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A pilot study for the association between KCNQ1 and hypertension with T2DM in Chinese Han population
Yuhao Han, Chao chen Abstract：
Background: KCNQ1 has been associated with the susceptibility of hypertension and Type 2 diabetes mellitus. However, there are few researches detecting the association between KCNQ1 and hypertension complicated with T2DM in Chinese population. Therefore, we conduct a preliminary experiment to identify the shared genetic etiology of hypertension with T2DM.
Methods: We recruited 94 diabetic patients complicated with hypertension and 103 local healthy controls. The diagnosis of hypertension complicated with T2DM based on relevant criteria. The peripheral blood and clinical characteristics from both subjects are collected from Beijing Hospital. We genotyped the SNPs rs2237892 and rs2237895 by PCR-HRM and RFLP method. The statistical analysis was conducted by genetic models and clinical phenotype strata.
Results：1. We identified a novel haplotype involved rs2237892 and rs2237895 associated with the hypertension complicated with T2DM patients (p=1.6×10-6; OR= 2.62; 95% CI:1.742-3.954). 2. Through genotype-phenotype analysis by metabolic parameters strata in case group, a. the heterozygous carriers of AA and AC of rs2237895 were associated with total cholesterol level (p=0.01, OR=0.03, 95%CI: 0.0026-0.4293). b. The homozygous carriers of CC of rs2237892 showed the association between triglyceride level and females (p=0.03, OR=14.40; 95% CI: 1.375-150.814). c. The heterozygous carriers of CT and TT of rs2237892 were associated with the diastolic blood pressure in males (p=0.021; OR= 0.30; 95% CI: 0.305-0.098). 3. However, our results have not reached statistical significance level of the association between rs2237892 or rs2237895 and hypertension with T2DM separately.
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Conclusion: we identified a novel risk haplotype with dose-effect, which are associated with hypertension complicated with T2DM in Chinese population. Furthermore, we also obtain the supporting evidences from clinical parameters that showed the KCNQ1 genetic variants associated with the metabolic phenotypes of hypertension with T2DM. Key words: hypertension complicated with T2DM, risk haplotype, dose-effect Introduction:
Diabetes severely impacts on human health; it is an important issue worldwide. According to the International Diabetes Federation(IDF), it is estimated that the number of T2DM patients would grow up to 592 million from 387 million, during the period between 2014 and 2035(Xu et al. 2013). Among the Chinese population, about 114 million individuals were disturbed by diabetes in 2013, and the Type 2 diabetes mellitus occupied the main position among all of the different types of diabetes(Hu 2011, Zhang et al. 2015).
T2DM is the consequence of joint action by multiple genetic factors and others like diet, smoking and lifestyle. To be specific, genes play a more significant role in developing the T2DM compared with other reasons. During the past decades, GWAS (Genome-wide association studies) help us to uncover numerous T2D susceptibility loci, such as C2CD4A/B、CDC123、FTO、HNF1B、KCNQ1, and these loci have been verified in several ethnics(Cui et al. 2011, Zhang et al. 2012, Kong et al. 2016). Although be with the different functions and unclear pathogenic mechanism, most of the risk genes are relevant with the impaired β-cell function and insulin secretion. The T2DM often coexist with other complications, like dyslipidemia and obesity, while the hypertension is the most common one among all of them. More importantly, when complicated with T2DM, the hypertension becomes an essential risk factor for inducing the cardiovascular (CVD) and coronary heart disease (Chan and Cockram 1997, Thomas et al. 1999, Alsafar et al. 2015).
During the past decades, numerous studies concentrated on the KCNQ1, and most of these studies reported a significant association between T2DM and KCNQ1 (Chen et al. 2010, Qian et al. 2015, Gao et al. 2016). But the results remained
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inconsistent(Diabetes Genetics Initiative of Broad Institute of et al. 2007, Zeggini et al. 2007). KCNQ1 encodes a potassium voltage-gated channel, which is critical for the cardiac action potential(Barhanin et al. 1996, Oliveras et al. 2014, Liin et al. 2015). Furthermore, the KCNQ1 can also affect the insulin secretion. The jeopardized KvLQT1 channel may be able to facilitate insulin secretion (Ullrich et al. 2005). Recently, several researches reported that the SNPs from KCNQ1, containing rs2237892, rs2237895 and rs2237897, in a 40-kb linkage disequilibrium (LD) region in 15th intron were strongly associated with T2DM, increased blood pressure and the impairment of insulin secretion in several ethnics, such as East Asian population, Chinese Han population in Wen Zhou and Caucasian populations(Unoki et al. 2008, Yasuda et al. 2008, Tan et al. 2009, Zhang, Wang et al. 2015).
