- Email: [email protected]
TG haplotype in the LRP8 is associated with myocardial infarction in south Indian population
Accepted Manuscript TG haplotype in the LRP8 is associated with myocardial infarction in south Indian population
Muhammed Asif, Shivarama Mohammed S. Mustak
Bhat,
Sheikh
Nizamuddin,
PII: DOI: Reference:
S0378-1119(17)30870-3 doi:10.1016/j.gene.2017.10.037 GENE 42256
To appear in:
Gene
Received date: Revised date: Accepted date:
14 June 2017 15 September 2017 11 October 2017
Please cite this article as: Muhammed Asif, Shivarama Bhat, Sheikh Nizamuddin, Mohammed S. Mustak , TG haplotype in the LRP8 is associated with myocardial infarction in south Indian population. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Gene(2017), doi:10.1016/j.gene.2017.10.037
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT TG Haplotype in the LRP8 Is Associated with Myocardial Infarction in South Indian population Muhammed Asif ‡, Shivarama Bhat‡, Sheikh Nizamuddin†, Mohammed S Mustak††
PT
‡ Department of Anatomy, Yenepoya Medical College and Hospital, Mangalore-575018, Karnataka, India
RI
†Centre for cellular and Molecular Biology, Hyderabad, Telangana, India
SC
††Department of Applied Zoology, Mangalore University, Mangalagangothri-574199,
NU
Mangalore, India
MA
Correspondence Dr Mustak MS
D
Department of Applied Zoology,
Karnataka, India
PT E
Mangalore University, Mangalagangothri-574199
CE
Email: [email protected]
AC
Phone: +91-824-2287373; +91-9743289671
1
ACCEPTED MANUSCRIPT Abstract Myocardial infarction (MI) is a complex multifactorial cardiovascular disease. India experiences a much greater burden of MI, also suggesting an experimental increase of this burden in the future. The absolute reasons for MI are context dependent and differ with different geographical settings. Several reports indicate that SNPs that are associated with
PT
certain diseases in other populations may not be associated with Indian population. It is,
RI
therefore, important to validate the association of SNPs. Low density lipoprotein receptor
SC
related protein 8 (LRP8) gene plays central role in human lipoprotein metabolism as it facilitates the clearance of bad cholesterol LDL, VLDL from plasma and is reported to be
NU
associated with MI in the western population. However, this gene has not been studied in the South Indian population. We aim to test the role of the LRP8 gene variants correlating with
MA
the lipid profile in MI patients in South Indian population. We sequenced regions of SNPs rs10788952, rs7546246, rs2297660 and rs5174 of LRP8 in 100 MI patients and 100 age-
D
matched controls. Our result revealed a total of 4 variations. None of the SNPs were
PT E
significantly associated with MI (p>0.973). Interestingly, haplotype based association analysis showed TG and CG of rs10788952 and rs7546246 significantly associated with MI
CE
(p<0.01 and p<0.00005) and in particular, haplotype TG was positively correlated with the risk of MI, as this increased the LDL and total cholesterol level in MI patients in south
population.
AC
Indians. Our results suggest that haplotype TG is a risk factor for MI in South Indian
Key words: Myocardial infarction, LRP8 gene, SNP, Haplotype analysis, association study, lipid profile
2
ACCEPTED MANUSCRIPT 1. Introduction Myocardial infarction (MI) is a multifactorial disease and it has been projected that by 2020 MI would be the leading cause of death and disability worldwide (Lloyd-Jones et al., 2009). Despite changing lifestyle and invention of pharmacologic approaches, MI continues to be a
PT
principal cause of death in many countries (Braunwald, 1997; Breslow, 1997). Thus the magnitude of this disease represents the importance of identifying genetic and environmental
RI
factors in their pathogenesis. Even though MI has a genetic basis the precise underlying
SC
genetic cause remains controversial (Mayer et al., 2007). Recently Genome wide linkage and GWAS approaches are used to lead the identification of several candidate genes in the
NU
chromosome loci for MI (Peden and Farrall, 2011). Epidemiological studies revealed that
MA
several genetic variants that are associated with lipid metabolism might increase the risk of MI (Yamada et al., 2002). Both genome wide association studies and candidate-gene approach identified a number of novel chromosome loci or genes that are associated with MI
PT E
D
(Ganesan et al., 2016). Low density lipoprotein receptor 8 (LRP8) gene is localized on chromosome 1, which is of 85,786 base pairs (bp) in size and consist of 22 exons (Gene ID: 7804). LRP8 is a cell surface protein that plays roles in both signal transduction and receptor
CE
mediated endocytosis of low density lipoprotein (LDL-C), very low density lipoprotein
AC
(VLDL-C) for lysosomal degradation and controls the lipoprotein homeostasis in plasma. LRP8 is expressed in endothelial cells, platelets, heart and muscles (Kim et al., 1996). Studies showed that dysfunction of lipid metabolism plays a key role in the development of MI by decreased HDLC, apolipoprotein A-I (apoA-I) levels and increased low density lipoprotein (LDLC) level (Kuo et al., 1998; Boersma et al., 2003; Thygesen et al., 2007; Reitz et al., 2010), total cholesterol (TC) and triglycerides (TG) (Karthikeyan et al., 2009). The elevated level of LDL-C is shown to be a high risk factor for coronary artery diseases from many 3
ACCEPTED MANUSCRIPT studies (Woo et al., 1993). Since, Indian populations are unique in their origin and are practicing endogamy for the past few decades, it can be expected in Indians to have a unique set of mutations which lead to several diseases (cardiac disease) in particular (Dhandapany et al., 2009; Reich et al., 2009; Nizamuddin et al., 2015; Selvi Rani et al., 2015). Among the fastest growing non-communicable diseases, cardiovascular diseases (CVDs) are expected to
PT
cause the largest number of mortality and morbidity within India (Chauhan and Aeri, 2013).
RI
Indians have unique lipid profile characterized by high triglycerides, low HDL-C, and
SC
increased LDL-C and VLDL levels (Hoogeveen et al., 2001). LRP8 play a major role in LDL-C, VLDL metabolism and this gene possess several SNPs (rs5174, rs10788952,
NU
rs2297660, rs7546246) that have been reported to be associated with plasma LDL, VLDL levels. Mutation rs5174/R952Q in LRP8 is shown to be associated with LDL induced platelet
MA
aggregation causing the development of atherosclerosis or atherothrombosis found in American, Caucasian and Italian population (Korporaal et al., 2004; Johns et al., 2005; Shen
D
et al., 2013). Reports indicate that SNPs or haplotypes that are associated with certain
PT E
diseases in the western population may not be associated with Indian population (Sharma and Ganguly, 2005; Rosenberg et al., 2010) . There is a need for identification of the loci/ gene
CE
for MI in Indian population compared to the western population. LRP8 gene has not been analyzed in South Indian population. Hence, the study was aimed to investigate whether
AC
LRP8 gene variation influences MI in South Indian population. Further, we also examined the association between LRP8 genetic variants with lipid levels with risk of MI. 2. Materials and methods 2.1 Sample details The study subjects were composed of 100 acute MI and 100 age and ethnically matched healthy controls from South India, of which 80% individuals are sporadic and late onset (age 4
ACCEPTED MANUSCRIPT group 58.94 ± 9.65) in present study. Blood samples were collected from K S Hegde and Yenepoya hospitals, Mangalore, India. The diagnosis of MI was based on typical electrocardiographic (ECG) changes; increase in the serum activities of enzymes such as creatine kinase, aspartate aminotransferase, and the concentration of troponin T, confirmed by the panel of cardiologists. Angiographically proved coronary artery disease after MI, those
PT
who underwent CABG (coronary artery bypass graft) after MI and acute coronary syndrome
RI
with MI were also considered. Control individuals were ethnically matched control and free
SC
from MI, as determined by medical history, clinical examinations or ECG. The mean age of healthy controls was 65.27 ± 10.30 years while that of patients was 58.94 ± 9.65 years. In MI
NU
patients 20% were tobacco chewers, while it was 8% among the healthy controls. The
MA
demographic details of the individuals included in the study are given in Table 1. 2.2 Sample collection and DNA isolation
D
The blood samples were collected from MI patients admitted in the cardiac care unit. The
PT E
blood samples of healthy controls from the same ethnic background without MI based on the electrocardiograph were collected. 10 ml of intravenous blood sample of both cases and
CE
controls were collected in the EDTA vaccutainer, after obtaining informed written consent. Genomic DNA isolation was done by using standard protocol (Miller et al., 1988). This study
AC
followed the principles outlined in the Declaration of Helsinnki (WMA World Medical Association Declaration of Helsinki) and approved by Institutional Ethics Committee of Yenepoya University and Nitte University, Mangalore, India. The serum lipid profile was measured within the first 24 hrs of the onset of symptoms of MI. The serum total cholesterol, HDL cholesterol, and triglyceride levels were measured on automated clinical chemistry machine in the clinical lab. All the blood samples were 6hr fasting samples.
