Associations of lipid levels susceptibility loci with coronary artery disease in Chinese population

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1 Wng et l. Lipids in Helth nd Disese (2015) 14:80 DOI /s RESEARCH Open Access Associtions of lipid levels susceptibility loci with coronry rtery disese in Chinese popultion Xue-bin Wng 1, Y-di Hn 1, Ning-hu Cui 2, Ji-ji Go 1, Jie Yng 1, Zhu-ling Hung 1, Qing Zhu 3* nd Fng Zheng 1* Abstrct Bckground: Recent genome-wide ssocition studies (GWAS) hve identified severl single nucleotide polymorphisms (SNPs) tht were ssocited with blood lipid levels in Cucsins. This study investigted whether these loci influenced lipid levels nd whether they were ssocited with the risk of coronry rtery disese (CAD) nd its ngiogrphic severity in Chinese popultion. Methods: Six SNPs were genotyped in 1100 CAD cses nd 1069 controls using the high-resolution melting (HRM) method. Coronry therosclerosis severity ws ssessed by the vessel scores nd the Gensini scoring system. Results: Among the 6 SNPs nd the genetic risks scores (GRS), the minor lleles of HNF1A rs (odd rtio (OR) = 1.18, 95 % confidence intervl (CI) , P = 0.006) nd MADD-FOLH1 rs (OR = 1.20, 95 % CI , P = 0.002) s well s the GRS (P = ) were significntly ssocited with incresed risk of CAD fter flse discovery rte (FDR) correction. The vessel (P = 0.013) nd Gensini scores (β =0.113,P=0.002)differedmong CAD ptients with different SNP rs C > T genotypes. The multiple liner regression nlyses using n dditive model reveled tht the minor llele C of SNP rs (β =0.060,P=0.001)ndtheGRS(β =0.033,P= ) were significntly ssocited with incresed totl cholesterol (TC) levels, the minor llele A of SNP rs (β =-0.024, P = 0.007) nd the GRS (β = , P = 0.004) were significntly ssocited with decresed high-density lipoprotein cholesterol (HDL-c) levels. Conclusions: The present study demonstrted tht SNPs rs , rs nd the GRS were significntly ssocited with lipid levels nd the risk of CAD in Chinese popultion. Furthermore, the llele C of SNP rs incresed the odds of coronry therosclerosis severity. Keywords: Coronry rtery disese, HNF1A rs , MADD-FOLH1 rs , Gensini scores Bckground Coronry rtery disese (CAD), one of the most common crdiovsculr disese [1], is ssocited with high morbidity nd mortlity nd remins one of the most common cuses of deth globlly [2]. A min underlying pthology of CAD is therosclerosis, process of cumultive deposition of lipoproteins in the rteries supplying blood to the hert tht eventully leds to impired or bsent blood supply nd myocrdil infrction (MI) [3]. Atherosclerosis hs numerous genetic nd environmentl risk fctors [4], * Correspondence: zhuqingdr@163.com; zhengfng@whu.edu.cn 3 Deprtment of Gsteroenterology, Provincil Hospitl Affilited to Shndong University, Jinn , Shndong, Chin 1 Center for Gene Dignosis, Zhongnn Hospitl of Wuhn University, Wuhn , Hubei, Chin Full list of uthor informtion is vilble t the end of the rticle nd bnormlities of plsm lipids nd lipoproteins re heritble risk fctors for CAD, with heritbility estimtes rnging from % for totl cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-c), high-density lipoprotein cholesterol (HDL-c) nd % for CAD [5]. Recently, independent genome-wide ssocition studies (GWAS) hve identified severl loci tht influence blood lipid levels nd CAD risk in Cucsins [6 11]. However, the ssocitions between these novel single nucleotide polymorphisms (SNPs), lipid levels nd the risk of CAD were not well estblished in Chinese popultion. Otherwise, few studies hve exmined the utility of genetic risk scores (GRS) to identify Chinese subjects t incresed CAD risk [12, 13] Wng et l. This is n Open Access rticle distributed under the terms of the Cretive Commons Attribution License ( which permits unrestricted use, distribution, nd reproduction in ny medium, provided the originl work is properly credited. The Cretive Commons Public Domin Dediction wiver ( cretivecommons.org/publicdomin/zero/1.0/) pplies to the dt mde vilble in this rticle, unless otherwise stted.

