Investigation of KIF6 Trp719Arg in a Case-Control Study of Coronary Artery Disease in Western Indians

Abstract

Background: A single-nucleotide polymorphism Trp719Arg (rs20455T>C) in the kinesin-like protein 6 (KIF6), which is a protein involved in intracellular transport, has been shown to predict increased coronary artery disease (CAD) risk and event reduction during statin therapy. Aim: In the current study, we have evaluated the association of the variant Trp719Arg with CAD/non fatal myocardial infarction (MI) in Western Indians. Methods: Genotyping for Trp719Arg was done by an allele-specific real-time assay in 227 cases with confirmed CAD and 150 controls. Results: We have found that the KIF6 719 Arg carriers were not at a significantly higher risk for CAD/non-fatal MI in this case-control study of an Indo-European population from Western India (Unadjusted odds ratio [OR] 0.767 95% confidence interval [CI] 0.573-1.027 for 719Arg carriers). When the genotypes were further tested to determine association with prevalent myocardial infarction as the event versus CAD, no association was seen in a univariate analysis (MI vs. CAD OR 0.804 95% CI 0.543-1.189; MI vs. Controls OR 0.702 95% CI 0.482-1.021). Conclusion: In summary, carriers of the KIF6 719Arg allele were not at increased risk of CAD/non-fatal MI in a case-control study of Indians (Indo-Europeans) living in Western India.

Introduction

The Indian population accounts for among the largest incidences of coronary artery disease (CAD); also, deaths due to cardiovascular disease occur 5-10 years earlier than in the West (Yusuf et al., 2004; Gupta, 2005). The risk factors such as age, blood pressure, cigarette smoking, total cholesterol (TC), high-density lipoprotein cholesterol, diabetes status, and so on were seen to have a similar collective impact as in other countries (Joshi et al., 2007). This has raised the possibility that South Asians exhibit a special susceptibility for CAD that is not explained by traditional risk factors. Genetic polymorphisms contributing to CAD incidence are another type of emerging risk factor under investigation (Morrison et al., 2007; Samani et al., 2007; Talmud et al., 2008).

Recently, a common single-nucleotide polymorphism (SNP) Trp719Arg (rs20455T>C) in kinesin-like protein 6 (KIF6), a member of the KIF9 family of kinesins, has been reported to be associated with CAD risk. The variable 719 residue of KIF6 is located in the tail domain, a domain shown to bind cargo molecules and to regulate motor activity and microtubule binding in other kinesins (Woehlke and Schliwa, 2000). The role of the KIF6 kinesin in cardiovascular disease is not known; however, KIF6 is expressed in coronary arteries (King et al., 2005).

In several large studies, including the cholesterol and recurrent events trial (CARE), West of Scotland coronary prevention study (WOSCOPS) (Iakoubova et al., 2008b), women health study (Shiffman et al., 2008a), atherosclerosis risk in communities (Bare et al., 2007), and the cardiovascular health study (Shiffman et al., 2008b), the KIF6 719Arg (rs20455C) variant has been associated with increased risk of CAD and differential reduction of coronary events from statin therapy. In a meta-analysis of seven prospective studies, carriers of the 719Arg variant had more than a 20% increased risk of CAD, compared with noncarriers (Li et al., 2010).

However, no association between the KIF6 Trp719Arg polymorphism and CAD was observed in three case-control studies of the predominant Caucasian population (WTCCC, 2007; Stewart et al., 2009; Assimes et al., 2010). Similarly, carriers of the KIF6 719Arg allele were not at increased risk of non-fatal myocardial infarction (MI) in a case-control study of a Costa Rican population (Bare et al., 2010). In recent times, even the purported association of the KIF6 Trp719Arg variant with statin response has become debatable. In the Heart Protection Study (HPS) (Hopewell et al., 2011), which included 18,348 patients randomized to 40 mg simvastatin or placebo, no impact of the KIF6 genotype with statin response was seen. Similarly, in the recently completed Justification for the Use of Statins in Primary Prevention: An Intervention Trial Evaluating Rosuvastatin (JUPITER) trial, rosuvastatin was equally effective at reducing cardiovascular event rates among both carriers and non-carriers of the KIF6 719Arg allele (Ridker et al., 2011).

