神经管畸形患者携带的CASP9基因错义突变对蛋白功能及细胞凋亡的影响

发布时间：2018-04-21 14:43

本文选题：神经管畸形 + 凋亡　；参考：《北京协和医学院》2017年博士论文

【摘要】：研究背景神经管畸形(Neural tube defects,NTDs)是由遗传因素和环境因素交互作用所致的多因素复杂疾病。遗传因素主要由涉及细胞分化、增殖、凋亡和极性等生物学过程相关的基因变异所引起,环境因素主要包括母体叶酸、血糖和维甲酸水平异常等。其中已有研究发现母体血清叶酸水平缺乏能够增加胎儿NTDs发病率。凋亡是机体发育过程中不可或缺的生物学过程。体外胚胎培养发现小鼠的颅部神经管闭合需要正常的细胞凋亡,尤其在闭合交界区。此外也有研究报道凋亡通路基因缺陷会导致小鼠颅部NTDs的发生,其中包括抗凋亡基因Bcl10,及促凋亡基因Apaf1、Casp9和Casp3等。鉴于凋亡基因在小鼠神经管闭合中的关键作用,本研究拟在人类NTDs患者中调查凋亡基因的遗传学贡献以及该遗传因素与叶酸环境因素之间的相互影响。研究目的1.探讨凋亡相关基因对人类NTDs的发生是否有贡献。2.验证在人类NTDs中发现的凋亡基因错义突变是否影响凋亡功能。研究方法通过靶基因二代测序的方法,选取355例NTDs患者和225例地域匹配的对照人群,对14个凋亡相关基因的外显子区和高度保守区进行测序。通过ClustalX、SIFT和PolyPhen2分析软件对筛查出的错义突变进行保守性、氨基酸理化性质变化和功能破坏性预测等生物信息学分析,以预测错义突变可能发生的功能变化。构建野生型和突变型质粒。以NE-4C和HEK293T细胞系为工具细胞,建立瞬转CASP9突变细胞模型。通过 real-time PCR、western blot、caspase activity 检测和 Annexin V-FITC/PI双染方法对生物学分析预测可能具有功能破坏性的错义突变在分子水平和细胞水平进行凋亡功能研究。同时探讨在低叶酸条件下错义突变对凋亡的影响。研究结果1.在NTDs患者中筛查到28次凋亡相关基因错义突变发生,对照组中发现4次错义突变,具有统计学差异(P0.05),错义突变在NTDs患者中显著富集。其中CAASP9基因发生错义突变所占的比例为25%(7/28)。2.CASP9基因的 4 个错义突变(p.G66A、p.R173C、p.R191G 和 p.Y251C)发生在7个NTDs患者中,其中有4个患者携带p.R191G突变。p.G66A和p.Y251C错义突变所在的位点氨基酸高度保守,而p.R173C和p.R191G所在位点氨基酸保守性较差。错义突变p.G66A位于CASP9的CARD蛋白功能结构域中,p.R173C、p.R191G和p.Y251C位于催化功能结构域中,p.Y251C临近结构域中的催化半胱氨酸位点。对错义突变的蛋白进行SIFT和PolyPhen2预测,结果每个突变至少有一种预测方法提示具有功能破坏性。根据NTDs患者中的突变复现频率、氨基酸位置、保守性和生物信息学分析,预测CASP9错义突变p.G66A、p.R191G和p.Y251C可能影响生物学功能。3.在外源过表达野生或突变型CASP9质粒构建的NE-4C和HEK293T瞬转细胞系中,CASP9p.Y251C突变pro-CASP9和cleaved-CASP9的蛋白表达水平明显降低,该突变的CASP9凋亡酶活性也明显降低。4.在分子水平实验中发现CASP9 p.Y251C突变减弱内源性凋亡通路中cleaved-CASP3和cleaved-PARP的水平,降低下游CASP3凋亡酶的活性。在细胞水平实验中也发现CASP9p.Y251C突变导致凋亡细胞减少。5.在叶酸缺乏条件下,CASP9 p.R191G和p.Y251C突变与野生型相比降低CASP9和CASP3凋亡酶活性,减少凋亡细胞数量。结论我们发现凋亡相关基因错义突变在人类NTDs患者中富集,对其中的CASP9基因错义突变进行功能验证,发现错义突变明确影响蛋白质功能,引起明显的细胞凋亡功能缺陷,并受到低叶酸环境影响,从而可能在NTDs发生的病理机制中发挥重要的作用。
[Abstract]:Background Neural tube defects (NTDs) is a multi factor complex disease caused by interaction of genetic and environmental factors. Genetic factors are mainly caused by genetic variations related to biological processes such as cell differentiation, proliferation, apoptosis and polarity. The environmental factors mainly include maternal folic acid, blood sugar and retinoic acid water. It has been found that the deficiency of the maternal serum folate level can increase the incidence of fetal NTDs. Apoptosis is an indispensable biological process in the development of the body. In vitro embryo culture shows that the closure of the cranial nerve canal in the mouse needs normal apoptosis, especially in the closed junction area. Furthermore, there are also reports of apoptosis. Pathway gene defects can lead to the occurrence of NTDs in the cranium of mice, including anti apoptotic gene Bcl10, and apoptosis gene Apaf1, Casp9 and Casp3. In view of the key role of apoptotic gene in the closure of neural tube in mice, this study is intended to investigate the contribution of the apoptotic gene in human NTDs patients and the genetic factors and the folic acid environment. The study aims 1. to investigate whether apoptosis related genes contribute to the occurrence of human NTDs..2. verifies whether the missense mutation of the apoptotic gene found in human NTDs affects the apoptosis function. The method selected the two generation of target genes to select 355 NTDs patients and 225 geographical matched controls, to 14 The exons and highly conserved regions