2 Wng et l. Lipids in Helth nd Disese (2015) 14:80 Pge 2 of 11 The locus rs on chromosome 11p11.2, ws first identified s strongly lipid-ssocited locus ccording to GWAS in Cucsins [6]. This study lso identified SNP rs s novel lipid-ssocited locus locted in intron of the dynein xoneml hevy chin 11 (DNAH11) gene [6]. SNP rs , which is locted 4.3 kb downstrem of the ABO blood group (ABO) gene, ws lso found to be ssocited with lipid levels in GWAS of Europens [10]. Then lrge-scle ssocition nlysis in individuls of Europen descent identified the following three vrints ssocited with lipid levels nd CAD risk: SNPs rs , rs nd rs , which locted in or ner the heptocyte nucler fctor 1-α (HNF1A) gene, N-cetyltrnsferse 2 (NAT2) gene nd SPT2, Suppressor of Ty, domin contining 1 (SPTY2D1) gene, respectively [11]. In this study, we imed to exmine the ssocitions of these 6 lipid-ssocited vrints (individully nd in combintion) with lipid levels nd CAD risk in Chinese popultion. Additionlly, cross-sectionl study on the ssocitions between these six SNPs nd the severity of coronry therosclerosis hs been conducted. Mterils nd methods Study popultion Study prticipnts were recruited from Zhongnn hospitl of Wuhn University nd Asi Hert Hospitl between Jnury 2011 nd October The present cse control study involved 1100 CAD cses nd 1069 non-cad controls. CAD cses were dignosed bsed on 50 % luminl stenosis in t lest one mjor coronry rteries or their mjor brnches by stndrd coronry ngiogrphy. Non-CAD controls were the subjects without detectble stenosis by coronry ngiogrphy nd helthy popultion controls without dignosis of CAD, hypertension nd dibetes mellitus (DM) by routine physicl exmintions. Otherwise, to ssess the effects of these six SNPs on lipid levels in our control group, subjects who were undergoing lipid-lowering mediction or dyslipidemi were lso excluded from the controls. Fsting concentrtions of the plsm glucose, C-rective protein (CRP), TC, TG, LDL-c nd HDL-c were mesured using stndrd methods [14]. Other clinicl dt collected from study prticipnts included ge, sex, history of smoking, lcohol intke, body mss index (BMI), weight sttus (overweight nd obesity sttus), hypertension, dibetes mellitus, dyslipidemi nd phrmcologicl therpy (including lipid/glucose/blood pressure lowering drug tretment) sttus. BMI ws defined s weight divided by height in squre meters (kg/ m 2 ). Overweight nd obesity were defined s BMI 25 nd 30, respectively [15]. Hypertension ws dignosed bsed on usge of ongoing therpy for hypertension, systolic blood pressure (SBP) of 140 mmhg or distolic blood pressure (DBP) of 90 mmhg [16]. DM ws defined s ongoing therpy for dibetes or fsting plsm glucose (FPG) levels of 7.0 mmol/l, or with plsm glucose levels of 11.1 mmol/l [17]. This study ws pproved by ethnics committee of Zhongnn hospitl of Wuhn University nd met the declrtion of Helsinki. Scoring of coronry ngiogrm Coronry ngiogrms were scored ccording to the vessel nd Gensini scores. The vessel scores were defined s the number of vessels hving 50 % stenosis. In the Gensini scoring system [18], the nrrowing of the coronry rtery lumen is scored 1 for 0 25 % stenosis, 2 for %, 4 for %, 8 for %, 16 for % nd 32 for 100 %. Ech stenosed segment ws then weighted from 0.5 to 5, depending on the functionl significnce of the re supplied by tht segment. These scores were multiplied by the coefficient defined for ech coronry rtery nd segment, nd the results were then summed. The scores were independently ssessed by two experienced interventionl crdiologists who were blinded to the procedurl dt nd clinicl outcomes. The κ for inter-observer vribility tht ws used to estimte the vessel nd Gensini scores were 0.98, 0.88, respectively, wheres the κ for intr-observer vribility tht were 0.99, 0.93, respectively. Any disgreements regrding the scores were resolved