Original Article Association Study of BUD13-ZNF259 Gene Rs964184 Polymorphism and Hemorrhagic Stroke Risk

Int J Clin Exp Med 2015;8(12):22503-22508 www.ijcem.com /ISSN:1940-5901/IJCEM0016028

Original Article Association study of BUD13-ZNF259 gene rs964184 polymorphism and hemorrhagic stroke risk

Shengjun Zhou, Jikuang Zhao, Zhepei Wang, Keqin Li, Sheng Nie, Feng Gao, Jie Sun, Xiang Gao, Yi Huang

Department of Neurosurgery, Ningbo First Hospital, Ningbo Hospital of Zhejiang University, Ningbo 315010, Zhe- jiang, China Received September 11, 2015; Accepted December 3, 2015; Epub December 15, 2015; Published December 30, 2015

Abstract: We aimed to evaluate the association of rs964184 of BUD13-ZNF259 gene with the risk of hemorrhagic stroke (HS). A total of 138 HS cases and 587 controls were recruited for the association of rs964184 of BUD13- ZNF259 gene with the risk of HS. Tm shift PCR was used for genotyping. We were unable to find the association of rs964184 of BUD13-ZNF259 gene with the risk of HS (P>0.05). Significant difference was found in the TG level among the three genotypes (CC: 1.51±1.02; CG: 1.68±1.10; GG: 1.90±1.11, P=0.036). The TG level showed strong correlation with rs964184 genotypes (P=0.010, correlation=0.101). Significantly higher TC, HDL-C, and LDL-C levels were observed in the case group. And no difference was found in the TG, ApoA-I, ApoB. Our case-control study supported the significant association between rs964184 genotype and the blood TG concentration, although we were unable to find association between BUD13-ZNF259 rs964184 and the risk of HS in Han Chinese.

Keywords: Hemorrhagic stroke, BUD13-ZNF259, SNP, rs964184, TG

Introduction torin HS [8]. The single nucleotide polymorphism (SNP) at rs688 in the low-density lipopro- More than a million people suffer from stroke tein receptor (LDLR) isassociated with primary worldwide every year [1]. Among the Chinese intracranial hemorrhage, and the risk of HS is population, the incidence of brain hemorrhage increased 73% among the carriers of the rs688- comprises 20-40% of the overall incidence of TT in the population [9]. Collectively, these stud- stroke [2], which is 9%-18% higher than many ies indicate that SNPs in lipid-related genes western countries [3]. Hemorrhagic stroke (HS) play an important role in the pathological pro- is an intraparenchymal hemorrhage due to rup- cess of HS. tured intracranial vasculature, and its pathogenesis is very complex. Recent studies have The underlying pathologies of HS and coronary shown that the occurrence of stroke is theresul- heart disease share similarities. For example, tof genetic and environmental interactions, but atherosclerosis plays an important role in the the exact mechanism is unclear [4]. The patho- initiation process of HS [10]. Therefore, gene logical process of HS involves vascular amyloi- mutations that are implicated in coronary heart dosis or arterial sclerosis [5]. Increasing evi- disease may also be involved in HS. The dence has shown the role of novel geneticdiag- rs964184 mutation in the zinc finger protein nostic markers in the prevention of HS. Several gene BUD13-ZNF259 on chromosome 11q23.3 studies have pointed out that polymorphisms in has been shown to affect lipid levels in the lipid-related genes are closely associated with blood and thus increase the risk of coronary HS. For example, APOE polymorphism affects heart disease [11]. Therefore, this mutation the vascular deposition of amyloid-β, which is may also participate in the disease develop- associated with amyloidosis HS [6, 7]; lipopro- ment of HS tein lipase (LPL) anomalies are associated with diseases such as hyperlipidemia, atherosclero- Currently, there are no studies focus on the sis, and stroke [8], and the G allele mutation in relationship between BUD13-ZNF259 gene the Hind III site of LPL may be a protective fac- polymorphism and the risk of HS. Here, we rs964184 and hemorrhagic stroke risk

heart disease, cancer, severe liver or kidney diseases, and autoimmune diseases.

