Association between STK11 Gene Polymorphisms and Coronary

Research ArticleAssociation between STK11 Gene Polymorphisms and CoronaryArtery Disease in Type 2 Diabetes in Han Population in China

Xiaowei Ma Ge Bai Difei Lu Linjuan Huang Jianwei Zhang Ruifen Deng Shan DingNan Gu and Xiaohui Guo

Department of Endocrinology Peking University First Hospital Beijing China

Correspondence should be addressed to Xiaowei Ma mxiaowei1967sinacom

Received 10 December 2016 Accepted 24 January 2017 Published 28 February 2017

Academic Editor Hiroshi Okamoto

Copyright copy 2017 Xiaowei Ma et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Background Recent studies indicated that the Serine threonine kinase 11 (STK11) which is a key regulator of the AMP-activatedprotein kinase (AMPK) plays a crucial role in cardiovascular systemThis study aimed to investigate whether genetic variations inthe STK11 gene affect the risk of coronary artery disease (CAD) in Chinese type 2 diabetics Methods 5 haplotype-tagging singlenucleotide polymorphisms (SNPs) were selected and 288 CAD-positive cases and 159 CAD-negative controls with type 2 diabeteswere genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay Results The carriersof minor allele A at rs12977689 had a higher risk of CAD compared to the homozygotes of CC (OR = 1572 95 CI = 1039ndash2376119901 = 0035) and the difference was still significant after adjustment for the other known CAD risk factors (OR1015840 = 1184 95 CI1015840 =1036ndash1353 1199011015840 = 0013)Conclusion Genetic variability at STK11 locus is associated with CAD risk in type 2 diabetes in the Chinesepopulation

1 Introduction

The rapid increase in prevalence of coronary artery diseasehas been a major health challenge worldwide especially intype 2 diabetic patients A number of studies showed that thediabetic patients had a 2ndash4-fold greater risk of developingCAD than those of nondiabetics [1] In addition to theimportant contribution of environmental factors includingchanges in dietary patterns and lifestyle genetic determinantsplay a major role in the individual susceptibility to coronaryartery disease (CAD)

Plenty of prior studies have proved that adiponectin anadipocyte-secreted adipocytokine has the antidiabetic andantiatherogenic effects [2ndash4] partly by evoking nitric oxide(NO) production and endothelium-dependent vasodilata-tion via activation of AMP-activated protein kinase (AMPK)[5 6]

Serine threonine kinase 11 (STK11) is a key regulator of afamily of kinases including AMPK It was initially discoveredbecause of its role as a tumor suppressor and mutated in anumber of cancers (Peutz-Jeghers Syndrome lung and cervi-cal cancer) [7] But it is becoming clear that STK11 has a wide

range of biological roles that reach far beyond that Recentlyconvincing evidence has been presented that the endothelialnitric oxide synthase (eNOS) activation depends on STK11mediated by adiponectin pathway [8] And increasing studieswere focused on its crucial role in protecting cardiac musclesfrom damage during ischemia [9 10]

However despite the potential importance of STK11activity in terms of regulating cardiovascular systems veryfew studies had been conducted on STK11 gene and geneticvariants in heart diseases especially in coronary arterydisease We hypothesized that genetic variants in STK11 genemight affect the CAD risk through impacting adiponectinsignaling pathway To confirm the hypothesis we investigatedthe CAD-positive and CAD-negative Han patients with type2 diabetes in China

2 Materials and Methods

21 Subjects The Chinese Han individuals with type 2 dia-betes were studied All patients and controls were recruitedamong type 2 diabetic patients who underwent cardiac

HindawiJournal of Diabetes ResearchVolume 2017 Article ID 6297087 6 pageshttpsdoiorg10115520176297087

2 Journal of Diabetes Research

Table 1 Characteristics of study population

Group (number) CAD (288) Control (159) 119901 valueMale () 652 452 0000lowast

Age (y) 6378 plusmn 910 6208 plusmn 1008 0070

DM duration (y) 818 plusmn 731 829 plusmn 647 0878

Hypertension () 778 712 0150

BMI (kgm2) 2595 plusmn 340 2577 plusmn 384 0640

HbA1c () 736 plusmn 150 712 plusmn 147 0193

FBG (mmolL) 723 plusmn 256 679 plusmn 186 0074

TG (mmolL) 162 plusmn 094 181 plusmn 132 0105

TCHO (mmolL) 409 plusmn 102 455 plusmn 095 0000lowast

HDL (mmolL) 098 plusmn 029 109 plusmn 033 0001

LDL (mmolL) 247 plusmn 084 271 plusmn 082 0009lowast

Smokers () 493 352 0010lowast

TG triglycerides TC total cholesterol LDL-C low density lipoprotein-cholesterol HDL-C high density lipoprotein-cholesterolContinuous variables were expressed as mean plusmn standard deviation 119901 valuesof continuous variables were calculated by independent samples 119905-testCategorical variables were expressed as percentage 119901 values were obtainedby 1205942 test lowast refers to the significant difference between the CAD and controlgroups

catheterization at Peking University First Hospital (PUFH)from 2007 to 2010 Type 2 diabetes was defined by 1999World Health Organization criteria [11] which stipulatesfasting plasma glucose ge 70mmolL andor 2-hour plasmaglucose ge 111mmolL or casual plasma glucose (randomblood sugar) ge 111mmolL Type 1 diabetic patients andsubjects with active inflammatory conditions autoimmunediseases malignancies using of immunosuppressive drugsand known hematological disorders were excluded from thestudy