by consensus. SNP selection nd genotyping We first looked for previously reported SNPs ssocited with lipid levels from GWAS with repliction evidence in Cucsin popultions [6, 7, 10, 11]. Then, SNPs tht hve been ssocited with CAD risk in Chinese (such s SORT1 rs [19], CILP2 rs [19], DOCK7rs [20], etc.) were excluded. Finlly, to derive dequte power (>80 %), only SNPs with minor llele frequencies (MAF) >15 % in Chinese individuls in the HpMp dtbse were selected, including HNF1A rs , MADD-FOLH1 rs , NAT2 rs , DNAH11 rs , ABO rs , SPTY2D1 rs (Tble 1). Genomic DNA ws isolted from peripherl blood white cells using the phenol/chloroform method. SNPs were genotyped using LightScnner 96 High Resolution Melt (HRM) system (Idho Technology, Slt Lke City, UT, USA). PCR rection for genotyping ws performed in totl of 10 μl PCR volume contining 1 μl of LC green dye, 5 pmol of ech primer, 25 ng of genomic DNA, 2 μl of 10 PCR buffer with 1.5 mmol/l MgCl 2, 2 mmol of deoxynucleotide triphosphtes, nd 1 unit of Tq polymerse. HRM nlysis is employed post- PCR to thermlly denture the smll mplicons nd mesure the subtle differences in melting temperture

3 Wng et l. Lipids in Helth nd Disese (2015) 14:80 Pge 3 of 11 Tble 1 Chrcteristics of 6 SNPs in our study SNP Chr Position Nerby gene Risk llele RAF HWE (P vlue) Cse Control Cse Control rs q HNF1A C rs p MADD-FOLH1 A rs p NAT2 G rs p DNAH11 C rs q ABO T rs p SPTY2D1 C Chr chromosome, RAF risk llele frequency, HWE Hrdy-Weinberg equilibrium Informtion for chromosome position is bsed on NCBI genome build 38.2 (Tm) between different genotypes [21]. Wild-type nd homozygous mutnt smples re distinguished by Tm shifts. Heterozygous smples re best distinguished from homozygous, not by Tm, but by ltered curve shpe (Fig. 1). Genotyping cll rtes for ll 6 SNPs were > 99 %. Primer detils nd product lengths were shown in Additionl file 1. For ech SNP, totl of 24 cses nd controls were rndomly selected to be sequenced, nd the genotypes were confirmed. DNA sequence nlysis ws performed with forwrd nd/or reverse primers using the BigDye Termintior v3.1 Cycle Sequencing Kits on n ABI PRISM 3100 genetic Anlyzer (Applied Biosystems, Foster City, CA, USA). Sttisticl nlyses Continuous vribles with norml distribution were expressed s men ± SD, nd differences between groups were compred by the Student s t-tests. Vribles with skewed distributions were ln-trnsformed before nlyses. Ctegoricl vribles were represented s percentges nd were tested by the χ 2 tests, which were lso used to test for devition of genotype distributions from Hrdy-Weinberg equilibrium (HWE). In the cse control nlyses, the llele frequencies in cses nd controls were compred using the χ 2 tests. The genotypic ssocitions of 6 SNPs with CAD risk were ssessed by the logistic regression nlyses under different models of inheritnce (dditive, recessive nd dominnt) fter djusting for ge, sex, smoking, lcohol intke, weight sttus, hypertension, type 2 dibetes, dyslipidemi nd phrmcologicl therpy covrites. The weighted GRS of CAD were the weighted sum cross three significnt (uncorrected) SNPs (rs , rs nd rs ) combining the odds rtios (ORs) nd doses of risk lleles bsed on n dditive model [22, 23]. The effects of SNPs on plsm lipid levels were ssessed by the multiple liner regression nlyses under n dditive model. The weighted GRS of lipid levels were lso ssessed s the sum of doses of the risk lleles weighted by the β coefficients t the polymorphisms [24, 25]. For CAD crosssectionl study, the vessel scores were compred mong the genotypes of six SNPs using the liner-by-liner ssocition χ 2 test nd the χ 2 test. Otherwise, the ssocitions between the Gensini scores nd six SNPs were ssessed by treting the Gensini scores s quntittive trits (the multiple liner regression) nd using medin cse control methods (the logistic regression) [26, 