Clinical observation index

Information regardingage, sex, smoking and drinking, diabetes, hypertension and other clinical data were registered following the hospital- ization of patients. Venous blood was collected for the detection of biochemical markers, including triglycerides (TG), total cholesterol (TC), high-density lipoprotein (HDL-C), low-density lipoprotein (LDL-C), apolipo proteinA (ApoA-I), and apolipo proteinB (ApoB). Lipid testing was performed using automatic biochemical analyzers (Olympus AU2700, Japan), in which TG and TC were detected by the dual- wave length end-point method, HDL-C and LDL-C were detected by direct homogenous assay and ApoA-I and ApoB were determined by Figure 1. Genotyping results for the rs964184 using end-point turbidimetric assay [12]. the Tm shift PCR. SNP genotyping investigated the potential relationship between Genomic DNA extraction was performed using the SNP rs964184 in BUD13-ZNF259 and the automated nucleic acid extraction instru- blood lipid concentrations, and evaluated for ment (Lab-Aid 820, Xiamen City, China). Tm- an association with HS in a case-control study shift genotyping methods were used for BUD13- of individuals in the Han population in Ningbo, ZNF25 rs964184 point mutation genotyping Zhejiang Province. [13]. Primers were designed using the Primer 5.0 software and the optimal SNP primer with Materials and methods Tm values between 59-62°C and length between 15-22 bp was selected [13]. The se- Research subjects quences of the primers for the BUD13-ZNF259 rs964184 mutation were as follows: long prim- This study was approved by the Medical Ethics ers 5’-gcgggcagggcggcTCACCATCTGATGTACTG- Committee of the Ningbo First Hospital; all sub- TTTTCCTc-3’; short primers 5’-gattaccgTCACC- jects provided written informed consent. Case ATCTGATGTACTGTTTTCCTg-3’; generic primer and control subjects were recruited from 5’-TTCATGGAACTTGAAGTCTAGTGGGGA-3’. Ro- Ningbo First Hospital. The case group (n=138) cheLightCycler480 PCR instrument (Roche consisted of patients hospitalized in the Diagnostics, Germany) was used for PCR ampli- Department of Neurosurgery for spontaneous fication. The genotypes were determined by the intracerebral hemorrhage between September melting curve analysis using the analysis soft- 2011 and December 2013. Of these, 86 were ware from the quantitative PCR instrument male and 52 were female, with an average age (Figure 1). of 53.62±16.70. All cases were confirmed by CT or MRI. Stroke cases caused by cardiac or Statistical analysis trauma drugs were excluded. The control population consisted of samples and physical exam- Data analyses were performed using SPSS ination data from 587 randomly selected nor- 16.0 software. The lipid concentrations mal individuals inthe Ningbo First Hospital between the two study groups were compared during the same period of time. Of these, 331 by the t-test. The lipid concentrations between were male and 256 were female with an aver- different genotypes were analyzed byone-way age age of 58.74±10.22. All selected research ANOVA. Pearson correlation was used to deter- subjects were Han Chinese in the Ningbo, mine the association between lipoproteins and Zhejiang province and were free of congenital genotypes. Hardy-Weinberg equilibrium (HWE)

