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16,551 bp，10,346 bp，4,563 bp和187,067 bp；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TGFβ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/ENaC BSI=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lgG1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。

Part 1 Application of NGS in screening for pathogenic mutations in 125 Chinese patients with Marfan syndrome

  Marfan syndrome (MFS) is a pleiotropic connective tissue disease inherited as an autosomal dominant trait, due to mutations in the FBN1 gene encoding fibrillin 1. It is an important protein of the extracellular matrix that contributes to the final structure of a microfibril. Few cases displaying an autosomal recessive transmission are reported in the world. The FBN1 gene, which is made of 66 exons, is located on chromosome 15q21.1.

  Objective: Study the mutation spectrum of Chinese MFS; The MFS genotype-phenotypic correlation analysis; Identify new pathogenic genes responsible for MFS.

  Subjects and methods: 1. Subjects: 125 Chinese patients with MFS and their family members; 2. Methods: Ion torrent PGM sequencing: 125 patients were sequenced by Ion torrent PGM sequencing platform. Linkage analysis: STR markers near the FBN1 gene of the MFS families were genotyped and linkage analysis was performed. For the families showling linkage with FBN1, the FBN1 gene exons were re-sequenced. For those not linked with FBN1, whole exome sequencing was performed. A twin MFS family was performed exome sequencing. MLPA: MLPA (Multiples ligation-dependent probe amplification) was performed in patients for whom the pathogenic mutations were not detected by PGM sequencing; qPCR was performd to shorten the upstream and downstream flanking sequence of the breakpoint; Long-Range PCR was performed for breakpoints flanking sequence amplification; Total RNA was extracted from aortic tissue of the patient with FBN1 exon 48-53 deletion, cDNA synthesis and transcription level analysis were performed. Genotype-phenotypic correlation analysis was performed.

  Results: Ion torrent PGM sequencing results: In 96 out of 125 patients, 100 (likely) pathogenic variants were identified with 55 novel. 97 (97.0%) pathogenic mutations were located in the FBN1 gene, 2 (2.0%) were located in the TGFBR1 gene, and one (1%) in TGFBR2. Amone 100 variants, 55(55%) are missense mutations, 11 (11.0%) are splice site mutations, 11 (11.0%) are frameshift mutations and 15 (15.0%) are nonsense mutations. Unexpectedly, of 125 patients, one patient (0.8%) was compound heterozygous and 2 patients (1.6%) had 2 variants in one fibrillin-1 (FBN1) allele .  In family 50, the linkage analysis showed tight linkage with the FBN1 gene, and a frameshift deletion mutation was found bysequencing the entire FBN1 gene. Family 58 didn’t show linkage withFBN1, and a SMAD3 mutation was detected. Four gross deletions were identified in FBN1: exon 6, exons 48-53, exons 49-50, and exons 1-36 deletions. Three deletions (exon 6, exons 48-53, and exons 49-50) were predicted to be in-frame deletions; the remaining deletion (exons 1-36) was a out-of-frame deletion and nonsense mediated mRNA decay was expectedso that loss of one allele of the FBN1 gene will be predicted. The breakpoints of these 4 deletions were cloned, and sequencing of the fragments junctions revealed deleted sizes of 16,551 base pairs (bp), 10,346 bp, 4,563 bp, and 187,067 bp respectively. By analyzing the transcription levels from patients’ RNA samples available, the deletion of exons 48-53 was confirmed to be in-frame. This deletion removed several calcium-binding EGF domain(s) and/or TB domain(s) of fibrillin-1. Clinical features of three patients with in-frame deletions differed from those of one patient with exon1 deletion of FBN1. Genotype-phenotypic correlation analysis: The clinical features were characterized and compared, and we found that the patients with splice site mutations and those with mutations involving cysteines had a higher prevalence of ectopia lentis; aortic dissection occurred more frequently in patients with protein-truncating mutations. The compound heterozygous patients and patients with double variants in one FBN1 allele exhibited mild classical MFS features. Patient with p.C123G are associated with rare common carotid artery dissection. Genotype-phenotype analysis in patients with large deletions, gross deletions of FBN1 exons results in variable phenotypes of MFS, but tends to cause severe Neonatal MFS.