27]. Using the flse discovery rte (FDR) method [28], multiple testing correction for the ssocitions with lipid levels, CAD risk nd coronry therosclerosis severity were conducted seprtely. The P FDR vlue ws clculted by multiplying its P vlue by the number of tests performed nd then divided by the rnk order of ech P vlue (where rnk order 1 is ssigned to the smllest P vlue). An FDR of 0.05 ws used s criticl vlue to ssess whether P FDR vlue ws significnt. Sttisticl nlyses were conducted by SPSS 17.0 (SPSS, Inc., Chicgo, Illinois, USA). Power nlysis ws crried out using Power nd Smple Size Progrm 3.0 (Vnderbilt University, Nshville, TN, USA). Results Chrcteristics of the study popultion Clinicl chrcteristics of the study popultion re listed in Tble 2. Age nd gender distributed similrly between the two groups. Blood pressure (BP), plsm concentrtions of glucose, TC, TG, CRP levels, BMI nd the prevlence of overweight nd obesity were significntly higher, while HDL-c levels were significntly lower in the cse group thn those of the control group. There were no significnt differences for LDL-c levels nd the rte of smoking nd lcohol intke between the two groups. The genotypes of 6 SNPs were ll in HWE (P > 0.05, Tble 1). Associtions with lipid levels We investigted the ssocitions between six novel SNPs nd lipid levels in 1069 helthy control subjects (Tble 3). Under n dditive model djusted for ge, sex, lcohol intke, smoking nd weight sttus, the minor llele C of HNF1A rs ws significntly ssocited with incresed TC levels (β = 0.060, SE = 0.018, P = 0.001) nd

4 Wng et l. Lipids in Helth nd Disese (2015) 14:80 Pge 4 of 11 Fig. 1 HRM plots for different genotypes of six SNPs. The normlized melting peks re given in the left column, nd the normlized melting curves re given in the right column. Arrows indicte the genotypes. The representtive HRM plots of HNF1A rs , MADD-FOLH1 rs , NAT2 rs , DNAH11 rs , ABO rs , SPTY2D1 rs re shown in, b, c, d, e nd f, respectively

5 Wng et l. Lipids in Helth nd Disese (2015) 14:80 Pge 5 of 11 Tble 2 Clinicl chrcteristics of prticipnts in the cse control study Chrcteristics CAD ptients (n = 1100) Controls (n = 1069) Age, yers 59.9 ± ± Mle, n (%) 666 (60.5) 659 (61.6) Current smoking, n (%) 415 (37.7) 373 (34.9) Alcohol intke, n (%) 409 (37.2) 387 (36.2) BMI, kg/m ± ± 3.35 <0.001 Overweight, n (%) 341 (31.0) 278 (26.0) Obesity, n (%) 183 (16.6) 99 (9.3) <0.001 Hypertension, n (%) 647 (58.8) 0 (0) <0.001 DM, n (%) 357 (32.5) 0 (0) <0.001 Hyperlipidemi, n (%) 234 (21.3) 0 (0) <0.001 Antihypertensive 601 (54.6) 0 (0) <0.001 tretment, n (%) Glucose-lowering 323 (29.4) 0 (0) <0.001 tretment, n (%) Lipid-lowering tretment, n (%) 226 (20.5) 0 (0) <0.001 SBP, mmhg ( ) ( ) <0.001 DBP, mmhg 83.5 ( ) 80.7 ( ) <0.001 FPG, mmol/l 6.04 ( ) 4.97 ( ) <0.001 TC, mmol/l 4.32 ± ± 0.42 <0.001 TG, mmol/l 1.53 ± ± 0.36 <0.001 LDL-c, mmol/l 2.78 ± ± HDL-c, mmol/l 1.22 ± ± 0.22 <0.001 CRP, mmol/l 4.54 ± ± 1.78 <0.001 Gensini scores 22.3 (21.2, 23.5) Dt re expressed s men ± SD or geometric men (95 % confidence intervl) CAD coronry rtery disese, BMI body mss index, DM dibetes mellitus, SBP systolic blood pressure, DBP distolic blood pressure, FPG fsting plsm glucose, TC totl cholesterol, TG triglyceride, LDL-c low-density lipoprotein cholesterol, HDL-c high-density lipoprotein cholesterol, CRP C-rective protein decresed HDL-c levels (β = , SE = 0.009, P = 0.046); The minor llele A of MADD-FOLH1 rs ws ssocited with decresed HDL-c levels (β = , SE = 0.009, P = 0.007). In ddition, the lipid levels GRS bsed on n dditive model showed positive ssocitions with incresed TC levels (β = 0.033, SE = 0.009, P = ) nd decresed HDL-c levels (β = , SE = 0.005, P = 0.004). After FDR correction for multiple testing, the sttisticl ssocitions for SNP rs with TC levels (P FDR = 0.014), SNP rs with HDL-c levels (P FDR = 0.049) nd the GRS with TC (P FDR =0.010) nd HDL-c levels (P FDR = 0.037) remined significnt. Associtions with the risk of CAD Among the 6 SNPs, there were significnt differences in the llele frequencies between CAD cses nd controls P for HNF1A rs (OR = 1.18, 95 % CI = , P = 0.006), MADD-FOLH1 rs (OR = 1.20, 95 % CI = , P = 0.002) nd NAT2 rs (OR = 1.15, 95 % CI = , P = 0.024) (Tble 4). After correction for multiple testing, the minor lleles of SNPs rs (P FDR = 0.030) nd rs (P FDR = 0.025) were still significntly ssocited with incresed risk of CAD. Bsed on MAF of 43.0 % in our control group nd type I error of 0.05, the recruited smples could provide 84.1 % power to detect genetic effect with n llelic OR of 1.20 for SNP rs For SNP rs , our popultion hd power of 84.5 % to detect the ssocition with CAD risk ssuming n llelic OR of 1.20 nd MAF of 48.1 % in our control group. To explore the potentil inheritnce ptterns, three models of inheritnce including dditive, dominnt nd recessive models were explored for ech SNP (Tble 4). Results from the logistic regression nlyses indicted tht SNPs rs nd rs were significntly ssocited with the risk of CAD under both dditive (OR = 1.25, 95 % CI = , P = 0.008, P FDR = for SNP rs ; OR = 1.22, 95 % CI = , P = 0.012, P FDR = for SNP rs ) nd dominnt models (OR = 1.45, 95 % CI = , P = 0.004, P FDR = for SNP rs ; OR = 1.50, 95 % CI = , P = 0.003, P FDR = for SNP rs ) fter djusting for ge, sex, current smoking, lcohol intke, weight sttus, hypertension, type 2 dibetes, dyslipidemi nd phrmcologicl therpy covrites. Significnt genotypic ssocition ws identified between SNP rs nd CAD risk under recessive model (OR = 1.31, 95 % CI = , P = 0.029), but it ws not sufficient robust to withstnd the FDR correction (P FDR = 0.081). Associtions between other three SNPs nd CAD risk were not significnt in neither llelic nor genotypic ssocition nlyses. To exmine the cumultive effect of three ssocited SNPs (rs , rs nd rs ) on the risk of CAD, the weighted GRS ws clculted. The men GRS of CAD cses ws significntly higher thn tht of controls (P = , P FDR = ). Moreover, the logistic regression nlyses reveled tht subjects with the top quintile GRS were ssocited with 1.89-fold incresed risk of CAD compred with those hving the low quintile GRS (fter djusting for 9 covrites, Fig. 2 nd Additionl file 2). Angiogrphic severity of coronry therosclerosis The ssocitions between six SNPs nd the ngiogrphic severity in CAD cses were evluted by investigting the vessel nd Gensini scores. According to the number of significntly ffected vessels, we observed dosedependent effect of genotypes of SNP rs on the vessel scores (χ 2 test for liner-by-liner ssocition: P = 0.013; χ 2 test:p=0.002,p FDR = 0.012, Tble 5).

6 Tble 3 Associtions of 6 SNPs with lipid levels in 1069 helthy control subjects SNP Chr Locus Risk/non-risk llele TG (mmol/l) TC (mmol/l) LDL-c (mmol/l) HDL-c (mmol/l) β (SE) P P FDR β (SE) P P FDR β (SE) P P FDR β (SE) P P FDR rs HNF1A C/A (0.016) (0.018) (0.015) (0.009) rs MADD-FOLH1 A/G (0.015) (0.018) (0.015) (0.009) rs NAT2 G/A (0.016) (0.018) (0.015) (0.009) rs DNAH11 C/T (0.015) (0.018) (0.015) (0.009) rs ABO T/C (0.038) (0.044) (0.036) (0.022) rs SPTY2D1 C/T (0.015) (0.018) (0.014) (0.009) GRS (0.008) (0.009) (0.006) (0.005) SNP TC TG LDL-c HDL-c β (SE) P P FDR β (SE) P P FDR β (SE) P P FDR β (SE) P P FDR rs (0.018) (0.015) (0.015) (0.009) rs (0.018) (0.015) (0.015) (0.009) rs (0.018) (0.016) (0.015) (0.009) rs (0.018) (0.015) (0.015) (0.009) rs (0.022) (0.019) (0.018) (0.011) rs (0.018) (0.015) (0.015) (0.009) GRS (0.009) (0.008) (0.006) (0.005) Itlic vlues re sttisticlly significnt fter FDR correction Chr chromosome, TC totl cholesterol, TG triglyceride, LDL-c low-density lipoprotein cholesterol, HDL-c high-density lipoprotein cholesterol; β (SE), effect size (stndrd error) P-vlue from Benjmini-Hochberg method control for flse discovery rte (FDR) Wng et l. Lipids in Helth nd Disese (2015) 14:80 Pge 6 of 11

