Single Nucleotide Polymorphism rs17173608 in the Chemerin Encoding Gene: Is It a Predictor of Insulin Resistance and Severity of Coronary Artery Disease in Non-Obese Type 2 Diabetes?

(1) Background: Chemerin, or the RARRES2 (Retinoic Acid Receptor Responder 2) gene, is found to be associated with an increased incidence of insulin resistance, endothelial dysfunction, type 2 diabetes (T2D), and coronary artery disease (CAD). This study investigates associations of RARRES2 rs17173608 with insulin resistance and the severity of CAD in non-obese T2D patients in relation to the clinical and genetic factors. (2) Methods: A total of 300 patients with T2D and CAD were recruited in this study. The associations of insulin resistance and the severity of CAD with RARRES2 rs17173608 and clinical factors were assessed. The genotyping procedures were performed using the TaqMan method. The significant associations (p ≤ 0.05) from preliminary tests were employed to carry out the secondary analysis. (3) Results: RARRES2 rs17173608 (TT, TG, and GG polymorphisms in the preliminary analysis; TG and GG polymorphisms in a secondary analysis) was associated with insulin resistance and the severity of CAD in both the preliminary and secondary analysis (all p-values were < 0.05). Additionally, in the secondary analysis, FPG and ACEI were also associated with insulin resistance and the severity of CAD (all p-values were < 0.05). (4) Conclusion: From the preliminary findings, rs17173608 is a significant predictor of insulin resistance and the severity of CAD.


Introduction
Chemerin, also known as retinoic acid receptor responder protein 2 (RARRES2) and tazarotene50 induced gene 2 [1], is a chemoattractant protein involved in the pathogenesis of metabolic syndrome [2]. It is secreted in the liver, acts as a chemotactic agent, and is highly stimulated by elements of the innate immune system, such as plasmacytoid dendritic cells and macrophages [2]. Chemerin induces angiogenesis of endothelial cells and results in endothelial dysfunction [3]. Besides which, it can activate inflammatory response and oxidative stress in adipose tissue, results in insulin resistance and further enhance endothelial dysfunction [4]. Endothelial dysfunction and insulin resistance cause the progression of type 2 diabetes (T2D) and atherosclerotic coronary artery disease (CAD).
The CAD is classified as severe when the atherosclerotic plaques narrow down the vessels with stenosis by more than 50% [5]. In a study by Dahpy et al., serum chemerin levels were higher in patients with T2D and metabolic syndromes than nondiabetics and patients without metabolic syndromes [4]. There is growing evidence showing a relationship between coronary atherosclerosis and chemerin. Several cross-sectional studies showed an independent correlation between chemerin concentrations and CAD. Chemerin is moderately heritable, with 16-25% of variations ascribed to genetic factors [5]. • Insulin resistance: HOMA-IR value of more than 7.17 from the ROC curve is categorised as insulin resistance (IR) whilst less than 7.17 is categorised as insulin-sensitive (IS).

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The severity of CAD: one, two, or three arteries narrowed by 50% of stenosis. One vessel = single-vessel disease (SVD), two vessels = double-vessel disease (DVD), three vessels = triple-vessel disease (TVD). TVD is the most severe type of vessel disease (3 major vessels involved) [5]. • Obesity: BMI more than or equal to 30 kg/m 2 [15]. Multivariate tests: involved a set of multiple variables that were significant from the univariate tests. p values were obtained for each of the variables involved by running the binary/multinomial analysis as a group. Table 2 shows the demographic and clinical data of the study population. The number of males was higher in T2D patients with CAD, and CAD-only patients. The average age of patients was higher in the CAD-only group compared to the other two groups. Indian patients were more prone to T2D with or without CAD, in comparison with Malay and Chinese patients. Patients with only CAD had a lower BMI than T2D patients with CAD and T2D-only patients. Most of the patients had a family history of T2D and CAD.

