Investigation of LPA sequence variants rs6415084,rs3798220 with conventional coronary artery disease in Iranian CAD patients

Abstract

BACKGROUND:

Conventional coronary artery disease (CAD) is the leading cause of morbidity and mortality in the general population. In recent years, multiple CAD promising risk factors have been reported and used for risk stratification. Lipoprotein(a) [LPA] concentration in plasma was shown associated with CAD risk and LPA genetic variants in different ethnic groups remains less clear.

METHODS:

We obtained data from 100 affected patients with established CAD and 100 healthy controls. We tested Body mass index (BMI), total cholesterol level (TC), systolic blood pressure (SBP), diastolic blood pressure (DBP), low-density lipoprotein (LDL), high-density lipoprotein (HDL), triglycerides (TG), fasting blood sugar (FBS) and two LPA (rs10455872 and rs3798220 SNPs) between cases and healthy controls. TaqMan SNP genotyping assays were performed to detect variants.

RESULTS:

Obtained data for BMI, TC, SBP, DBP, and LDL have significantly difference between two groups. Individually, the single SNPs were not associated with CAD in different analysis models. Also there was no significant difference in the incidence of CAD among cases carrying different genotypes of the two variants in LPA with $p>$ 0.05.

DISCUSSION:

In this study patients with CAD, lipoprotein(a) concentrations and genetic variants showed no associations and we conclude that these variables are not useful risk factors to predict progression to disease is Iranian population. However, the prevalence and association of LPA SNPs with size of LPA and isoforms are highly variable and genetic background-specific.

CONCLUSION:

Our data did not indicate a relationship between genomic LPA variants (rs10455872 and rs3798220) and subsequent cardiovascular events in Iranian CAD patients. We did not confirm the association of the theses SNPs with CAD in our samples of Iranian patients. For the studied variants, our finding is consistent with reports which showed the lack of this genetic association in other populations.

Keywords

Cardiovascular disease genome-wide association lipoprotein(a)

1. Introduction

Coronary artery disease is defined as a silently chronic progressive disease and usually symptoms of affected patient start appearing by the time. Also it is known as the leading cause of death worldwide according to latest WHO reports. Risk factors like age, gender, race, hypertension, blood lipids, diabetes, weight, smoking and environmental factors have been the regarded for risk prediction of CAD behind genetic background. The prevalence of disease in South Asians is higher than other regions of word and middle and low income countries have the more affected population in comparing with high income countries [1, 2]. Due to the main role of high-density lipoproteins (HDL) in the pathogenesis of atherosclerosis, many studies have been done on the antiatherogenic potential of HDL particles for several years. However, circulating HDL particles may not clearly reflect the antiatherogenic potential of different HDL subpopulations because of heterogeneous groups of particles such as lipid, protein or cholesterol content and function [3]. Their apolipoprotein (ApoA) composition are regarded to classification between those that have ApoA-I without ApoA-II [lipoprotein (Lp) A-I] and those that have both ApoA-I and ApoA-II (LpA-I/A-II). The physiological function of lipoprotein(a) is still remained unknown and one copy of the LpA glycoprotein is connected by a disulfide bridge LDL-like core containing apolipoprotein B [4, 5]. It seems that LpA-I promotes cell cholesterol efflux, while LpA-I/A-II cannot it. Also, LpA is known as likely causal risk factor for myocardial infarction, stroke, peripheral arterial disease1,2 and calcific aortic valve stenosis. Transcription speed of circulating plasma Lp(a) are largely genetically regulated in several levels including biochemical influences on transcription factors, sequence variants in LPA single nucleotide polymorphisms (SNPs) and the inter or intra individual heterogeneity in kringle IV type 2 (KIV2) isoform repeats [6, 7].

To this day, relation between increased LPA level and cardiac problems in patients with CAD has been less studied. Furthermore, risk might be modified by single nucleotide polymorphisms in LPA gene and promoter region. Two common LPA variant, rs10455872 (intronic non-coding) and rs3798220 (missense variant Ile4399Met in the apolipoprotein(a) protease-like domain), responsible for much of the difference in LPA concentrations, and are associated with the risk of incident myocardial infarction. We aimed to investigate systematically whether lipoprotein(a) and two LPA SNPs are associated with CAD and disease severity in Iranian population.