Hence, the Trp719Arg variant is currently a source of controversy, but nevertheless, it is an important marker, because not only has it been associated with increased CAD risk but it has also been associated with response to statins (Iakoubova et al., 2008a, 2008b, 2010; Shiffman et al., 2008a, 2008b). Replication of the predictive ability of this marker is needed in other population-based samples.

There are very limited data available regarding whether the KIF6 719Arg allele is associated with CAD in other ethnic populations. Therefore, we asked whether the KIF6 719Arg allele is associated with CAD or MI risk in a select Indian population (Indo Europeans).

Materials and Methods

Subjects

The study was performed on a total of 377 unrelated individuals that consisted of 227 patients with CAD confirmed by coronary angiography: >50% stenosis in one or more arteries and stable or unstable angina and 150 controls: examined clinically and investigated by electrocardiography and treadmill stress test to exclude CAD. Informed consent was obtained from all the subjects. The study is in accordance with the Helsinki declaration and was approved by the local ethical committee. A detailed case-record form pertaining to information on demographics, medical history, and coronary risk factors such as presence of diabetes, hypertension, smoking, lifestyle, and current medication was completed for each participant through personal interviews and through perusal of their medical records.

Blood specimens were collected by veni-puncture after an overnight fast of 12 h using the vacutainer system from Becton Dickinson in the anticoagulant ethylenediaminetetraacetic acid (EDTA) as well as plain bulb for serum. Serum, EDTA plasma samples were separated by centrifugation; and aliquots were preserved at −20°C till analysis.

Biochemical parameters

The laboratory parameters, namely serum TC triglyceride and high-density lipoprotein-cholesterol levels, were determined by routine enzymatic endpoint methods (X Imola; Randox Laboratories). Low-density lipoprotein-cholesterol and very low density lipoprotein (VLDL) cholesterol were calculated according to Friedwald's formula. Serum apolipoproteins A1 and B were measured by the Immunoturbidimetric immunoassay (Daytona; Randox Laboratories).

Genotyping

The KIF6 Trp719Arg genotypes were determined for all study participants using an allele-specific real-time polymerase chain reaction Taqman genotyping assay. The DNA was standardized to 10 ng/mL.

Statistical analysis

Allele frequency was calculated as the number of occurrences of the test allele in the population divided by the total number of alleles. Any deviation of the genotype frequencies from the Hardy-Weinberg equilibrium was assessed by Fischer's exact test. Chi-square tests were used for comparison of binary variables across groups. The nonparametric Kolmogorov-Smirnov test was used as a test of normality for the quantitative variables, and the values were subsequently log-transformed to normalize distribution. To determine risk for CAD, odds ratios (OR) were determined by both univariate analysis and multivariate analysis adjusting for other covariables. Routine statistical analysis was carried out with the SPSS v 15 software (SPSS), and GraphPad. StatMate 2.0 (GraphPad software) was used to determine the power of the study. 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 the main genetic effect between 1.5 and 1.2, our study design can reach >85% power when the relative risk is 1.5% and 50% when it is 1.2.

Results

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<0.01), presence of family history (p<0.0001), diabetes (p<0.0001), and hypertension (p<0.0001) in cases versus controls. Genotypic and allelic frequencies of the rs20455 SNP are shown in Table 2. No significant departure from the Hardy-Weinberg equilibrium was seen as assessed by the Fischer's exact test. A higher frequency of the rs20455 C allele was seen in controls as compared with cases (0.51 vs. 0.45). No statistically significant differences were seen between the allelic frequency in cases and controls (OR 0.767; 95% confidence interval [CI] 0.573-1.027; p=0.087) in the univariate analysis (Table 2). When the genotypes were further tested to determine association with prevalent myocardial infarction as the event versus CAD, no association was seen in a univariate analysis (Table 3). No association of the genotypes with severity of CAD or age was also seen (data not shown).

Table 1.

