Correlation between SCAP Genetic Polymorphism and Coronary Artery Disease in a Han population in Xinjiang, China

SREBP cleavage-activating protein (SCAP) plays a vital role in the modulation of cholesterol homeostasis, and cholesterol dysregulation is tightly associated with coronary artery disease (CAD). To investigate the correlation of the genetic polymorphism of SCAP with CAD, we conducted a case-control study of 528 CAD patients (case group) and 483 age- and sex- matched subjects from whom CAD was excluded (control group). Three tagSNPs (rs147215799, rs17079634 and rs59586735) in SCAP gene were genotyped in all participants, the genotype and allele frequencies of which were compared between two groups to determine their associations with CAD. We found rs17079634 showed signicant difference in genotype distribution between the case and control group (P=0.016). The difference was most prominent in a dominant model (TT vs. CT + CC, P=0.004). After adjustment for confounding factors, the difference remained statistically signicant (OR =1.363, 95% condence interval [CI]:1.022~1.818, P=0.035). Whereas no signicant associations of the other two SNPs with CAD were observed (P=0.393 for rs147215799 and 0.303 for rs59586735, respectively (cid:0) . We drew conclusion that the SCAP genetic polymorphism rs17079634 was associated with CAD.


Introduction
Coronary artery disease (CAD) is a leading cause of morbidity and mortality worldwide, and its incidence is still rapidly increasing, especially in China, meanwhile the burden arising from it in terms of mortality and nancial cost is increasingly huge [1,2] . The etiology of CAD is extremely complex and there are still abundant problems awaiting resolution. Overall, numerous studies have indicated that CAD is a complicated polygenic disease and a result of interaction between an individual's genetic composition and a variety of environmental risk factors [3,4] . Some traditional risk factors for CAD such as hypercholesterolemia and so on have been well-established. Besides these, many genetic alterations have been demonstrated to be associated with CAD and may contribute to CAD susceptibility [5,6] . However, most of the heritability of CAD remain unexplained, indicating that additional susceptibility loci await identi cation [7,8] .
As an important macromolecule involved in the development and progression of atherosclerosis which acts as major pathophysiologic events, cholesterol level in vivo is modulated by genetic and environmental factors [9] , among them the sterol regulatory element binding protein (SREBP)-SREBP cleavage-activating protein (SCAP) pathway plays a crucial role. Being a major component in this pathway, SCAP binds with SREBPs through its carboxyl-terminal domain to forge SCAP-SREBP complex [10] . Working with insulin induced gene proteins (INSIGs), the complex transfers SREBPs from the endoplasmic reticulum to the Golgi apparatus via feedback regulation of cholesterol levels, nally affecting the synthesis of lipid [11][12][13] .
Given the central role of the SREBP-SCAP pathway in the regulation of cholesterol, the variations in the SCAP locus might affect the development and progression of atherosclerosis, thereby contributing to the susceptibility of CAD. Thus far, several studies has investigated the association of genetic variants in SCAP gene with coronary heart disease [14][15][16] . However, the SNPs involved in these studies were mainly limited to rs12487736 and the results were at odds with each other. To further systematically evaluate the association of genetic polymorphism in SCAP with CAD, here we chose three tagSNPs in SCAP and conducted a case-control study in a Han population in Xinjiang, China.

Materials And Methods
Ethical approval of the study protocol Most patients in the case group had received standardized treatment for coronary heart disease, including lipid-lowering drugs. All of subjects in the control group had not taken lipid-lowering drugs.
Smoking was de ned as currently smoking cigarettes. Diabetes mellitus was de ned as: classic symptoms of hyperglycemia or hyperglycemic crisis plus elevated plasma glucose, includes FPG ≧ 7.0mmol/L, or 2h PG ≧ 11.1mmol/L during OGTT, or a random plasma glucose ≧ 11.1mmol/L; In the absence of unequivocal symptoms of hyperglycemia, diagnosis requires repeating the glucose measurement on another day.

SNP selection and Genotyping
Blood samples were taken by using anticoagulant ethylene diamine tetraacetic acid (EDTA) tube, and standardized phenol-chloroform method was used to extract genomic DNA from peripheral leukocytes.

Statistical analyses
Statistical analyses were carried out using SPSS version 22.0 (SPSS, Chicago, IL). The Hardy-Weinberg equilibrium was assessed by Chi-square test [19] . Continuous variables are expressed as means ± SD in case of normal distribution and as the median (interquartile range) in case of non-normal distribution. Differences in the categorical variables, such as the frequencies of smoking, hypertension, diabetes, and genotypes were analyzed using the chi-square test. After adjusting confounding variables, general linear model analysis was undertaken to test the association between SCAP genotypes and CAD. In addition, a two-tailed P-value less than 0.05 was considered to be statistically signi cant. Table 1 showed the clinical characteristics of CAD patients (n = 528) and control subjects(n = 483).