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Table 1. The comparison of characters between HS (P>0.05, Table 2). The similar relation- and control groups# ships were found in the subgroup analysis Characters Control (587) HS (138) P according to hypertension and diabetes (P>0.05, Table3). Age 58.74±10.22 53.62±16.70 0.0001 Gender (male) 331 86 0.517 The comparisons of the clinical data in the TG (mmol/L) 1.58±0.98 1.57±1.31 0.695 3 genotypes were shown in Table 4. TC (mmol/L) 4.38±1.02 4.91±1.22 <0.0001 Significant difference was found in the TG HDL-C (mmol/L) 1.15±0.30 1.23±0.30 0.001 level among the three genotypes (CC: LDL-C (mmol/L) 2.55±0.86 2.84±0.82 0.001 1.51±1.02; CG: 1.68±1.10; GG: 1.90± ApoA (g/L) 1.03±0.23 1.04±0.22 0.848 1.11, P=0.036). However, the concentrations of TC, HDL-C, LDL-C, ApoA-I, and ApoB (g/L) 0.80±0.47 0.76±0.23 0.402 ApoB, showed no significant differences #: There were 35 patients with smoking or drinking, 58 patients with hypertension and 14 patients with diabetes. The P values were between genotypes (P>0.05). adjusted by age, gender, smoking, drinking, diabetes, and hypertension. A further correlation analysis was performed to assess the association between lipoproteins and BUD13-ZNF259 was analyzed by the Arlequin software (version rs964184 genotypes (Table 5). The results 3.5, Bern, Switzerland) [14]. The genotype dis- showed significant correlations between the tribution and allele frequencies of the control rs964184 genotype and TG (P=0.010, correla- group and the HS group were analyzed by the tion=0.101) and HDL-C (P=0.038, correla- Chi-square test. The association between the tion=0.082). Furthermore, we also found sig- BUD13-ZNF25 rs964184 and HS was analyzed nificant correlations between the concentrations by using the additive model (C vs G), the domi- of all lipids (P<0.05), except that between nant model (CC vs CG+GG) and the recessive HDL-C and ApoB (P=0.541). model (CC+CG vs GG) [15]. Power and Sample Size Calculation software (version3.0.43, Discussion Nashville, USA) was used to determine the statistical power [16]. The adjusted calculation The metabolism of blood lipidsis a unique risk was analyzed though SPSS package with binary predictor for cardiovascular and cerebrovascu- logistic regression. All the P values were adjust- lar diseases. Studies have demonstrated that ed by age, gender, smoking, drinking, diabetes, the serum ApoA level in normal young people and hypertension. P<0.05 was considered to can reflect their risk for cardiovascular and be statistically significant. cerebrovascular diseases [17]. In the African- American population, high blood concentra- Results tions of LDL-C and TG were shown to be significantly associated with the risk of intracerebral As shown in Table 1, the age was significantly hemorrhage [18]. Isley’s research [19] found different between the HS group and the control that high concentrations of TC and LDL-C could group (P=0.0001). The concentrations of TC, increase the risk of atherosclerosis. In our HDL-C and LDL-C in the HS group were higher study, we found that the blood concentrations than those of the control group (adjusted of TC, HDL-C, and LDL-C in the HS patients were P<0.005), where as the differences in the TG, significantly higher than those in the control ApoA-I, and ApoB concentrations between the group, which suggested that the pathogenesis two groups were not statistically significant of HS was likely to have originated from (P>0.005). atherosclerosis.

The distribution of the genotypes and alleles The BUD13-ZNF259 gene, located on chromo- was shown in Table 2. No departure of HWE some 11q23, was the subject of cell culture was observed in both the HS and control groups and animal studies. It was highly expressed in (P>0.05). No significant associations were the brain tissue of rats fed a high-calorie diet found between BUD13-ZNF259 rs964184 and [20]. High concentrations of BUD13-ZNF259 in HS risk among the additive model (P>0.05), neuronal cells could increase the level of hydro- dominant model (P>0.05) and recessive model gen peroxide and lead to cell death [20].