The prevalence of hypertension and T2DM in Chinese Han population remains constantly increasing, but the etiologic reasons of this phenomenon are still unclear. As mentioned above, It has been revealed that the development of T2DM and hypertension is associated with genetic factors, while the KCNQ1, in the past decades, was constantly reported that may has crucial function in the process of developing either the T2DM or the hypertension. By our knowledge, only few studies focused on investigating the association between KCNQ1 gene and hypertension in the Chinese Han individuals without T2DM.(An et al. 2016, Li et al. 2016). And some studies specialized KCNQ1 genetic varies associated with T2DM in Chinese population (Ma et al. 2015, Kong, Xing et al. 2016, Zhou et al. 2016). However, there are no researches for detecting the association between KCNQ1 and hypertension complicated with T2DM in Northern Chinese population. We speculate that, in terms of etiology and genetic, there may exist the similar factors that would triggering the hypertension with T2DM. Hence, we focused on the KCNQ1 designing a case-control preliminary experiment with small sample size in Northern Chinese Han population in order to identifying whether the hypertension complicated with T2DM shared the same genetic etiology.
Materials and Methods
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Study participants and clinical characteristics
The total number of recruited individuals in this study is 197, including 94 of case and 103 of local healthy control. All of the participants belong to the Northern Chinese Han population. The 1999 World Health Organization(WHO) criteria (FPG≧7.0 mmol/l; 2-h OGTT≧11.1 mmol/L ) was used to diagnose the T2DM, the other reported types of diabetes such as type 1 diabetes and secondary diabetes were precluded. The hypertension was defined by systolic blood pressure≧140 mmHg and/or diastolic blood pressure≧90 mmHg. All of the peripheral blood samples were provided by Beijing Hospital from July 2016 to January 2017, moreover, all the control data were obtained from the 1000 Genomes (http://asia.ensembl.org/index.html).
Age, gender, body mass index (BMI), systolic blood pressure (SBP), diastolic blood pressure (DBP), fasting plasma glucose (FPG), total cholesterol(TC), triglyceride(TG), HDL-C, LDL-C, HbA1c were measured for all participants. The measurement of age, gender, SBP and DBP by means of standard methods. The blood sample of the candidates was used to measure the level of FPG, TC, TG, HDL-C, LDL-C and HbA1c. The formula of BMI calculation was body weight/height2 (kg/m2)
Genomic DNA isolation
The genomic DNA was extracted from the peripheral blood samples. After destruct the leukocytes and the histone using the lysis buffer, the genomic DNA was directly isolated by utilizing the phenol-chloroform. The needed solution in DNA isolation including <1> lysis buffer: 250ml KCL(1M)，240ml Tris-HCL(1m, pH=8.0)，2.25ml Twen-20 and 2.25ml NP-40，filtration sterilization; <2> phenol-chloroform solution: 250ml Tris-phenol mixed with 250ml chloroform. The genomic DNA was dissolved in the Milli-Q water (MilliporeSigma, Darmstadt, Germany).
Genotyping
PCR-HRM method was used for genotyping. The length of the fragment spanning the rs2237892 loci contains 54-bp, and it
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was amplified for the further HRM analysis using. The HRM analysis was performed on the LightScanner high resolution melting HRM System (Idaho Technology Inc, Salt Lake City, USA). The DNA sequence containing rs2237892 loci was downloaded from the dbSNP database with the accession No.NG_008935.1. The primer for amplifying the PCR fragment was designed against the obtained DNA sequence (primer sequence: Forward：5’-TTTTTCTTTCACAGGACTTTGCCAC-3’; Reverse：5’-TTTTTGAGTTTCTAGGCCCCTCAC-3’). The PCR program was 95℃，3min；95℃，30sec；59.5℃，20sec；72℃, 6sec；72℃，30sec with 40 cycles. After the amplifying step, the PCR products was heated to 95℃ for 30 sec and then cooled to 24℃for 2 min twice. The HRM analysis was then performed with temperature ranging from 65-95℃, ascending at 0.3℃/s. The LightScanner Call IT was utilized to generate the HRM curve (Figure. 1).
The genotyping of rs2237895 was achieved by using the Restriction Fragment Length polymorphism (RFLP). The DNA sequence spanning the rs2237895 loci was also obtained from the dbSNP database with accession No. NG_008935.1. The total length of the PCR products was 732-bp, and the primer details were forward-5’-ATTCTTCAGGCAGCTCACCC-3’, Reverse：5’-CTACCAGACCCATCCCCTCA-3’. The PCR program was 95℃ 3min，95℃ 30sec，59.5℃ 30sec，72℃ 45sec, 72℃ 1min 20sec. The restriction enzyme Ava I(TAKARA, Dalian, China) was used to process the PCR products, and the reaction requirement, including time and temperature, was 37℃ over night. The length of CC, AA and AC genotype was 273bp+459bp, 732bp, 732bp+459bp+273bp respectively (Figure. 1).
Statistical analysis
SHEsis (Shi and He 2005, Li et al. 2009) platform was used to calculate the genotype distribution and allele frequencies, and it was also utilized to perform the haplotype analysis, and the results of genotype distribution and allele frequencies were exhibited as number(%). The Hardy-Weinberg equilibrium in control group was assessed by Chi-square test. The clinical characteristics was listed as mean±standard deviations in different genotype categories(Table. 1). SPSS 17(SPSS, Chicago, IL, USA) was used to process the clinical parameters, and the difference between the genotype categories was compared by
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