CPA1基因和SPINK1外显子突变在慢性胰腺炎中的致病作用及其机制研究

发布时间：2018-04-25 05:13

本文选题：CPA1 + 特发性慢性胰腺炎　；参考：《第二军医大学》2017年博士论文

【摘要】：第一部分中国汉族人群中CPA1功能性突变与特发性慢性胰腺炎关联的鉴定研究研究背景和目的慢性胰腺炎(chronic pancreatitis,CP)是一种胰腺组织进行性炎症病变,反复发作,病程长,严重影响患者的生活质量。目前,对慢性胰腺炎发病机制比较公认的观点是,遗传因素、环境因素及两者的相互作用共同参与了慢性胰腺炎的发生发展。在过去的20年间,随着分子遗传学研究的逐步深入,遗传因素在慢性胰腺炎发病机制中占据了愈发重要的位置,发现阳离子胰蛋白酶原基因、阴离子胰蛋白酶原基因、囊性纤维化跨膜转导调节因子基因、Kazal 1型丝氨酸蛋白酶抑制剂基因、糜蛋白酶原C基因等在慢性胰腺炎发病或保护机制起重要作用。2013年,来自德国的一项研究报道了CPA1功能性突变与非酒精性慢性胰腺炎的发病相关,同时来自欧洲其他地区的实验数据以及亚洲的两个小样本研究数据(日本、印度)均支持这一结果。然而,为了证实某一致病基因确与疾病发生有关,通常需要进行独立的、不同人种的重复实验。特别是考虑到上述研究中CPA1功能性突变在不同人种间的发生及分布情况存在很大差异,亚洲人群仅有两个小样本的研究数据,以及中国人群慢性胰腺炎易感基因和位点可能有别于欧洲人种的情况,进行独立的、大样本的中国人群CPA1功能性突变鉴定研究是非常必要的。本研究的目的是探究中国汉族人群中CPA1功能性突变与特发性慢性胰腺炎的关联,进一步充实中国慢性胰腺炎患者的遗传特征数据,为慢性胰腺炎的精准医疗打下基础。研究方法本实验纳入了1112名中国汉族特发性慢性胰腺炎患者以及1580名汉族对照人群,采用目标区域二代测序的方法对其进行CPA1全外显子以及外显子/内含子交界处序列的测序研究,并利用Sanger测序法对发现的CPA1突变进行验证。对新发现的CPA1突变,进行CPA1酶活性及分泌情况的功能研究。研究结果1、本实验在中国汉族特发性慢性胰腺炎患者及汉族对照人群中共发现18个CPA1罕见突变,其中11个突变为首次报道。2、经过功能研究分析,证实有五个突变为功能受损性突变,分别是p.Glu100Lys,p.Arg240Gln,p.Gly225Ser,p.His306Tyr,p.Glu380Lys。3、在中国汉族人群中,无论是CPA1罕见突变(患者:20/1112(1.8%)vs.对照:24/1580(1.52%);P=0.573),还是CPA1功能受损性罕见突变(患者:3/1112(0.27%)vs.对照:2/1580(0.13%);P=0.410),均与特发性慢性胰腺炎的发生无关。结论1、本试验首次重复研究了CPA1功能突变与特发性慢性胰腺炎的关系,是目前样本量最大的、单一人种的针对CPA1编码序列及CPA1外显子/内含子交界处序列的测序研究。2、本研究首次报道了中国汉族人群中CPA1功能性突变与特发性慢性胰腺炎无显著关联。此结论与欧洲数据以及亚洲小样本研究数据不一致,这可能是由于人种遗传因素特异性所致。3、CPA1功能性突变与慢性胰腺炎的关联性在不同人种间存在差异,进一步证明了探究本国人群遗传因素具有重要意义。第二部分SPINK1基因外显子区域突变对前体m RNA剪接影响的功能研究研究背景和目的胰腺分泌胰蛋白酶抑制剂基因(pancreatic secretory trypsin inhibitor,SPINK1)是研究最早、最广泛的慢性胰腺炎易感基因之一,SPINK1的功能丧失性突变在慢性胰腺炎的发病过程中起重要作用。目前已报道了32个SPINK1外显子区域突变,其致病机理研究主要集中于错义突变是否影响了蛋白功能。有研究指出基因外显子区域突变可以通过产生新的剪接位点,或通过改变与剪接发生密切相关的多种顺势作用元件,如剪接增强子、剪接沉默子等,影响前体m RNA剪接功能。同时,越来越多的研究报道包括同义突变在内的外显子区域突变可通过影响前体m RNA剪接的方式增加患病风险,更有研究预测,在分布于外显子末端的致病SNPs突变中,有20-45%的突变可能影响剪接功能。此外,minigene模型是常用的分析基因突变对前体m RNA剪接影响的模型。但minigene舍弃了基因的部分内含子或外显子区域,可能因此掩盖了远端内含子、外显子对待研究区域剪接的影响,或改变m RNA二级结构,降低其稳定性。本研究的主要目的是使用SPINK1全长基因研究模型,探究SPINK1外显子区域突变是否对前体m RNA剪接产生影响,进一步鉴定SPINK1外显子区域突变的致病性,明确疾病易感突变的致病机理。同时,通过对比SPINK1全长基因研究模型与SPINK1 minigene研究模型的实验结果,进一步探讨minigene模型给研究结果带来的可能偏差。研究方法本课题针对已报道的32个SPINK1外显子区域突变进行研究。构建野生型及突变型SPINK1全长基因及minigene载体质粒,转染HEK293T细胞,通过RT-PCR方法研究突变对基因转录本的影响,并用一代测序检测转录本序列;通过Q-PCR的方法研究突变对m RNA相对表达量的影响。利用Alamut软件对SPINK1外显子突变进行影响剪接功能的预测分析。研究结果1、RT-PCR结果显示SPINK1外显子突变未导致异常转录本产生。2、Q-PCR结果显示SPINK1突变c.27del C(70.5±9.8,P=0.0092)、c.29GA(80.0±4.4,P=0.0029)、c.98_99ins A(71.9±1.9,P=0.0015)、c.190AG(82.2±4.1,P=0.0170)、c.199CT(77.1±10.1,P=0.0070)、c.203AG(87.4±7.0,P=0.0365)可致SPINK1 m RNA相对表达量降低。3、c.27del C、c.98_99ins A产生提前终止密码子,NMD抑制剂放线菌酮作用4小时后,可致携带c.27del C突变的m RNA表达量回升(126.5±2.2,P=0.0023),对c.98_99ins A无影响(107.2±18.4,P=0.3606)。4、Alamut软件预测有7个突变可能对原有剪接位点产生影响,c.190AG等10个突变可能产生新的剪接位点;预测c.199CT、c.98_99ins A等12个突变可能降低ESEs活性,有8个突变可能增强ESEs活性。5、对比SPINK1全长基因模型与SPINK1 minigene模型的研究结果发现,两种模型的RT-PCR结果相似,均可以显示c.194+2TC突变导致的SPINK1异常转录本,且外显子其它突变对SPINK1转录本并无影响。但Q-PCR结果有所不同:minigene模型的研究显示c.110AG(64.1±2.0,P=0.001)、c.137TA(131.1±7.9,P=0.0211)、c.194GA(44.5±6.4,P=0.0044)可影响m RNA的表达量,而全长基因模型研究未提示突变改变m RNA表达量;c.190AG在两种模型中的研究结果相反,全长基因模型研究显示该突变可降低SPINK1 m RNA的相对表达量,而在minigene模型的研究中,该突变增加m RNA的相对表达量(121.4±2.0,P=0.0030)。结论1、本研究进一步鉴定了SPINK1外显子区域突变的致病性,明确了疾病易感突变的致病机理。2、常用剪接分析模型minigene存在一定缺陷,建议使用全长基因模型进行研究分析。
[Abstract]:The first part of the study of the association between CPA1 functional mutation and idiopathic chronic pancreatitis in Chinese Han population, background and objective chronic pancreatitis (chronic pancreatitis, CP) is a progressive inflammatory disease of the pancreatic tissue, recurrent attacks, long course of disease, and serious influence on the quality of life of the patients. It is generally accepted that genetic factors, environmental factors, and their interaction are involved in the development of chronic pancreatitis. In the past 20 years, with the gradual deepening of molecular genetic research, genetic factors have occupied a more important position in the pathogenesis of chronic pancreatitis, and the discovery of