In agreement with the Helsinki Declaration all of total447 unrelated subjects gave informed consent to participatein this study The CAD-positive cases (119899 = 288) weredefined as those who had a stenosis gt 50 in at least onemajor coronary artery or their main branches The CAD-negative type 2 diabetic patients (119899 = 159) had a coronarystenosis le 50 with cardiac catheterization or spiral com-puter tomography (CT) coronary angiography [12]

Other demographic data with known cardiovascular riskfactors including age gender bodymass index (BMI) fastingplasma glucose lipid profile and the histories of hypertension(ge14090mmHg or receiving antihypertensive therapies) andsmoking (ldquoeverrdquo or ldquoneverrdquo ldquoeverrdquo defined as having smokedmore than 1 cigarette per day for more than 1 year) werecollected for all the subjects (Table 1)

22 Ethical Approval of the Study Protocol Thestudy protocoland informed consent procedures were approved by theresearch ethics committees of PUFH

23 Genotyping Genomic DNA was extracted from leuko-cytes obtained from 2mL EDTA-coated peripheral bloodsamples All 5 haplotype-tagging SNPs at STK11 locus with 10exons spanning 21 kb on chromosome 19p133 were selectedin this study from CHB data in HapMap Phase II database

R27 (httpswwwhapmaporg) (1199032 lt 08 MAF ge 005)including rs6510599 (CgtT) rs35369365 (AgtG) rs9282860(CgtT) rs3764640 (GgtT) and rs12977689 (AgtC) All of these5 SNPs are located in introns

These 5 SNPs were typed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP)assay with the genotyping success rates of 85ndash95 andrepeatability rates of 98ndash100 Directly DNA sequencingwas used to further confirm the genotypes for each SNP for 1percent of the cases and controls with the concordance ratesbetween RFLP and DNA sequencing of average 933

24 Statistical Analysis The associations between CAD andgenotypes were analyzed by a 1205942 test and multiple stepwiseregression analysis was utilized to adjust for potential con-founders The clinical and laboratory data were expressed asmean plusmn SD or percentage Qualitative variables were com-pared using 1205942 test and quantitative variables using inde-pendent samples 119905 test The Hardy-Weinberg equation wasemployed to determine the proportion of each genotypeAdjusted odd ratios (ORs) and 95 confidence intervals (95CIs) were calculated The null hypothesis was rejected when119901 was considered significant at lt005 Statistical analysis wasperformed using the SPSS software (version 160 forwindowsUSA) Haploview (version 42 USA) was used for haplotypeanalysis Haplotype frequencies were analyzed by 1205942 test andhaplotype block was defined by confidence interval methodin the software

3 Results

31 Clinical Characteristics Comparing with the controlgroup patients with CAD were more likely to be men orsmokers Lower plasma lipids levels were found in CADgroup which may be due to the fact that the clinical lab-oratory profile was after clinical intervention (Table 1)

32 Genotype Distributions No significant deviation fromthe Hardy-Weinberg equilibrium was observed in controls atall 5 SNPs (119901 gt 005)

All the 5 SNPs had similar allele frequencies in the twogroups (Table 2) At rs12977689 the carriers of major alleleA (AC+AA) had a 15-fold increased CAD risk compared tohomozygotes of CC (OR = 1572 95 CI = 1039ndash2376 119901 =0035) After adjustment for the other known cardiovascularrisk factors (age gender duration of diabetes BMI plasmalipid profile smoking andhypertension status) the trendwasconsistent (adjusted OR1015840 = 1184 95 CI1015840 = 1036ndash1353 1199011015840= 0013) (Table 3) At another SNP rs35369365 there was asignificant association of AG genotype with CAD revealing a18-fold higher risk (adjusted OR1015840 = 1848 95 CI1015840 = 1056ndash3236 1199011015840 = 0032) In comparison with the carriers of AAgenotype those carrying minor allele G (AG+GG) had thetendency of an increase in CAD risk after adjustment for theknown cofounders (1199011015840 = 0069) (Table 3)

33 Haplotype Analysis The 1199032 values and the linkage dise-quilibrium (LD) plot between SNPs of all patients in the study

Journal of Diabetes Research 3

Table 2 Distributions of alleles at STK11 in CAD-positive and CAD-negative subjects

