The Association Of The Polymorphisms On9p21with Peripheral Arterial Disease In Diabetics And Exploration On The Interaction Of The Loci And Other Factors

Peripheral arterial disease (PAD) is a kind of complicated chronic disease,caused by multiple genetic and environmental factors, with high risk ofcardiovascular events, cardiovascular mortality, and all-cause mortality. Therisk of PAD was increased in the patients with type2diabetes mellitus, with amagnified risk of early-onset and faster progression compared with that in thepopulation without diabetes. At present, due to lack of remarkably clinicalsymptom in the PAD patients with diabetes, the PAD patients with diabetes areunderdiagnosed.In the patients with diabetes, a lot of non-inherited factors (such as age,smoking, dyslipidaemia, hypertension, high glucose and diabetes duration)played an important role in the prevalence of PAD. Epidemiological studiesshowed the prevalence of PAD was different in different ethical populationwith diabetes, suggesting the inherited factors played the important role in theprevalence of PAD in the diabetes.Presently, numerous genome-wide studies have shown that variants onchromosome9p21are closely associated with increased risk of atheroscleroticdiseases including coronary artery disease (CAD), myocardial infarction andstroke, with two representative single nucleotide polymorphisms (SNPs)rs10757274and rs10757278on9p21linked particularly with CAD. Moreover,both loci are also significantly associated with ischaemic stroke. PAD is acommon form of atherosclerotic disease that has a similar aetiology andpathogenesis to CAD and ischaemic stroke. According to our knowledge, onlyfew studies investigated the association of9p21with PAD. However, no studyexplored the association of9p21with PAD in diabetics.Therefore, the present study recruited the participates with Type2 diabetes from Tangshan Gongren hospital investigated the prevalence andnon-inherited factors of PAD in the patients with diabetes in Tangshan, andexplored the association of the polymorphisms on9p21with PAD and theinteraction of9p21and non-inherited factors on PAD in diabetics, whichcould provided evidence for prevention, therapy and improving of prognosisof PAD with diabetes.Part-1Prevalence and non-inherited risk factors of peripheral arterialdisease in Han patients with type2diabetes in TangshanObjective：To investigated the prevalence and non-inherited risk factorsof peripheral arterial disease in the patients with type2diabetes mellitus inTangshan.Methods：Tangshan Gongren hospital were randomly chosed from threethird-grade class-A hospital by cluster sampling. This study included1389Han patients with type2diabetes aged≥45years (250patients with PAD and1139patients without PAD). For all study participants, data regardingdemographic characteristics and medical history were collected byquestionnaire interview or review of medical records. The height, body weightand ankle-brachial index (ABI) were measured. Serum total cholesterol (TC),triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), low-densitylipoprotein cholesterol (LDL-C) levels, and glycosylated hemoglobin (HbA1c)were determined. Comparisons of parameters were tested by Student’s t-test,Mann-Whitney U-test or chi-square test. Logistic regression analysis was doneto estimate odds ratios (ORs) and their corresponding95%confidenceintervals. Statistical analyses were performed using SPSS16.0.Results: In this study the prevalence of PAD was18.0%(95%CI，16.0%-20.0%) in the diabetic patients (250PAD patients) in Tangshan. Therewere significant differences in age, systolic blood pressure (SBP), diabetesduration, TG, HDL-C, HbA1c, smoking, hypertension, CAD and cerebralinfarction (CI) between the PAD patients and non-PAD patients (p＜0.05).There were no significant differences in sex, body mass index (BMI), diastolicblood pressure (DBP), TC and LDL-C between the PAD patients and non-PAD patients (p＞0.05). The trend for the increase of the prevalence ofPAD according to age was statistically significant in diabetics (χ~2趋势=128.423,p＜0.01). Age was the independent risk factor of PAD (OR,1.084;95%CI,1.065-1.104; p＜0.01) by multivariate logistic regression analysis (Thevariates