马凡综合征和支气管扩张致病突变研究

发布时间：2018-05-11 12:06

本文选题：马凡综合征 + Ion　；参考：《北京协和医学院》2017年博士论文

【摘要】：研究内容一 125例中国马凡综合征患者致病基因突变筛查马凡综合征(Marfan syndrome,MFS)是一种常染色体显性遗传的结缔组织病,在人群中发病频率为1/3000-1/5000。通过高通量测序对MFS相关基因FBN1、TGFBR1、TGFBR2进行检测,是对患者进行明确诊断,指导手术及药物治疗的有效手段。研究目的:绘制中国MFS人群突变谱;进行中国MFS基因型表型关联分析;为中国MFS患者提供明确诊断、指导治疗、预后评估及遗传咨询;对未筛查到致病变异的家系进行全外显子组测序(WES)以期发现新的可能致病基因。研究对象和方法:1.研究对象:125例中国MFS患者及家系成员;2.研究方法:(1)高通量二代测序:通过Ion torrent测序平台对125例患者进行靶基因测序,测序Panel(靶基因引物库)中包含3个明确的MFS致病基因;对二代测序结果进行筛选;并对所筛选到的结果进行Sanger测序验证;(2)家系分析:对通过Ion torrent PGM测序未发现疑似致病变异的有家族史的患者进行了 STR marker家系连锁分析,确定连锁区域。对致病基因连锁在FBN1基因的家系,进行FBN1基因重测序或整个FBN1基因测序;对于FBN1基因不连锁的家系,进行全外显子组测序分析,以期发现新的致病基因。对一个类马凡的双胞胎家系进行全外显子组测序分析。(3)MLPA检测大片段插入/缺失突变:对未发现致病变异患者进行多重连接依赖式探针扩增技术(MLPA)检测患者是否携带FBN1基因DNA大片段的插入/缺失突变;通过定量PCR的方法对MLPA结果进行验证,并且通过步移法来缩短断裂点上下游的序列;Long Range PCR(长片段PCR)进行扩增和测序,确定具体断裂点;提取FBN1基因48-53号外显子缺失的患者血管组织RNA,进行转录水平分析;(4)对高通量测序筛选和MLPA检测到的变异进行基因型表型关联性分析。实验结果:1.高通量测序结果:在125例患者中,96例患者发现100个疑似的致病变异,其中55个为新突变(55.0%);97个(97.0%)疑似的致病变异位于FBN1基因,2个(2.0%)位于TGFBR1基因,1个(1.0%)位于TGFBR2基因;所有变异中包含有34个(34.0%)半胱氨酸的错义突变;21个(21.0%)非半胱氨酸错义突变;11个(11.0%)剪接位点突变;11个(11.0%)移码突变和15个(15.0%)无义突变;携带两个疑似致病变异的患者:发现3例患者携带两个疑似的致病变异;对家系成员测序分析,发现其中1例患者为复合杂合,而另2例为的两个致病变异发生在同一等位基因;2.家系研究:在一个家系中发现FBN1基因附近marker与疾病表型连锁,通过对FBN1基因重测序发现41号外显子发生移码插入突变;与FBN1不连锁家系,经过医学外显子组基因高通量测序,发现位于SMAD3基因突变;发现CKB基因为双胞胎家系候选致病基因。3.基因组重排检测结果:通过MLPA检测,共发现有4例患者分别携带有FBN1基因的6号,48-53号,49-50号和1-36号外显子的大片段缺失,通过长片段PCR(Long Range PCR)进行长片段扩增和测序,确定该4例患者分别缺失了16,551bp,10,346bp,4,563 bp和 187,067bp;通过分析患者血管组织mRNA水平发现缺失的48-53号为非移码缺失缺失,其后的mRNA在体内保持稳定不被降解,由此推测6号和49-50号外显子缺失可能也为非移码缺失。而1-36号外显子缺失突变范围包含FBN1基因上游11.5kb,使缺失的等位基因不能正常转录翻译。4.基因型表型相关性分析:发现携带半胱氨酸错义突变的患者和携带剪接位点突变的患者与携带截短突变的患者相比,更容易患晶状体脱位,两例色氨酸突变为精氨酸的患者失明。心血管方面,携带截短(PTC)突变的患者相较于剪接位点突变的患者更容易患主动脉夹层。携带FBN1基因p.C123G变异患者表现为罕见的颈总动脉夹层。5.结合以往研究报道对大片段缺失的患者进行基因型—表型分析发现,大片段非移码缺失突变患者表型取决于缺失位置及片段大小,但倾向于严重新生儿型MFS。大片段缺失(FBN1)引起单倍剂量不足导致的MFS,症状多表现为经典的MFS症状。结论:1.本研究通过PGM测序技术,检测到中国MFS人群中103个疑似的致病突变,其中有55个为新突变,丰富了 MFS的基因突变谱;2.携带FBN1基因剪接位点突变和半胱氨酸错义突变的患者相对于携带PTC的患者更倾向于患晶状体脱位;而PTC变异导致的患者,更倾向于患主动脉夹层;携带FBN1基因PTC突变的患者与携带TGFBR1/2基因突变的患者表型类似,均表现为不易患晶状体异位,易患主动脉夹层。提示MFS患者眼部异常与原纤维蛋白-1结构变化相关,主动脉夹层与TGFp信号通路改变相关。3.MLPA是检测MFS大片段缺失/重复的有效手段,携带非移码缺失突变的患者倾向于表现为新生儿MFS,单倍剂量不足引起的MFS多表现为经典型;4.复合杂合变异也能够导致MFS,对马凡综合征的产前筛查具有重要提示作用。研究内容二成人支气管扩张致病基因筛查及病因学研究支气管扩张(Bronchiectasis,简称支扩)是一种常见的气道炎症疾病,因反复气道感染和炎症造成支气管和细支气管管腔异常扩张,关于该病尤其是非囊性纤维化支气管扩张(non-CF bronchiectasis,NCFB)的病因、发病机制及疾病进展过程等的相关研究国内仍较匮乏。支气管扩张是一种异质性很强的疾病,目前对该病的治疗手段有限,严重影响着患者的生活质量。目前对支气管扩张的病因学评估分类研究仅能解释约50%的患者。患者的病因学明确有助于患者的管理,改善患者治疗方法以及巩固长期疗效。研究目的:研究中国成人支气管扩张患者病因组成。从遗传学角度揭示支气管扩张患者的遗传易感性,为支气管扩张患者病因学分类提供新的标准。研究对象和方法:1.研究对象:知情同意基础上收集192例中国成人支气管扩张患者以及100例确诊无肺部疾病的正常对照个体外周血;2.研究方法:(1)192例支气管扩张患者的病因学分类;(2)高通量测序:通过Ion torrent PGM测序平台对192例支扩患者以及100例正常个体进行靶基因Panel测序;测序完成后数据通过wANNOVAR网站注释VCF格式的原始文件;对二代测序结果进行常规筛选,保留可能致病的变异位点;通过致病性软件对变异位点进行致病性判断;对所筛选到的结果进行Sanger测序验证,根据变异类型进行分类统计;(3)对192例患者及100例正常对照进行CFTR基因8号内含子区域(TG)m Tn重复序列的PCR扩增,使用ABI3730型号的DNA测序分析仪器测序后分析(TG)m Tn重复次数;(4)分析CFTR基因双等位基因突变和CFTR/ENaC基因双重杂合变异,以及分析CFTR基因V470M多态;筛选囊性纤维化(PCD)相关致病基因的纯合变异,复合杂合突变;比较192例患者与100例正常个体的PCD相关致病基因的突变频率。(4)基因型—表型分析,统计携带DNAH5,DNAH11双等位基因突变和CFTR/ENaC双重杂合变异的患者的表型,比较各个基因突变导致的患者的支气管扩张严重程度综合评分(BSI值)。(5)对发现致病变异的患者进行病因学统计,并统计遗传因素在支气管扩张发病中所占的比例。实验结果:(1)本研究对中国成人支气管扩张患者人群进行了病因学分类,发现31.8%患者为特发性,27.6%的患者为感染后发病,13.0%的患者为免疫缺陷,4.2%的患者为哮喘,3.1%的患者为胃食管反流,其他类型为8.3%。