7 Tble 4 Allelic nd genotypic ssocitions of 6 SNPs with CAD risk in our cse control study SNP Genotype Frequency (n) Allelic comprison Additive model Dominnt model Recessive model Cses Controls OR (95 % CI) P P FDR OR (95 % CI) P P FDR OR (95 % CI) P P FDR OR (95 % CI) P P FDR rs AA (1.05, 1.33) (1.06, 1.45) (1.13, 1.86) (0.93, 1.61) C vs A AC CC rs GG (1.07, 1.36) (1.05, 1.43) (1.15, 1.97) (0.91, 1.52) A vs G AG AA rs AA (1.02, 1.30) (0.99, 1.36) (0.82, 1.45) (1.03, 1.66) G vs A AG GG rs TT (0.96, 1.22) (0.92, 1.25) (0.97, 1.47) (0.87, 1.46) T vs C TC CC rs CC (0.83, 1.11) (0.84, 1.15) (0.83, 1.20) (0.64, 1.49) T vs C TC TT rs TT (0.92, 1.17) (0.92, 1.21) (0.88, 1.36) (0.85, 1.31) C vs T TC CC Itlic vlues re sttisticlly significnt fter FDR correction OR (95 % CI), odds rtio (95 % confidence intervl) P-vlue from Benjmini-Hochberg method control for flse discovery rte (FDR) Wng et l. Lipids in Helth nd Disese (2015) 14:80 Pge 7 of 11

8 Wng et l. Lipids in Helth nd Disese (2015) 14:80 Pge 8 of 11 Fig. 2 GRS ctegories nd risk for CAD. Blck nd gry brs represent the subjects of cse nd control in ech quintile, respectively. Solid dots depict the respective odds rtios for CAD risk when different quntiles compred with quintile 1 For SNP rs , the geometric men (95 % CI) of the Gensini scores were 19.4 ( ) in the genotype AA crriers, 23.3 ( ) in the genotype AC crriers nd 23.6 ( ) in the genotype CC crriers, respectively. When we nlyzed the Gensini scores s quntittive trits (Tble 6), the minor llele C of SNP rs ws significntly ssocited with higher Gensini scores fter djusting for 9 covrites (β = 0.113, SE = 0.036, P = 0.002, P FDR = 0.012). When the llele frequencies of ech SNP in the higher nd lower Gensini scores of ptients were compred in cse control design (Tble 6), significnt ssocition ws obtined gin between SNP rs nd the Gensini scores (OR = 1.30, 95 % CI = , P = 0.003, P FDR = 0.018). Otherwise, there were no significnt ssocitions between other five SNPs nd the severity of CAD s ssessed both by the vessel nd Gensini scores. Tble 5 Associtions of 6 SNPs with the vessel scores in the 1100 CAD ptients Discussion In this study, we hve demonstrted tht two SNPs (HNF1A rs , MADD-FOLH1 rs ) nd the SNP Disesed vessel scores (%) χ 2 test Liner-by- liner χ 2 test χ 2 vlue P P FDR χ 2 vlue P rs AA 126 (33.9) 95 (32.3) 97 (22.4) AC 161 (43.3) 131 (44.6) 234 (53.9) CC 85 (22.8) 68 (23.1) 103 (23.7) rs GG 81 (21.8) 68 (23.1) 90 (20.7) AG 190 (51.1) 160 (54.4) 212 (48.9) AA 101 (27.1) 66 (22.5) 132 (30.4) rs AA 64 (17.2) 53 (18.0) 78 (18.0) AG 165 (44.4) 132 (44.9) 213 (49.1) GG 143 (38.4) 109 (37.1) 143 (32.9) rs TT 93 (25.1) 77 (26.3) 89 (20.6) TC 179 (48.2) 142 (48.5) 247 (57.0) CC 99 (26.7) 74 (25.2) 97 (22.4) rs CC 230 (61.8) 186 (63.3) 272 (62.7) TC 121 (32.5) 94 (31.9) 140 (32.3) TT 21 (5.7) 14 (4.8) 22 (5.0) rs TT 97 (26.0) 86 (29.2) 130 (30.0) TC 181 (48.7) 139 (47.3) 214 (49.3) CC 94 (25.3) 69 (23.5) 90 (20.7) Itlic vlues re sttisticlly significnt fter FDR correction P-vlue from Benjmini-Hochberg method control for flse discovery rte (FDR)

9 Wng et l. Lipids in Helth nd Disese (2015) 14:80 Pge 9 of 11 Tble 6 Associtions of 6 SNPs with the ln-trnsformed Gensini socre in the 1100 CAD ptients under dditive model SNP (risk llele) Quntittive trit ssocition Medin cse control ssocition β (SE) P b P FDR c Risk llele frequency (%) High scores Low scores OR (95 % CI) P b P FDR c rs (C) (0.037) (1.09, 1.53) rs (A) (0.038) (0.82, 1.16) rs (G) (0.037) (0.93, 1.31) rs (T) (0.038) (0.89, 1.26) rs (T) (0.045) (0.86, 1.29) rs (C) (0.037) (0.75, 1.05) Itlic vlues re sttisticlly significnt fter FDR correction β (SE) effect size (stndrd error), OR (95 % CI) odds rtio (95 % confidence