5
ACCEPTED MANUSCRIPT 2.3 Genotyping The reference genomic sequence of LRP8 (ENSG0000157193) was obtained from the Ensemble database (asia.ensembl.org). The primers were designed to amplify coding region (UTR, exon and exon-intron boundaries) of LRP8 using Primer3 web version 4.0. PCRs
PT
(polymerase chain reactions) were performed using Gene Amp 9700 (Applied Biosystems, Foster City, USA) using Emerald Amp GT PCR master mix (TaKaRa) according to the
RI
manufacturer’s protocol. After PCR, Amplicons were size fractionated using 2% agarose gel,
SC
stained with ethidium bromide and observed under UV transilluminator. Subsequently, amplicons were treated with Exo-SAP (USB Corp., USA), and sequenced using a BigDye
NU
terminator (v3.1) cycle sequencing kit (Applied Biosystems, Foster City, USA) on an ABI
MA
3730XL DNA analyzer. Sequences obtained were assembled with the reference sequences using AutoAssembler software (Applied Biosystems, Foster City, USA). Variations observed
D
were noted for further analysis.
PT E
2.4 Statistical analysis
Allele and genotype frequencies were calculated by allele counting method. Single marker
CE
and haplotype based association analysis were carried out by Plink (Purcell et al., 2007) and Haploview software (Barrett et al., 2004). The haplotypes block was defined using
AC
confidence interval of Gabriel et al. (2002) method in Haploview. For both single marker and haplotype association analysis, Chi-square test was used. The p value < 0.05 were considered for statistical significance in all analysis. To explore the Hardy-Weinberg equilibrium (HWE), we consider genotype distribution in control samples and only those variants having HWE p value >0.05 were utilized in further association analysis. Linear regression analysis was also performed to explore the effect of the mutation on blood lipid levels using glm function of R. 6
ACCEPTED MANUSCRIPT 3. Results We have investigated the exons, exon-intron boundaries and UTR of LRP8 in 100 individuals with MI and 100 ethnically matched controls. We genotyped four SNPs within LRP8 (rs5174, rs10788952, rs2297660, rs7546246) of which two were in introns [rs7546246(A/G),
PT
rs10788952 (C/T)] and one was synonymous rs2297660 (C/A); one was nonsynonymous in 3’UTR [rs5174 (G/A)] (Table 2 and Fig 1).
RI
It is well known that spurious association can arise due to sample level substructure or
SC
genotyping error, and can be detected with Hardy-Weinberg equilibrium (HWE) test. In
NU
common practice, those SNPs, for which HWE is violated, should be removed in association analysis. However, we did not find any violation of HWE equilibrium for all 4 variants
MA
(p>0.05) (Table 3). None of the markers [rs5174 (p=0.6889), rs229766 (p=0.9255), rs107889 (p=0.0802) and rs7546246 (p=0.9729)] was significantly associated with MI in South Indian
D
population by single marker association study. Since we did not observe the single marker
PT E
SNP association; we proceeded for haplotype based association analysis using the Haploveiw software.
CE
3.1 Haplotype based Association Analysis
AC
In order to identify the haplotype in LRP8, haplotype analysis was carried out for genotyping data of SNPs (rs5174, rs10788952, rs2297660, rs7546246) and it was analyzed for their association with MI. Using Gabriel et al (2002) confidence intervals method, we observed single haplotype block (Fig 2) in our dataset. Only 2 SNPs (rs10788952 and rs7546246) were present in the block and have normalized coefficient of linkage disequilibrium or D’ >0.8. Further, we estimated haplotypes within the block (TG, CG, TA and CA) in our cohort and
7
ACCEPTED MANUSCRIPT performed association analysis. Intriguingly, we found haplotype TG (p=0.0155) and CG (p=0.00005) were significantly associated with risk of MI in South Indian patients (Table 4). 3.2 Effect of haplotype on blood lipid profile We have measured the blood profile of cases and controls by automated clinical chemistry
PT
machine and the mean values of TC, HDL, LDL, VLDL, triglycerides are 204.4 mg/dl, 39.4 mg/dl, 118.1 mg/dl, 27.7 mg/dl, 145.8 mg/dl respectively in the MI patients of South Indian
RI
population as shown in Table 1. The maximum, minimum and median values of total
SC
cholesterol, HDL, LDL, VLDL and triglycerides of MI are depicted in Table 5.
NU
Further, to explore the effect of mutation of the LRP8 gene on blood lipid levels, we performed the linear regression analysis against the total cholesterol (TC), LDL, HDL, VLDL
MA
and triglycerides in the MI cases (Table 6). Since CG was absent in the MI cases, we could analyze only TG haplotype. Interestingly, we observed that TG haplotype was significantly
D
associated with LDL (p=0.00607) and cholesterol level (p=0.0344), whereas HDL (p>0.523),
PT E
VLDL (p>0.772), triglycerides (p>0.883) were not significantly associated with MI. The haplotype CA (0.0241) with negative beta (-20.6) value were also seen to be significantly
CE
associated with LDL. This finding suggests that subjects with TG haplotype, are having significantly higher level of LDL and TC, comparative to other subjects with TA and CA
AC
haplotypes. It also suggests that rs10788952-rs7546246 (TG) haplotype might be increasing the risk of MI through increasing the level of LDL cholesterol. 4. Discussion MI is caused by both genetic and environmental factors (Walter and Zeiher, 2000). LRP8 plays a central role in human lipoprotein metabolism as it facilitates the clearance of bad cholesterol LDL, VLDL from plasma via endocytosis (Kim et al., 1996; Waterworth et al., 8
ACCEPTED MANUSCRIPT 2010). The previous study on the nonsynonymous mutation R952Q/rs5174 of LRP8 had shown a significant association in MI patients and not in control group (Shen et al., 2007; Lieb et al., 2008; Martinelli et al., 2009; Shen et al., 2013; Shen et al., 2014a). Considering the important role of LRP8 in lipid metabolism, we investigated the association of genetic variants of the LRP8 and lipid profile with the risk of myocardial infarction in patients from
PT
south India. In the current study, we did not observe a significant association of SNP variant
RI
(rs5174, rs10788952, rs2297660, and rs7546246) with the MI in South Indians; however, we
SC
found significant haplotype association TG (P=0.0155) and CG (P=0.00005) with MI in rs10788952 and rs7546246. A genome wide association analysis conducted on European
NU
descent Americans, Italian Caucasian and Korean population analyzed over 521786 markers and found fifty susceptibility SNPs were associated risk of an incident of MI (Shen et al.,
MA
2014a). Shen et al. 2014 observed that the genetic variant R952Q/rs5174 in the LRP8 gene was associated with familial and early-onset MI in European-descent Americans and Italian
D
Caucasian populations, but not with the sporadic and late onset form of the disease (Shen et
PT E
al., 2014a). Another study showed a significant association of SNP R952Q/ rs5174 of LRP8 with plasma triglyceride (222±16.6) level in 358 probands of Gene Quest Family cohort with
CE
familial and premature CAD and MI. Further, this study was replicated in independent Gene Bank cohort of 134 patients with premature MI (Shen et al., 2012). However, we did not find
AC
association of R952Q/rs5174 with MI. Different ancestry and clinicopathologic features (sporadic and late onset) of MI patients of present study, might be the reason of insignificant association. Interestingly, when analyzing our result by regression analysis, we found that haplotype TG positively (<0.006) associated with total cholesterol and LDL-C level in MI. Considering the significantly higher frequency of TG in MI patients comparative to controls, it can be concluded that TG haplotype increases the risk of MI in South Indian through increasing the bad cholesterol (LDL-C) level. 9
ACCEPTED MANUSCRIPT Our present study in lipid profile of MI patients showed an increase in TC (204.4 ± 49.4), LDL (118.1 ± 39.9), VLDL (27.7 ± 14.3) and triglycerides (145.8 ± 65.8) and decrease in HDL (39.4 ± 12.3) when compared to control, which is align with other population (Kumar and Sivakanesan, 2009). On the other hand, our result on genetic variants (rs5174, rs10788952, rs2297660, and rs7546246) was not found to be significantly associated with MI
Therefore, we investigated on
RI
the single marker based association with lipid profile.
PT
against the western Caucasian population (Shen et al., 2014a). Hence we could not perform
SC
haplotype based association analysis which resulted in presence of haplotype TG and CG are significantly associated with MI compared to controls. It is possible that individuals having
NU
mutation at both genetic loci (rs10788952: C>T and rs7546246: A>G) are having higher genetic risk of MI, comparative to mutation at single locus only and hence, more power to
MA
detect association in haplotype association analysis. This can be the reason; we found signal of association in haplotype based association analysis only. Haplotype CG was not present in
D
control, hence, we analyzed only TG which increases the LDL and total TC levels leading to
PT E
risk for the MI. To explore the effect of the mutation on blood lipid levels, linear regression analysis was performed. Interestingly we found TG haplotype association with LDL and TC
CE
levels. We observed the presence of haplotype CA was inversely correlated with LDL. Similarly, the study using the GeneQuest and Italian population, the risk haplotype TACGC
AC
in LRP8 had higher LDL levels was found with CAD and MI (Shen et al., 2014b). Further they showed TCCGC haplotype exists only in normal individuals and not in MI, where it acts as a protective role against MI (OR = 0.42–0.71) (Shen et al., 2013). In our study, high levels of triglycerides (145.81 ± 65.82) were observed in MI when compared to control (113.2 ± 32.05); however, no significant correlation between triglycerides levels and TG haplotype was observed.
10
ACCEPTED MANUSCRIPT In conclusion, we demonstrated that haplotype TG and CG of rs10788952 & rs7546246 are significantly associated with MI, whereas particularly presence of haplotype TG may significantly increase LDL and TC levels leading to MI. Further, our result showed that non synonymous variant R952Q /rs5174 were not observed in association with MI (sporadic and
PT
late-onset) in South Indian Population.