(2)通过CFTR基因和上皮钠离子转运蛋白基因(ENaC)的基因筛查,共发现3例患者携带位于CFTR基因的复合杂合突变,其中1例患者为IVS8 5T(TGmT5)纯合变异携带者;另外发现2例患者携带CFTR/ENaC基因双重杂合突变;CFTR基因V470M多态,在纯合M470/M470背景下,发现12例患者携带CFTR基因致病变异,正常对照组为1例,支扩组显著多于正常对照组(6.3%vs.1.0%,P=0.039);(3)PCD相关基因突变筛查:发现20例患者携带PCD相关基因双等位基因突变(10.4%,20/192),其中14例携带DNAH11基因双等位基因突变,4例携带DNAH5基因复合杂合突变,1例携带CCDC40基因复合杂合突变,1例携带HEATR2纯合突变。并且发现1例携带位于X染色体的RPGR基因突变;在100例正常个体中发现有3例携带位于DNAH11基因的双突变。(4)基因型—表型关联性分析:发现携带有DNAH5基因突变的患者相比携带DNAH11基因突变患者,其表型要严重(BSI平均值:8.8 vs.4.2);携带CFTR/ENaC基因双重杂合子的患者症状表型要比携带CFTR基因复合杂合的患者症状严重(CFTR/ENaCBSI=10 Vs CFTR/CFTR BSI=5.2)。(5)共有 26 例患者发现携带可能致病的致病变异,占到患者总人数的13.54%。这些患者中16例病因学为特发性,5例为结核感染后发病,2例为反流,1例为1gG1免疫缺陷,1例为哮喘和1例麻疹感染后发病。结论:本研究对中国成人支气管扩张患者人群进行了病因学分析,发现31.8%患者病因学为特发性,27.6%的患者为感染后发病。通过PGM测序分析支气管扩张患者,发现在CFTR基因纯合M470背景下,发生一个CFTR基因的致病变异,极可能导致支气管扩张;DNAH5基因突变导致的支扩要比DNAH11和CFTR基因突变导致的支扩症状严重;共有13.54%的患者发病可能是由遗传因素导致,本研究为支气管扩张病因学分类提供了新的标准,对支扩的预防及临床预后有着重要的意义。
[Abstract]:A 125 case of Marfan syndrome (MFS) is an autosomal dominant connective tissue disease in Chinese Marfan syndrome. The frequency of the disease is 1/3000-1/5000. through high throughput sequencing of the MFS related gene FBN1, TGFBR1, and TGFBR2, which is a definite diagnosis for the patients. Effective means of operation and drug therapy. Objective: to draw a mutation spectrum of Chinese MFS population; to conduct a Chinese MFS genotype phenotype correlation analysis; provide a clear diagnosis, guide treatment, prognostic evaluation and genetic counseling for Chinese MFS patients; a new exon sequence (WES) for a family that has not been screened for pathogenic variation (WES) is to be found in order to discover new Possible pathogenic genes. Research objects and methods: 1. subjects: 125 Chinese MFS patients and family members; 2. research methods: (1) high throughput two generation sequencing: the target gene was sequenced by the Ion torrent sequencing platform, and the sequencing of Panel (target gene primer Library) contained 3 clear MFS pathogenic genes; the two generation sequencing results were screened. The selected results were verified by Sanger sequencing; (2) family analysis: a STR marker family linkage analysis was carried out for patients with family history of a family history that did not find suspected pathogenic variation through the Ion torrent PGM sequencing. The linkage region was determined by the linkage of the pathogenic gene to the FBN1 based family, and the FBN1 gene resequencing or the whole FBN1 gene was carried out. Sequencing analysis, sequencing analysis of exon group for FBN1 gene unlinked families, in order to find new pathogenic genes. Sequence analysis of a total exon group for a twin family of Marfan. (3) MLPA detection of large fragment insertion / deletion mutation: multiple connection dependent probe amplification (MLPA) for patients with no pathogenic mutation (MLPA ) whether the patients carry the insertion / deletion mutation of the FBN1 gene DNA fragment, verify the MLPA results by quantitative PCR, and shorten the upstream and downstream sequences of the breakpoint by step method; Long Range PCR (long fragment PCR) amplifes and sequenced the specific breakpoints, and extracts the patients with the deletion of the exon 48-53 of the FBN1 gene. Vascular tissue RNA, transcriptional analysis; (4) genotype phenotype correlation analysis of high throughput