene, have also been associated with disease, similarly raising an interest in these sequences as key players in the mechanisms involved in its interactions with disease. Thus, since only changes involving the two coding variants, rs699 and rs4762, may be directly responsible for triggering alterations related to protein functional expression associated with disease, it can certainly be speculated that the mechanisms involving alterations at such loci are not directly ascribable to such changes in the AGT protein functional expression. Hence, the findings of associations for both coding and non-coding AGT variants with cardiovascular disease traits implicate various mechanisms possibly involving both protein functional changes and gene regulatory processes in CAD pathways. The potential role of the 3’UTR in disease is increasingly gaining acknowledgement, as it contains important elements, particularly the micro RNAs, regulating certain properties of the gene, such as mRNA maturation. While not much speculation can be derived from the current observations alone, it seems nonetheless reasonable to argue that such mechanisms might be related to gene regulatory processes. The findings also provide a working basis for the notion of the AGT 3’UTR-trait interactions as an important component of pathways leading to atherosclerosis. This notion thus advocates for further studies on this subject.

We therefore conclude that the AGT constitutes an independent risk gene for HTN, obesity and MI, which may be important in the manifestation of CAD associated with these disorders. Together with the discovery of several common causative haplotypes for these cardiovascular risk traits and CAD, these findings reaffirm the potential pleiotropic role of the AGT on disease pathways leading to atherosclerosis, possibly involving gene-disease interactive mechanisms.