included sex, age, BMI, smoking status, lipemia, HbA1c, diabetesduration, hypertension, CAD and CI, with the same to the following.). Therewere no significant difference in prevalence of PAD between male and female(χ~2=0.412, p=0.521), and the sex was not associated with PAD in diabetics（OR=1.428；95%CI:0.926-2.203；p=0.080), using multivariate logisticregression analysis. There were no significant difference in prevalence of PADbetween the diabetics with BMI≥25and those with BMI＜25(χ~2=0.190,p=0.663), and the BMI was not associated with PAD in diabetics (OR=1.063；95%CI:0.779-1.450；p=0.701), using multivariate logistic regression analysis.There were significant difference in prevalence of PAD between smokers andnon-smokers in diabetics (χ~2=8.048, p=0.005), and the smoking wassignificantly associated with the increased risk of PAD in diabetics (OR=1.831;95%CI:1.232-2.721; p=0.003), using multivariate logistic regression analysis.Low HDL-C and high HbA1c level were the independent risk factors for PADin diabetics (OR=1.419,95%CI:1.021-1.971, p=0.037; OR=1.495,95%CI:1.034-2.161, p=0.033; respectively), using multivariate logistic regressionanalysis. The trend for the increase of the prevalence of PAD according todiabetes duration was statistically significant in diabetics (χ~2趋势=32.249, p＜0.01). The diabetes duration≥15years were the independent risk factors forPAD (OR=2.017,95%CI:1.365-2.981, p＜0.01) using multivariate logisticregression analysis, compared with the diabetes duration＜15years. Therewere significant difference in prevalence of PAD between the diabetics withhypertension and those without hypertension (χ~2=13.813, p＜0.01), and thehypertension was significantly associated with the increased risk of PAD(OR=1.559；95%CI:1.140-2.133; p=0.005), using multivariate logisticregression analysis. There were significant difference in prevalence of PADbetween the diabetics with CAD or CI and those without CAD or CI (p＜0.01), and the CAD and CI were significantly associated with the increased risk ofPAD in diabetics (OR=2.060，95%CI:1.479-2.868，p＜0.01；OR=1.962，95%CI:1.385-2.780，p＜0.01；respectively), using multivariate logisticregression analysis.Conclusion： In this study, the prevalence of PAD was18%(95CI,16%-20%) in the diabetics aged≥45years in Tangshan. The age,smoking, low HDL-C, high HbAIc, diabetes duration, hypertension, CAD andCI were significantly associated with the increased risk of PAD.Part-2The association of the polymorphisms on9p21with peripheralarterial disease in the patients with type2diabetesObjective：To explore the association of the polymorphisms on9p21with peripheral arterial disease in the patients with type2diabetes mellitus.Methods：The study randomly recruited the participates with diabetesaged≥45years (250cases and252age-matched controls), who were attendingthe Second Department of Endocrinology in Tangshan Gongren hospital.Genomic DNA was isolated using a QIAamp DNA mini kit. Genotyping ofrs10757274and rs10757278was performed by matrix-assisted laserdesorption/ionization time-of-flight mass spectrometry (MALDI–TOF–MS).Comparisons of parameters were tested by Student’s t-test,Mann-Whitney U-test or chi-square test. Logistic regression analysis was doneto estimate odds ratios (ORs) and their corresponding95%confidenceintervals. Statistical analyses were performed using SPSS16.0. TheHardy–Weinberg equilibrium for rs10757274and rs10757278was analyzedusing Haploview, version4.0.Results:(1) Both rs10757274and rs10757278were in Hardy–Weinbergequilibrium in both groups in the population with diabetes mellitus (p＞0.05).(2) There were significant differences in genotype distribution ofrs10757274and rs10757278between the PAD patients and non-PAD patientsin the diabetics (p＜0.05).(3) Rs10757274was the independent risk factors for PAD in diabetics in additive model (OR=1.537,95%CI:1.169-2.020, p＜0.01) after adjustmentfor sex, age, smoking, hypertension, dyslipidaemia, HbA1c and diabetesduration. Moreover, the AG genotype of rs10757274increased1.6folds riskof PAD (95%CI:1.002-2.414, p＜0.05) and GG genotype of this locusincreased2.4folds risk of PAD (95%CI:1.359-4.086, p＜0.01). Similarly,rs10757278was also the independent risk factors for PAD in diabetics inadditive model after adjustment for other factors (OR=1.493，95%CI：1.140-1.955，p＜0.01). Moreover, GG genotype of this locus increased2.2folds risk of PAD (95%CI:1.304-3.855, p＜0.01).