General Characteristics of Cases and Controls

General characteristics	Cases (n=227)	Controls (150)
Age (years)	55±11	50±11
Sex (male)	89	70
Smoking Status
Smoker (upto 10 cigs/day)(%)	22^a	13
Ex-smoker (%)	18.5^b	6
Alcohol (%)	18	18
Hypertension (%)	50^b	18
Diabetes (%)	39^b	11
Family history(%)	50^b	13
Plasma cholesterol (mg/dL)	164.8±52.9	192.8±35.3
HDL-cholesterol (mg/dL)	38.9±13.3	40.8±11
LDL-cholesterol (mg/dL)	98.1±45.8	125±30.2
VLDL-cholesterol (mg/dL)	30.5±19.4	30±17.6
Triglycerides (mg/dL)	151.9±96.4	152±88
Single vessel disease (%)	39	NA
Double vessel disease (%)	8	NA
Triple vessel disease (%)	53	NA
Myocardial Infarction (%)	39	NA

p<0.01.

p<0.0001.

HDL, high density lipoprotein; LDL, low density lipoprotein; VLDL, very low density lipoprotein; NA, not applicable.

Table 2.

Genotype and Allelic Frequency in Cases and Controls

	Genotype frequencies n (%)			Major	Minor	OR ^a T versus C (allelic)	95% CI p-value	OR ^a CC versus TT	95% CI p-value	OR ^a CT versus TT	95% CI p-value
KIF6 Trp719Arg	TT	TC	CC	T	C
Cases (n=227)	70 (31)	111 (49)	46 (20)	0.55	0.45	0.767	0.573-1.027	0.586	0.323-1.064	0.654	0.396-1.018
Controls (n=150)	33 (22)	80 (53)	37 (25)	0.49	0.51		0.087		0.095		0.104

Unadjusted OR derived using Cochran-Mantel-Haenszel statistics. Allele frequencies were estimated by direct counting.

OR, odds ratio; 95% CI, 95% confidence interval.

Table 3.

Genotype and Allelic Frequency and Association of KIF6 Trp719Arg in Coronary Heart Disease (Non MI) Versus MI Cases and MI Versus Controls

	Allele frequency
Outcome	T	C	OR	95% CI	p-Value
CAD	0.52	0.48
MI	0.57	0.43	0.804^a	0.543-1.189^a	0.318^a
Controls	0.49	0.51	0.702^b	0.482-1.021^b	0.070^b

MI versus CAD (non-MI).

MI versus controls.

CAD, coronary heart disease; MI, myocardial infarction.

Discussion

To the best of our knowledge, this is the first article that reports the frequency of this variant in the select Indian population. The minor allele frequency KIF6 719Arg in our study is 0.51. The KIF6 (719Arg) allele frequency reported in the HapMap database differs between different populations (European 0.358, Han Chinese 0.556, Japanese 0.386, Sub Saharan African 0.908). The allelic frequency in our study when compared with these is similar to that seen in the Chinese population, higher than Caucasians and Japanese, and lower than the Sub-Saharan African population. The Costa-Rican population, an admixture of three populations, Southern Europeans, Amerindians, and West Africans, has a minor allele frequency of 0.345 (Bare et al., 2010), which is much lower than that seen in our study.

We have found that the KIF6 719 Arg carriers were not at a significantly higher risk for CAD/non-fatal MI in this case-control study of an Indo-European population from Western India either in the univariate (Unadjusted OR 0.767 95% CI 0.573-1.027 for 719Arg carriers) or in the multivariate analysis after adjustment of traditional risk factors; rather, it was associated with a trend toward reduced risk. However, the same cohort has also been analyzed with regard to the 9p21 variant, where a significant association was found with CAD (manuscript communicated). Strangely, in the current study, a higher frequency of the risk allele C allele was seen in controls (0.51 vs. 0.45) as compared with cases; our controls are not on any lipid lowering medications. Further, when the cases were sub-grouped into those with angiographically defined CAD and those who have suffered non-fatal MI, 719Arg carriers with MI did not show significance when compared both with controls as well as with CAD only.