sequencing screening and MLPA detected mutations. Results: 1. high throughput sequencing results: among 125 patients, 96 patients found 100 suspected mutations, 55 of which were new mutations (55%); 97 (97%) suspected ectopia. In the FBN1 gene, 2 (2%) are located in the TGFBR1 gene and 1 (1%) are in the TGFBR2 gene; all the variations contain 34 (34%) cysteine missense mutations; 21 (21%) non cysteine missense mutations; 11 (11%) splice site mutations; 11 (11%) code mutation and 15 (15%) nonsense mutations; carry a patient with suspected pathogenic variation: 3 patients were found to carry two suspected mutations; 1 of them were complex heterozygous and two of the other 2 were in the same allele in the other 2 cases. 2. families found that the FBN1 gene was linked to the marker disease phenotype in a family and found 41 by resequencing the FBN1 gene. The exons of the exons were inserted into the mutation, and the gene mutation was found in the SMAD3 gene by high throughput sequencing of the exon gene of the FBN1, and the CKB gene was the result of the.3. genome rearrangement of the candidate gene for the twin family of the twins: the total of 4 patients with the FBN1 gene, 48-53, respectively, were detected by MLPA. The large fragment deletion of 49-50 and 1-36 exons was amplified and sequenced by long fragment PCR (Long Range PCR). The 4 patients were determined to be missing 16551bp, 10346bp, 4563 BP, and 187067bp, and the deletion of the missing 48-53 was found to be a non graft deletion by analyzing the mRNA level of the patient's vascular tissue, and the subsequent mRNA was preserved in the body. The deletion of the 6 and 49-50 exons may also be a non transcoding deletion, and the deletion mutation range of the exon 1-36 of the exon 1-36 contains the upstream 11.5kb of the FBN1 gene, making the deletion of the allele non normal transcriptional translation.4. genotype phenotype correlation analysis: the patients carrying the cysteine missense mutation and the carrying scissors are found. Patients with truncated mutations are more likely to suffer from lens dislocation than those with truncated mutations, two patients with tryptophan mutation to arginine blindness. Cardiovascular, patients carrying truncated (PTC) mutations are more likely to suffer from aortic dissection than patients with mutations in the splice site. Patients with the FBN1 gene p.C123G variation are rare. The genotypic phenotypic analysis of the common carotid artery dissection.5. combined with previous reports on patients with large segment deletion found that the phenotype of the large segment of the non shift code deletion mutation depends on the missing location and size of the fragment, but tends to be the MFS with multiple symptoms of the severe neonatal type MFS. deletion (FBN1). The classic MFS symptoms. Conclusion: 1. this study detected 103 suspected mutations in the Chinese MFS population by PGM sequencing technology, of which 55 were new mutations and enriched the gene mutation spectrum of MFS; 2. patients with FBN1 gene splicing and cysteine missense mutations were more prone to crystalline than those with PTC. The patients with PTC mutation tend to suffer from aortic dissection, and the patients carrying the FBN1 gene PTC mutation are similar to those with the TGFBR1/2 