SC
RI
Conflict of interest statement
NU
All of the authors declare that there is no conflict of interest.
MA
Acknowledgements
We thank all the patients, cardiologists and nursing staff who helped us with this study. M A
D
acknowledges the support of Yenepoya University, Nitte University and Mangalore
PT E
University, Mangalore, India. The authors acknowledge Dr. K.Thangaraj Senior Scientist, CCMB-Hyderabad, Prof. Subramanyam K, cardiologist Nitte University, Prof Chakrapani
AC
CE
M, Physician KMC, for their constant support and encouragement.
11
ACCEPTED MANUSCRIPT References Barrett, J.C., Fry, B., Maller, J. and Daly, M.J., 2004. Haploview: analysis and visualization of LD and haplotype maps. Bioinformatics 21, 263-265. Boersma, E., Mercado, N., Poldermans, D., Gardien, M., Vos, J. and Simoons, M.L., 2003. Acute myocardial infarction. The Lancet 361, 847-858.
PT
Braunwald, E., 1997. Cardiovascular medicine at the turn of the millennium: triumphs, concerns, and
RI
opportunities. New England Journal of Medicine 337, 1360-1369.
Breslow, J.L., 1997. Cardiovascular disease burden increases, NIH funding decreases. Nature
SC
medicine 3, 600-601.
NU
Chauhan, S. and Aeri, B.T., 2013. Prevalence of cardiovascular disease in India and it is economic impact-A review. International Journal of Scientific and Research Publications 3, 1-5.
MA
Dhandapany, P.S., Sadayappan, S., Xue, Y., Powell, G.T., Rani, D.S., Nallari, P., Rai, T.S., Khullar, M., Soares, P. and Bahl, A., 2009. A common MYBPC3 (cardiac myosin binding protein C) variant associated with cardiomyopathies in South Asia. Nature genetics 41, 187-191.
D
Gabriel, S.B., Schaffner, S.F., Nguyen, H., Moore, J.M., Roy, J., Blumenstiel, B., Higgins, J.,
PT E
DeFelice, M., Lochner, A. and Faggart, M., 2002. The structure of haplotype blocks in the human genome. Science 296, 2225-2229.
CE
Ganesan, M., Nizamuddin, S., Katkam, S.K., Kumaraswami, K., Hosad, U.K., Lobo, L.L., Kutala, V.K. and Thangaraj, K., 2016. c.* 84G> A Mutation in CETP Is Associated with Coronary
AC
Artery Disease in South Indians. PloS one 11, e0164151. Hoogeveen, R.C., Gambhir, J.K., Gambhir, D., Kimball, K.T., Ghazzaly, K., Gaubatz, J.W., Vaduganathan, M., Rao, R.S., Koschinsky, M. and Morrisett, J.D., 2001. Evaluation of Lp [a] and other independent risk factors for CHD in Asian Indians and their USA counterparts. Journal of Lipid Research 42, 631-638. Johns, D.G., Ao, Z., Willette, R.N., Macphee, C.H. and Douglas, S.A., 2005. Role of p38 MAP kinase in postcapillary venule leukocyte adhesion induced by ischemia/reperfusion injury. Pharmacological research 51, 463-471. 12
ACCEPTED MANUSCRIPT Karthikeyan, G., Teo, K.K., Islam, S., McQueen, M.J., Pais, P., Wang, X., Sato, H., Lang, C.C., Sitthi-Amorn, C. and Pandey, M., 2009. Lipid profile, plasma apolipoproteins, and risk of a first myocardial infarction among Asians: an analysis from the INTERHEART Study. Journal of the American College of Cardiology 53, 244-253. Kim, D.-H., Iijima, H., Goto, K., Sakai, J., Ishii, H., Kim, H.-J., Suzuki, H., Kondo, H., Saeki, S. and
PT
Yamamoto, T., 1996. Human apolipoprotein E receptor 2 A novel lipoprotein receptor of the low density lipoprotein receptor family predominantly expressed in brain. Journal of
RI
Biological Chemistry 271, 8373-8380.
SC
Korporaal, S.J., Relou, I.A., van Eck, M., Strasser, V., Bezemer, M., Gorter, G., van Berkel, T.J., Nimpf, J., Akkerman, J.-W.N. and Lenting, P.J., 2004. Binding of low density lipoprotein to
Biological Chemistry 279, 52526-52534.
NU
platelet apolipoprotein E receptor 2′ results in phosphorylation of p38MAPK. Journal of
MA
Kumar, A. and Sivakanesan, R., 2009. Serum lipid profile abnormality in predicting the risk of myocardial infarction in elderly normolipidaemic patients in South Asia: a case-controlled
D
study. Internet J Altern Med 6, 6.
PT E
Kuo, Y.-M., Emmerling, M.R., Bisgaier, C.L., Essenburg, A.D., Lampert, H.C., Drumm, D. and Roher, A.E., 1998. Elevated low-density lipoprotein in Alzheimer's disease correlates with brain Aβ 1–42 levels. Biochemical and biophysical research communications 252, 711-715.
CE
Lieb, W., Zeller, T., Mangino, M., Götz, A., Braund, P., Wenzel, J.J., Horn, C., Proust, C., Linsel-
AC
Nitschke, P. and Amouyel, P., 2008. Lack of association of genetic variants in the LRP8 gene with familial and sporadic myocardial infarction. Journal of molecular medicine 86, 11631170.
Lloyd-Jones, D., Adams, R., Carnethon, M., De Simone, G., Ferguson, T.B., Flegal, K., Ford, E., Furie, K., Go, A. and Greenlund, K., 2009. Heart disease and stroke statistics—2009 update a report from the American Heart Association Statistics Committee and Stroke Statistics Subcommittee. Circulation 119, e21-e181.
13
ACCEPTED MANUSCRIPT Martinelli, N., Olivieri, O., Shen, G.-Q., Trabetti, E., Pizzolo, F., Busti, F., Friso, S., Bassi, A., Li, L. and Hu, Y., 2009. Additive effect of LRP8/APOER2 R952Q variant to APOE ε2/ε3/ε4 genotype in modulating apolipoprotein E concentration and the risk of myocardial infarction: a case-control study. BMC medical genetics 10, 41. Mayer, B., Erdmann, J. and Schunkert, H., 2007. Genetics and heritability of coronary artery disease
PT
and myocardial infarction. Clinical Research in Cardiology 96, 1-7.
human nucleated cells. Nucleic acids research 16, 1215.
RI
Miller, S., Dykes, D. and Polesky, H., 1988. A simple salting out procedure for extracting DNA from
SC
Nizamuddin, S., Govindaraj, P., Saxena, S., Kashyap, M., Mishra, A., Singh, S., Rotti, H., Raval, R., Nayak, J. and Bhat, B., 2015. A novel gene THSD7A is associated with obesity. International
NU
Journal of Obesity 39, 1662.
Peden, J.F. and Farrall, M., 2011. Thirty-five common variants for coronary artery disease: the fruits
MA
of much collaborative labour. Human molecular genetics, ddr384. Purcell, S., Neale, B., Todd-Brown, K., Thomas, L., Ferreira, M.A., Bender, D., Maller, J., Sklar, P.,
D
De Bakker, P.I. and Daly, M.J., 2007. PLINK: a tool set for whole-genome association and
PT E
population-based linkage analyses. The American Journal of Human Genetics 81, 559-575. Reich, D., Thangaraj, K., Patterson, N., Price, A.L. and Singh, L., 2009. Reconstructing Indian population history. Nature 461, 489-494.
CE
Reitz, C., Tang, M.-X., Schupf, N., Manly, J.J., Mayeux, R. and Luchsinger, J.A., 2010. Association
AC
of higher levels of high-density lipoprotein cholesterol in elderly individuals and lower risk of late-onset Alzheimer disease. Archives of neurology 67, 1491-1497. Rosenberg, N.A., Huang, L., Jewett, E.M., Szpiech, Z.A., Jankovic, I. and Boehnke, M., 2010. Genome-wide association studies in diverse populations. Nature reviews. Genetics 11, 356. Selvi Rani, D., Nallari, P., Dhandapany, P.S., Rani, J., Meraj, K., Ganesan, M., Narasimhan, C. and Thangaraj, K., 2015. Coexistence of digenic mutations in both thin (tpm1) and thick (myh7) filaments of sarcomeric genes leads to severe hypertrophic cardiomyopathy in a South Indian FHCM. DNA and cell biology 34, 350-359. 14
ACCEPTED MANUSCRIPT Sharma, M. and Ganguly, N.K., 2005. Premature coronary artery disease in Indians and its associated risk factors. Vascular health and risk management 1, 217. Shen, G.-Q., Girelli, D., Li, L., Olivieri, O., Martinelli, N., Chen, Q., Topol, E.J. and Wang, Q.K., 2013. Multi-allelic haplotype association identifies novel information different from singleSNP analysis: A new protective haplotype in the LRP8 gene is against familial and early-
PT
onset CAD and MI. Gene 521, 78-81. Shen, G.-Q., Girelli, D., Li, L., Rao, S., Archacki, S., Olivieri, O., Martinelli, N., Park, J.E., Chen, Q.
RI
and Topol, E.J., 2014a. A Novel Molecular Diagnostic Marker for Familial and Early-Onset
SC
CAD and MI in the LRP8 Gene. Circulation: Cardiovascular Genetics, CIRCGENETICS. 113.000321.
