Association of APOA1-75G/A and +83C/T polymorphic variation with acute coronary syndrome patients in Kashmir (India)

Introduction: Acute coronary syndrome (ACS) comes under the ambit of cardiovascular disease.APOA-1 gene plays a vital role in lipid metabolism and has been observed to have plausible role in ACS. This cross sectional case-control study was conducted to evaluate association between APOA 1-75G/A(rs1799837), +83C/T (rs5069) genotypes and risk for ACS. Methods: The current case-control study that included confirmed 90 ACS cases and 150 healthy controls were genotyped for APOA 1-75 G/A and +83 C/T by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLF) method. Results:APOA 1-75G/A distribution of genotypes/alleles among cases and controls was seen proportionally same with no association to ACS (P = 0.5). APOA 1+83 C/T variants showed protective effect with ACS where variant TT genotype presented more in controls (12%) than cases (1.6%) (P = 0.004) and likewise variant ‘T’ allele was found more in controls than ACS cases (9.4% vs.28.5% respectively: P < 0.05). Further, significantly high difference of CT genotype was seen among cases and controls 15% vs. 33% respectively (P = 0.002). The overall distribution of different haplotypes showed a marked difference in GT when compared with GC between cases and controls (P = 0.0001). Conclusion: The study shows that TT genotype and variant T allele of APOA 1 +83 C/T depicted a protective role with respect to ACS whereas APOA 1-75G>A showed no relation. Haplotype GT was observed to significantly over-represent in controls with its protective effect in ACS as against wild type haplotype GC.


Introduction
Acute coronary syndrome (ACS) is one among several conditions of cardiovascular disease. 1 ACS is believed to determine clearly the disease progression associated with myocardial ischemia and includes unstable angina and myocardial infarction (MI) under its ambit. 2 Symptomatically ACS comprises a continuous intensity from non-ST segment elevation MI (NSTEMI) to STsegment elevation MI (STEMI). The Indian origin from the Asian subcontinent population accounts for a fifth of all worldwide deaths from CHD that includes ACS and overall 7 million deaths occur as per the estimates each year together due to CAD and ACS. 3,4 ACS comprising about half of the global burden is now considered to be the prime cause of deaths in the Asia-Pacific region. 4 ACS is a multi-faceted problem that results due to genetic variations, environmental effect, dietary habits, and lifestyle. It is known that ACS often results from the compound effects of altered multiple genes where in most cases it is not clear whether ACS is inherited in a dominant or recessive manner. Currently the LDL receptor (LDLR) gene, 5 ApoB-100 gene, [6][7][8][9] ARH gene 10 and ABCA1, 11,12 have been identified as disease causing genes for familial hypercholesterolemia and Tangier disease, a risk factor for ACS.
The APOA1 -75 G/A and +83 C/T gene polymorphic variations is comprehensively investigated and found to be associated with other disease as well. Bi Hong et al 13 investigated the role of APOA1 -75 G/A and +83 C/T SNPs and found APOA1-75 A allele to have a lower risk of CAD. Also, a pilot study in North India 14 found association of APOA1 +83 C/T in patient with MI. In fact, it is strongly believed that above APOA1 polymorphic sequence variations have an important role in a host of other diseases like Alzheimer's, breast cancer and schizophrenia etc. The genetic nature of variability accounts for nearly 50% in the plasma HDL cholesterol concentration and among many one such sequence variants is within the APOA1 gene, where a guanine to adenine transition occurs 75 base pairs upstream from the start of transcription (-75G/A) and other site (first intron) of the APOA1 gene results with transition of cytosine to thymine at +83bp (+83 C/T). Although ACS is the most prevalent disease throughout the world yet our population (Kashmir province, North India) is also heavily burdened with the same disease. The overall prevalence of coronary artery disease in our region as calculated by different diagnostic procedures stands at 7.54% where its frequency in males is higher 7.80% versus 6.63% in females. 15 Keeping the well-recognized role of APOA1 gene in ACS in many populations of the world but no study till date has been done on APOA1 gene SNPs with respect to ACS in Kashmiri population. Thus with an increasing number of ACS cases being reported here with lack of any significant study, we designed our study with an aim to demonstrate the association between various genotypes of APOA1 -75 G/A, +83 C/T and ACS.

Material and Methods
The current cross-sectional case-control study was conducted at Advanced Center for Human Genetics, Sheri-I-Kashmir Institute of Medical Sciences (SKIMS), North India. A total of 240 subjects were included in the study comprising of 90 ACS patients and 150 healthy controls free from any disease who visited the hospital for general checkups. Consent information was duly sought from each ACS patient and healthy controls. Clinical parameters were recorded according to the given proforma. The inclusion criteria of ACS patients included: ACS with or without undergoing percutaneous coronary intervention, ST elevation myocardial infarction, non-ST elevation myocardial infarction, unstable angina. The exclusion criteria was history of bleeding diathesis, stroke less than 3 months platelet count<70 000/mm 3 , hematocrit <30%

Extraction of genomic DNA and polymerase chain reaction for amplification
The blood samples of ACS patients and healthy controls were subjected to extraction of genomic DNA by common phenol-chloroform method as well as kit based DNA Extraction (Zymo Research Corporation, USA). The quality and quantity was determined by absorbance at 260nm and 280nm in a Spectrophotometer or by running on 1% agarose gel. The isolated DNA was stored at -20C• until for further analysis.

Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP)
PCR-RFLP was performed to genotype the APOA1-75 G/ A and +83 C/T polymorphic sequence variants using 5-10 units of restriction endonuclease enzyme MspI (NEB, New England Biolabs), to digest amplified product overnight at 37°C. The digested product was subjected to electrophoresis in a 3% agarose gel followed by ethidium bromide staining and ultraviolet illumination to identify the genotypes. MspI restriction site at APOA1 -75 bp (G allele) and at +83 bp (C allele) digests into 209, 113, 45 and 66bp. The lack of the restriction site at-75 (A allele) and +83(T allele) displays 3 products of 209, 179, 45bp and one single product 254bp respectively ( Figure 1).
For quality control and reproducibility of results, two independent researchers randomly selected 10% samples from both control and cases for genotyping.

Statistical analysis
IBM Statistics SPSS software (Version-23) was used for statistical evaluation. The cases and controls were compared using the Chi-square test for various categorical parameters, likegender and age of the demographic variables. A goodness-of-fit Chi-square test was used to analyze if the polymorphisms were in Hardy-Weinberg equilibrium between cases and controls. Odds ratios (OR) were used as estimates of the relative risk, and 95% confidence intervals (CI) were calculated to estimate the association between certain genotypes or other related

Results
The clinico-pathological characteristics of ACS patients and controls are listed in Table 1. The cases and controls were frequency matched in terms of their age, gender, and smoking status. In this study ratio of the cases, male: female was 3:1 (67 versus 23 respectively).The mean age was 60 ± 15 years for cases and 55 ± 15years for the controls.
Of the total number of cases and controls, 61(72.6%) and 115(77%) were smokers respectively. In the study groups, no significant sex-related or smoking status differences were observed between the cases and controls (P > 0.05).
In cohort group, 81.4% ACS cases had hypertension. ACS STEMI group numbered 71% as compared to 22.2% while as preMI included 19.1% versus 80.9% with no pre MI history. The demographic features of the rest of parameters for ACS patients are recorded in Table 1.
Overall, the distribution of genotypes for APOA1-75G/A among cases and controls was seen almost proportionally the same in all the genetic models performed that showed no significant difference among two groups (P > 0.05; Table 2).When classified further into groups, no major difference in frequency of APOA1-75G/A genotype was found in age, gender or patients with different smoking status as against the control group (P > 0.05; Table 3). When the patients were classified with respect to different pathologies, like hypertension, although variant genotype GA+AA was found more often in hypertensive (77.3%) than non-hypertensive (22.7%) group, yet the difference was insignificant, OR = 0.63 (C.I:0.15-2.5; P = 0.3). Despite the differences in the frequency of APOA1-75G/A genotypes among different parameters of ACS, none of them could achieve the significant association (P > 0.05) ( Table 3).
When the patients were classified as cohort with respect to different pathologies as shown in Table 3, no significant differences were observed within different parameters with respect to variant genotypes of APOA1 +83 C/T.
Haplotypic analysis was performed to demonstrate the pattern of linkage disequilibrium and few haplotypes were found with frequencies more than 5% among both cases and controls and those with <1% frequency was not considered. Table 4 shows the frequencies for the estimated 2-marker haplotypes among patients and controls. The haplotype observed in cases and controls with highest frequency as G/C haplotype that accounted for 40.0% in cases and 27.4% in controls of the SNPs studied haplotypes (APOA1-75G/A andAPOA1 +83 C/T). Haplotype GT showed a marked difference when compared with wild type GC between cases and controls (P < 0.0001) as depicted in Table 4.