the cationic trypsinogen gene, The anionic trypsinogen gene, the cystic fibrosis transmembrane transduction regulator gene, the Kazal 1 serine protease inhibitor gene, and the chymotrypsinogen C gene play an important role in the pathogenesis or protection of chronic pancreatitis,.2013 years. A study from Germany reported that the CPA1 functional mutation and non-alcoholic chronic pancreatitis were reported. Experimental data from other regions of Europe, as well as two small sample data in Asia (Japan, India), support this result. However, in order to confirm that a pathogenic gene is associated with the disease, an independent, different race repeat experiment is usually needed. Especially considering the CPA1 function in the above study. There are great differences in the occurrence and distribution of sexual mutation among different species. There are only two small sample data in Asian population, and the susceptibility genes and loci of chronic pancreatitis in Chinese population may be different from those of European ethnicity. It is necessary to identify the functional mutation of CPA1 in large samples of Chinese people. The purpose of this study was to explore the association of CPA1 functional mutations in Chinese Han population with idiopathic chronic pancreatitis and to further enrich the genetic data of patients with chronic pancreatitis in China, and to lay the foundation for the precise medical treatment of chronic pancreatitis. The study included 1112 patients with chronic chronic pancreatitis in Han Chinese. The sequence of CPA1 total exon and exon / intron junction sequence of 1580 Han control population was sequenced by two generation of target region, and the CPA1 mutation was verified by Sanger sequencing. The function of the CPA1 enzyme activity and secretion of the newly discovered CPA1 mutation was studied. 1, in this experiment, 18 rare CPA1 mutations were found in Chinese Han idiopathic chronic pancreatitis and Han control population, of which 11 mutations were first reported.2. After functional analysis, five mutations were identified as p.Glu100Lys, p.Arg240Gln, p.Gly225Ser, p.His306Tyr, p.Glu380Lys.3, respectively, in China. In the Han population, whether a rare CPA1 mutation (patients: 20/1112 (1.8%) vs. control: 24/1580 (1.52%), P=0.573), or a rare mutation of CPA1 function (patients: 3/1112 (0.27%) vs. control: 2/1580 (0.13%); P=0.410), is not related to the occurrence of idiopathic chronic pancreatitis. Conclusion 1, this trial repeated the first repeated study of CPA1 function mutation and idiopathic The relationship of chronic pancreatitis is the largest sample size at present. The sequence of CPA1 coding sequence and CPA1 exon / intron junction sequence of single human race was sequenced.2. This study was the first to report that there was no significant association between CPA1 functional mutation and idiopathic chronic pancreatitis in Chinese Han population. This conclusion is with European data and Asian samples. The data of this study are inconsistent, which may be due to the genetic factor specificity of.3, and the correlation between CPA1 functional mutation and chronic pancreatitis is different between different people, and further proves the significance of exploring the genetic factors of the population in the country. The second part of the SPINK1 gene exon mutation affects the splicing of the precursor m RNA Functional research background and objective pancreatic secretory trypsin inhibitor (SPINK1) is one of the earliest and most extensive chronic pancreatitis susceptible genes. The loss of function of SPINK1 plays an important role in the pathogenesis of chronic pancreatitis. There are 32 reports of SPINK1 outside of the pathogenesis of chronic pancreatitis. It is pointed out that the mutation of the exon region can be produced by producing new splice sites, or by changing a variety of homeopathic elements closely related to