NU
Shen, G.-Q., Girelli, D., Li, L., Rao, S., Archacki, S., Olivieri, O., Martinelli, N., Park, J.E., Chen, Q. and Topol, E.J., 2014b. A novel molecular diagnostic marker for familial and early-onset
MA
coronary artery disease and myocardial infarction in the LRP8 gene. Circulation: Cardiovascular Genetics 7, 514-520.
D
Shen, G.-Q., Li, L., Girelli, D., Seidelmann, S.B., Rao, S., Fan, C., Park, J.E., Xi, Q., Li, J. and Hu,
PT E
Y., 2007. An LRP8 variant is associated with familial and premature coronary artery disease and myocardial infarction. The American Journal of Human Genetics 81, 780-791. Shen, G.Q., Li, L. and Wang, Q.K., 2012. Genetic Variant R952Q in LRP8 is Associated with
CE
Increased Plasma Triglyceride Levels in Patients with Early‐Onset CAD and MI. Annals of
AC
human genetics 76, 193-199. Thygesen, K., Alpert, J.S. and White, H.D., 2007. Universal definition of myocardial infarction. Journal of the American College of Cardiology 50, 2173-2195. Walter, D.H. and Zeiher, A.M., 2000. Genetic risk factors for myocardial infarct. Herz 25, 7-14. Waterworth, D.M., Ricketts, S.L., Song, K., Chen, L., Zhao, J.H., Ripatti, S., Aulchenko, Y.S., Zhang, W., Yuan, X. and Lim, N., 2010. Genetic variants influencing circulating lipid levels and risk of coronary artery disease. Arteriosclerosis, thrombosis, and vascular biology 30, 2264-2276.
15
ACCEPTED MANUSCRIPT Woo, J., Ho, S., Wong, S., Woo, K., Tse, C., Chan, K., Kay, C., Mak, W., Cheung, K. and Lam, C., 1993. Lipids, lipoproteins and other coronary risk factors in Chinese male survivors of myocardial infarction. International journal of cardiology 39, 195-202. Yamada, Y., Izawa, H., Ichihara, S., Takatsu, F., Ishihara, H., Hirayama, H., Sone, T., Tanaka, M. and Yokota, M., 2002. Prediction of the risk of myocardial infarction from polymorphisms in
AC
CE
PT E
D
MA
NU
SC
RI
PT
candidate genes. New England Journal of Medicine 347, 1916-1923.
16
ACCEPTED MANUSCRIPT Figures legends: Fig 1. Observed variations and its location in LRP8 gene mRNA. ENSG00000157193 was utilized to represent the physical location of variants Fig 2. LD analysis of LRP8 structure defined by four SNPs (rs5174, rs2297660, rs10788952,
PT
rs7546246) within LRP8 regions. Strong LD was detected between rs10788952 and rs7546246 with LRP8 spanning 0kb size. The pair wise correlation between SNPs was
RI
measured as D’ and shown (x100) in each diamond. Red to white color gradient indicates the
AC
CE
PT E
D
MA
NU
SC
magnitude of pair wise LD, the region from higher to lower values of LD.
17
RI
PT
ACCEPTED MANUSCRIPT
AC
CE
PT E
D
MA
NU
SC
Fig. 1
18
PT E
D
MA
NU
SC
RI
PT
ACCEPTED MANUSCRIPT
AC
CE
Fig. 2
19
ACCEPTED MANUSCRIPT Table 1: Demographic and lipid profiles of patients and controls with MI MI
Control
Number of samples
100
100
Linguistic affiliation
Dravidian (Kannadiga)
Dravidian (Kannadiga)
Age (years) at sampling (mean±S.D.)
58.94 ± 9.65
65.27 ± 10.30
Tobacco
20%/80%
10% /90%
Alcohol
35%/65%
Hypertension
32%/68%
Family history (Y/N)
15%/85%
Total cholesterol (mg/dl) (mean±S.D.)
204.4 ± 49.4
HDL (mg/dl) (mean±S.D.)
39.4 ± 12.3
47.4 ± 8.1
LDL (mg/dl) (mean±S.D.)
118.1 ± 39.9
88.1 ± 19.6
TG (mg/dl) (mean±S.D.)
RI
6.66% /93.33%
SC
NU
MA
VLDL (mg/dl) (mean±S.D.)
PT
Traits
NA Nil 175.7 ± 31.9
27.7 ±14.3
23.3 ± 8.7
145.8 ± 65.8
113.2 ± 32.05
AC
CE
PT E
D
S.D.- Standard deviation; (mg/dl) – Milligram/deciliter
20
ACCEPTED MANUSCRIPT Table 2: Genotype and allele frequency distributions of SNPs in LRP8 among cases and controls Allele
MI
Control
P - value
HWE - Value
rs7546246
GG
0
26
0.9729
0.6039
GA
31
11
AA
53
2
GG
63
31
AG
26
9
AA
0
CC
56
CA
24
AA
0
CC
49
29
CT
36
9
0
2
0.9255
0.5587
0.0802
1
RI
SC
NU
MA
9
D
TT
PT E
rs10788952
29
CE
rs22977660
0.09119
0.6889
3
AC
rs5174
PT
SNP
21
1
ACCEPTED MANUSCRIPT Table 3: Single marker association study Assoc-
Case, Control-
Case, Control-
SNPs
Association Chi-square
HWE P-value
Ratio-Counts
Frequencies
P-value
rs5174
G
146:26,68:14
0.849,0.829
0.16
0.6889
0.09119
rs2297660
C
23:135,11:67
0.146,0.141
0.009
0.9255
0.5587
rs10788952
T
36:134,9:67
0.212,0.118
3.062
rs7546246
A
136:32,63:15
0.810,0.808
0.001
AC
CE
PT E
D
MA
NU
SC
RI
PT
Allele
22
0.0802
1
0.9729
0.6039
ACCEPTED MANUSCRIPT Table 4: Haplotype based association study Haplotype
F_A
F_U
ChiSq
DF
P
SNPs
H1
TG
0.189
0.06757
5.859
1
0.0155
rs10788952|rs7546246
H1
CG
0
0.1216
20.73
1
0.00005
rs10788952|rs7546246
H1
TA
0.01829
0.04054
1.027
1
0.3109
rs10788952|rs7546246
H1
CA
0.7927
0.7703
0.1521
1
0.6965
rs10788952|rs7546246
AC
CE
PT E
D
MA
NU
SC
RI
PT
Locus
23
ACCEPTED MANUSCRIPT Table 5: Cholesterol levels in MI patients of South India.
Min Median Max
TC
HDL
LDL
VLDL
TG
86
22
43
11
41
210.5
38
108
23.5
134.5
343
93
241
81
404
PT
TC – Total cholesterol; HDL – High density lipoprotein; LDL – Low density lipoprotein;
AC
CE
PT E
D
MA
NU
SC
RI
VLDL – Very low density lipoprotein; TG - Triglycerides
24
ACCEPTED MANUSCRIPT Table 6: Effect of haplotype on blood lipid level: linear regression analysis
BP1
BP2
SNP1
5373818 2
2
2
3
3
3
SNP2
HAP
F
BETA
LDL
P
BETA
P
HDL
1
1
1
rs1078895
53738100
0.19 rs7546246
3
2
5373818
rs1078895
3
2
5373818
rs1078895
TG
25.2
0.00
C S U
0.03
-1.89
BET
P
0.52
rs7546246
53738100
D E
rs7546246 2
TA
CA
0.02
N A
-26.6
-14.9
-20.6
-60
M
0.79
0.04
0.27
P
C C
P A
0.77 0.98
0.88 2.32
2
-8.91
3
-
0.70
-
0.60
3.37
2
20.9
8
0.89
0.79
0.24
0.19
0.02
3.13
0.28 0.46
T P E
Triglyceride BET
A
6
53738100
3
24.4
VLDL
T P
BETA
I R
CHR
NHAP
NSNP
TC
0.95 9
SNP – Single nucleotide polymorphism; NSNP - synonymous SNP; NHAP-Number of haplotype associated Polymorphism ; CHR – chromosome; BP1 –
A
basepair 1; BP2 – basepair 2; HAP – haplotype; F – frequency; TC – total cholesterol; LDL – low density lipoprotein; HDL – high density lipoprotein; VLDL – very low density lipoprotein
25
ACCEPTED MANUSCRIPT
Abbreviations
MA
HDLC - High density lipoprotein cholesterol apoA-I - Apolipoprotein A-I
PT E
TG- Triglycerides
D
TC- Total cholesterol
NU
VLDL C- very low density lipoprotein cholesterol
CE
CVDs - CardioVascular diseases
AC
HWE- Hardy-Weinberg equilibrium
26
RI
LDL C - low density lipoprotein cholesterol
SC
LRP8- Low density lipoprotein receptor related protein 8
PT
MI - Myocardial infarction
ACCEPTED MANUSCRIPT Highlights
Myocardial infarction (MI) is multifactorial and caused by imbalance in the blood lipid levels.
LRP8 plays central role in human lipoprotein metabolism as it facilitates the clearance of
PT
SC
RI
bad cholesterol LDL, VLDL from plasma.
Evaluated the TG and CG Haplotypes in the LRP8 gene and found to be associated with
Haplotype TG is significantly associated increase in LDL and total cholesterol (TC)
MA
NU
Myocardial Infarction in South Indian population.
PT E
Non synonymous variant rs5174 associated with MI in western population were not
CE
associated in South Indian Population.