Our results corroborate the findings of the Ottawa Heart Genomic Study (OHGS), a case-control study that investigated the association between KIF6 719Arg and MI in a population with angiographically defined CAD (Stewart et al., 2009). The OHGS data do not support the hypothesis that Caucasian carriers of the 719Arg allele of KIF6 have increased risk of CAD or MI. Moreover, data from the WTCCC (2007) also showed no association of rs20455 with CAD (trend p=0.897). Since 71% of cases in the WTCCC had an MI, it is also doubtful whether rs20455 SNP associates with MI in this cohort.

Similarly, the study on the Costa-Rican population (Bare et al., 2010) found that KIF6 719Arg carriers were not at a significantly higher risk for non-fatal MI in this study after adjustment for traditional risk factors or admixture (OR=1.12; 95% CI, 0.98-1.28). Though in the study, heterozygotes of the KIF6 Trp719Arg variant were at an increased risk of non-fatal MI, the adjusted OR was 1.16 (95% CI, 1.01-1.34), but according to the authors would not be significant after a multiple-testing correction.

In the placebo group of the Prospective Study of Pravastatin in the Elderly at Risk (PROSPER) cohort, too, carriers of the KIF6 719Arg variant were not at an increased risk for fatal or non-fatal coronary events compared with non-carriers adjusted hazard ratio 1.06 (95% CI 0.86-1.30; p=0.59) (Iakoubova et al., 2010).

In another large replication study (Assimes et al., 2010) on a total of 17,000 cases and 39,369 controls of European descent as well as a modest number of South Asians, African Americans, Hispanics, East Asians, and admixed cases and controls, none of the 19 studies demonstrated an increased risk of CAD in carriers of the 719Arg allele compared with noncarriers. No increase in the risk of CAD among 719Arg carriers was also seen in the subset of Europeans with early-onset disease.

These results are in contrast to five prospective studies (Iakoubova et al., 2008a, 2008b, 2010; Shiffman et al., 2008a, 2008b) where the 719Arg carriers had up to 55% increased risk for CAD events. Also, in the CARE (Iakoubova et al., 2008b) and WOSCOPS trial (Iakoubova et al., 2008b), KIF6 carriers on pravastatin therapy had a 5.6 and 1.9-fold greater event reduction as compared with non-carriers. Among carriers, the absolute risk reduction by pravastatin was 4.89% (95% CI 1.81%-7.97%) in the CARE trial and 5.49% (95% CI 3.52%-7.46%) in the WOSCOPS trial. Further, it was also seen that the KIF6 carriers received greater benefit from 80 mg atorvastatin, compared with 40 mg pravastatin, than did noncarriers in the Pravastatin or Atorvastatin Evaluation and Infection Therapy trial (Iakoubova et al., 2008a). In the PROSPER trial, an association between carriers and previous vascular disease was observed; only carriers of KIF6 719Arg with previous vascular disease received substantial and significant reduction of coronary events from statin therapy: absolute risk reduction was 6.3% in carriers versus 1.2% non-carriers (Iakoubova et al., 2010).

However, recently, the association of KIF6 Trp719Arg variant with statin response has also become arguable. The HPS which involved considerably greater incidence of vascular events was able to demonstrate that statin therapy produces similar proportional reductions in the risk of major vascular events among KIF6 carriers and non-carriers (23% and 24% respectively) and also that these benefits are highly significant (Hopewell et al., 2011). Also, in the JUPITER trial, the KIF6 genotype had no impact on rosuvastatin efficacy, an almost identical reduction in the primary endpoint (major vascular event) was seen among carriers (hazard ratio [HR] 0.61, 95% CI 0.43-0.87) as among non-carriers (HR 0.59, 95% CI 0.39-0.88) (p-interaction=0.90) allele (Ridker et al., 2011).

In summary, the carriers of the KIF6 719Arg allele were not at an increased risk of CAD/non-fatal MI in a case-control study of Indians (Indo-Europeans) living in Western India. Our results corroborate the findings of other case-control studies and are in contrast to the results of prospective studies on this variant. Further multi-centric studies are warranted especially in the Indian population to address the association between carriers of the KIF6 719Arg and MI and also to determine why there are differences between this and other case-control studies and the previously reported prospective studies.

Footnotes

Disclosure Statement

No competing financial interests exist.

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