CDKN2BAS gene polymorphisms and the risk of intracranial aneurysm in the Chinese population

This study was supported by Zhujiang Hospital of Southern Medical University ethics com- mittee, all the participants reported themselves to be of Han residents, have signed informed consent. Zhujiang Hospital is located in developed areas of China—Guangzhou, where the people from other provinces of China accounts for a large part of city population, and popul- ation flow is also very frequent, so the subjects of this study are representative. All particip- ants exclude genetic history, family history and history of malignancy, controlling the age between 18 and 80 years.

Within 14 months, this study recruited 200 patients with sporadic IAs, the median age was 52.69 ± 11.50 years. Patients with IAs diagnosed by computed tomography angiography(CTA), magnetic resonance angiography(MRA) or digital subtraction angiography(DSA) has at least one aneurysm. For the diagnosis of hemorrhage caused by aneurysms, especially ruptured IAs, not only combined with clinical symptoms, but also confirmed by the DSA results eventually. All cases with confirmed IAs based on DSA were retrospectively reviewed by 2 independent doctors, a neuroradiologist and a neurosurgeon, they are all experienced in the diagnosis of aneurysms. All patients in the study underwent either craniotomy or interventional therapy to confirm the correct diagnosis of IAs again.

During the same period, 200 blood donors form a control group for the study, with a median age of 49.99 ± 13.00. Considering the prevalence of IAs vary between 0.2 and 9% of the population, this study does not recruit volunteers from the outside, control participants were patients(such as cerebral hemorrhage, cerebral trauma, epilepsy) from the Department of Neurosurgery of Zhujiang Hospital, as most of them have imaging data(computed tomography(CT), magnetic resonance imaging(MRI), MRA) and clinical information excluded the presence of aneurysms. Controls were eliminate diseases not only including polycystic kidney disease, Malaysia’s syndrome, systemic lupus erythematosus (SLE), etc., but also severity of neurological diseases.

The subjects fully considered the balance and comparable of the baseline data during collection process, genomic DNA was extracted from Peripheral whole blood use kit, using agarose gel electrophoresis to detect the DNA integrity, ensure the follow-up test smoothly.

Through foreign research literatures found the highest positive sites correlation with IAs, they are rs1333040, rs6475606, rs10757272; and using genetic database Hapmap (http://​hapmap.​org), Haploview (http://​www.​broad.​mit.​edu/​mpg/​haploview) found the tagSNP loci of CDKN2BAS in Chinese population, including rs3217992, rs974336, rs3217986, rs1063192; so the seven sites as this research objects, SNP genotyping by PCR-LDR in Shanghai Biowing Applied Biotechnology Co., Ltd. The seven SNPs primer sequences and PCR length are given in Table 1; these SNPs probe sequences and LDR length are read in Table 2. PCR and LDR manipulated as previous describe [18], Firstly, PCR amplification of SNP sites in 20ul master mix, containing 2ul 1 × buffer, 0.6ul Mg++, 2ul dNTPs, 0.2ul Taq polymerase, 4ul 1 × Q-solution, 0.4ul primer mix and 9.8ul ddH²O, after thoroughly incorporated, take 19ul partial shipments in PCR reaction tube and add 1ul genomic DNA of samples, set the following program on Gene Amp PCR system (model 9600, Perkin Elmer): denaturing at 95 °C for 15 min; then denaturing at 94 °C for 30 s, annealing at 56 °C for 1 min, extension at 72 °C for 1 min, repeat 35 cycles; also need to extend 7 min under 72 °C temperature. Secondly, LDR performed to further amplification in 10ul volume of Multiplex LDR mixture, before operation LDR add the same as volume ddH²O to PCR amplification product, the LDR mixture containing resultant PCR product of 1ul, 1ul probe mix, 0.05ul Taq DNA ligase, and 6.95ul ddH²O, after thoroughly incorporated, take 9ul partial shipments in PCR reaction tube and add 1ul PCR amplification product, set LDR conditions: initial denaturing at 95 °C for 2 min, followed by 35 cycles of denaturing at 94 °C for 30 s, and annealing at 50 °C for 2 min, mix 1ul of LDR product with 0.6ul ROX(ABI.GS-500), after denaturing at 95 °C for 2 min, in the ice cold snap, following capillary electrophoresis, meanwhile applying Genemapper(ABI, Inc.) for data analysis and genotyping.

The clinical information of the study participants was summarized in Table 3. Female patients with IAs accounted for 61.5%, is 1.60 times of male patients, and consistent with other related reports [18, 23]. The baseline data balanced between the experimental group and the control group, including gender, smoking, drinking, hypertension and diabetes. Patients with IAs also make specific statistic about aneurysm number and position. There were 90 patients with IAs located in the posterior communicating artery and internal carotid artery, 41 patients in the anterior cerebral artery and 32 patients in posterior cerebral artery. The numbers of patients with one aneurysm, two aneurysms, three aneurysms, more than 3 aneurysms are 130, 35, 21, 14 respectively.

We randomly selected two loci of the Genemapper as shown in Fig. 1. The genotype and allele frequency of cases and controls was presented in Table 4. The control group completely accorded with Hardy-Weinberg equilibrium and objects included in the study could represent the crowd finely. The difference in seven sites of genotype and allele frequency was compared with chi-square or Fisher’s exact tests. The genotype frequency of rs6475606 show significant difference between cases and controls(X² = 10.961, P = 0.004), There also existed difference among groups when compared frequency of TT with CC and CT genotypes combined (X² = 9.008, OR 1.831 [95% confidence interval 1.232—2.723], P = 0.003), the C allele showed reduction the risk of IAs(X² = 4.784, OR 0.710 [95% confidence interval 0.522—0.966], P = 0.029). The distribution of the variants rs1333040 was significantly different in genotype frequency among cases and controls(X² = 8.545, P = 0.014), and compared frequency of TT with CC and CT genotypes combined, the results were significant(X² = 7.295, OR 1.722 [95% confidence interval 1.159—2.558], P = 0.007).

The study aimed to assess the correlation between CDKN2BAS SNP genotypes and clinical presentation(such as hemorrhage, epilepsy, aneurysm location and size) of aneurysms(Table 5). Statistical analysis indicated that both of rs1333040 and rs6475606 were associated with hemorrhage. The C allele of rs1333040 reduced the incidence of hemorrhage. The C allele of rs6475606 was overrepresented in controls which controversial with it was the risk of hemorrhage(X² = 9.626, OR 2.002 [95% confidence interval 1.286—3.119], P = 0.002); and rs13330 40 also related to aneurysm size(P < 0.000), the C allele was the risk factor of IAs with size < 3 cm(X² = 7.358, OR 2.020 [95% confidence interval 1.207—3.380], P = 0.007). The study did not find that CDKN2BAS SNP genotypes was associated with epilepsy and aneurysm location.