Discussion
Apolipoprotein A1, a protein in humans is encoded by the APOA1 gene that has a vital role in lipid metabolism.    APOA1 is a major protein ingredient of HDL and a relatively abundant plasma protein. 17 In the APOA1 gene -75 G/A and +83C/T, play an important role in lipid metabolism. [18][19][20] Regarding ACS, the studies have been very limited to reach the conclusive remark. A cross sectional case-control study was conducted to observe the role of APOA1 gene -75 G/A and +83C/T variations with respect to ACS in our population (Kashmir province, North India). In our report, APOA1 -75 G/A, the frequency of three genotypes of APOA1-75 G/A SNP among ACS patients and the controls revealed no significant differences. Similar scenario has been published by Yan Ding et al, 21 that reported lack of association for APOA1-75 G/A in ACS. Further, APOA1 -75 G/A and ACS risk was not proved in European population with 98.6%, 1.3% and 0.1% genotypic frequency in cases and 99.2%, 0.8% and 0% in controls. 22 It is worthwhile to mention that the frequency of APOA1-75 G/A varies considerably among different ethnic regions as can be seen among three studies which show quite different genotypic differences in both cases and controls. In our study frequency of variant A allele APOA1 -75 G/A showed no association which is supported by similar scenario reported by Yan et al. 21 On the contrary, a study in Australian population, 18,19 found that the presence of the variant A allele increased the severity of ACS, a finding that is in stark contrast to our study and one more report carried earlier. 21 In this study, combined genotype (GA+AA) was found comparatively higher in smokers of control group (controls: 82.9% versus cases 78.3%). This is in discordance with the study by Morgan et al. 23 Also, the combined genotype (GA+AA) was found more often in hypertensive in accordance with the study performed in the Chinese population. 13 Interestingly, in contrast to our study, Morgan et al, 23 has shown an association between the APOA1-75G/A polymorphism and diabetes mellitus (DM). The possible reasons for this contradiction are unclear though these could be due to different geographical regions. It must be stressed that even among populations of similar ethnicity and gene pool, environmental factors such as diet, stress and physical inactivity compound to an individual's composite risk for ACS in inexplicable proportions.
In our study, APOA1 +83C/T showed a protective role with respect to the development of ACS where the frequencies of different genotypes showed significant differences between cases and controls. The frequency of homozygous variant genotype TT allele was observed significantly higher in controls than cases (12% versus 1.6% respectively). This clearly demonstrates a significant association of APOA1+83 C/T variant genotype with ACS in our population. APOA1 +83 variant T allele was found to be higher in controls (0.34 versus 0.12) than cases and clearly demonstrates that T allele has uniquely a protective role for ACS in our population. In contrast to our findings, Morgan et al, 23 found no association between ACS and APOA1 +83 C/T gene polymorphism. The proposed protective role of APOA1 +83 variant T allele as found in our study is substantiated by an investigation by Wang et al. who found T allele associated with increased levels of HDL and APOA1, an evidence that suggests protective role over ACS. 19 One of the possible explanations for this transition is that APOA1 +83 reside in the 5′ region of the APOA1 gene. The MspI site at APOA1+83bp embedded with CpG island is known to be hypermethylated in non-expressing cells but demethylated in cells expressing APOA1. It is possible that T substitution at this site may lead to further demethylation, resulting in more cells expressing the gene.
Further, in our study APOA1 +83 C/T variations was associated with ACS in men. This is partially in accordance with an earlier study that found no association in men and women. 23 Our study found no significant association of ACS was found with hypertension and diabetes. Our results are in accordance with another study conducted earlier. 23 On the basis of cardiovascular events pre-MI, STEMI, NSTEMI and USA, no association with APOA1 +83 C/T was found in contradiction with the study by Morgan et al. 23 Discrepancies in the findings of these studies with our report could be attributed to differences in the genetic susceptibility between different ethnic groups. Also, the APOA1 gene locus resides in a cluster with CIII and AIV loci, which could attribute to linkage disequilibrium with different APOA1 alleles in some but not in other populations.
The putative role of APOA1 -75 G/A and +83 C/T has been studied in various diseases across the globe where prominent diseases that have been complied in this study to highlight the implication and role of this sequence variation include hypertension, 24 renal cancer, 15 Alzheimer's disease, 22 CAD, 13 and all the diseases have shown distinct but varied associations. This augments for the need to study the role of APOA1 in more diseases.
Further, the effect of a single polymorphic nucleotide variation is questionable to lead to an outcome in the study of multifaceted diseases. Therefore, the amalgamation of different genetic variants in the set of connections of same loci strengthens their efficiency to improve the analytical influence for complex diseases. 25,26 To demonstrate whether APOA1−75 G/A and +83 C/T polymorphic variants could translate a synergistic effect in the development or progression of ACS, we looked at their collective impact to examine the inclination to the disease. The most frequently implicated haplotype was GT variant haplotype that showed a significant protective impact on ACS as compared to wild type haplotype GC. Our study showed the haplotypes at two restriction sites as more informative and demonstrated a significant association together to confer a protective role in ACS. In consistence with our data, there a few reports that have shown more or less the same trend, 27,28 implicating that combining both polymorphic sites of APOA1 −75 G/A and +83 C/T sites as more informative and significantly portray the combined effect with almost twice the amount of phenotypic variation in plasma APOA1compared to single RFLP site. This study shows that haplotype analysis in the APOAl gene is very important to display its functional significance in ACS.

Conclusion
The study concludes that APOA1 -75G/A has no association with ACS but its other related polymorphic variant T allele and TT genotype of +83 C/T was observed to confer a protective effect with respect to the same condition. Our report further implies that a particular haplotype (GT) of APOA1 gene is highly implicated and possibly can act as low penetrance genotypes in the predilection to ACS. These findings are subject to be validated in large sample cohort studies to decide the course of ACS management at molecular perspective.