SNPs Alleles CAD Control OR 95 CI 119901119899 () 119899 ()

rs6510599 C 324 (593) 157 (565) 1125 0840ndash1507 0455T 222 (407) 121 (435)

rs35369365 G 182 (355) 96 (328) 1172 0866ndash1586 0320A 330 (645) 204 (672)

rs9282860 T 76 (137) 40 (139) 1014 0672ndash1532 1000C 478 (863) 248 (861)

rs3764640 G 176 (359) 104 (382) 1104 0813ndash1500 0531T 314 (641) 168 (618)

rs12977689 C 341 (651) 199 (706) 1287 0941ndash1759 0117A 183 (349) 83 (294)

Table 3 Association between CAD and SNPs at STK11 locus in different genetic model

SNPs Genotype CAD Control OR 95 CI 119901 OR1015840 95 CI1015840 1199011015840119899 = 288 119899 = 159

rs3764640 TT 98 (400) 53 (390) 1155 0660ndash2020 0665 1019 0445ndash2331 0964GG+GT 147 (600) 83 (610) 1 1

rs6510599 CC+CT 233 (853) 116 (835) 1055 0680ndash1603 0913 1104 0840ndash1452 0477TT 40 (147) 23 (165) 1 1

rs35369365 GG+GA 160 (625) 83 (550) 1365 0908ndash2054 0144 1275 0981ndash1656 0069AA 96 (375) 68 (450) 1 1

rs9282860 CC 204 (736) 105 (729) 1038 0659ndash1635 0908 1002 0965ndash1040 0926TT+TC 73 (264) 39 (271) 1 1

rs12977689 AA+AC 161 (615) 71 (504) 1572 1039ndash2376 0035lowast 1184 1036ndash1353 0013lowastCC 101 (385) 70 (496) 1

OR1015840 95 CI1015840 1199011015840 After adjustment for age sex duration of diabetes and hypertension BMI smoking and plasma lipid status lowast refers to the significantdifference between the CAD and control groups

Table 4 Haplotype analyses in patients with CAD and controlsubjects

SNPs Haplotype Frequency119901

1 2 3 CAD ControlA C C 0505 0524 0585

1 rs35369365G C A 0342 0300 0217

2 rs9282860A T C 0123 0128 0827

3 rs12977689G C C 0019 0036 0138

Haplotypes with frequency gt 005 were estimated using Haploview software119901 values were calculated by chi-squared test

populationwere shown and 3 out of 5 SNPswere in a LDblock(Figure 1) Of all the haplotypes constructed by rs35369365rs9282860 and rs12977689 in an order of their physical posi-tions there were total 4 common haplotypes with frequenciesof gt5 But none of the 4 common haplotypes at STK11 genewas associated with CAD risk in the type 2 diabetic patients(Table 4)

rs3764640

rs6510599

rs35369365

rs928286

0

rs12977689

30 33 8 7

10 10 84

20 35

10

1 2 3 4 5Block 1 (6kb)

Figure 1 The 1199032 values and the linkage disequilibrium (LD) plotbetween SNPs at STK11 locusThe color of the squares indicated theintensity of the linkage between SNPs The 1199032 values were showedin the squares The triangular block circled the LD plot (confidenceinterval method)


Table 5 Characteristics of different genotype at rs12977689

Genotype (number) AA+AC (232) CC (171) 119901 valueBMI (kgm2) 2627 plusmn 336 2558 plusmn 359 0060




TCHO (mmolL) 420 plusmn 108 433 plusmn 097 0252


LDL (mmolL) 249 plusmn 081 263 plusmn 090 0145


TG triglycerides TC total cholesterol LDL-C low density lipoprotein-cholesterol HDL-C high density lipoprotein-cholesterolContinuous variables were expressed as mean plusmn standard deviation 119901 valuesof continuous variables were calculated by independent samples 119905-testCategorical variables were expressed as percentage 119901 values were obtainedby 1205942 test

34 SNP rs12977689 and BMI In further analysis we foundSNP rs12977689 may also affect BMI The carriers of riskallele A at rs12977689 had a tendency of a higher BMI levelcompared to homozygotes of CC (1199011015840 = 0060) (Table 5)

4 Discussion

The kinase STK11 is required for activation of AMP-activatedprotein kinase It normally functions in a large proteincomplex including pseudokinase STRAD (Serinethreonineprotein kinase 20 related adaptor) and the scaffolding MO25(mouse protein 25) [13] Under basal conditions STK11is localized in the nucleus while APPL1 (adaptor proteincontaining PH domain PTB domain and leucine zippermotif 1) and inactive AMPK in the cytosol Adiponectininduces the translocation of STK11 from the nucleus intocytosol in association with the binding of APPL1 to AdipoR(the membrane receptor of adiponectin) followed by thephosphorylation of AMPK [14 15] at a specific threonineresidue (Thr172) on 120572 subunit [16 17]