Table 1
Lipoprotein(a) SNPs and genotypes in this study

Gene	Locus	SNPs	Function	Position (kb)b	Alleles
LPA	6q26–27	rs10455872	Intron	161010118	A/G
		rs3798220	Missense	160880877	T/C

Table 2

Comparison of clinical between cases and healthy controls

Characteristics	Cases		Controls		$p$ -value
BMI (kg/m ${}^{2}$ )	26.8	$\pm$ 3.63	35.3	$\pm$ 7.18	35.3	$\pm$ 7.18
SBP	126.57	$\pm$ 17.76	153.76	$\pm$ 28.36	153.76	$\pm$ 28.36
DBP	77.33	$\pm$ 11.65	88.17	$\pm$ 13.2	88.17	$\pm$ 13.2
TC (mg/dL)	166.89	$\pm$ 54.62	194.77	$\pm$ 34.45	194.77	$\pm$ 34.45
TG (mg/dL)	148.14	$\pm$ 79.76	154.41	$\pm$ 95.27	154.41	$\pm$ 95.27
HDL (mg/dL)	47.48	$\pm$ 21.94	48.48	$\pm$ 12.53	48.48	$\pm$ 12.53
LDL (mg/dL)	88.19	$\pm$ 40.62	112.71	$\pm$ 24.19	112.71	$\pm$ 24.19
FBS (mg/dL)	110.26	$\pm$ 36.66	105.09	$\pm$ 33.08	105.09	$\pm$ 33.08

Table 3

Different models of rs10455872 genotype frequency

Model	Genotype	Case	Control	OR	95% Cl	$p$ -value
Co dominant	AA	96 (96%)	95 (95%)	1	Reference	0.73
	AG	4 (4%)	5 (5%)	1.26	0.32–4.84
	GG	0 (0%)	0 (0%)	1.23	0.93–1.71
Dominant	AA	96 (96%)	95 (95%)	1	Reference	0.73
	AG-GG	4 (4%)	5 (5%)	1.26	0.32–4.84
Recessive	AA-AG	100 (100%)	100 (100%)	1	Reference	0.99
	GG	0 (0%)	0 (0%)	0.99	0.66–1.48
Over dominant	AA-GG	96 (96%)	95 (95%)	1	Reference	0.73
	AG	4 (4%)	5 (5%)	1.26	0.32–4.84

Table 4

The RAFs in cases versus controls for rs10455872 variant

Allelic	Case	Control	OR	95% Cl	$p$ -value
frequency
A	196	195	1	(Reference)	0.736
G	4	5	1.26	0.32–4.84

Table 5

Different models of rs3798220 genotype frequency

Model	Genotype	Case	Control	OR	95% Cl	$p$ -value
Co dominant	TT	98 (98%)	100 (100%)	1	Reference	0.15
	TC	2 (2%)	0 (0%)	1.23	0.95–1.76
	CC	0 (0%)	0 (0%)	2.34	1.24–4.15
Dominant	TT	98 (98%)	100 (100%)	1	Reference	0.15
	CC-TC	2 (2%)	0 (0%)	1.23	0.95–1.76
Recessive	TT-TC	100 (100%)	100 (100%)	1	Reference	0.99
	CC	0 (0%)	0 (0%)	0.93	0.62–1.84
Over dominant	CC-TT	98 (98%)	100 (100%)	1	Reference	0.15
	CT	2 (2%)	0 (0%)	1.23	0.95–1.76

Table 6

The RAFs in cases versus controls for rs3798220 variant

Allelic	Case	Control	OR	95% Cl	$p$ -value
frequency
A	196	195	1	(Reference)	0.156
G	4	5	1.24	0091–1.62

2. Material and methods

The case-control study was conducted in the Department of Medical Genetics. Study was approved by the Institutional Ethical review committees of Shahid Beheshti. A total of 100 subjects aged $\geqslant$ 40 years who were stable and were due to undergo coronary angiography with angio score $\geqslant$ 50. The participants had an episode of left sided chest pain and were advised angiography by consultant cardiologists after assessing. Subjects who displayed $>$ 50% stenosis in one coronary artery on angiography were selected in case group. Those patients who had a history of heart disease by birth, angina, previous MI, diabetes mellitus, infectious, autoimmune diseases, hyperlipidemia (familial), arthritis along with those not giving informed or written consent were excluded in the study. Moreover, 100 sex and age matched healthy controls that were declared to be disease free on angiography were included. Also subjects with acute or chronic ailment or taking anti-inflammatory drugs, high systolic blood pressure more $>$ 140, high diastolic blood pressure more $>$ 90, diabetes mellitus with FBS $>$ 126, cholesterol more $>$ 220 was not included in the control group. General physical examination including detailed history and physical examination was performed by a medical practitioner. Ten ml blood sample was taken from the femoral vein in the EDTA tubes. Also five ml whole blood was collected into EDTA tube to extract genomic DNA using Gene All kit (Gene All, Korea).