(4) Rs10757274was still the independent risk factors for PAD indiabetics in additive model, further excluding the patients with macrovasculardiseases with adjustment for the traditional factors (OR=1.476,95%CI:1.057-2.061, p＜0.05). Moreover, GG genotype of this locus increased2.2folds risk of PAD (95%CI：1.127-4.289，p＜0.05).Similarly, rs10757278werestill the independent risk factors for PAD in diabetics in additive model afteradjustment for other factors (OR=1.437,95%CI:1.034-1.997; p＜0.05).Moreover, GG genotype of this locus increased2.1folds risk of PAD (95%CI：1.084-4.020，p＜0.05).Conclusion: Rs10757274and rs10757278on9p21were significantlyassociated with the increased risk of PAD in diabetics.Part-3The interaction of the polymorphisms on9p21and environmentalfactors on peripheral arterial disease in the patients with diabetesObjective：To explore the interaction of rs10757274and rs10757278andenvironmental factors on peripheral arterial disease in the patients withdiabetes mellitus.Methods: The study population with diabetes was same to those in Part-2.Comparisons of parameters were tested by Student’s t-test, Mann-WhitneyU-test or chi-square test. Logistic regression analysis was done to estimateodds ratios (ORs) and their corresponding95%confidence intervals.Statistical analyses were performed using SPSS16.0.Results: The following OR were estimated by multivariate logistic regression analysis with adjusting for age (continuous variable), sex, smoking,dyslipidaemia, hypertension, HbA1c and diabetes duration, with excludingthe variate itself (not including the age).(1)The interaction of age and the polymorphisms of rs10757274andrs10757278In the diabetics aged＜65years, rs10757274was associated with theincreased risk of PAD in additive model (OR=2.840,95%CI:1.579-5.109, p＜0.05). In the diabetics aged≥65years, rs10757274was not associated withthe increased risk of PAD in additive model. No significant interactions werefound between the locus and age (p=0.090). Similarly, no significantinteractions were found between the AG and GG genotype of locus and age (pall＞0.05).In the diabetics aged＜65years, rs10757278was associated with theincreased risk of PAD in additive model (OR=2.371，95%CI：1.334-4.213，p＜0.05); In the diabetics aged≥65years, rs10757278was not associated withthe increased risk of PAD in additive model. No significant interactions werefound between the locus and age (p=0.241). Similarly, no significantinteractions were found between the GG genotype of the locus and age(p=0.266).(2)The interaction of sex and the polymorphisms of rs10757274andrs10757278In the male diabetics, rs10757274was associated with the increased riskof PAD in additive model (OR=1.770，95%CI：1.159-2.701，p＜0.05). In thefemale diabetics, rs10757274was not associated with the increased risk ofPAD in additive model. No significant interactions were found between thelocus and sex (p=0.388). Similarly, no significant interactions were foundbetween the GG genotype of the locus and sex (p=0.382).In the male diabetics, rs10757278was associated with the increased riskof PAD in additive model (OR=1.665，95%CI：1.096-2.528，p＜0.05). In thefemale diabetics, rs10757278was not associated with the increased risk ofPAD in additive model. No significant interactions were found between the locus and sex (p=0.521). Similarly, no significant interactions were foundbetween the GG genotype of the locus and sex (p=0.458).(3)The interaction of smoking and the polymorphisms of rs10757274andrs10757278In the non-smoker in diabetics, rs10757274was associated with theincreased risk of PAD in additive model (OR=1.710，95%CI：1.203-2.433，p＜0.05). In the smoker in diabetics, rs10757274was not associated with theincreased risk of PAD in additive model. No significant interactions werefound between the locus and smoking (p=0.378). Similarly, no significantinteractions were found between the GG genotype of the locus and smoking(p=0.353).In the non-smoker in diabetics, rs10757278was associated with theincreased risk of PAD in additive model (OR=1.724，95%CI：1.214-2.447，p＜0.05). In the smoker in diabetics, rs10757278was not associated with theincreased risk of PAD in additive model. No significant interactions werefound between the locus and smoking (p=0.231). Similarly, no significantinteractions were found between the GG genotype of the locus and smoking(p=0.236).