Correlation of tumor necrosis factor receptor superfamily 13B variation with sporadic intracranial aneurysm and clinical characteristics in Han Chinese populations☆
Pengfei Wu, Anhua Wu, Yunjie Wang

Department of Neurosurgery, First Affiliated Hospital of China Medical University, Shenyang  110001, Liaoning Province, China
Pengfei Wu☆, Doctor, At-tending physician, Depart-ment of Neurosurgery, First Affiliated Hospital of China Medical University, Shen-yang  110001, Liaoning Province, China

Corresponding author: An-hua Wu, Doctor, Professor, Department of Neurosurgery, First Affiliated Hospital of China Medical University, Shenyang   110001, Liaon-ing Province, China
wuanhua@hotmail.com

Abstract
BACKGROUND: Inflammatory reaction correlates with sporadic intracranial aneurysm (IA). Varia-tion of tumor necrosis factor receptor superfamily 13B (TNFRSF13B), an inflammatory mediator receptor, may associate with IA.
OBJECTIVE: To explore the relationship between TNFRSF13B gene and sporadic IA, as well as the clinical characteristics of sporadic IA.
DESIGN, TIME AND SETTING: Case-control study of genetic association was performed at the Experimental Technology Center of China Medical University from November 2006 to January 2008.
PARTICIPANTS: A total of 367 patients with IA, confirmed by three-dimensional computed tomo-graphy angiography, magnetic resonance angiography, digital subtraction angiography, and neuro surgery, were admitted to the Department of Neurosurgery, First Affiliated Hospital of China Medical University from 2006 to 2007, and were selected as the case group. All patients were Han, with no family history of IA. In addition, a total of 396 non-IA patients were selected as control subjects.
METHODS: Peripheral vein blood was harvested to extract whole blood genomic DNA. Genotyping and TNFRSF13B single nucleotide polymorphism (SNP) rs11078355 G>A allele polymorphisms were determined by polymerase chain reaction-restriction fragment length polymorphism. The rela-tionship of TNFRSF13B SNP rs11078355 G>A polymorphisms to IA and IA clinical characteristics were analyzed using the chi-square and two-sided test.
MAIN OUTCOME MEASURES: TNFRSF13B SNP rs11078355 G>A genotype distribution.
RESULTS: In the IA patients, TNFRSF13B SNP rs11078355 G>A genotype frequency was sig-nificantly increased (X2 = 16.306, odds ratio = 1.881, 95% confidence interval = 1.382-2.560, P < 0.001). In IA patients aged ≥ 65 years, the frequency of TNFRSF13B SNP rs11078355 GA + AA genotype was significantly greater than the GG genotype (X2 = 26.604, odds ratio = 5.248, 95% confidence interval = 2.662-10.345, P < 0.001).
CONCLUSION: The TNFRSF13B gene may associate with sporadic IA in Han Chinese populations. In elderly patients, allele A may be an independent risk factor for IA, in addition to senile diseases, such as hypertension and diabetes mellitus.
Key Words: intracranial aneurysm; single nucleotide polymorphism; tumor necrosis factor receptor superfamily 13B gene

INTRODUCTION

Intracranial aneurysm (IA) is a multigenic disease[1-8]. Studies have shown that single nucleotide polymorphism (SNP) highly cor-relates with IA occurrence[9]. Inflammatory reactions have also been shown to correlate with IA[10-11], which is a direct reaction to IA rupture and bleeding, and may even exist prior to IA rupture[10]. Tumor necrosis factor receptor superfamily 13B (TNFRSF13B) is located at 13q32-34[12], and studies have demonstrated that TNFRSF13B variations result in amino acid replacement and even-tually common variable immunodefi-ciency[13-14]. Moreover, heterozygotic TNFRSF13B variation has been shown to associate with common variable immunode-ficiency[15-16]. IA is complicated by immu-nological diseases, hereditary connective tissue disease, systemic disease (Marfan syndrome), type I neurofibromatosis, and Ehlers-Danlos syndrome[17]. However, the relationship between the TNFRSF13B gene and IA remains unclear.
A preliminary study has shown that the TNFRSF13B gene may be an IA suscepti-bility gene in Japanese populations[9]. However, the role of TNFRSF13B in IA in Chinese populations 
remains uncertain.
The present study analyzed the SNP rs11078355 G>A of the TNFRSF13B gene in 367 sporadic IA Han Chinese patients and 396 Han Chinese control subjects to de-termine the relationship between TNFRSF13B and IA, as well as the IA clinical characteristics. In addition, TNFRSF13B SNP rs11078355 G>A polymorphisms and risk factors for sporadic IA (hypertension and diabetes mellitus) were analyzed.