AC
D
levels in MI patients and suggests being risk factor for MI in south Indian population.
27
Muhammed Asif, Shivarama Mohammed S. Mustak
Bhat,
Sheikh
Nizamuddin,
PII: DOI: Reference:
S0378-1119(17)30870-3 doi:10.1016/j.gene.2017.10.037 GENE 42256
To appear in:
Gene
Received date: Revised date: Accepted date:
14 June 2017 15 September 2017 11 October 2017
Please cite this article as: Muhammed Asif, Shivarama Bhat, Sheikh Nizamuddin, Mohammed S. Mustak , TG haplotype in the LRP8 is associated with myocardial infarction in south Indian population. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Gene(2017), doi:10.1016/j.gene.2017.10.037
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT TG Haplotype in the LRP8 Is Associated with Myocardial Infarction in South Indian population Muhammed Asif ‡, Shivarama Bhat‡, Sheikh Nizamuddin†, Mohammed S Mustak††
PT
‡ Department of Anatomy, Yenepoya Medical College and Hospital, Mangalore-575018, Karnataka, India
RI
†Centre for cellular and Molecular Biology, Hyderabad, Telangana, India
SC
††Department of Applied Zoology, Mangalore University, Mangalagangothri-574199,
NU
Mangalore, India
MA
Correspondence Dr Mustak MS
D
Department of Applied Zoology,
Karnataka, India
PT E
Mangalore University, Mangalagangothri-574199
CE
Email: [email protected]
AC
Phone: +91-824-2287373; +91-9743289671
1
ACCEPTED MANUSCRIPT Abstract Myocardial infarction (MI) is a complex multifactorial cardiovascular disease. India experiences a much greater burden of MI, also suggesting an experimental increase of this burden in the future. The absolute reasons for MI are context dependent and differ with different geographical settings. Several reports indicate that SNPs that are associated with
PT
certain diseases in other populations may not be associated with Indian population. It is,
RI
therefore, important to validate the association of SNPs. Low density lipoprotein receptor
SC
related protein 8 (LRP8) gene plays central role in human lipoprotein metabolism as it facilitates the clearance of bad cholesterol LDL, VLDL from plasma and is reported to be
NU
associated with MI in the western population. However, this gene has not been studied in the South Indian population. We aim to test the role of the LRP8 gene variants correlating with
MA
the lipid profile in MI patients in South Indian population. We sequenced regions of SNPs rs10788952, rs7546246, rs2297660 and rs5174 of LRP8 in 100 MI patients and 100 age-
D
matched controls. Our result revealed a total of 4 variations. None of the SNPs were
PT E
significantly associated with MI (p>0.973). Interestingly, haplotype based association analysis showed TG and CG of rs10788952 and rs7546246 significantly associated with MI
CE
(p<0.01 and p<0.00005) and in particular, haplotype TG was positively correlated with the risk of MI, as this increased the LDL and total cholesterol level in MI patients in south
population.
AC
Indians. Our results suggest that haplotype TG is a risk factor for MI in South Indian
Key words: Myocardial infarction, LRP8 gene, SNP, Haplotype analysis, association study, lipid profile
2
ACCEPTED MANUSCRIPT 1. Introduction Myocardial infarction (MI) is a multifactorial disease and it has been projected that by 2020 MI would be the leading cause of death and disability worldwide (Lloyd-Jones et al., 2009). Despite changing lifestyle and invention of pharmacologic approaches, MI continues to be a
PT
principal cause of death in many countries (Braunwald, 1997; Breslow, 1997). Thus the magnitude of this disease represents the importance of identifying genetic and environmental
RI
factors in their pathogenesis. Even though MI has a genetic basis the precise underlying
SC
genetic cause remains controversial (Mayer et al., 2007). Recently Genome wide linkage and GWAS approaches are used to lead the identification of several candidate genes in the
NU
chromosome loci for MI (Peden and Farrall, 2011). Epidemiological studies revealed that
MA
several genetic variants that are associated with lipid metabolism might increase the risk of MI (Yamada et al., 2002). Both genome wide association studies and candidate-gene approach identified a number of novel chromosome loci or genes that are associated with MI
PT E
D
(Ganesan et al., 2016). Low density lipoprotein receptor 8 (LRP8) gene is localized on chromosome 1, which is of 85,786 base pairs (bp) in size and consist of 22 exons (Gene ID: 7804). LRP8 is a cell surface protein that plays roles in both signal transduction and receptor
CE
mediated endocytosis of low density lipoprotein (LDL-C), very low density lipoprotein
AC
(VLDL-C) for lysosomal degradation and controls the lipoprotein homeostasis in plasma. LRP8 is expressed in endothelial cells, platelets, heart and muscles (Kim et al., 1996). Studies showed that dysfunction of lipid metabolism plays a key role in the development of MI by decreased HDLC, apolipoprotein A-I (apoA-I) levels and increased low density lipoprotein (LDLC) level (Kuo et al., 1998; Boersma et al., 2003; Thygesen et al., 2007; Reitz et al., 2010), total cholesterol (TC) and triglycerides (TG) (Karthikeyan et al., 2009). The elevated level of LDL-C is shown to be a high risk factor for coronary artery diseases from many 3
ACCEPTED MANUSCRIPT studies (Woo et al., 1993). Since, Indian populations are unique in their origin and are practicing endogamy for the past few decades, it can be expected in Indians to have a unique set of mutations which lead to several diseases (cardiac disease) in particular (Dhandapany et al., 2009; Reich et al., 2009; Nizamuddin et al., 2015; Selvi Rani et al., 2015). Among the fastest growing non-communicable diseases, cardiovascular diseases (CVDs) are expected to
PT
cause the largest number of mortality and morbidity within India (Chauhan and Aeri, 2013).
RI
Indians have unique lipid profile characterized by high triglycerides, low HDL-C, and
SC
increased LDL-C and VLDL levels (Hoogeveen et al., 2001). LRP8 play a major role in LDL-C, VLDL metabolism and this gene possess several SNPs (rs5174, rs10788952,
NU
rs2297660, rs7546246) that have been reported to be associated with plasma LDL, VLDL levels. Mutation rs5174/R952Q in LRP8 is shown to be associated with LDL induced platelet
MA
aggregation causing the development of atherosclerosis or atherothrombosis found in American, Caucasian and Italian population (Korporaal et al., 2004; Johns et al., 2005; Shen
D
et al., 2013). Reports indicate that SNPs or haplotypes that are associated with certain
PT E
diseases in the western population may not be associated with Indian population (Sharma and Ganguly, 2005; Rosenberg et al., 2010) . There is a need for identification of the loci/ gene
CE
for MI in Indian population compared to the western population. LRP8 gene has not been analyzed in South Indian population. Hence, the study was aimed to investigate whether
AC
LRP8 gene variation influences MI in South Indian population. Further, we also examined the association between LRP8 genetic variants with lipid levels with risk of MI. 2. Materials and methods 2.1 Sample details The study subjects were composed of 100 acute MI and 100 age and ethnically matched healthy controls from South India, of which 80% individuals are sporadic and late onset (age 4
ACCEPTED MANUSCRIPT group 58.94 ± 9.65) in present study. Blood samples were collected from K S Hegde and Yenepoya hospitals, Mangalore, India. The diagnosis of MI was based on typical electrocardiographic (ECG) changes; increase in the serum activities of enzymes such as creatine kinase, aspartate aminotransferase, and the concentration of troponin T, confirmed by the panel of cardiologists. Angiographically proved coronary artery disease after MI, those
PT
who underwent CABG (coronary artery bypass graft) after MI and acute coronary syndrome
RI
with MI were also considered. Control individuals were ethnically matched control and free
SC
from MI, as determined by medical history, clinical examinations or ECG. The mean age of healthy controls was 65.27 ± 10.30 years while that of patients was 58.94 ± 9.65 years. In MI
NU
patients 20% were tobacco chewers, while it was 8% among the healthy controls. The
MA
demographic details of the individuals included in the study are given in Table 1. 2.2 Sample collection and DNA isolation
D
The blood samples were collected from MI patients admitted in the cardiac care unit. The
PT E
blood samples of healthy controls from the same ethnic background without MI based on the electrocardiograph were collected. 10 ml of intravenous blood sample of both cases and
CE
controls were collected in the EDTA vaccutainer, after obtaining informed written consent. Genomic DNA isolation was done by using standard protocol (Miller et al., 1988). This study
AC
followed the principles outlined in the Declaration of Helsinnki (WMA World Medical Association Declaration of Helsinki) and approved by Institutional Ethics Committee of Yenepoya University and Nitte University, Mangalore, India. The serum lipid profile was measured within the first 24 hrs of the onset of symptoms of MI. The serum total cholesterol, HDL cholesterol, and triglyceride levels were measured on automated clinical chemistry machine in the clinical lab. All the blood samples were 6hr fasting samples.
5
ACCEPTED MANUSCRIPT 2.3 Genotyping The reference genomic sequence of LRP8 (ENSG0000157193) was obtained from the Ensemble database (asia.ensembl.org). The primers were designed to amplify coding region (UTR, exon and exon-intron boundaries) of LRP8 using Primer3 web version 4.0. PCRs
PT
(polymerase chain reactions) were performed using Gene Amp 9700 (Applied Biosystems, Foster City, USA) using Emerald Amp GT PCR master mix (TaKaRa) according to the
RI
manufacturer’s protocol. After PCR, Amplicons were size fractionated using 2% agarose gel,
SC
stained with ethidium bromide and observed under UV transilluminator. Subsequently, amplicons were treated with Exo-SAP (USB Corp., USA), and sequenced using a BigDye
NU
terminator (v3.1) cycle sequencing kit (Applied Biosystems, Foster City, USA) on an ABI
MA
3730XL DNA analyzer. Sequences obtained were assembled with the reference sequences using AutoAssembler software (Applied Biosystems, Foster City, USA). Variations observed
D
were noted for further analysis.