Hardy-Weinberg Equilibrium Test for SNP rs17173608
The SNP rs17173608 did not deviate from the Hardy-Weinberg Equilibrium (p > 0.05) in T2D patients with CAD (p = 0.98), T2D-only (p = 0.92), and CAD-only (p = 0.22) groups. Thus, the SNP was included in the subsequent analyses.

Associations of Clinical Factors with rs17173608
From the preliminary statistical analysis, among the OHA-and insulin-combination treatments, biguanides and insulin were associated with rs17173608. Among the monotherapy treatments, biguanides were associated with rs17173608 in T2D patients with CAD. Sulphonylureas and CCBs were found to be associated with rs17173608 in the T2D-only group. Meanwhile, FPG, FPI, and A1C levels were significantly associated with rs17173608 in CAD-only groups. Among all these significant associations, the association of FPG with rs17173608 was the strongest, with an OR value of 6.053. Table 3 demonstrates the associations of clinical factors with rs17173608 in the study population.

Associations of rs17173608 with Insulin Resistance and Severity of CAD
Preliminary associations of rs17173608 with insulin resistance and the severity of CAD are presented in Table 4, Figures 1 and 2. The preliminary results showed significant correlations between rs17173608 and insulin resistance and the severity of CAD in T2D patients with CAD and CAD-only groups. Secondary analysis using binary and multinomial logistic regression was carried out to find out the associations.

Associations of rs17173608 with Insulin Resistance and Severity of CAD.
Preliminary associations of rs17173608 with insulin resistance and the severity of CAD are presented in Table 4, Figures 1 and 2. The preliminary results showed significant correlations between rs17173608 and insulin resistance and the severity of CAD in T2D patients with CAD and CAD-only groups. Secondary analysis using binary and multinomial logistic regression was carried out to find out the associations.

Association Models of Insulin Resistance and Severity of CAD in Correlation with rs17173608 and Clinical Factors (Secondary Analysis)
From the secondary analysis, FPG (p value for IR= 0.047, DVD= 0.017, TVD= 0.032), ACEI (p value for IR = 0.024, DVD = 0.017, TVD = 0.025), rs17173608 TG polymorphism (p value for IR = 0.015, DVD = 0.028, TVD = 0.036), and rs17173608 GG polymorphism (p value for IR = 0.012, DVD = 0.042, TVD = 0.048) were associated with insulin resistance and the severity of CAD. Tables 5 and 6 and Figures 3 and 4 show the secondary analysis of insulin resistance and the severity of CAD in correlation with rs17173608 and clinical factors. Appendix A contains a summary of the findings of this study.

Association Models of Insulin Resistance and Severity of CAD in Correlation with rs17173608 and Clinical Factors (Secondary Analysis)
From the secondary analysis, FPG (p value for IR = 0.047, DVD = 0.017, TVD = 0.032), ACEI (p value for IR = 0.024, DVD = 0.017, TVD = 0.025), rs17173608 TG polymorphism (p value for IR = 0.015, DVD = 0.028, TVD = 0.036), and rs17173608 GG polymorphism (p value for IR = 0.012, DVD = 0.042, TVD = 0.048) were associated with insulin resistance and the severity of CAD. Tables 5 and 6, Figures 3 and 4 show the secondary analysis of insulin resistance and the severity of CAD in correlation with rs17173608 and clinical factors. Appendix A contains a summary of the findings of this study.