SUBJECTS AND METHODS

Design
Case-control study of genetic association.
Time and setting
The present experiment was performed at the Experi-mental Technology Center of China Medical University from November 2006 to January 2008.
Subjects
Case group: a total of 367 sporadic IA patients admitted to the Department of Neurosurgery, First Affiliated Hos-pital of China Medical University between 2006 and 2007, with an average age of (53 ± 10) years, were selected. IA was confirmed by digital subtraction angiography, 3D computed tomography (CT) angiography or magnetic resonance (MR) angiography, as well as neurosurgery. Patients exhibiting other intracranial pathological changes, as confirmed by digital subtraction angiography, 3D CT angiography or MR angiography, or not confirmed by neurosurgery, were excluded.
Control group: a total of 396 patients, with an average age of (52 ± 13) years and no history of IA, were selected from the First Affiliated Hospital of China Medical Uni-versity between 2006 and 2007.
All participants were Han Chinese and were not related. Informed consent was obtained for blood sample analy-ses. All experimental procedures were in accordance with the Administrative Regulations on Medical Institution, formulated by the State Council of the People's Republic of China[18].
Methods
Genomic DNA extraction
A total of 2 mL venous blood was harvested and anti-coagulated with ethylenediaminetetraacetic acid. Genomic DNA was extracted from peripheral blood using the potassium iodide method, quantified by ultraviolet spectrophotometry (Eppendorf, Germany), and stored at –20 °C.
TNFRSF13B SNP polymorphism, as detected by po-lymerase chain reaction (PCR)
TNFRSF13B SNP rs11078355 G>A genotypes were determined by PCR-restriction fragment length poly-morphism. Primers were designed using PRIMER5.0 (http://csupharmacol.com/Soft/ShowSoft.asp?SoftID=15), and synthesized by Applied Takara Biotechnology, China. The primers were 5′-TCC CTC CCT GAC CCC CAT T-3′ (forward) and 5′-GGA CCC CCG ACC CCA CTT G-3′ (reverse), and the amplified fragment length was 155 bp.
A total of 5 μL genomic DNA was amplified in 20 μL of reaction mixture containing 2 μL 10 × PCR buffer, 1 μL of each primer, 1.6 μL dNTP, and 0.1 μL Taq DNA poly-merase (Applied Takara Biotechnology, Dalian, China). PCR amplification was performed using a thermal cycler (GeneAmp PCR System model 9700, Applied Biosys-tems, USA): initial denaturation at 94 °C for 7 minutes, followed by 35 cycles of denaturation at  94 °C for 30 seconds, annealing at 50 °C for 30 seconds, and exten-sion at 72 °C for 30 seconds. The reaction was termi-nated by a final extension at 72 °C for 7 minutes. The PCR products were electrophoresed on a 2% agarose gel to observe product purity and quantity.
According to SNP rs11078355 data from the National Center for Biotechnology Information Database (http://www.ncbi.nlm.nih.gov/sites/entrez?db=snp&cmd=search&term=rs11078355), the restriction enzymes sites were selected using New England Biochemical Labora-tory (http://www.neb.com/nebecomm/default.asp). The restriction enzymes (Van91 I) were identified and syn-thesized (Applied Takara Biotechnology, Dalian, China). A total of 10 μL PCR product was placed into  20 μL reaction mixture containing 2 μL 10 × L buffer, 7 μL sterile water, and 1 μL restriction enzyme. The samples were incubated in a water bath at 37 °C overnight. The prod-ucts were identified by 2% agarose gel electrophoresis. Genotype analysis and identification were performed using the blind method.
Main outcome measures
TNFRSF13B SNP rs11078355 G>A genotype distribu-tion.
Statistical analysis
Allele frequency was predicted using the Hardy-Weinberg equilibrium law. Genotype distribution of SNP rs11078355 G>A of TNFRSF13B between cases and controls was analyzed using SPSS 13.0 software (SPSS, Chicago, IL, USA). All data were subjected to the chi-square test and two-sided test. Odds ratio (OR) was calculated, and confidence interval (CI) was 95%. A P value < 0.05 was considered statistically significant.

RESULTS

Quantitative analysis of participants
All 367 IA patients and 396 controls were included in the final analysis.
Baseline data
The 763 participants were Han Chinese from the Chi-nese mainland (Table 1). Age distribution of patients is shown in Figure 1.
Of the 367 IA patients, 236 (64.3%) were 40–59 years old, and there were significantly more female patients than male patients (X2 = 17.225, OR = 1.848, 95% CI = 1.381-2.474, P < 0.001). The case group patients dis-played higher levels of fasting blood glucose and blood pressure (X2 = 86.001, OR = 4.121, 95% CI = 3.034-5.597, P < 0.001; X2 = 27.687, OR = 2.167,   95% CI = 1.622-2.896, P < 0.001). However, there was no significant difference in age distribution between cases and controls.

