Ⅰ型胶原蛋白α2基因是中国东北地区颅内动脉瘤发生的祸首？

doi:10.3969/j.issn.1673-5374.2013.05.008

摘要/Abstract

摘要：

对中国东北地区散发颅内动脉瘤和其他神经系统肿瘤患者的外周血进行Ⅰ型胶原蛋白α2基因多态性分析，限制性片段长度多态性聚合酶链反应检测发现，两组Ⅰ型胶原蛋白α2基因多态位点rs42524 G>C基因型频率差异显著。在Hunt and Hess分级大于3级的颅内动脉瘤患者中，GC+CC基因型频率明显高于GG基因型，说明颅内动脉瘤患者Ⅰ型胶原蛋白α2基因单核苷酸多态性位点rs42524 G>C基因型与颅内动脉瘤明显相关。由此认为，Ⅰ型胶原蛋白α2基因有可能是中国东北地区颅内动脉瘤的易感基因之一，其基因多态位点rs42524等位基因C可能是导致颅内动脉瘤患者病情加重的重要危险因素。

关键词: 神经再生, 临床实践, 颅内动脉瘤, Ⅰ型胶原蛋白, 基因, 单核苷酸多态性, 限制性片段长度多态性聚合酶链反应, 中国东北, 蛛网膜下腔出血, 易感性, 危险因素

Abstract:

In this study, we investigated whether a single nucleotide polymorphism (rs42524 G > C) in the type I alpha 2 collagen gene was associated with sporadic ruptured intracranial aneurysm or its clinical characteristics in patients from Northeast China. Genotyping of the rs42524 G > C polymorphism was carried out using a polymerase chain reaction-restriction fragment length polymorphism assay. The data showed that the frequency of the rs42524 GC + CC genotype was significantly higher than the GG genotype among intracranial aneurysm patients whose Hunt and Hess grading scale was > 3. In addition, the rs42524 G > C genotype was found to have a statistically significant association with intracranial aneurysm risk. These findings indicate that the type I alpha 2 collagen gene may be involved in a predisposition to intracranial aneurysm in the Northeast Chinese population. Crucially, the rs42524 C allele may be an important risk factor for increased severity of the condition in patients with ruptured intracranial aneurysms.

Key words: neural regeneration, clinical practice, intracranial aneurysm, type I collagen gene, single nucleotide polymorphism, polymerase chain reaction-restriction fragment length polymorphism assay, susceptibility, risk factors, neuroregeneration

. Ⅰ型胶原蛋白α2基因是中国东北地区颅内动脉瘤发生的祸首？[J]. 中国神经再生研究(英文版), 2013, 8(5): 445-451.

Pengfei Wu, Bo Li, Anhua Wu, Yunjie Wang. Is type I alpha 2 collagen gene responsible for intracranial aneurysm in Northeast China?[J]. Neural Regeneration Research, 2013, 8(5): 445-451.

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引言

材料方法

Design

A case-control study.

Time and setting

Experiments were performed in the Central Laboratory of China Medical University from March 2007 to January 2008.

Subjects

The subjects were unrelated individuals from the Northeast of China. Case group: the trial recruited 367 sporadic intact intracranial aneurysm patients and 396 control subjects. The patients comprised 128 men and 239 women who had been diagnosed by digital subtraction angiography, three-dimensional computed tomography angiography, or magnetic resonance angiography. Diagnoses for these patients were confirmed by neurosurgical operations performed at the Department of Neurosurgery, First Affiliated Hospital of China Medical University in China. Control group: 396 controls, consisting of 197 men and 199 women, were randomly selected among people admitted to the same hospital during the same time period. These patients had tumors of the nervous system but no intracranial aneurysm as determined by digital subtraction angiography, three-dimensional computed tomography angiography or magnetic resonance angiography.

All subjects were informed about the program and its potential risks before starting and informed consent was obtained according to the Administration Regulations of Medical Institution, formulated by the State Council of the People's Republic of China^[32].

All subjects agreed to ulnar vein blood samples being taken; these were collected in anticoagulant test tubes prior to analysis. Detailed information and categorization of risk factors including gender, age, blood pressure and fasting blood glucose is shown in Table 3.

Two milliliter samples were collected from the ulnar vein of each subject, after which DNA was extracted using potassium iodide, and qualified by ultraviolet spectrophotometry. The type I alpha 2 collagen gene single nucleotide polymorphism rs42524 G > C genotypes were determined using a PCR-restriction fragment length polymorphism assay^[33]. The primers were designed using Primer 5.0 (Applied Takara Biotechnology, Dalian, China), and then synthesized. The sequences of these primers are shown in Table 4.

Type I alpha 2 collagen gene single nucleotide polymorphism rs42524 detected by PCR-restriction fragment length polymorphism

Five nanograms of genomic DNA was amplified in a 20 μL reaction volume containing 10 × PCR buffer, 100 nM of each primer, 160 nM of each dNTP, and 0.1 U of AmpliTaq Gold DNA Polymerase (Applied Takara Biotechnology, Dalian, China). The PCR conditions were as follows: initial denaturation at 94°C for 7 minutes, followed by 35 cycles of denaturation at 94°C for 30 seconds, annealing at 53°C for 30 seconds, and extension at 72°C for 30 seconds, and a final extension at 72°C for 7 minutes. Thermocycling was conducted using the GeneAmp PCR System (model 9700, Applied Biosystems, USA). PCR products were analyzed using 2% agarose gels to determine their purity.

The National Center of Biotechnology Information database (http://www.ncbi.nlm.nih.gov) was used to determine the restriction endonuclease cutting site for the rs42524 single nucleotide polymorphism. On the basis of this information, Bsa JI was selected using the New England Biochemical Laboratory Website (http://www.neb.com). Bsa JI was obtained from Applied Takara Biotechnology (Dalian, China). Ten nanograms of each type I alpha 2 collagen gene PCR product was added to a 20 μL reaction mixture containing 10 × buffer K and 100 nM Bsa JI. Reactions were incubated in a 37°C water-bath overnight. The reaction products were run on 2% agarose gels. Samples with homozygous C alleles produced 128 bp bands; homozygous G alleles produced 107 and 21 bp bands, whilst heterozygotes produced bands of 128, 107 and 21 bp in length.

Statistical analysis

Data were analyzed with SPSS 13.0 statistical software (SPSS, Chicago, IL, USA). Numerical data were analyzed using a two-sided fourfold table chi-square test. A level of P < 0.05 was considered a statistically significant difference.