Discussion
Insulin resistance and the severity of CAD were postulated to come independently from "common soil". The demographical and clinical factors associated with insulin resistance may be associated with CAD patients. The study by Arambewela et al. found that the prevalence of T2D with CAD was higher in males than females [29], and the study by Krishnan et al. found that the prevalence of CAD in non-diabetic CAD patients was higher in males than females [30]. Gender differences exist because, regardless of T2D, males develop CAD 7 to 10 years earlier than females [31]. Females are more likely to develop CAD as a result of massive hormonal changes following menopause [32].
Similarly, more males than females were found in T2D patients with CAD and CADonly patients in our study. Nonetheless, females were diagnosed with T2D at a higher rate than males in T2D-only patients. The National Health and Morbidity Survey (NHMS) 2019 found that the prevalence of T2D was higher in females than in males [33]. Several studies have found that the prevalence of T2D and CAD varies across ethnic groups living in the same country. It has been postulated to be associated with environmental and genetic factors. Indian patients had the highest prevalence in our study, followed by Malay and Chinese patients in the T2D with CAD and T2D-only groups. Tee and Yap [34] discovered similar results in the Malaysian population [34]. This could be because Indians have a higher inherent risk of T2D and CAD in Malaysia [34].
FPG and A1C levels were higher in T2D patients with CAD than in T2D patients without CAD in a previous study [35]. As a result, the findings of the studies contradicted our

Discussion
Insulin resistance and the severity of CAD were postulated to come independently from "common soil". The demographical and clinical factors associated with insulin resistance may be associated with CAD patients. The study by Arambewela et al. found that the prevalence of T2D with CAD was higher in males than females [29], and the study by Krishnan et al. found that the prevalence of CAD in non-diabetic CAD patients was higher in males than females [30]. Gender differences exist because, regardless of T2D, males develop CAD 7 to 10 years earlier than females [31]. Females are more likely to develop CAD as a result of massive hormonal changes following menopause [32].
Similarly, more males than females were found in T2D patients with CAD and CADonly patients in our study. Nonetheless, females were diagnosed with T2D at a higher rate than males in T2D-only patients. The National Health and Morbidity Survey (NHMS) 2019 found that the prevalence of T2D was higher in females than in males [33]. Several studies have found that the prevalence of T2D and CAD varies across ethnic groups living in the same country. It has been postulated to be associated with environmental and genetic factors. Indian patients had the highest prevalence in our study, followed by Malay and Chinese patients in the T2D with CAD and T2D-only groups. Tee and Yap [34] discovered similar results in the Malaysian population [34]. This could be because Indians have a higher inherent risk of T2D and CAD in Malaysia [34].
FPG and A1C levels were higher in T2D patients with CAD than in T2D patients without CAD in a previous study [35]. As a result, the findings of the studies contradicted our findings. Meanwhile, LDL-c levels in T2D patients without CAD were higher than in T2D patients with CAD and CAD patients without T2D [35]. The results for FPI and HOMA-IR in the same study contradicted our findings, as the values were higher in T2D patients without CAD [35]. BMI is said to have an effect on glycaemic control in people with diabetes and other metabolic disorders. The BMIs of T2D-only patients were higher than the other two groups in this study, which may have resulted in an increase in FPG, A1C, LDL-c, FPI, and HOMA-IR levels in T2D patients only when compared to the other groups of patients.
Furthermore, previous studies were conducted on obese individuals, yielding contradictory results [35]. In a previous study, CAD patients had higher hs-CRP, HDL-c, and total cholesterol levels, which were comparable to the findings of our study. [36]. rs17173608 is a RARRES2 gene variant, and to the best of our knowledge, this is the first study to associate rs17173608 with demographic and clinical factors of insulin resistance and severity of CAD. In the preliminary associations, some elements were discovered to be associated with rs17173608. Gender was associated with rs17173608. rs17173608 was associated with increased visceral fat mass in non-obese subjects in one study [37]. Men tend to store more fat in their visceral depots. As a result, the association of rs17173608 with the male gender is most likely due to gender-related body fat distribution [38]. Furthermore, according to Table 2, the majority of T2D patients with CAD were not receiving biguanide treatment (Yes = 47, No = 103).
As a result of the significant association of rs17173608 with biguanides in preliminary analysis, it was hypothesised that the administration of biguanides to these patients would reduce the expression of RARRES2 and thus chemerin. In the T2D-only group, however, many patients were on sulphonylureas. Sulphonylureas are the second-line drugs in T2D management, and the use is associated with an adverse vascular function, which results in endothelial dysfunction and cardiovascular events [39]. SNP rs17173608 was associated with insulin resistance in our study and previous studies [36]. Besides this, RARRES2 SNP rs17173608 was associated with the severity of CAD in the study by Lachine et al. [40]. Our findings were consistent with previous studies, despite differences in the study population and pharmacological treatments. Secondary analyses were performed to determine the additional association of rs17173608 polymorphisms (TT, TG, and GG) with insulin resistance and the severity of CAD in T2D patients with CAD. The secondary analysis revealed a significant association of rs17173608 polymorphisms (TG and GG) with insulin resistance and the severity of CAD.