gene mutation, all of which are not susceptible to ectopic lens ectopic and dissection of the aorta, suggesting that the abnormalities of the eyes in MFS patients are associated with the changes in the structure of the fibrin -1, and the aortic dissection and TG Fp signaling pathway change related.3.MLPA is an effective means to detect the deletion / repetition of the large MFS fragments. The patients with non graft deletion mutations tend to show MFS in the newborn, and the MFS of a single dose of low dose of MFS is a classic type; 4. complex heterozygous variation can also lead to MFS, which has an important hint for the prenatal screening of Marfan syndrome. Study two adult bronchiectasis gene screening and etiology study of bronchiectasis (Bronchiectasis, abbreviated bronchiectasis) is a common airway inflammation disease, resulting in abnormal bronchiolotracheal dilatation due to repeated airway infection and inflammation, and the disease, especially the non cystic fibrosis bronchiectasis (non-CF bron) The etiology, pathogenesis, and progression of chiectasis, NCFB, are still scarce in China. Bronchiectasis is a very heterogeneous disease, and the treatment of this disease is limited, which seriously affects the quality of life of the patients. At present, the classification of the etiology assessment of bronchiectasis can only explain about 50% of the patients. The etiology of the patients is clearly helpful to the management of the patients, to improve the patient's treatment and to consolidate the long-term effect. Research objectives: To study the etiology of bronchiectasis in Chinese adults. The genetic susceptibility of bronchiectasis patients is revealed from the genetic perspective. Image and method: 1. subjects: on the basis of informed consent, 192 cases of Chinese adult bronchiectasis and 100 cases of normal controlled peripheral blood without lung disease were collected; 2. study methods: (1) the etiological classification of 192 cases of bronchiectasis; (2) high throughput test: 192 patients with bronchiectasis through Ion PGM sequencing platform The target gene Panel sequencing was carried out in 100 normal individuals; the original data were annotated by the wANNOVAR website by the sequencing of the target gene, and the two generation sequencing results were routinely screened to preserve the potentially pathogenic mutation sites; the pathogenic software was used to determine the pathogenicity of the ectopic sites, and the selected results were sequenced by Sanger sequencing. (3) PCR amplification of the CFTR gene 8 intron (TG) m Tn repeat sequence was carried out in 192 patients and 100 normal controls, and DNA sequencing analysis instrument of ABI3730 model was used for sequencing analysis (TG) m Tn repetition, and (4) analysis of the double allele mutation of CFTR gene and dual heterozygosity of CFTR/ENaC genes. Mutation, and analysis of the CFTR gene V470M polymorphism; screening the homozygous variation and complex heterozygous mutation of the cystic fibrosis (PCD) related pathogenic genes; comparing the mutation frequency of the PCD related pathogenic genes in 192 patients and 100 normal individuals. (4) genotype phenotype analysis, statistics carrying DNAH5, DNAH11 double allele mutation and CFTR/ENaC double heterozygosity. The