The STK11-dependent AMPK activation could blockIKK-NF-120581B signaling thus it could inhibit proinflamma-tory cytokines and adhesion molecules production and alsoreduce interleukin-18-mediated endothelial cell death [18]Adiponectin-stimulated STK11-AMPK signaling results inenhancement of eNOS and in turn production of nitric oxideis increasedwhich could cause the promotion of vasodilationinhibition of platelet aggregation and proliferation of vas-cular smooth muscle [19] In addition STK11-AMPK phos-phorylation inactivates acetyl-CoA carboxylase resulting infatty acid oxidation [20] In general since STK11 has beenshown to be an activator of AMPK as above it may indirectlycontribute to a major part of the cellular effects of AMPKand therefore plays a crucial role in antiatherosclerosis andcoronary protection

Recently accumulating studies focused on the physio-logical function of STK11 in cardiovascular system It wasdemonstrated that STK11 is critical in maintaining normal

cardiac function under aerobic condition or during recoveryafter ischemia [9] After ischemia reperfusion dominant-negative AMPK overexpression inhibited cardiac functionthrough the suppression of glucose uptake and fatty acid 120573-oxidation in cardiac myocytes [20ndash22] Also the decrease ofAMPK leads to the reduction of subsequent endothelial NOproduction and further impairs endothelium-based vasodi-lation Conversely Italian researchers found that myocardialperfusion in chronic diabetic mice was improved by theupregulation of STK11 and AMPK signaling [23] This evi-dence indicated STK11 might also contribute to the myocar-dial perfusion in a diabetic model

Besides STK11 appears to have more biological effects incardiovascular system other than catalyzing AMPK Inter-estingly the study on STK11 knock-out mice showed thatthemice exhibited cardiac hypertrophy and dysfunction witha decrease in capillary density of both atria and ventricleswhich was consistent with significant lower mRNA andprotein levels of vascular endothelial growth factor [24] Sim-ilarly a report from United Kingdom indicated that deletionof STK11 in the endothelial lining of blood vessels induced adecline in TGF120573 (transforming growth factor 120573) productionwhich was activated independent of AMPK by STK11 andresulted in fragile and distorted vessels characterized by lossof the supporting vascular smooth muscle cells which werenecessary for stabilization and maturation [25] The aboveevidence indirectly implies that STK11 may contribute toindividual susceptibility to angiogenesis and this effect isindependent of AMPK activation The mechanism could beexplained that the endothelial cells were relying on TGF120573-based communication between endothelium and smoothmuscles to recruit smoothmuscle cells into the newly formedblood vessels And the role of STK11 in this process appearedto regulate TGF120573 availability to the nearby smooth muscleand mesenchymal cells [7] Taken together changes in theexpression of STK11 gene or in other words in its functionsmay affect cardiac and vascular endothelial function

So we postulated that the genetic variability in the STK11gene could affect the susceptibility to CAD in type 2 diabeticpatients in China To test the hypothesis we carried out acase-control study to assess the potential effect of STK11 geneon CAD in Chinese Han population with type 2 diabetesWe carefully designed and implemented this study Firstwe recruited subjects from type 2 diabetes patients as theCAD as the diabetic population have a higher morbidity andmortality of CAD and it is clinically important to screen andto prevent this microvascular complication at the early stageSecond we carefully selected the CAD cases by using a widelyaccepted criterion And moreover we used HapMap websiteto choose tagging SNPs and these 5 SNPs in our study covered100 of the STK11 gene Finally to minimize confoundingfactors we performed logistic regression to adjust age BMIsex duration of diabetes and hypertension smoking andplasma lipid status

In this study we found that there was an associationbetween SNPs at STK11 locus and individual susceptibility toCAD in the Chinese type 2 diabetes mellitus (DM) patientsThe risk of CAD for the carriers of allele A of rs12977689 was16 times as high as for the noncarriers (119901 = 0035) And the


association after adjustment was consistent which suggestedthat rs12977689 might influence the CAD risk independentof the other known cardiovascular risk factors in the ChineseHan type 2 DM patients Meanwhile rs35369365 at STK11locus might be also associated with CAD susceptibility inthe subjects with type 2 DM with borderline statisticalsignificance after adjustment As of date none of the studieson genetic variations at STK11 locus was reported amongCAD subjects with or even without type 2 DM

STK11 plays a key role in cellular metabolism In theadiponectin pathway the phosphorylation of AMPK bySTK11 leads to inactivation of acetyl CoA carboxylase result-ing in fatty acid oxidation [26] Furthermore STK11 genehas been demonstrated to have a relationship with insulinsensitivity [27]

As a result to investigate the mechanism of the STK11gene influencing the CAD morbidity we assessed the asso-ciation between the SNP rs12977689 and status of BMIfasting plasma glucose lipid profile and the histories ofhypertension As we have found there may also be anassociation between SNP rs12977689 and body mass indexwith a borderline significant difference which may indirectlyimply that genetic variability of STK11 gene may affect theindividual susceptibility to CAD via regulating metabolism