intervl) CAD ptients were clssified into two groups ccording to their Gensini scores using the medin s cutoff point: >20.5 for the high scores group nd 20.5 for the low scores group. b P vlues were obtined using the multivrite liner nd logistic regression nlyses with ge, sex, smoking, lcohol intke, weight sttus, hypertension, dibetes nd dyslipidemi nd phrmcologicl therpy sttus s covrites. c P-vlue from Benjmini-Hochberg method control for flse discovery rte (FDR) GRS were ssocited with lipid levels in Chinese popultion. The present study lso indicted tht these two SNPs nd the GRS were ssocited with CAD risk fter correction for multiple testing, nd this is the first investigtion demonstrting tht SNP rs ws significntly ssocited with the ngiogrphic severity of coronry therosclerosis. SNP rs on chromosome 11p11.2 showed relible evidence for ssocitions with HDL-c levels nd the risk of CAD in our study, which ws consistent with the results from previous GWAS in Cucsins [6]. However, nother ssocition study including 727 Gungxi Hn popultion demonstrted tht this locus ws significntly ssocited with TC, TG, HDL-c nd LDL-c levels [29]. To our knowledge, the frequencies of G llele in the Gungxi Hn subjects were significnt lower thn those in our Hubei control subjects (43.7 % vs 51.9 %) nd lso much lower thn those in the HpMp CHB dtbse (43.7 % vs 56.5 %), suggesting tht the Chinese popultion my not be geneticlly similr. Aprt from genetic bckground, such s smple size nd different sttisticl method, my lso contribute to the discrepncies mong our study nd other studies in Chinese popultion. The role tht this SNP might ply in lipoprotein metbolism is still uncler now. SNP rs is ssigned s MADD-FOLH1 locus of chromosome 11p, nd the two genes flnking the locus, MAP-kinse ctivting deth domin (MADD) gene nd folte hydrolse 1(FOLH1) gene hve not been implicted in lipid metbolism. However, the liver X receptors lph vrint 1 (LXRA) gene, n orphn member of the nucler receptor tht regulte pthwys centrl to lipid homeostsis [30, 31], is locted just 0.5 kb telomeric of MADD gene. Moreover, polymorphisms of this gene hve been ssocited with lipid levels nd CAD risk in Cucsins [32]. So whether SNP rs is functionl one, or just tgging one, needs to be determined by further functionl studies. Another SNP, rs , which is non-synonymous SNP in the coding region of HNF1A gene, hs been reported to be ssocited with TC, LDL-c levels nd CAD risk in GWAS of Cucsins [11]. Reiner et l. lso found significnt ssocitions of the minor llele C with higher LDL-c levels nd incresed risk of CAD in the two cse control smples from Africn -Americns nd Cucsins [33]. In our study, the minor llele C of SNP rs ws ssocited with higher TC levels nd incresed risk of CAD, but not ssocited with LDL-c levels in Chinese popultion. One possible reson of these differences is tht our smple size is not enough to detect the ssocition becuse of the subtle effect size of the individul vrint on LDL-c levels. Another explntion is tht different linkge disequilibrium ptterns existed in different popultions. After ll, number of studies hve suggested tht there my be genetic differences between the determinnts of lipid profiles in different ethnicities [34, 35]. HNF1A gene, encoded the trnscription fctor heptocyte nucler fctor 1-α, which regulted the trnscription of numerous genes involved in lipid trnsport nd metbolism [36]. SNP rs is locted within the HNF1A dimeriztion domin tht hs been ssocited with decresed in vitro trnscriptionl ctivity of downstrem trget gene promoters [37]. Therefore, this vrint my influence multiple therosclerosisrelted genes or their plsm products through effects on HNF-1α structure or function [33]. The Gensini scoring system is well-used method for mesuring the severity of coronry therosclerosis, which is the primry pthophysiologicl process underlying CAD [38]. Becuse our 1100 Gensini scores were in ccordnce with norml distribution fter ln-trnsformtion, we performed quntittive trit ssocition nd medin cse