PT E
2.4 Statistical analysis
Allele and genotype frequencies were calculated by allele counting method. Single marker
CE
and haplotype based association analysis were carried out by Plink (Purcell et al., 2007) and Haploview software (Barrett et al., 2004). The haplotypes block was defined using
AC
confidence interval of Gabriel et al. (2002) method in Haploview. For both single marker and haplotype association analysis, Chi-square test was used. The p value < 0.05 were considered for statistical significance in all analysis. To explore the Hardy-Weinberg equilibrium (HWE), we consider genotype distribution in control samples and only those variants having HWE p value >0.05 were utilized in further association analysis. Linear regression analysis was also performed to explore the effect of the mutation on blood lipid levels using glm function of R. 6
ACCEPTED MANUSCRIPT 3. Results We have investigated the exons, exon-intron boundaries and UTR of LRP8 in 100 individuals with MI and 100 ethnically matched controls. We genotyped four SNPs within LRP8 (rs5174, rs10788952, rs2297660, rs7546246) of which two were in introns [rs7546246(A/G),
PT
rs10788952 (C/T)] and one was synonymous rs2297660 (C/A); one was nonsynonymous in 3’UTR [rs5174 (G/A)] (Table 2 and Fig 1).
RI
It is well known that spurious association can arise due to sample level substructure or
SC
genotyping error, and can be detected with Hardy-Weinberg equilibrium (HWE) test. In
NU
common practice, those SNPs, for which HWE is violated, should be removed in association analysis. However, we did not find any violation of HWE equilibrium for all 4 variants
MA
(p>0.05) (Table 3). None of the markers [rs5174 (p=0.6889), rs229766 (p=0.9255), rs107889 (p=0.0802) and rs7546246 (p=0.9729)] was significantly associated with MI in South Indian
D
population by single marker association study. Since we did not observe the single marker
PT E
SNP association; we proceeded for haplotype based association analysis using the Haploveiw software.
CE
3.1 Haplotype based Association Analysis
AC
In order to identify the haplotype in LRP8, haplotype analysis was carried out for genotyping data of SNPs (rs5174, rs10788952, rs2297660, rs7546246) and it was analyzed for their association with MI. Using Gabriel et al (2002) confidence intervals method, we observed single haplotype block (Fig 2) in our dataset. Only 2 SNPs (rs10788952 and rs7546246) were present in the block and have normalized coefficient of linkage disequilibrium or D’ >0.8. Further, we estimated haplotypes within the block (TG, CG, TA and CA) in our cohort and
7
ACCEPTED MANUSCRIPT performed association analysis. Intriguingly, we found haplotype TG (p=0.0155) and CG (p=0.00005) were significantly associated with risk of MI in South Indian patients (Table 4). 3.2 Effect of haplotype on blood lipid profile We have measured the blood profile of cases and controls by automated clinical chemistry
PT
machine and the mean values of TC, HDL, LDL, VLDL, triglycerides are 204.4 mg/dl, 39.4 mg/dl, 118.1 mg/dl, 27.7 mg/dl, 145.8 mg/dl respectively in the MI patients of South Indian
RI
population as shown in Table 1. The maximum, minimum and median values of total
SC
cholesterol, HDL, LDL, VLDL and triglycerides of MI are depicted in Table 5.
NU
Further, to explore the effect of mutation of the LRP8 gene on blood lipid levels, we performed the linear regression analysis against the total cholesterol (TC), LDL, HDL, VLDL
MA
and triglycerides in the MI cases (Table 6). Since CG was absent in the MI cases, we could analyze only TG haplotype. Interestingly, we observed that TG haplotype was significantly
D
associated with LDL (p=0.00607) and cholesterol level (p=0.0344), whereas HDL (p>0.523),
PT E
VLDL (p>0.772), triglycerides (p>0.883) were not significantly associated with MI. The haplotype CA (0.0241) with negative beta (-20.6) value were also seen to be significantly
CE
associated with LDL. This finding suggests that subjects with TG haplotype, are having significantly higher level of LDL and TC, comparative to other subjects with TA and CA
AC
haplotypes. It also suggests that rs10788952-rs7546246 (TG) haplotype might be increasing the risk of MI through increasing the level of LDL cholesterol. 4. Discussion MI is caused by both genetic and environmental factors (Walter and Zeiher, 2000). LRP8 plays a central role in human lipoprotein metabolism as it facilitates the clearance of bad cholesterol LDL, VLDL from plasma via endocytosis (Kim et al., 1996; Waterworth et al., 8
ACCEPTED MANUSCRIPT 2010). The previous study on the nonsynonymous mutation R952Q/rs5174 of LRP8 had shown a significant association in MI patients and not in control group (Shen et al., 2007; Lieb et al., 2008; Martinelli et al., 2009; Shen et al., 2013; Shen et al., 2014a). Considering the important role of LRP8 in lipid metabolism, we investigated the association of genetic variants of the LRP8 and lipid profile with the risk of myocardial infarction in patients from
PT
south India. In the current study, we did not observe a significant association of SNP variant
RI
(rs5174, rs10788952, rs2297660, and rs7546246) with the MI in South Indians; however, we
SC
found significant haplotype association TG (P=0.0155) and CG (P=0.00005) with MI in rs10788952 and rs7546246. A genome wide association analysis conducted on European
NU
descent Americans, Italian Caucasian and Korean population analyzed over 521786 markers and found fifty susceptibility SNPs were associated risk of an incident of MI (Shen et al.,
MA
2014a). Shen et al. 2014 observed that the genetic variant R952Q/rs5174 in the LRP8 gene was associated with familial and early-onset MI in European-descent Americans and Italian
D
Caucasian populations, but not with the sporadic and late onset form of the disease (Shen et
PT E
al., 2014a). Another study showed a significant association of SNP R952Q/ rs5174 of LRP8 with plasma triglyceride (222±16.6) level in 358 probands of Gene Quest Family cohort with
CE
familial and premature CAD and MI. Further, this study was replicated in independent Gene Bank cohort of 134 patients with premature MI (Shen et al., 2012). However, we did not find
AC
association of R952Q/rs5174 with MI. Different ancestry and clinicopathologic features (sporadic and late onset) of MI patients of present study, might be the reason of insignificant association. Interestingly, when analyzing our result by regression analysis, we found that haplotype TG positively (<0.006) associated with total cholesterol and LDL-C level in MI. Considering the significantly higher frequency of TG in MI patients comparative to controls, it can be concluded that TG haplotype increases the risk of MI in South Indian through increasing the bad cholesterol (LDL-C) level. 9
ACCEPTED MANUSCRIPT Our present study in lipid profile of MI patients showed an increase in TC (204.4 ± 49.4), LDL (118.1 ± 39.9), VLDL (27.7 ± 14.3) and triglycerides (145.8 ± 65.8) and decrease in HDL (39.4 ± 12.3) when compared to control, which is align with other population (Kumar and Sivakanesan, 2009). On the other hand, our result on genetic variants (rs5174, rs10788952, rs2297660, and rs7546246) was not found to be significantly associated with MI
Therefore, we investigated on
RI
the single marker based association with lipid profile.
PT
against the western Caucasian population (Shen et al., 2014a). Hence we could not perform
SC
haplotype based association analysis which resulted in presence of haplotype TG and CG are significantly associated with MI compared to controls. It is possible that individuals having
NU
mutation at both genetic loci (rs10788952: C>T and rs7546246: A>G) are having higher genetic risk of MI, comparative to mutation at single locus only and hence, more power to
MA
detect association in haplotype association analysis. This can be the reason; we found signal of association in haplotype based association analysis only. Haplotype CG was not present in
D
control, hence, we analyzed only TG which increases the LDL and total TC levels leading to
PT E
risk for the MI. To explore the effect of the mutation on blood lipid levels, linear regression analysis was performed. Interestingly we found TG haplotype association with LDL and TC
CE
levels. We observed the presence of haplotype CA was inversely correlated with LDL. Similarly, the study using the GeneQuest and Italian population, the risk haplotype TACGC
AC
in LRP8 had higher LDL levels was found with CAD and MI (Shen et al., 2014b). Further they showed TCCGC haplotype exists only in normal individuals and not in MI, where it acts as a protective role against MI (OR = 0.42–0.71) (Shen et al., 2013). In our study, high levels of triglycerides (145.81 ± 65.82) were observed in MI when compared to control (113.2 ± 32.05); however, no significant correlation between triglycerides levels and TG haplotype was observed.
10
ACCEPTED MANUSCRIPT In conclusion, we demonstrated that haplotype TG and CG of rs10788952 & rs7546246 are significantly associated with MI, whereas particularly presence of haplotype TG may significantly increase LDL and TC levels leading to MI. Further, our result showed that non synonymous variant R952Q /rs5174 were not observed in association with MI (sporadic and
PT
late-onset) in South Indian Population.
SC
RI
Conflict of interest statement
NU
All of the authors declare that there is no conflict of interest.