Although the SNPs we found associated with CAD riskin the study population are all placed in intronic regionsit is already known that intronic SNPs may regulate geneexpression and alternative splicing [28 29]Therefore intronSNPs may affect the expression levels of STK11 influencingthe AMPK activation and affecting the angiogenesis of themyocardium In addition it seems more likely that theseSNPs rs12977689 and rs35369365 are genetic markers whichmay be in high linkage disequilibrium with neighboringfunctional SNPs in the kinase gene region thereby potentiallyaffecting the structure andor catalytic activity of the enzyme

The studied subjects were the diabetic patients who tookcardiac catheterization or spiral CT coronary angiographyprocedure in PUFH Multiple studies confirmed the highcorrelation of the spiral CT coronary angiography withcardiac catheterization a gold standard for CAD diagnosiswith its specificity of 95 plusmn 39 sensitivity of 823 plusmn 34false-negative rate at 5 plusmn 43 and false-positive rate at79 plusmn 67 [30] As of spiral CT for CAD diagnosis inPUFH it had the specificity of 97 sensitivity of 73ndash80false-negative rate at 26 and false-positive rate at 59 [12]Therefore in order to minimize the effect that the recruitedsubjects alone incur in the case-control study CAD-positivegroup was comprised of the diabetic patients with CADdiagnosed only based on cardiac catheterization and thecontrol group was defined on either cardiac catheterizationor spiral CT coronary angiography

We are conscious that there are some limitations inthis study The sample size was not large enough and thusthe statistical power for some SNPs with lower frequenciesof minor allele was not strong enough to make negativeconclusions The clinical features were not perfectly matchedbetween the case and control groups which might bias the

results And further function study should be completed onthe genetic variants at STK11 locus

This study only provided preliminary evidence about theassociation of genetic variations of STK11 with developmentofCAD inT2DMsubjects inChina Further studies should bedone to confirm the findings in a large patient series and evenanumber of ethnics and races A larger number of the patientsshould be continuously recruited to reduce the variationsThe mechanisms by which the genetic variability in the genecontributes to the CAD risk should be further investigatedAlso our findings should be confirmed in prospective studiesbefore STK11 polymorphisms can be applied to predict therisk of CAD in type 2 DM patients in the Chinese Hanpopulation

Abbreviations

STK11 Serine threonine kinase 11AMPK AMP-activated protein kinaseCAD Coronary artery diseaseSNP Single nucleotide polymorphismPCR-RFLP Polymerase chain reaction-restriction

fragment length polymorphismNO Nitric oxideeNOS Endothelial nitric oxide synthasePUFH Peking University First HospitalCT Computer tomographyBMI Body mass indexDNA Deoxyribonucleic acidEDTA Ethylenediaminetetraacetic acidMAF Minor allele frequencyOR Odd ratioCI Confidence intervalLD Linkage disequilibriumSTRAD Serinethreonine protein kinase 20 related

adaptorMO25 Mouse protein 25APPL1 Adaptor protein containing PH domain

PTB domain and leucine zipper motif 1IKK I120581B kinaseNF-120581B Nuclear factor 120581BTGF120573 Transforming growth factor 120573DM Diabetes mellitus

Competing Interests

The authors declare that they have no competing interests

Acknowledgments

The study was granted by National 973 project(2006CB503903 2006CB503908) and home-country fundsin ChinaThe authors are grateful to all participants involvedin this studyThey also thank their colleagues inDepartmentsof Cardiology Radiology and Statistics at First HospitalPeking University


References

[1] W B Kannel andD LMcgee ldquoDiabetes and cardiovascular riskfactors the Framingham StudyrdquoCirculation vol 59 no 1 pp 8ndash13 1979

[2] N Ouchi S Kihara Y Arita et al ldquoAdipocyte-derived plasmaprotein adiponectin suppresses lipid accumulation and classA scavenger receptor expression in human monocyte-derivedmacrophagesrdquo Circulation vol 103 no 8 pp 1057ndash1063 2001

[3] W-S Yang C-Y Jeng T-J Wu et al ldquoSynthetic peroxi-some proliferator-activated receptor-120574 agonist rosiglitazoneincreases plasma levels of adiponectin in type 2 diabeticpatientsrdquo Diabetes Care vol 25 no 2 pp 376ndash380 2002

[4] Y Okamoto Y Arita M Nishida et al ldquoAn adipocyte-derivedplasma protein adiponectin adheres to injured vascular wallsrdquoHormone andMetabolic Research vol 32 no 2 pp 47ndash50 2000

[5] X Mao C K Kikani R A Riojas et al ldquoAPPL1 binds toadiponectin receptors and mediates adiponectin signalling andfunctionrdquo Nature Cell Biology vol 8 no 5 pp 516ndash523 2006