splicing, such as splicing enhancer, splicing silencer, etc., affecting the precursor m RNA At the same time, more and more studies have reported that the exons region mutation, including synonymous mutations, can increase the risk of the disease by affecting the m RNA splicing of the precursor, and more research predicts that the mutation of the 20-45% can affect the splicing function in the pathogenetic SNPs mutation distributed at the exon end of the exons. Furthermore, the minigene model is commonly used. An analysis of the effects of gene mutation on the splicing of the precursor m RNA. But minigene abandons the partial intron or exons of the gene, which may mask the distal intron, the exons treat the study area splicing, or change the m RNA two structure to reduce its stability. The main purpose of this study is to use the SPINK1 full length gene. To investigate whether the mutation of the SPINK1 exon region affects the splicing of the precursor m RNA, further identifying the pathogenicity of the mutation in the exon of SPINK1, and clarification of the pathogenicity of the susceptibility mutation of the disease. At the same time, by comparing the experimental results of the SPINK1 full-length gene research model with the SPINK1 minigene model, the minigene is further explored in minigene. The possible deviation of the model to the results of the study. The research method is aimed at 32 reported mutations in the SPINK1 exons. To construct wild and mutant SPINK1 full length gene and minigene vector plasmid, transfect HEK293T cells, and study the effect of mutation on the gene transcript by RT-PCR method, and use one generation sequencing test. Transcriptional sequence; study the effect of mutation on the relative expression of M RNA by means of Q-PCR. Use Alamut software to predict the splicing of SPINK1 exon mutation. Results 1, RT-PCR results show that SPINK1 exons mutation does not cause the abnormal transcriptional transcript to produce.2, Q-PCR results show SPINK1 mutation c.27del C (70.5 + 9.8). =0.0092), c.29GA (80 + 4.4, P=0.0029), c.98_99ins A (71.9 + 1.9, P=0.0015), c.190AG (82.2 + 4.1, P=0.0170), c.199CT (77.1 + 10.1, P=0.0070), c.203AG (87.4 + 7, P=0.0070). The m RNA expression of.27del C mutation increased (126.5 + 2.2, P=0.0023), and had no effect on c.98_99ins A (107.2 + 18.4, P=0.3606).4. Alamut software predicted that 7 mutations could affect the original splice site, c.190AG and other 10 mutations may produce new splice sites, and 12 mutations such as pretest c.199CT and 12 may reduce the activity of 8. There are 8 The mutation may enhance the ESEs activity.5. Compared with the results of the SPINK1 full-length gene model and the SPINK1 minigene model, the results of the RT-PCR results of the two models are similar, and all the SPINK1 abnormal transcripts caused by the c.194+2TC mutation can be displayed, and the other mutations of the exons have no effect on the SPINK1 transcript. But the Q-PCR results are different: minigene modulus The study showed that c.110AG (64.1 + 2, P=0.001), c.137TA (131.1 + 7.9, P=0.0211), c.194GA (44.5 + 6.4, P=0.0044) could affect the expression of M RNA, while the full length gene model did not suggest that the mutation changed the m RNA expression; c.190AG in the two models had the opposite results, and the full length gene model study showed that the mutation could reduce SPINK1 In the study of minigene model, the relative expression of M RNA was increased (121.4 + 2, P=0.0030). Conclusion 1. This study further identified the pathogenicity of the mutation in the exons of SPINK1, clarified the pathogenicity mechanism of the susceptibility mutation of the disease, and the common splicing analysis model minigene has some defects, suggesting the use of the whole system. The long gene model was studied and analyzed.

【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R576

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