Strength and Limitations
An essential strength of this study was the homogenous sample of individuals. All the samples were non-obese individuals. This enabled researchers to draw conclusions about the impact of rs1717308 in a non-obese T2D population with and without CAD. Furthermore, this was the first study to associate rs17173608 with both insulin resistance and the severity of CAD. The main constraint was the small sample size. Nonetheless, the samples were chosen using very strict inclusion and exclusion criteria in order to reduce the impact of confounding variables that could interfere with the outcome of this association study.

Conclusions
Among Malaysia's three major ethnic groups, Indians had the highest prevalence of insulin resistance and the severity of CAD in the study population. The preliminary and secondary analyses revealed that the TG and GG polymorphisms of rs17173608 were significantly associated with insulin resistance and the severity of CAD. Thus, the findings indicate that rs17173608 is a significant predictor of insulin resistance and the severity of CAD.
Author Contributions: S.P. was responsible for clinical sample collection, laboratory work, interpretation of data and writing the whole manuscript. H.Z.H. and W.A.W.A. involved in the conceptual design of the study and revised the manuscript critically for important intellectual content. All authors have read and agreed to the published version of the manuscript.

Conflicts of Interest:
The authors have no other relevant affiliations or financial involvement with any organisation or entity with a financial interest in or financial conflict with the subject matter of materials discussed in the manuscript apart from those disclosed. No writing assistance was utilised in the production of this manuscript. Table A1. Summary points.

Chemerin and RARRES2
• Chemerin and its coding gene RARRES2, involved in the pathogenesis of metabolic syndrome, induce angiogenesis and activate an inflammatory response.

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The increased level of chemerin and expression of RARRES2 results in insulin resistance, leading to T2D and atherosclerotic CAD.

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Several studies have been conducted on the association of SNP rs17173608 with insulin resistance or atherosclerotic CAD.

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Endothelial dysfunction is the main culprit that results in insulin resistance and progression of atherosclerosis.

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The CAD is severe when the atherosclerotic plaques narrow down the vessels with more than 50% of stenosis.

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In our study, we included the patients with more than 50% of atherosclerotic stenosis, and we classified the patients as having SVD, DVD, and TVD (the most severe type of CAD) Demographic and clinical factors • Age, gender, ethnicity, BMI, and family history of T2DM and CAD were the demographic factors assessed in our study. Indians had the highest prevalence of insulin resistance and severity of CAD than Malays and Chinese. • Laboratory investigations, comorbidities, and pharmacological treatments were the clinical factors we studied.

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We excluded some of the comorbidities such as obesity and asthma, as these diseases may influence the outcome of the study. rs17173608 • rs17173608 is a SNP of RARRES2 gene. • Some studies showed a significant correlation between rs17173608 and T2D whilst a significant proportion of previous evidence exhibiting a correlation between rs17173608 and CAD.

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Our study is the first study explicitly associating rs17173608 with both insulin resistance and severity of CAD in non-obese T2D patients. Conclusion

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TG and GG polymorphisms of rs17173608 were significantly associated with insulin resistance and the severity of CAD. • rs17173608 is a significant predictor of insulin resistance and the severity of CAD in non-obese T2D patients.