[6] K K Y Cheng K S L Lam Y Wang et al ldquoAdiponectin-induced endothelial nitric oxide synthase activation and nitricoxide production are mediated by APPL1 in endothelial cellsrdquoDiabetes vol 56 no 5 pp 1387ndash1394 2007

[7] B Y Shorning and A R Clarke ldquoLKB1 loss of function studiedin vivordquo FEBS Letters vol 585 no 7 pp 958ndash966 2011

[8] B Thors H Halldorsson and G Thorgeirsson ldquoENOS activa-tion mediated by AMPK after stimulation of endothelial cellswith histamine or thrombin is dependent on LKB1rdquo Biochimicaet Biophysica ActamdashMolecular Cell Research vol 1813 no 2 pp322ndash331 2011

[9] N Jessen H-J Koh C D Folmes et al ldquoAblation of LKB1in the heart leads to energy deprivation and impaired cardiacfunctionrdquo Biochimica et Biophysica Acta vol 1802 no 7-8 pp593ndash600 2010

[10] K Sakamoto E Zarrinpashneh G R Budas et al ldquoDeficiencyof LKB1 in heart prevents ischemia-mediated activation ofAMPK1205722 but not AMPK1205721rdquo American Journal of Physiology -Endocrinology and Metabolism vol 290 no 5 pp E780ndashE7882006

[11] K G M M Alberti and P Z Zimmet ldquoDefinition diagnosisand classification of diabetesmellitus and its complications Part1 diagnosis and classification of diabetes mellitus provisionalreport of aWHO consultationrdquoDiabetic Medicine vol 15 no 7pp 539ndash553 1998

[12] Y Gong and Y Huo ldquoClinical application of the 64-slice CTindetection of coronary artery stenosisrdquo Chinese Journal ofInterventional Cardiology vol 6 article 1 2005

[13] E Zeqiraj B M Filippi M Deak D R Alessi and D M F VanAalten ldquoStructure of the LKB1-STRAD-MO25 complex revealsan allosteric mechanism of kinase activationrdquo Science vol 326no 5960 pp 1707ndash1711 2009

[14] X Fang R Palanivel J Cresser et al ldquoAn APPL1-AMPK signal-ing axis mediates beneficial metabolic effects of adiponectin inthe heartrdquo American Journal of PhysiologymdashEndocrinology andMetabolism vol 299 no 5 pp E721ndashE729 2010

[15] L Zhou S S Deepa J C Etzler et al ldquoAdiponectin activatesAMP-activated protein kinase in muscle cells via APPL1LKB1-dependent and phospholipase CCa2+Ca2+calmodulin-dependent protein kinase kinase-dependent pathwaysrdquo Journalof Biological Chemistry vol 284 no 33 pp 22426ndash22435 2009

[16] D G Hardie ldquoNew roles for the LKB1rarrAMPK pathwayrdquoCurrent Opinion in Cell Biology vol 17 no 2 pp 167ndash173 2005

[17] M C Towler and D G Hardie ldquoAMP-activated protein kinasein metabolic control and insulin signalingrdquo Circulation Re-search vol 100 no 3 pp 328ndash341 2007

[18] QWuWDeng B Chen L Xia andZ Liang ldquoEffect of BMSCstransplantation on cardiac function of diabetes mellitus ratsrdquoChinese Journal of Reparative and Reconstructive Surgery vol23 no 10 pp 1241ndash1245 2009

[19] V A Morrow F Foufelle J M C Connell J R Petrie GW Gould and I P Salt ldquoDirect activation of AMP-activatedprotein kinase stimulates nitric-oxide synthesis in human aorticendothelial cellsrdquo Journal of Biological Chemistry vol 278 no34 pp 31629ndash31639 2003

[20] D D J Habets W A Coumans M El Hasnaoui et al ldquoCrucialrole for LKB1 to AMPK1205722 axis in the regulation of CD36-mediated long-chain fatty acid uptake into cardiomyocytesrdquoBiochimica et Biophysica Acta - Molecular and Cell Biology ofLipids vol 1791 no 3 pp 212ndash219 2009

[21] D Nagata and Y Hirata ldquoThe role of AMP-activated proteinkinase in the cardiovascular systemrdquo Hypertension Researchvol 33 no 1 pp 22ndash28 2010

[22] T Horie K Ono K Nagao et al ldquoOxidative stress inducesGLUT4 translocation by activation of PI3-KAkt and dualAMPK kinase in cardiac myocytesrdquo Journal of Cellular Physi-ology vol 215 no 3 pp 733ndash742 2008

[23] C Kusmic A LrsquoAbbate G Sambuceti et al ldquoImproved myocar-dial perfusion in chronic diabetic mice by the up-regulation ofpLKB1 and AMPK signalingrdquo Journal of Cellular Biochemistryvol 109 no 5 pp 1033ndash1044 2010