2.1 Genetic associations

The recently published data from the Genome-wide association studies (GWAS) and CARDIoGRAMplus- C4D consortium was regarded for selection of risk SNPs that about 50 SNPs from 45 different loci were reported to be associated with CAD [8]. Some of SNPs were developed as a CAD risk panel in 2010 and we have selected the CAD risk SNPs rs3798220 and rs10455872 (Table 1). Primer designing was done by online Oligo Analyzer software. Samples were genotyped using a TaqMan assay (Applied Biosystems) on a LightCycler 96 instrument (Roche, Germany) at 95 ${}^{\circ}$ C for 15 min, and then 40 cycles of 95 ${}^{\circ}$ C for 15 seconds and 60 ${}^{\circ}$ C for 1 minute by standard protocols. The reagent volumes for TAQMAN genotyping assays per tube was DNA template about 100 ng, probe 1 pmol/ $\mu$ l, each of primers1 pmol/ $\mu$ l, 4 $\mu$ l of 10 $\times$ PCR buffer and 13 $\mu$ l of distilled water. Positive carrier status was defined as heterozygosity or homozygosity for the minor alleles of rs3798220, rs10455872, or both. Confirmation for the different genotype of certain samples randomly has been done sequencing.

2.2 Statistical analysis

Data analysis was performed using standard SPSS software version-23 (SPSS Inc, Chicago, IL, USA) and R v3.0.3. The continuous variables like blood lipid levels were compared between two groups using independent sample student $t$ -test. The categorical variables such as risk allele frequencies (RAFs) of all SNPs and Hardy-Weinberg equilibrium (HWE) equation were assessed for by a $\chi$ 2 goodness of fit test. Also the association of the SNPs with CAD as a binary variable was compared using binary logistic regression. The CAD status was regarded as dependent variable and gene score as covariate.

3. Results

3.1 Characteristics of study participants

The patients of CAD had mean $\pm$ SD age of 11 $\pm$ 111 years comprising 50 males and 50 females and the healthy controls had mean $\pm$ SD age of 22 $\pm$ 222 years (50 males and 50 females). The healthy controls were lower diabetic and hypertensive, smoking rate was also higher in cases than controls. Body mass index (BMI), total cholesterol level (TC), systolic blood pressure (SBP), diastolic blood pressure (DBP) and low-density lipoprotein (LDL) were significantly higher, whereas, high-density lipoprotein (HDL), triglycerides (TG) and fasting blood sugar (FBS) did not differ significantly between two groups (Table 2). The genotypic distribution of the two CAD SNPs in different models along with their odds ratios and risk allele frequencies are shown in Tables 3 to 6 respectively. The association of RAFs with CAD was analyzed by deriving their odds ratios (OR). For all the models studied for SNP rs10455872, the odds for CAD association were greater than 1 (except in recessive model) but the association was not statistically significant in between our samples. Individually, the RAFs of the rs10455872 variant were lower in CAD cases compared to controls, but the difference was not only statistically significant (Table 3). No significant association was detected between the rs3798220 SNP with risk of CAD or its characteristics under allele or alternative genotype models ( $p>$ 0.05). Also, the risk allele frequencies were not significantly greater in the patients compared to the controls ( $p<$ 0.05). Therefore, this variation is not a risk allele of the development of CAD in Iranian population. The RAFs in cases versus controls, the $p$ -values for statistical difference between the frequencies and confidence interval (CI) are demonstrated in Table 6. We found no association between studied LPA SNPs.