10 Wng et l. Lipids in Helth nd Disese (2015) 14:80 Pge 10 of 11 control ssocition nlyses for the six SNPs. For both nlyses, the ssocitions between the Gensini scores nd SNP rs were significnt even fter FDR correction. Moreover, we lso observed dose-dependent effect of genotypes of this polymorphism on the vessel scores. Although the exct biologicl mechnism of these ssocitions remined to be explored, our study provided evidence tht SNP rs my contribute to the etiology of the severity of coronry therosclerosis nd plyed n importnt role in the therosclerotic process. To combine the reltively smll effects of individul genes nd to better consider the ctul effect of ech SNP on the trit [22], we clculted the weighted GRS bsed on the three significnt (uncorrected) SNPs. The results indicted tht ech dditionl risk llele ws ssocited with incresed TC levels nd decresed HDL-c levels even fter multiple testing. More importntly, for weighted GRS of CAD, quintile 5 hd 1.89 times incresed odds of CAD s compred to quintile 1, suggesting potentil predictive effect on CAD risk. Limittions of our study merit considertion. First, the retrospective design of this study hs inherent drwbcks nd precludes cusl inferences [39]. Second, the smple size of the present study my not enough either to detect mrginl effect from very low penetrnce SNPs or to identify significnt ssocitions of the effect in different genetic model nlyses. Third, the GRS were bsed on SNPs tht were significntly ssocited with CAD risk in our study, nd this process my resulted in the possible exggertion of risk prediction [22]. Becuse the effect sizes of SNPs were clculted nd tested in the sme cohort. Finlly, due to lck of clinicl dt, we hve not exmined the effects of SNPs nd the GRS on more biomrkers such s lipoprotein (), polipoprotein A1 nd polipoprotein B, nd on imging mesures, such s the extent of coronry therosclerosis (the Sullivn Extent scores) nd crotid intim-medi thickness (CIMT). Conclusions In summry, in cse control study with 1100 CAD ptients nd 1069 controls, we hve identified tht two SNPs (HNF1A rs , MADD-FOLH1 rs ) s well s the GRS were significntly ssocited with TC, HDL-c levels nd CAD risk, nd correltion my exist between the HNF1A rs nd severity of coronry therosclerosis. Functionl studies re required to explore the mechnisms governing these effects. Additionl files Additionl file 1: Amplifiction primers utilized in the genotyping. (XLS 19 kb) Additionl file 2: Cumultive effects of ssocited vrints on the risk of CAD. (XLS 18 kb) Competing interests The uthors declre tht they hve no competing interests. Authors contributions XBW undertook genotyping. XBW nd FZ drfted the mnuscript. YDH nd NHC collected blood smples nd extrcted DNA. JY nd ZLH helped with genotyping nd contributed to the cquisition of clinicl dt. QZ nd FZ prticipted in the design. All uthors red nd pproved the finl mnuscript. Acknowledgement This study ws supported by grnts from the Ntionl Nturl Science Foundtion of Chin ( nd ). Author detils 1 Center for Gene Dignosis, Zhongnn Hospitl of Wuhn University, Wuhn , Hubei, Chin. 2 Deprtment of Clinicl Lbortory, Children s Hospitl of Zhengzhou, Zhengzhou , Henn, Chin. 3 Deprtment of Gsteroenterology, Provincil Hospitl Affilited to Shndong University, Jinn , Shndong, Chin. Received: 10 Februry 2015 Accepted: 10 July 2015 References 1. Go AS, Mozffrin D, Roger VL, Benjmin EJ, Berry JD, Blh MJ, et l. Executive summry: hert disese nd stroke sttistics 2014 updte: report from the Americn Hert Assocition. Circultion. 2014;129: Miller CL, Anderson DR, Kundu RK, Riesdn A, Nurnberg ST, Diz R, et l. 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