Identification of target gene fragment
Absorbance of extracted DNA was 0.8. Agarose gel electrophoresis revealed a 155-bp homozygote allele G (Figure 2).

Enzyme restriction results for TNFRSF13B
The restriction enzyme (Van91 I) results revealed a sin-gle 155-bp band for the homozygote allele G, 81-bp and 74-bp bands for the homozygote allele A, and 155-bp, 81-bp, and 74-bp bands for the heterozygote (Figure 3).

TNFRSF13B SNP rs11078355 G>A genotype and al-lele distribution
Distribution of TNFRSF13B SNP rs11078355G>A poly-morphisms in cases and controls were consistent with the Hardy-Weinberg equilibrium (P < 0.001). The TNFRSF13B SNP rs11078355 G>A polymorphism genotype is shown in Table 2.
Comparison of TNFRSF13B SNP rs11078355G>A genotype between case and control groups suggested a correlation with IA in the Han Chinese population (X2= 16.306, OR = 1.881, 95% CI = 1.382-2.560, P < 0.001), which suggested that the AA/GA genotype was a risk factor for sporadic IA.

TNFRSF13B genotype and IA clinical characteristics
The association between the SNP rs11078355G>A genotype and clinical features was evaluated in IA pa-tients. Gender, blood pressure, and fasting blood glu-cose did not correlated with the SNP rs11078355G>A genotype (P > 0.05). However, frequency of the TNFRSF13B SNP rs11078355 GA+AA genotype was > the GG genotype in IA patients aged ≥ 65 years (X2 = 26.604, OR = 5.248, 95% CI = 2.662-10.345, P < 0.001, Table 3).

DISCUSSION

SNP is the most frequent type of polymorphism in the human genome, and some SNPs can influence gene function and result in disease. SNPs are useful for un-derstanding individual phenotypic discrepancies and susceptibility to disease, especially complex diseases. Therefore, identification of polymorphic sites in DNA sequences associated with common diseases is impor-tant for understanding the etiology of human dis-eases[19-20].
Sporadic IA has been detected in all age groups, but is more frequent in people aged 40–49 years[21]. In the present study, IA incidence increased in patients aged 40–59 years (64.3%), peaked in patients aged 50–59 years, and decreased after 70 years, indicating that ad-vanced age increases pathogenicity of other risk fac-tors[22-23]. Moreover, subject data revealed that suscepti-bility was greater in female patients, and fasting blood glucose and blood pressure highly correlated with IA, which was consistent with previous results[9, 24-25].
In addition, the relationship between TNFRSF13B SNP rs11078355 G>A polymorphism and sporadic IA was analyzed in a Han Chinese population. Results showed significant frequency differences in the TNFRSF13B SNP rs11078355 G>A genotype between IA patients and controls (P < 0.001, OR = 1.881). This suggested a cor-relation between SNP and sporadic IA in Han Chinese; the TNFRSF13B gene may be associated with IA in Han Chinese, which was consistent with previous results[9].
Moreover, the role of TNFRSF13B gene in IA clinical characteristics was investigated. There was no signifi-cant difference between frequency of TNFRSF13B SNP rs11078355 GA+AA genotype and GG genotype in blood pressure or fasting blood glucose levels among IA pa-tients. However, the frequency of TNFRSF13B SNP rs11078355 GA+AA genotype was > the GG genotype in IA patients aged ≥ 65 years, indicating that the TNFRSF13B SNP rs11078355 A allele may lead to onset of IA in the elderly. The A allele could be an independent risk factor for IA in addition to diseases of the elderly, such as hypertension and diabetes mellitus. The in-creased risk of IA in AA carriers of the SNP rs11078355 polymorphism may be a result of local or peripheral in-flammation, which could partially contribute to IA etiopa-thogenesis in aged individuals.
There were some limitations to the study. The relatively small sample size could have resulted in a less precise estimation of the association between TNFRSF13B polymorphisms and IA susceptibility. Additionally, the participants were from mainland China. Therefore, these results may not apply to other populations.
In conclusion, sporadic IA occurred primarily in individuals aged 40–59 years, and peaked in the group aged 50–59 years. In addition, females are more susceptible to IA than males, and fasting blood glucose and hypertension are significantly associated with IA. Moreover, an association between the TNFRSF13B gene and IA was initially determined in a Han Chinese population. The A allele could be an independent risk factor for IA in the elderly, in addition to other diseases of the elderly. However, other races and nations could possess different susceptibility genes[26]. Therefore, further studies are needed in different populations, and a larger sample size is required to validate our results.

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