4. Discussion

To this day epidemiological studies have proposed that genetic factors are more likely to influence the development of CAD than environmental factors. Several earlier observational studies, GWAS and more recent meta-analysis studies have evaluated the association between polymorphisms located in different genomic regions and the risk of premature CAD [9]. Despite the critical roles of plasma lipoproteins in the initiation and development of atherosclerosis, controversial scientific evidences have seen for the causal associations of lipoproteins with coronary artery disease (CAD) [10]. In the recent years, LPA has gained great attention as a heritable genetic CAD risk factor and reasonable therapeutic target. This molecule is made of an LDL particle and the glycoprotein apolipoprotein(a) [apo(a)] which is linked to the apolipoprotein B from LDL by a single disulfide bond [11]. It is an extremely atherogenic lipoprotein that is expressed in the liver but the site and mechanism of catabolism is discussed controversially. To date no specific receptor has been detected but several reports mention to a role of the kidney in Lp(a) catabolism [11, 12]. The LPA gene locus is under strong genetic control and specially SNPs including a highly polymorphic copy number variation of the so called kringle IV repeats at this locus have a pronounced influence on Lp(a) expression level. Despite these findings, the role of LPA variations in CAD risk remains controversial. Moreover, recent genetic studies suggest the need for multiple replications in distinct populations for finding differences in the allelic frequencies, Lp(a) levels and association with CAD according to ethnic groups. Two SNPs rs10455872 and rs3798220 in the LPA gene have been regarded more than others to influence Lp(a) levels and increase CAD risk. To date, different groups attended to assess the genotyping of these genetic variants for diagnostic purpose in CAD affected population. However, the minor allele frequencies of rs10455872 and rs3798220 SNPs have been reported only 7 and 2 %, respectively [13]. In addition, the allelic frequencies might be different for various ethnicities. For example, Africans did not have rs3798220 and K-IV repeat number in. Also, in East and Southeast Asians, allele frequencies have shown from 2.9 to 11.6%, and were very low (0.15%) in CAD patient and healthy controls from India [13].

In this scenario, the main aim of our work was to investigate the association of the LPA rs10455872 and rs3798220 polymorphisms on CAD in Iranian patients. The two LPA polymorphisms targeted in this study are some of the most important genetic markers for CAD. The frequencies of the rs10455872 G and rs3798220 C variant alleles were 2% and 1%, respectively. These variant alleles were not associated with CAD risk in our population. We did no confirm significant results for the targeted variation in our population. CAD is a multi-factorial phenotype, and different genetic polymorphisms combined with environmental factors may cause in phenotypic variability; therefore, factors that end to cardiovascular events in CAD are complex. For the LPA rs10455872 polymorphism, we did not observe significant association with coronary lesions. Results from other studies also did not report a relationship between this LPA variant and CAD [14, 15]. Li et al. showed that patients with CAD have not increased rate for carrying LPA risk alleles and they did not diagnose difference between affected people with CAD and control group [16]. However, rs10455872 strongly demonstrated prevalent CAD (per allele odds ratio $=$ 2.33, 95% CI $=$ 1.67–3.33, $p=$ 1.75 $\times$ 10 ${}^{-9}$ ) [9].

Similar to results of our study, Santos et al. did not found that the rs3798220 polymorphism strongly predicted prevalent CAD (per allele OR $=$ 1.09, 95% CI $=$ 0.67–1.76, $p=$ 0.73) [17]. Furthermore, Anderson et al. studying patients with coronary angiography, did not detected an association between rs3798220 and CAD (OR 1.99, 95% CI 0.99–4.00, $p=$ 0.065) [9]. Also assessing the rs3798220 polymorphism in Chinese Han CAD patients did not support previous reports in a relationship between this genetic LPA variant and CAD [16]. In contrast to our study, the rs3798220 has previously been reported to have an association with the Lp(a) level and the risk of coronary disease [18, 19, 20]. A possible hypothesis for the lack of these associations in our study could be explained by the difference of ethnicity between rs3798220 and rs10455872 frequency identified in the Iranian patients compared with some studies with patients predominantly from European descent. Also it is clear that other work with larger sample size must be planned to be performed in Iranian population.

5. Conclusions

Taken together with previous and present studies, our data did not confirmed the association of the LPA variants rs6415084, rs3798220 a with CAD in Iranian CAD patients; however, other studies showed the lack of this genetic association and confirm these findings.

Footnotes

Abbreviations

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Investigation of LPA sequence variants rs6415084,rs3798220 with conventional coronary artery disease in Iranian CAD patients

Abstract

BACKGROUND:

METHODS:

RESULTS:

DISCUSSION:

CONCLUSION:

Keywords

1. Introduction

Table 1 Lipoprotein(a) SNPs and genotypes in this study

2.1 Genetic associations

2.2 Statistical analysis

3. Results

3.1 Characteristics of study participants

4. Discussion

5. Conclusions

Footnotes

Abbreviations

References

Table 1
Lipoprotein(a) SNPs and genotypes in this study