MA
Acknowledgements
We thank all the patients, cardiologists and nursing staff who helped us with this study. M A
D
acknowledges the support of Yenepoya University, Nitte University and Mangalore
PT E
University, Mangalore, India. The authors acknowledge Dr. K.Thangaraj Senior Scientist, CCMB-Hyderabad, Prof. Subramanyam K, cardiologist Nitte University, Prof Chakrapani
AC
CE
M, Physician KMC, for their constant support and encouragement.
11
ACCEPTED MANUSCRIPT References Barrett, J.C., Fry, B., Maller, J. and Daly, M.J., 2004. Haploview: analysis and visualization of LD and haplotype maps. Bioinformatics 21, 263-265. Boersma, E., Mercado, N., Poldermans, D., Gardien, M., Vos, J. and Simoons, M.L., 2003. Acute myocardial infarction. The Lancet 361, 847-858.
PT
Braunwald, E., 1997. Cardiovascular medicine at the turn of the millennium: triumphs, concerns, and
RI
opportunities. New England Journal of Medicine 337, 1360-1369.
Breslow, J.L., 1997. Cardiovascular disease burden increases, NIH funding decreases. Nature
SC
medicine 3, 600-601.
NU
Chauhan, S. and Aeri, B.T., 2013. Prevalence of cardiovascular disease in India and it is economic impact-A review. International Journal of Scientific and Research Publications 3, 1-5.
MA
Dhandapany, P.S., Sadayappan, S., Xue, Y., Powell, G.T., Rani, D.S., Nallari, P., Rai, T.S., Khullar, M., Soares, P. and Bahl, A., 2009. A common MYBPC3 (cardiac myosin binding protein C) variant associated with cardiomyopathies in South Asia. Nature genetics 41, 187-191.
D
Gabriel, S.B., Schaffner, S.F., Nguyen, H., Moore, J.M., Roy, J., Blumenstiel, B., Higgins, J.,
PT E
DeFelice, M., Lochner, A. and Faggart, M., 2002. The structure of haplotype blocks in the human genome. Science 296, 2225-2229.
CE
Ganesan, M., Nizamuddin, S., Katkam, S.K., Kumaraswami, K., Hosad, U.K., Lobo, L.L., Kutala, V.K. and Thangaraj, K., 2016. c.* 84G> A Mutation in CETP Is Associated with Coronary
AC
Artery Disease in South Indians. PloS one 11, e0164151. Hoogeveen, R.C., Gambhir, J.K., Gambhir, D., Kimball, K.T., Ghazzaly, K., Gaubatz, J.W., Vaduganathan, M., Rao, R.S., Koschinsky, M. and Morrisett, J.D., 2001. Evaluation of Lp [a] and other independent risk factors for CHD in Asian Indians and their USA counterparts. Journal of Lipid Research 42, 631-638. Johns, D.G., Ao, Z., Willette, R.N., Macphee, C.H. and Douglas, S.A., 2005. Role of p38 MAP kinase in postcapillary venule leukocyte adhesion induced by ischemia/reperfusion injury. Pharmacological research 51, 463-471. 12
ACCEPTED MANUSCRIPT Karthikeyan, G., Teo, K.K., Islam, S., McQueen, M.J., Pais, P., Wang, X., Sato, H., Lang, C.C., Sitthi-Amorn, C. and Pandey, M., 2009. Lipid profile, plasma apolipoproteins, and risk of a first myocardial infarction among Asians: an analysis from the INTERHEART Study. Journal of the American College of Cardiology 53, 244-253. Kim, D.-H., Iijima, H., Goto, K., Sakai, J., Ishii, H., Kim, H.-J., Suzuki, H., Kondo, H., Saeki, S. and
PT
Yamamoto, T., 1996. Human apolipoprotein E receptor 2 A novel lipoprotein receptor of the low density lipoprotein receptor family predominantly expressed in brain. Journal of
RI
Biological Chemistry 271, 8373-8380.
SC
Korporaal, S.J., Relou, I.A., van Eck, M., Strasser, V., Bezemer, M., Gorter, G., van Berkel, T.J., Nimpf, J., Akkerman, J.-W.N. and Lenting, P.J., 2004. Binding of low density lipoprotein to
Biological Chemistry 279, 52526-52534.
NU
platelet apolipoprotein E receptor 2′ results in phosphorylation of p38MAPK. Journal of
MA
Kumar, A. and Sivakanesan, R., 2009. Serum lipid profile abnormality in predicting the risk of myocardial infarction in elderly normolipidaemic patients in South Asia: a case-controlled
D
study. Internet J Altern Med 6, 6.
PT E
Kuo, Y.-M., Emmerling, M.R., Bisgaier, C.L., Essenburg, A.D., Lampert, H.C., Drumm, D. and Roher, A.E., 1998. Elevated low-density lipoprotein in Alzheimer's disease correlates with brain Aβ 1–42 levels. Biochemical and biophysical research communications 252, 711-715.
CE
Lieb, W., Zeller, T., Mangino, M., Götz, A., Braund, P., Wenzel, J.J., Horn, C., Proust, C., Linsel-
AC
Nitschke, P. and Amouyel, P., 2008. Lack of association of genetic variants in the LRP8 gene with familial and sporadic myocardial infarction. Journal of molecular medicine 86, 11631170.
Lloyd-Jones, D., Adams, R., Carnethon, M., De Simone, G., Ferguson, T.B., Flegal, K., Ford, E., Furie, K., Go, A. and Greenlund, K., 2009. Heart disease and stroke statistics—2009 update a report from the American Heart Association Statistics Committee and Stroke Statistics Subcommittee. Circulation 119, e21-e181.
13
ACCEPTED MANUSCRIPT Martinelli, N., Olivieri, O., Shen, G.-Q., Trabetti, E., Pizzolo, F., Busti, F., Friso, S., Bassi, A., Li, L. and Hu, Y., 2009. Additive effect of LRP8/APOER2 R952Q variant to APOE ε2/ε3/ε4 genotype in modulating apolipoprotein E concentration and the risk of myocardial infarction: a case-control study. BMC medical genetics 10, 41. Mayer, B., Erdmann, J. and Schunkert, H., 2007. Genetics and heritability of coronary artery disease
PT
and myocardial infarction. Clinical Research in Cardiology 96, 1-7.
human nucleated cells. Nucleic acids research 16, 1215.
RI
Miller, S., Dykes, D. and Polesky, H., 1988. A simple salting out procedure for extracting DNA from
SC
Nizamuddin, S., Govindaraj, P., Saxena, S., Kashyap, M., Mishra, A., Singh, S., Rotti, H., Raval, R., Nayak, J. and Bhat, B., 2015. A novel gene THSD7A is associated with obesity. International
NU
Journal of Obesity 39, 1662.
Peden, J.F. and Farrall, M., 2011. Thirty-five common variants for coronary artery disease: the fruits
MA
of much collaborative labour. Human molecular genetics, ddr384. Purcell, S., Neale, B., Todd-Brown, K., Thomas, L., Ferreira, M.A., Bender, D., Maller, J., Sklar, P.,
D
De Bakker, P.I. and Daly, M.J., 2007. PLINK: a tool set for whole-genome association and
PT E
population-based linkage analyses. The American Journal of Human Genetics 81, 559-575. Reich, D., Thangaraj, K., Patterson, N., Price, A.L. and Singh, L., 2009. Reconstructing Indian population history. Nature 461, 489-494.
CE
Reitz, C., Tang, M.-X., Schupf, N., Manly, J.J., Mayeux, R. and Luchsinger, J.A., 2010. Association
AC
of higher levels of high-density lipoprotein cholesterol in elderly individuals and lower risk of late-onset Alzheimer disease. Archives of neurology 67, 1491-1497. Rosenberg, N.A., Huang, L., Jewett, E.M., Szpiech, Z.A., Jankovic, I. and Boehnke, M., 2010. Genome-wide association studies in diverse populations. Nature reviews. Genetics 11, 356. Selvi Rani, D., Nallari, P., Dhandapany, P.S., Rani, J., Meraj, K., Ganesan, M., Narasimhan, C. and Thangaraj, K., 2015. Coexistence of digenic mutations in both thin (tpm1) and thick (myh7) filaments of sarcomeric genes leads to severe hypertrophic cardiomyopathy in a South Indian FHCM. DNA and cell biology 34, 350-359. 14
ACCEPTED MANUSCRIPT Sharma, M. and Ganguly, N.K., 2005. Premature coronary artery disease in Indians and its associated risk factors. Vascular health and risk management 1, 217. Shen, G.-Q., Girelli, D., Li, L., Olivieri, O., Martinelli, N., Chen, Q., Topol, E.J. and Wang, Q.K., 2013. Multi-allelic haplotype association identifies novel information different from singleSNP analysis: A new protective haplotype in the LRP8 gene is against familial and early-
PT
onset CAD and MI. Gene 521, 78-81. Shen, G.-Q., Girelli, D., Li, L., Rao, S., Archacki, S., Olivieri, O., Martinelli, N., Park, J.E., Chen, Q.
RI
and Topol, E.J., 2014a. A Novel Molecular Diagnostic Marker for Familial and Early-Onset
SC
CAD and MI in the LRP8 Gene. Circulation: Cardiovascular Genetics, CIRCGENETICS. 113.000321.