  Conclusion: In this study, 55 novel mutations were detected and that enriched the mutation spectrum of the MFS. By Genotype-phenotype correlation analysis, we found that the patients with splice site mutations and cysteine mutations were more likely to suffer lens ectopic than those patients with PTC (truncating mutation). Patients with PTC mutations were more likely to have aortic dissection phenotype. MLPA is an effective method for detecting large deletions in MFS patients. Patients with in-frame deletion mutations tend to suffer severe Neonatal MFS. Haploinsufficiency of FBN1 is responsible for the phenotypes in classic MFS. Compound heterozygous mutation can lead to MFS, which has important implications for prenatal screening of Marfan syndrome.

Part 2 Next-generation sequencing for identifying genetic variants in Chinese adults with bronchiectasis

  Defective host-defense, including impaired ciliary function or ultrastructure and mucus clearance, has been associated with bronchiectasis. However, whether genetic mutations related to host-defense defects (e.g. altered mucus properties, ciliary defects) are implicated in bronchiectasis pathogenesis remains unclear.

  Objective: To reveal the genetic susceptibility in patients with bronchiectasis, and to provide a new standard for the etiological classification of bronchiectasis.

  Subjects and methods: Subjects: Experimental study of 192 patients with bronchiectasis in China and 100 individuals without lung disease as normal control. Methods: Ion torrent PGM sequencing: 192 patients and 100 normal controls were sequenced by Ion torrent PGM sequencing platform. (TG)m Tn repeats genotyping: intron 8 of CFTR was amplified by PCR and analyzed by ABI3730 DNA Analyzer in 192 patients and 100 normal controls. Analysis of mutations in different genes: analysis compound heterozygous mutations of CFTR, trans-heterozygotes for CFTR/ENaC mutations, CFTR gene V470M polymorphism; PCD-related pathogenic homozygous and compound heterozygous mutations; Genotype-Phenotype correlation analysis: the patients with DNAH5, DNAH11, CFTR and CFTR/ENaC recessive pathogenic mutations were analyzed. Investigation of the etiology of patients with pathogenic mutations and study of the role of genetic factors in the pathogenesis of bronchiectasis.

  Results: 1. In this study, 192 cases of adult patients with bronchiectasis were classified by the etiology, the most common one is post-infectious (27.6%). 2. IVS8 5T were found in 13 patients, one patient with homozygous IVS8 5T, and 9 individuals carry IVS8 5T in normal control. 3. In the CFTR and ENaC gene, a total of 3 patients were found to have two mutations in CFTR, 2 patients carry CFTR/ENaC trans-heterozygotes mutations; 38 patients are homozygous for M470, 12 of them were found to carry another CFTR gene mutations, which is significantly higher than that in normal controls (6.3% vs. 1.0%, P = 0.039); Twenty patients were found to carry two mutations in PCD related pathogenic genes (10.4%, 20/192). 14 cases carry DNAH11 mutations, 4 cases carry DNAH5 mutations, one carries CCDC40 gene mutation, one carries HEATR2 homozygous mutation and 1 carries RPGR（X chromosome）mutation. Of the 100 normal subjects, 3 were found to carry two DNAH11 mutations. 4. Patients with two DNAH5 mutations were found to have a severe phenotype compared to patients with DNAH11 mutations (BSI mean: 8.8 vs. 4.2); patients with trans-heterozygotes for CFTR/ENaC mutations have more severe phenotype than those patients carrying two CFTR gene mutations (CFTR/ENaC BSI = 10 vs CFTR/CFTR BSI = 5.2).  5. A total of 26 patients were found to carry pathogenic mutations, which accounted for 13.54% of the total number of patients. They can be classified as following according to different etiology: 16 idiopathic, 5 post-tuberculosis, 2 gastroesophageal reflux, 1 lgG1 deficiency, 1 asthma and 1 measles.

  Conclusions: We have identified mutations mainly associated with CF and PCD in Chinese bronchiectasis patients. Homozygous M470 variant plus other CFTR mutants predispose to bronchiectasis. Some mutations (particularly DNAH5 mutation) are linked to greater bronchiectasis severity. A total of 13.54% of the patients were affected by genetic factors. This study provides a new standard for classification of bronchiectasis by etiology.