[24] Y Ikeda K Sato D R Pimentel et al ldquoCardiac-specific deletionof LKB1 leads to hypertrophy and dysfunctionrdquo Journal ofBiological Chemistry vol 284 no 51 pp 35839ndash35849 2009

[25] A Londesborough K Vaahtomeri M Tiainen et al ldquoLKB1in endothelial cells is required for angiogenesis and TGF120573-mediated vascular smooth muscle cell recruitmentrdquo Develop-ment vol 135 no 13 pp 2331ndash2338 2008

[26] M T Villarreal-Molina and B Antuna-Puente ldquoAdiponectinanti-inflammatory and cardioprotective effectsrdquo Biochimie vol94 no 10 pp 2143ndash2149 2012

[27] A Lopez-Bermejo M Dıaz E Moran F De Zegher and LIbanez ldquoA single nucleotide polymorphism in STK11 influencesinsulin sensitivity and metformin efficacy in hyperinsulinemicgirls with androgen excessrdquo Diabetes Care vol 33 no 7 pp1544ndash1548 2010

[28] A B Rose ldquoIntron-mediated regulation of gene expressionrdquoCurrent Topics in Microbiology and Immunology vol 326 pp277ndash290 2008

[29] L J Baier P A Permana X Yang et al ldquoA calpain-10 genepolymorphism is associated with reduced muscle mRNA levelsand insulin resistancerdquo Journal of Clinical Investigation vol 106no 7 pp R69ndashR73 2000

[30] SAchenbach TGieslerD Ropers et al ldquoDetection of coronaryartery stenoses by contrast-enhanced retrospectively electro-cardiographically-gated multislice spiral computed tomogra-phyrdquo Circulation vol 103 no 21 pp 2535ndash2538 2001

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Table 1 Characteristics of study population

Group (number) CAD (288) Control (159) 119901 valueMale () 652 452 0000lowast

Age (y) 6378 plusmn 910 6208 plusmn 1008 0070

DM duration (y) 818 plusmn 731 829 plusmn 647 0878


BMI (kgm2) 2595 plusmn 340 2577 plusmn 384 0640




TCHO (mmolL) 409 plusmn 102 455 plusmn 095 0000lowast


LDL (mmolL) 247 plusmn 084 271 plusmn 082 0009lowast

Smokers () 493 352 0010lowast

TG triglycerides TC total cholesterol LDL-C low density lipoprotein-cholesterol HDL-C high density lipoprotein-cholesterolContinuous variables were expressed as mean plusmn standard deviation 119901 valuesof continuous variables were calculated by independent samples 119905-testCategorical variables were expressed as percentage 119901 values were obtainedby 1205942 test lowast refers to the significant difference between the CAD and controlgroups

catheterization at Peking University First Hospital (PUFH)from 2007 to 2010 Type 2 diabetes was defined by 1999World Health Organization criteria [11] which stipulatesfasting plasma glucose ge 70mmolL andor 2-hour plasmaglucose ge 111mmolL or casual plasma glucose (randomblood sugar) ge 111mmolL Type 1 diabetic patients andsubjects with active inflammatory conditions autoimmunediseases malignancies using of immunosuppressive drugsand known hematological disorders were excluded from thestudy

In agreement with the Helsinki Declaration all of total447 unrelated subjects gave informed consent to participatein this study The CAD-positive cases (119899 = 288) weredefined as those who had a stenosis gt 50 in at least onemajor coronary artery or their main branches The CAD-negative type 2 diabetic patients (119899 = 159) had a coronarystenosis le 50 with cardiac catheterization or spiral com-puter tomography (CT) coronary angiography [12]

Other demographic data with known cardiovascular riskfactors including age gender bodymass index (BMI) fastingplasma glucose lipid profile and the histories of hypertension(ge14090mmHg or receiving antihypertensive therapies) andsmoking (ldquoeverrdquo or ldquoneverrdquo ldquoeverrdquo defined as having smokedmore than 1 cigarette per day for more than 1 year) werecollected for all the subjects (Table 1)

22 Ethical Approval of the Study Protocol Thestudy protocoland informed consent procedures were approved by theresearch ethics committees of PUFH

23 Genotyping Genomic DNA was extracted from leuko-cytes obtained from 2mL EDTA-coated peripheral bloodsamples All 5 haplotype-tagging SNPs at STK11 locus with 10exons spanning 21 kb on chromosome 19p133 were selectedin this study from CHB data in HapMap Phase II database

R27 (httpswwwhapmaporg) (1199032 lt 08 MAF ge 005)including rs6510599 (CgtT) rs35369365 (AgtG) rs9282860(CgtT) rs3764640 (GgtT) and rs12977689 (AgtC) All of these5 SNPs are located in introns

