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Frequency and association of disabled homolog 2-interacting protein (DAB2IP) variant rs7025486 G>A with coronary artery disease risk in Indian population

Genome wide association study has identified rs7025486 G>A polymorphism within DAB2IP (Disabled homolog 2-interacting protein) gene with increased risk of coronary heart disease (CAD). In this study we have determined the frequency and association of rs7025486 with CAD in Indians. The study was performed on 214 patients with CAD and 125 controls. The ‘AA’ genotype was associated with an increased risk in the CAD age group <50 yrs as compared to CAD age group>50 yrs (OR 3.149; P 0.034) and controls >50 yrs (OR 3.430; P 0.080). The risk allele (A) was significantly associated with premature CAD.


Introduction
In 2010, a Genome wide association study (GWAS) identified a sequence variant rs7025486 (G/A) within Disabled homolog 2-interacting protein (DAB2IP) to have a strong association with increased risk of vascular diseases like abdominal aortic aneurysm (AAA), pulmonary embolism, myocardial infarction (MI) and peripheral arterial disease (PAD) [1]. DAB2IP is a novel member of the Ras-GTPase activating protein family and acts as a tumor suppressor gene.
The minor allele (A) of rs7025486, present within intron-1 of DAB2IP gene on chromosome 9q33 was also found to be associated with 1.08-1.34 higher risk of coronary artery disease (CAD) [1,2]. CAD is a complex, multifactorial disease causing 3 million deaths per year in India alone [3,4].
Till date there is no information available on the frequency and association of DAB2IP rs7025486 G>A variant with CAD in Indians. The current study aims to address this lacunae by determining the frequency of this variant and its association with CAD/non-fatal MI in the Indian population.

Methods
The study population included a total of 339 unrelated individuals which consisted of 214 patients with CAD confirmed by coronary angiography: >50% stenosis in one or more arteries and stable or unstable angina and 125 controls: examined clinically and investigated by electrocardiography and tread mill test to exclude CAD. The study was approved by the local ethical committee and is performed in accordance with the Helsinki declaration. Blood specimens were collected using the vacutainer system from Becton Dickinson (Franklin Lakes, NJ USA).
Serum total cholesterol (TC) triglyceride (TG) and High Density Lipoproteincholesterol (HDL-C) levels, were determined by routine enzymatic endpoint methods (X Imola; Randox Laboratories Ltd, UK). Low density Lipoprotein-cholesterol (LDL-C) and VLDL cholesterol were calculated according to Friedwalds formula. Genomic DNA was extracted from EDTA whole blood using QIAamp1 DNA extraction kit (Qiagen, Germany).
DAB2IP (G/A) genotypes were determined by real-time PCR Taqman genotyping assay (Applied Biosystems).
Allele frequency were calculated, deviation of the genotype frequencies from the Hardy -Weinberg equilibrium (HWE) was assessed by Fischers exact test. Chi-square tests were used for comparison of binary variables across groups. Routine statistical analysis were carried out with the SPSS v 15 software (SPSS Inc., Chicago, IL). Under the significance level of P = 0.05, minor allele frequency between 0.25 and 0.40, assuming population disease prevalence between 5% and 10% and main genetic effect between 1.5 and 1.2, our study design can reach >85% power when the relative risk (RR) is 1.5 and 50% when it is 1.2. Table 1 displays means and standard deviations for the study subjects for relevant biochemical characteristics as well as risk factors. Statistically significant differences were seen in the smoking status (P < 0.005), presence of family history (P < 0.1), diabetes (P < 0.001) as well as hypertension (P < 0.001) in cases vs controls.

Results
The difference in the frequency of the rs7025486 (A) allele between cases and controls (0.31 vs 0.28), was not significant; OR = 1.157, CI-0.808-1.659, p-value-0.433 in the univariate analysis as well as in the multivariate analysis; OR = 1.42, CI = 0.85-2.35, p = 0.18 (Table 2a). However when the cases were sub-grouped based on age i.e > 50 years and < 50 years, almost 2-fold higher OR (OR 3.149 95% CI 1.181-8.389) with AA genotype was seen with CAD age group <50 yrs as compared to CAD age group >50 yrs along with statistically significant p-value (0.034) (Table 2b).

Discussion
CAD is a substantial and growing problem which has reached almost epidemic proportions in India [4]. This population based cohort study of CAD, determines for the first time the frequency and association of rs7025486 G/A SNP the risk of CAD in select Indian population. This sequence variant is important because it been associated with increased risk of CAD as well as other vascular diseases like AAA, PAD and MI [1].
The minor allele frequency of DAB2IP (A) allele in our study is 0.28, on comparing the allelic frequency in our population with that of other populations obtained from GWAS and HapMap database, it was seen to be almost similar to other populations (European 0.286, Japanese 0.215) [1,5].The HapMap reposition also has data on Asian-Sindhi population and the minor allele frequency of this variant was found to be 0.28 which is similar to the reported frequency in our population.
In the current study, higher frequency of the risk allele A was seen in cases (0.31 vs 0.28) as compared to controls. Both in the univariate and multivariate analysis a trend towards increased CAD risk was seen with the minor allele, albeit not reaching significance.
These findings are in agreement with a recent study where genotyping of 1386 CHD cases and 3532 controls was done, as well as meta-analysis using data from several studies such as the WTCCC [6], CVHS GWAS, Northwick Park Heart Study II (NPHS II), Simon Broome Study, HIFMECH, CABG and UDACS was performed [2,7,8]. The meta-analysis obtained an OR of 1.16, 95% CI 1.05-1.29 indicating statistically significant association of the SNP rs7025486 (A) with CAD. The rare allele was associated with 1.08-1.34 higher risk of CAD and interestingly, it was seen that in   each of the study groups a trend towards increased risk was seen, whereas only in the CABG (Coronary Artery Bypass Graft) a statistically significant increased risk with OR 1.2, 95% CI 1.03-1.46, P = 0.021 was seen [2]. Most interestingly in our study, the risk allele (A) of SNP rs7025486 conferred a significantly higher OR in the subset of subjects with premature CAD (CAD < 50 years) compared with CAD > 50 years. So it can be suggested that DAB2IP SNP could be a genetic marker for detection of premature CAD.
Though our current study has the limitation of sample size, but the similar frequency of this SNP with respect to other populations and a trend towards increased risk indicates that this can be an important marker even in our population. Larger studies are thus warranted selecting a sub-set of premature CAD cases in the Indian population

Funding source
No funding has been received for this study.