phenotypes of different patients were compared with the overall score of bronchiectasis severity (BSI) in patients with various gene mutations. (5) the etiological statistics of patients who were found to be found and the proportion of genetic factors in bronchiectasis were statistically analyzed. (1) this study was used in Chinese adults with bronchiectasis. The etiological classification was carried out in 31.8% patients, 27.6% of the patients were infected after infection, 13% of the patients were immune deficiency, 4.2% of the patients were asthma, 3.1% of the patients were gastroesophageal reflux, and the other types were 8.3%. (2) gene screening by CFTR gene and epithelial sodium ion transfer egg Bai Jiyin (ENaC) gene screening, and 3 patients were found to carry the position. In the compound heterozygous mutation of the CFTR gene, 1 patients were IVS8 5T (TGmT5) homozygous carriers. In addition, 2 patients carried the CFTR/ENaC gene double heterozygous mutation, and the CFTR gene V470M polymorphism. In the homozygous M470/M470 background, 12 patients were found to carry the CFTR gene mutation, the normal control group was 1 cases, and the bronchiectasis group was significantly more than the normal group. The control group (6.3%vs.1.0%, P=0.039); (3) PCD related gene mutation screening: 20 cases were found to carry the PCD related gene mutation (10.4%, 20/192), of which 14 cases carried the DNAH11 gene double allele mutation, 4 cases carried the DNAH5 gene compound heterozygous mutation, 1 cases carried the CCDC40 gene complex heterozygous mutation, 1 cases carried the HEATR2 homozygous mutation. And 1 cases of RPGR gene mutations were found on the X chromosome; 3 cases were found to carry double mutations in the DNAH11 gene in 100 normal individuals. (4) genotype phenotype correlation analysis: it was found that patients carrying DNAH5 mutations were more serious than those with DNAH11 gene mutations (BSI mean: 8.8 vs.4.2), and CF with CF. The symptomatic phenotype of the TR/ENaC gene double heterozygote was more severe than the patient carrying the complex CFTR gene (CFTR/ENaCBSI=10 Vs CFTR/CFTR BSI=5.2). (5) a total of 26 patients were found to carry the possible pathogenetic variation, and 16 of the patients with the total number of patients were idiopathic and 5 were tuberculosis. 2 cases were reflux, 1 cases were 1gG1 immune deficiency, 1 cases were asthma and 1 cases of measles infection. Conclusion: This study analyzed the etiological analysis of Chinese adults with bronchiectasis, and found that 31.8% patients were idiopathic and 27.6% of the patients were infected with post infection. PGM sequencing was used to analyze bronchiectasis patients and found in CF In the context of TR gene homozygous M470, a pathogenetic variation of the CFTR gene may lead to bronchiectasis, and the extension of the DNAH5 gene mutation is more severe than that caused by the mutation of the DNAH11 and CFTR genes; a total of 13.54% of the patients may be caused by genetic factors, and this study provides the classification of the etiology of bronchiectasis. The new standard is of great importance to the prevention and prognosis of bronchiectasis.

【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】：R562.22;R596

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