These 5 SNPs were typed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP)assay with the genotyping success rates of 85ndash95 andrepeatability rates of 98ndash100 Directly DNA sequencingwas used to further confirm the genotypes for each SNP for 1percent of the cases and controls with the concordance ratesbetween RFLP and DNA sequencing of average 933

24 Statistical Analysis The associations between CAD andgenotypes were analyzed by a 1205942 test and multiple stepwiseregression analysis was utilized to adjust for potential con-founders The clinical and laboratory data were expressed asmean plusmn SD or percentage Qualitative variables were com-pared using 1205942 test and quantitative variables using inde-pendent samples 119905 test The Hardy-Weinberg equation wasemployed to determine the proportion of each genotypeAdjusted odd ratios (ORs) and 95 confidence intervals (95CIs) were calculated The null hypothesis was rejected when119901 was considered significant at lt005 Statistical analysis wasperformed using the SPSS software (version 160 forwindowsUSA) Haploview (version 42 USA) was used for haplotypeanalysis Haplotype frequencies were analyzed by 1205942 test andhaplotype block was defined by confidence interval methodin the software

3 Results

31 Clinical Characteristics Comparing with the controlgroup patients with CAD were more likely to be men orsmokers Lower plasma lipids levels were found in CADgroup which may be due to the fact that the clinical lab-oratory profile was after clinical intervention (Table 1)

32 Genotype Distributions No significant deviation fromthe Hardy-Weinberg equilibrium was observed in controls atall 5 SNPs (119901 gt 005)

All the 5 SNPs had similar allele frequencies in the twogroups (Table 2) At rs12977689 the carriers of major alleleA (AC+AA) had a 15-fold increased CAD risk compared tohomozygotes of CC (OR = 1572 95 CI = 1039ndash2376 119901 =0035) After adjustment for the other known cardiovascularrisk factors (age gender duration of diabetes BMI plasmalipid profile smoking andhypertension status) the trendwasconsistent (adjusted OR1015840 = 1184 95 CI1015840 = 1036ndash1353 1199011015840= 0013) (Table 3) At another SNP rs35369365 there was asignificant association of AG genotype with CAD revealing a18-fold higher risk (adjusted OR1015840 = 1848 95 CI1015840 = 1056ndash3236 1199011015840 = 0032) In comparison with the carriers of AAgenotype those carrying minor allele G (AG+GG) had thetendency of an increase in CAD risk after adjustment for theknown cofounders (1199011015840 = 0069) (Table 3)

33 Haplotype Analysis The 1199032 values and the linkage dise-quilibrium (LD) plot between SNPs of all patients in the study




rs6510599 C 324 (593) 157 (565) 1125 0840ndash1507 0455T 222 (407) 121 (435)

rs35369365 G 182 (355) 96 (328) 1172 0866ndash1586 0320A 330 (645) 204 (672)

rs9282860 T 76 (137) 40 (139) 1014 0672ndash1532 1000C 478 (863) 248 (861)

rs3764640 G 176 (359) 104 (382) 1104 0813ndash1500 0531T 314 (641) 168 (618)

rs12977689 C 341 (651) 199 (706) 1287 0941ndash1759 0117A 183 (349) 83 (294)











1 2 3 CAD ControlA C C 0505 0524 0585

1 rs35369365G C A 0342 0300 0217

2 rs9282860A T C 0123 0128 0827

3 rs12977689G C C 0019 0036 0138



rs3764640

rs6510599

rs35369365

rs928286

0

rs12977689

30 33 8 7

10 10 84

20 35

10

1 2 3 4 5Block 1 (6kb)














4 Discussion

















Abbreviations





Competing Interests


Acknowledgments



References




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















rs6510599 C 324 (593) 157 (565) 1125 0840ndash1507 0455T 222 (407) 121 (435)

rs35369365 G 182 (355) 96 (328) 1172 0866ndash1586 0320A 330 (645) 204 (672)

rs9282860 T 76 (137) 40 (139) 1014 0672ndash1532 1000C 478 (863) 248 (861)

rs3764640 G 176 (359) 104 (382) 1104 0813ndash1500 0531T 314 (641) 168 (618)

rs12977689 C 341 (651) 199 (706) 1287 0941ndash1759 0117A 183 (349) 83 (294)











1 2 3 CAD ControlA C C 0505 0524 0585

1 rs35369365G C A 0342 0300 0217

2 rs9282860A T C 0123 0128 0827

3 rs12977689G C C 0019 0036 0138



rs3764640

rs6510599

rs35369365

rs928286

0

rs12977689

30 33 8 7

10 10 84

20 35

10

1 2 3 4 5Block 1 (6kb)














4 Discussion

















Abbreviations





Competing Interests


Acknowledgments



References




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




























4 Discussion

















Abbreviations





Competing Interests


Acknowledgments



References




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

























Abbreviations





Competing Interests


Acknowledgments



References




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















References




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Documents

Association between STK11 Gene Polymorphisms and Coronary