of the apoptosis related genes were sequenced. By ClustalX, SIFT and PolyPhen2 analysis software, bioinformatics analysis of the screened missense mutations, amino acid physicochemical properties and functional destructive prediction were used to predict the possible functional changes in missense mutations. Type plasmids. Using NE-4C and HEK293T cell lines as tool cells, a transient CASP9 mutant cell model was established. A study of real-time PCR, Western blot, caspase activity detection and Annexin V-FITC/PI double staining method for the prediction of the possible functional disruptive missense mutation at the sub level and cell level At the same time, the effect of missense mutation on apoptosis under the condition of low folic acid was investigated. Results 1. the missense mutation of 28 apoptosis related genes was screened in NTDs patients, and 4 missense mutations were found in the control group, with statistical difference (P0.05). The missense mutation was significantly enriched in the NTDs patients. The ratio of the missense mutation of the CAASP9 gene was the ratio of the missense mutation in the control group. The 4 missense mutations (p.G66A, p.R173C, p.R191G and p.Y251C) of the 25% (7/28).2.CASP9 gene occurred in 7 NTDs patients, of which 4 were highly conserved by the amino acids of the loci where p.R191G mutation.P.G66A and p.Y251C missense mutations were located, while the amino acid conservatism of p.R173C and p.R191G sites was poor. In the functional domain of the CARD protein of ASP9, p.R173C, p.R191G and p.Y251C are located in the catalytic functional domain, the catalytic cysteine loci in the p.Y251C near the domain. The missense mutations are predicted by SIFT and PolyPhen2, and at least one prediction method for each mutation shows the functional destructiveness. According to the mutation in the NTDs patient The frequency, amino acid position, conservatism and bioinformatics analysis, the prediction of CASP9 missense mutations p.G66A, p.R191G and p.Y251C may affect the biological function.3. in the NE-4C and HEK293T transient cell lines constructed by exogenous overexpression of wild or mutant CASP9 plasmids, CASP9p.Y251C process pro-CASP9 and cleaved-CASP9 protein expression level The CASP9 apoptotic activity of the mutant was significantly reduced by.4. in the molecular level experiment, and the CASP9 p.Y251C mutation was found to reduce the level of cleaved-CASP3 and cleaved-PARP in the endogenous apoptotic pathway and reduce the activity of the downstream CASP3 apoptotic enzyme. In the cell level experiment, the CASP9p.Y251C mutation caused the apoptotic cells to reduce.5. in the leaves. In acid deficiency, CASP9 p.R191G and p.Y251C mutations reduce CASP9 and CASP3 apoptotic activity and decrease the number of apoptotic cells compared with the wild type. Conclusion we found that the missense mutation of apoptosis related genes is enriched in human NTDs patients, and the missense mutation of the CASP9 gene can be verified, and the missense mutation clearly affects the protein work. It can cause obvious defects in cell apoptosis and be affected by low folate environment, which may play an important role in the pathogenesis of NTDs.

【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R741

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