NU
Shen, G.-Q., Girelli, D., Li, L., Rao, S., Archacki, S., Olivieri, O., Martinelli, N., Park, J.E., Chen, Q. and Topol, E.J., 2014b. A novel molecular diagnostic marker for familial and early-onset
MA
coronary artery disease and myocardial infarction in the LRP8 gene. Circulation: Cardiovascular Genetics 7, 514-520.
D
Shen, G.-Q., Li, L., Girelli, D., Seidelmann, S.B., Rao, S., Fan, C., Park, J.E., Xi, Q., Li, J. and Hu,
PT E
Y., 2007. An LRP8 variant is associated with familial and premature coronary artery disease and myocardial infarction. The American Journal of Human Genetics 81, 780-791. Shen, G.Q., Li, L. and Wang, Q.K., 2012. Genetic Variant R952Q in LRP8 is Associated with
CE
Increased Plasma Triglyceride Levels in Patients with Early‐Onset CAD and MI. Annals of
AC
human genetics 76, 193-199. Thygesen, K., Alpert, J.S. and White, H.D., 2007. Universal definition of myocardial infarction. Journal of the American College of Cardiology 50, 2173-2195. Walter, D.H. and Zeiher, A.M., 2000. Genetic risk factors for myocardial infarct. Herz 25, 7-14. Waterworth, D.M., Ricketts, S.L., Song, K., Chen, L., Zhao, J.H., Ripatti, S., Aulchenko, Y.S., Zhang, W., Yuan, X. and Lim, N., 2010. Genetic variants influencing circulating lipid levels and risk of coronary artery disease. Arteriosclerosis, thrombosis, and vascular biology 30, 2264-2276.
15
ACCEPTED MANUSCRIPT Woo, J., Ho, S., Wong, S., Woo, K., Tse, C., Chan, K., Kay, C., Mak, W., Cheung, K. and Lam, C., 1993. Lipids, lipoproteins and other coronary risk factors in Chinese male survivors of myocardial infarction. International journal of cardiology 39, 195-202. Yamada, Y., Izawa, H., Ichihara, S., Takatsu, F., Ishihara, H., Hirayama, H., Sone, T., Tanaka, M. and Yokota, M., 2002. Prediction of the risk of myocardial infarction from polymorphisms in
AC
CE
PT E
D
MA
NU
SC
RI
PT
candidate genes. New England Journal of Medicine 347, 1916-1923.
16
ACCEPTED MANUSCRIPT Figures legends: Fig 1. Observed variations and its location in LRP8 gene mRNA. ENSG00000157193 was utilized to represent the physical location of variants Fig 2. LD analysis of LRP8 structure defined by four SNPs (rs5174, rs2297660, rs10788952,
PT
rs7546246) within LRP8 regions. Strong LD was detected between rs10788952 and rs7546246 with LRP8 spanning 0kb size. The pair wise correlation between SNPs was
RI
measured as D’ and shown (x100) in each diamond. Red to white color gradient indicates the
AC
CE
PT E
D
MA
NU
SC
magnitude of pair wise LD, the region from higher to lower values of LD.
17
RI
PT
ACCEPTED MANUSCRIPT
AC
CE
PT E
D
MA
NU
SC
Fig. 1
18
PT E
D
MA
NU
SC
RI
PT
ACCEPTED MANUSCRIPT
AC
CE
Fig. 2
19
ACCEPTED MANUSCRIPT Table 1: Demographic and lipid profiles of patients and controls with MI MI
Control
Number of samples
100
100
Linguistic affiliation
Dravidian (Kannadiga)
Dravidian (Kannadiga)
Age (years) at sampling (mean±S.D.)
58.94 ± 9.65
65.27 ± 10.30
Tobacco
20%/80%
10% /90%
Alcohol
35%/65%
Hypertension
32%/68%
Family history (Y/N)
15%/85%
Total cholesterol (mg/dl) (mean±S.D.)
204.4 ± 49.4
HDL (mg/dl) (mean±S.D.)
39.4 ± 12.3
47.4 ± 8.1
LDL (mg/dl) (mean±S.D.)
118.1 ± 39.9
88.1 ± 19.6
TG (mg/dl) (mean±S.D.)
RI
6.66% /93.33%
SC
NU
MA
VLDL (mg/dl) (mean±S.D.)
PT
Traits
NA Nil 175.7 ± 31.9
27.7 ±14.3
23.3 ± 8.7
145.8 ± 65.8
113.2 ± 32.05
AC
CE
PT E
D
S.D.- Standard deviation; (mg/dl) – Milligram/deciliter
20
ACCEPTED MANUSCRIPT Table 2: Genotype and allele frequency distributions of SNPs in LRP8 among cases and controls Allele
MI
Control
P - value
HWE - Value
rs7546246
GG
0
26
0.9729
0.6039
GA
31
11
AA
53
2
GG
63
31
AG
26
9
AA
0
CC
56
CA
24
AA
0
CC
49
29
CT
36
9
0
2
0.9255
0.5587
0.0802
1
RI
SC
NU
MA
9
D
TT
PT E
rs10788952
29
CE
rs22977660
0.09119
0.6889
3
AC
rs5174
PT
SNP
21
1
ACCEPTED MANUSCRIPT Table 3: Single marker association study Assoc-
Case, Control-
Case, Control-
SNPs
Association Chi-square
HWE P-value
Ratio-Counts
Frequencies
P-value
rs5174
G
146:26,68:14
0.849,0.829
0.16
0.6889
0.09119
rs2297660
C
23:135,11:67
0.146,0.141
0.009
0.9255
0.5587
rs10788952
T
36:134,9:67
0.212,0.118
3.062
rs7546246
A
136:32,63:15
0.810,0.808
0.001
AC
CE
PT E
D
MA
NU
SC
RI
PT
Allele
22
0.0802
1
0.9729
0.6039
ACCEPTED MANUSCRIPT Table 4: Haplotype based association study Haplotype
F_A
F_U
ChiSq
DF
P
SNPs
H1
TG
0.189
0.06757
5.859
1
0.0155
rs10788952|rs7546246
H1
CG
0
0.1216
20.73
1
0.00005
rs10788952|rs7546246
H1
TA
0.01829
0.04054
1.027
1
0.3109
rs10788952|rs7546246
H1
CA
0.7927
0.7703
0.1521
1
0.6965
rs10788952|rs7546246
AC
CE
PT E
D
MA
NU
SC
RI
PT
Locus
23
ACCEPTED MANUSCRIPT Table 5: Cholesterol levels in MI patients of South India.
Min Median Max
TC
HDL
LDL
VLDL
TG
86
22
43
11
41
210.5
38
108
23.5
134.5
343
93
241
81
404
PT
TC – Total cholesterol; HDL – High density lipoprotein; LDL – Low density lipoprotein;
AC
CE
PT E
D
MA
NU
SC
RI
VLDL – Very low density lipoprotein; TG - Triglycerides
24
ACCEPTED MANUSCRIPT Table 6: Effect of haplotype on blood lipid level: linear regression analysis
BP1
BP2
SNP1
5373818 2
2
2
3
3
3
SNP2
HAP
F
BETA
LDL
P
BETA
P
HDL
1
1
1
rs1078895
53738100
0.19 rs7546246
3
2
5373818
rs1078895
3
2
5373818
rs1078895
TG
25.2
0.00
C S U
0.03
-1.89
BET
P
0.52
rs7546246
53738100
D E
rs7546246 2
TA
CA
0.02
N A
-26.6
-14.9
-20.6
-60
M
0.79
0.04
0.27
P
C C
P A
0.77 0.98
0.88 2.32
2
-8.91
3
-
0.70
-
0.60
3.37
2
20.9
8
0.89
0.79
0.24
0.19
0.02
3.13
0.28 0.46
T P E
Triglyceride BET
A
6
53738100
3
24.4
VLDL
T P
BETA
I R
CHR
NHAP
NSNP
TC
0.95 9
SNP – Single nucleotide polymorphism; NSNP - synonymous SNP; NHAP-Number of haplotype associated Polymorphism ; CHR – chromosome; BP1 –
A
basepair 1; BP2 – basepair 2; HAP – haplotype; F – frequency; TC – total cholesterol; LDL – low density lipoprotein; HDL – high density lipoprotein; VLDL – very low density lipoprotein
25
ACCEPTED MANUSCRIPT
Abbreviations
MA
HDLC - High density lipoprotein cholesterol apoA-I - Apolipoprotein A-I
PT E
TG- Triglycerides
D
TC- Total cholesterol
NU
VLDL C- very low density lipoprotein cholesterol
CE
CVDs - CardioVascular diseases
AC
HWE- Hardy-Weinberg equilibrium
26
RI
LDL C - low density lipoprotein cholesterol
SC
LRP8- Low density lipoprotein receptor related protein 8
PT
MI - Myocardial infarction
ACCEPTED MANUSCRIPT Highlights
Myocardial infarction (MI) is multifactorial and caused by imbalance in the blood lipid levels.
LRP8 plays central role in human lipoprotein metabolism as it facilitates the clearance of
PT
SC
RI
bad cholesterol LDL, VLDL from plasma.
Evaluated the TG and CG Haplotypes in the LRP8 gene and found to be associated with
Haplotype TG is significantly associated increase in LDL and total cholesterol (TC)
MA
NU
Myocardial Infarction in South Indian population.
PT E
Non synonymous variant rs5174 associated with MI in western population were not
CE
associated in South Indian Population.
AC
D
levels in MI patients and suggests being risk factor for MI in south Indian population.
27