(4)The interaction of dyslipidaemia and the polymorphisms ofrs10757274and rs10757278In the diabetics without dyslipidaemia, rs10757274was associated withthe increased risk of PAD in additive model (OR=2.140，95%CI：1.103-4.154，p＜0.05). In the diabetics with dyslipidaemia, rs10757274was not associatedwith the increased risk of PAD in additive model. No significant interactionswere found between the locus and dyslipidaemia (p=0.099). Similarly, nosignificant interactions were found between the AG and GG genotype of thelocus and dyslipidaemia (p均＞0.05).In the diabetics without dyslipidaemia, rs10757278were associated withthe increased risk of PAD in additive model (OR=1.964，95%CI：1.091-3.537，p＜0.05). In the diabetics with dyslipidaemia, rs10757278was not associatedwith the increased risk of PAD in additive model. No significant interactions were found between the locus and dyslipidaemia (p=0.236).(5)The interaction of hypertension and the polymorphisms of rs10757274and rs10757278No significant interactions were found between rs10757274(additivemodel) and hypertension. No significant interactions were found between theAG and GG genotype of this locus and hypertension yet.In the diabetics with normal blood pressure, rs10757278was associatedwith the increased risk of PAD in additive model (OR=1.668，95%CI：1.093-2.545，p＜0.05). In the diabetics with hypertension, rs10757274was notassociated with the increased risk of PAD in additive model. No significantinteractions were found between the locus and hypertension (p=0.634).Similarly, no significant interactions were found between the GG genotype ofthe locus and hypertension (p=0.642).(6)The interaction of HbA1c and the polymorphisms of rs10757274andrs10757278In the diabetics with normal HbA1c level, rs10757274was not associatedwith the increased risk of PAD in additive model. In the diabetics with highHbA1c level, rs10757274was associated with the increased risk of PAD inadditive model (OR=1.501，95%CI：1.103-2.042，p＜0.05). No significantinteractions were found between the locus and high HbA1c level (p=0.942).Similarly, no significant interactions were found between the GG genotype ofthe locus and high HbA1c level (p=0.948).In the diabetics with normal HbA1c level, rs10757278was not associatedwith the increased risk of PAD in additive model. In the diabetics with highHbA1c level, rs10757278was associated with the increased risk of PAD inadditive model (OR=1.457，95%CI：1.076-1.973，p＜0.05). No significantinteractions were found between the locus and high HbA1c level (p=0.899).Similarly, no significant interactions were found between the GG genotype ofthe locus and high HbA1c level (p=0.907).(7)The interaction of diabetes duration and the polymorphisms ofrs10757274and rs10757278 In the diabetics with diabetes duration＜15years, rs10757274wasassociated with the increased risk of PAD in additive model (OR=1.584，95%CI：1.136-2.209，p＜0.05). In the diabetics with diabetes duration≥15years, rs10757274was not associated with the increased risk of PAD inadditive model. No significant interactions were found between the locus anddiabetes duration (p=0.870). Similarly, no significant interactions were foundbetween the AG and GG genotype of the locus and diabetes duration (p all＞0.05).In the diabetics with diabetes duration＜15years, rs10757278wasassociated with the increased risk of PAD in additive model (OR=1.528，95%CI：1.095-2.123，p＜0.05). In the diabetics with diabetes duration≥15years, rs10757278was not associated with the increased risk of PAD inadditive model. No significant interactions were found between the locus anddiabetes duration (p=0.857). Similarly, no significant interactions were foundbetween the GG genotype of the locus and diabetes duration (p=0.749).Conclusion： No significant interactions were found between thepolymorphisms of rs10757274and rs10757278and age, sex, smoking,dyslipidaemia, hypertension, HbA1c and diabetes duration for PAD in thediabetics.