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Table 2. Distribution comparison of BUD13-ZNF259 gene rs964184 polymorphism between HS and control groups Additive Dominant Recessive Genotype Allele Gender Group model model model CC/CG/GG C (%)/G (%) OR (95% CI) OR (95% CI) OR (95% CI) All HS (N=138) 87/44/7 218 (79.0)/58 (21.0) 1.06 1.04 1.2 Control (N=587) 376/186/25 938 (79.9)/236 (20.1) (0.77-1.46) (0.71-1.53) (0.51-2.84) Male HS (N=86) 51/29/6 131 (76.2)/41 (23.8) 1.37 1.38 1.83 Control (N=331) 221/97/13 539 (81.4)/123 (18.6) (0.92-2.05) (0.85-2.24) (0.68-4.98) Female HS (N=52) 36/15/1 87 (83.7)/17 (16.3) 0.69 0.68 0.24 Control (N=256) 155/89/12 399 (77.9)/113 (22.1) (0.39-1.21) (0.36-1.29) (0.03-1.85)

Table 3. Distribution comparison of BUD13-ZNF259 gene rs964184 polymorphism between HS and control groups by hypertension or diabetes Genptype Allele Additive model Dominant model Recessive model Group CC/GC/GG C (%)/G (%) OR (95% CI) OR (95% CI) OR (95% CI) Control (N=587) 376/186/25 938 (79.9)/236 (20.1) HSwith hypertension or diabetes (N=60) 36/21/3 93 (77.5)/27 (22.5) 1.15 (0.73-1.81) 1.19 (0.69-2.04) 1.18 (0.35-4.04) HS without hypertension or diabetes (N=78) 51/23/4 125 (80.1)/31 (19.9) 0.99 (0.65-1.50) 0.94 (0.57-1.55) 1.22 (0.41-3.59)

Table 4. The comparison of characteristicsamong the three geno- BUD13-ZNF259 rs964184 types# polymorphism was shown Characteristics CC CG GG P to associate with the risk Age 57.51±12.05 57.83±11.78 60.59±10.82 0.366 of cardiovascular diseases [21]. Gene variation at this Gender (male) 272 126 19 0.579 locus could influence blood TG (mmol/L) 1.51±1.02 1.68±1.10 1.90±1.11 0.036 lipid concentrations [22] TC (mmol/L) 4.51±1.06 4.43±1.13 4.58±1.06 0.625 and increase the risk of car- HDL-C (mmol/L) 1.18±0.30 1.15±0.30 1.09±0.32 0.137 diovascular and cerebrova- LDL-C (mmol/L) 2.63±0.86 2.55±0.85 2.65±0.78 0.417 scular diseases. Brautbar ApoA-I (g/L) 1.04±0.23 1.03±0.22 0.98±0.25 0.361 et al. [23] suggested that ApoB (g/L) 0.80±0.51 0.77±0.24 0.79±0.24 0.618 the rs964184-G carriers #: Results with P values less than 0.05 are in bold font. have higher HDL-C concentrations than the rs964184- C carriers. Zhang et al. [24] Table 5. Assessment of association between lipoproteins and geno- showed that the rs964184- types# G carriers have lower TC Characteristics Genotype TG TC HDL-C LDL-C ApoA-I ApoB and LDL-C concentrations. Genotype —— 0.01 0.715 0.038 0.531 0.265 0.426 Braun et al. [25] found th- TG 0.101 —— <0.001 0.001 0.001 0.001 <0.001 at significant correlation TC -0.014 0.356 —— <0.001 <0.001 <0.001 <0.001 between BUD13-ZNF259 HDL-C -0.082 -0.128 0.336 —— 0.012 <0.001 0.541 rs964184 and blood TG LDL-C -0.025 0.127 0.855 0.099 —— <0.001 <0.001 concentration. Zhang et al’s ApoA-I -0.044 0.128 0.356 0.641 0.163 —— <0.001 study [22] also found that ApoB -0.031 0.198 0.537 0.024 0.539 0.22 —— in the Tibetan population in #: Italic fond indicates correlation coefficient (r), bold fond indicatesP value. The P China, the rs964184-CC values were adjusted by age, gender, smoking, drinking, diabetes, and hypertension. carriers had the lowest serum TG concentration while the rs964184-GG BUD13-ZNF259 could also increase the sus- carriers had the highest concentration. Our ceptibility to oxidative stress, and increasethe comparative study could not find any correla- risk of brain disorders [20]. In humans, the tion between BUD13-ZNF259 rs964184 and

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