Characteristics of study participants
Between the two groups, there existed signi cant differences in following variables: hypertension, diabetes, cigarette smoking, as well as the serum concentrations of TC, HDL-C, LDL-C, ApoA and FPG (all P < 0.05). No signi cant differences were present in following variables between the two groups: ages, sex, serum concentrations of TG, ApoB and Lp(a) (all P > 0.05). It was worthwhile noting that serum LDL-C and TC level were signi cantly higher in control group than in CAD group, the reason for which was the result from the prevalent use of lipid-lowering drugs in CAD patients

SCAPgenotypes and alleles distributions between CAD group and control subjects
The genotypes and allelse distributions of three selected SNPs in SCAP gene were listed in Table 2. The distributions of SCAP genotypes for CAD group and control group were all in accordance with predicted Hardy-Weinberg equilibrium (H-WE) values. Of the three SNPs, rs17079634 was shown to be signi cantly associated with CAD, for its three genotypes, T/T, C/T and C/C, distributed signi cantly differently between CAD and control groups (P = 0.016)(shown in Table 2). If analyzed by speci c genetic models (indicated in Table 3), the differences were most prominent in dominant model (TT vs CC + CT, P = 0.008), followed by additional model (CT vs TT + CC, P = 0.02), whereas not present in recessive model (CC vs CT + TT, P = 0.27). Meanwhile, there also existed signi cant difference in the distributions of the two alleles (T and C) between the two groups (P = 0.008). However, genotype distributions of the remaining two SNPs didn't show different between the two groups (P = 0.393 for rs147215799 and 0.303 for rs59586735, respectively) (shown in Table 2). The association ofSCAPrs17079634 with serum lipid pro le in control subjects To explore whether this polymorphism rs17079634 affects serum lipid pro le, thereby leading to its association with CAD, we compared the serum lipid pro le among different genotypes of this SNP only in the control subjects given CAD patients had prevalently taken lipid-lowering medications such as statins. The three genotypes didn't show signi cant differences in serum lipid levels (P = 0.934 for TG, 0.910 for TC, 0.284 for HDL-C, 0.992 for LDL-C, 0.805 for ApoA , 0.468 for apoB and 0.353 for Lp(a), respectively) (data not shown), which suggested that other underlying mechanisms contributed to the association.
The association ofSCAPrs17079634 with CAD after adjustment for confounding factors To further investigate whether the association of rs17079634 with CAD resulted from the confounding factors, we performed multivariable logistic regression analysis, with the results showed in Table 4. After adjustment for confounding factors such as hypertension, cigarette smoking as well as the serum concentrations of glucose and apoA , the association remained signi cant in a dominant model (OR = 1.363, 95%CI:1.022 ~ 1.818, P = 0.035).

Discussion
In this observational, candidate gene association study among 1011 participants, we examined the association between genetic variants in the SCAP locus and CAD susceptibility, and found rs17079634 in SCAP gene was signi cantly correlated with CAD, speci cally, the frequencies of the CT genotype and C allele of SCAP rs17079634 were signi cantly higher in CAD patients than in control subjects, and still signi cant after the confounding factors were adjusted. This indicated that carriers of SCAP rs17079634 CT genotype or C allele have signi cantly increased risk of CAD.
Abundant studies have demonstrated that, as a complicated polygenic disease, CAD is a consequence of interaction between an individual's genetic composition and a variety of environmental risk factors. As to the former, the foundation for putative causative genes that may be involved in CAD is based on a candidate gene approach. Among the numerous impacts disposing CAD, lipid metabolism dysregulation is believed to play a crucial role, including abnomality in serum lipid pro le and excessive cholesterol accumulation in coronary artery contributing to atherosclerosis, etc. Hence, genes involved in cholesterol metabolism are reasonable candidates for CAD. Actually, much attention has been focused on the association of genetic polymorphism in related genes with CAD. Serving as SREBP chaperone, SCAP is the sterol sensing receptor directly interacting with and controlling SREBP transcription factor activation, thereby playing a pivotal role in the modulation of cholesterol and other lipids synthesis. Accordingly, abnormality in SCAP gene and its expression may lead to disturbed cellular cholesterol homeostasis. [20][21][22] There has been some studies investigating the correlation of SCAP genetic polymorphism with CAD, although the polymorphism involved in these studies is mainly rs12487736 variant and the results were inconsistent. Fan et al investigated the expression of SCAP in human atheroma and the association of its allelic variants with sudden cardiac death(SCD), concluded that SCAP rs12487736 may contribute to SCD in early middle-aged men [16] , but Chen et al didn't found rs12487736 was associated with premature coronary artery disease in a Chinese population [15] . In fact, as of 2020, neither the largest common variant association studies, nor the largest exome-sequencing-based rare, coding variant association studies has nominated SCAP as a genome-wide signi cant risk locus for CAD [23][24] . In our study, We drew positive conclusion, that is, rs17079634 in SCAP gene was signi cantly associated with CAD. We speculated that these existing studies were primarily based on European ancestry populations, and future studies in East Asian populations may nd relevant in them.
Given the crucial role of SCAP in homeostasis modulation of cholesterol and other lipids, it's natural for researchers to consider if serum lipid levels mediate the correlation between SCAP rs17079634 and CAD, in other words, rs17079634 was associated with serum lipid, thereby leading to its correlation with CAD.
To make out this, we performed association analysis between rs17079634 and serum lipid pro le only in control subjects (for in case group, lipid-lowering agents such as statins have been used prevalently and their serum lipid levels have been affected). We came to a negative conclusion, that is, SCAP rs17079634 was not associated with blood lipid levels. Another explanation is, the genetic alterations in SCAP may involve in pathogenesis of atherosclerosis through disturbed cholesterol metabolism and accumulation locally at coronary arteries, thereby participating in the development and progression of CAD. Further functional analysis is warranted to verify the hypothesis.
Taken together, we found SCAP rs17079634 was strongly associated with CAD, and the carriers of CT genotype or C allele may be at greater risk for CAD. However, several limitations of this study should be mentioned. First and foremost, our study enrolled solely Chinese Han subjects and can not draw ubiquitous conclusion. Second, sample size of the present study was relatively small, which may in uence statistical signi cance and power. Third, the conclusions drew in this work was based only on observational study. Overall, it is reasonable to conduct further association studies with larger sample, rational design and involvement of diverse ethnics with different genetic backgrounds to validate our results, moreover, functional tests should also be put on the agenda to elucidate its underlying molecular mechanisms.