头颈部副神经节瘤患者琥珀酸脱氢酶基因致病突变研究

【作者】 查洋；

【导师】 高志强； 亓放； 姜鸿； 吕威；

【作者基本信息】 中国协和医科大学 ， 耳鼻咽喉科学， 2010， 博士

【摘要】 头颈部副神经节瘤是一种少见肿瘤疾病,好发于颈动脉体、颈静脉窝、鼓室和迷走神经等部位。近年来研究发现,副神经节瘤与琥珀酸脱氢酶(succinate dehydrogenase, SDH)亚单位的编码基因突变相关。为掌握中国人群头颈部副神经节瘤患者中琥珀酸脱氢酶基因的突变特点以及对疾病的影响,本研究搜集家族性和散发头颈部副神经节瘤患者DNA样本和临床资料,运用分子生物学技术和遗传学研究方法,对患者的SDH基因突变进行了相关研究。本研究共分为四个部分：第一部分头颈部副神经节瘤患者临床资料和DNA样本的搜集本部分研究工作查阅自2002年至今在我院经手术治疗后病理证实为副神经节瘤患者病历,设计临床信息登记表,联系患者进行回访,并邀请其参与本研究。在获得知情同意后,详细完善临床资料及家系情况,获取患者血样并提取DNA备用。对近期手术的患者以及研究阶段新入院的患者,在复查随访和术后阶段亦纳入本研究中。同时,从病理科调取所有患者的肿瘤组织石蜡包埋肿瘤组织标本,提取DNA备用。共搜集头颈部副神经节瘤患者血样34例,石蜡包埋组织样本83例(含34例已获取血样的患者)。提取了34例患者的血样DNA(男性9例、女性25例)和30例患者(男性13例、女性17例)的石蜡包埋组织样本DNA。第二部分琥珀酸脱氢酶基因点突变检测本部分研究对64例头颈部副神经节瘤患者的DNA样本进行了SDHB、SDHC、SDHD和SDHAF2基因突变检测。根据实验需要设计SDHB、SDHC和SDHD基因各外显子扩增引物(SDHAF2使用文献引物),经PCR扩增后行序列分析。BLAST序列对比找出SDH基因突变,并通过基因突变数据库对比、突变类型分析、突变位点物种保守性分析等方法,判断其致病性。对特殊突变通过PCR扩增子质粒克隆及单克隆测序分析进行进一步验证。本部分研究发现48.5%(31／64)的患者携带有SDHB (10例)、SDHC (3例)或SDHD(18例)基因突变,3者的检出率分别为16%、5%和28%,未发现有患者携带SDHAF2基因突变。其中发现了9种新突变,部分结果已提交至SDH突变数据并可在线查询。34例获取了静脉血样的患者中,61.7%(21／34)携带有SDH基因突变(SDHB7例,SDHC 3例,SDHD 11例)；30例检测肿瘤石蜡包埋组织样本DNA的患者中,仅33.3%(10／30)发现携带有SDH基因突变(SDHB 4例,SDHD 7例),小于静脉血样组,p＜0.005。6例家族性患者均携带SDHD基因突变,占携带SDHD突变的患者总数的33%(6／18),6例多发肿瘤患者亦均为SDHD突变携带者,其中3例为家族性发病患者。2例恶性肿瘤患者均携带有SDH突变,SDHB和SDHD各1例。第三部分琥珀酸脱氢酶基因大片段缺失检测本研究对PCR扩增直接测序未发现有突变的33例患者运用多重连接依赖式探针扩增技术对SDH基因的各外显子进行了拷贝数异常检测。未发现有患者的SDH基因拷贝数有异常,说明无染色体重排引起的SDH基因区域大片段缺失。第四部分多发突变的单体型分析和创始者效应分析对SDHD基因中出现的多发突变,通过构建各家系的微卫星单体型并将各患者的单体型进行比较,发现携带SDHD c.3G>C突变的4例患者中,有3个患者有相同的微卫星等位基因组合。在75例正常人群中统计了各等位基因的频率,判断他们共享的等位基因组合在正常人群中的概率仅2×10-8～5×10-5,4个不相关的家系同时携带此组合的几率则更小,因此考虑该基因突变为创始者效应所致。总结：研究发现64例头颈部副神经节瘤患者中48.5%携带有SDH基因突变,包括10例SDHB、3例SDHC和18例SDHD基因突变,其中9种为新突变。没有发现患者携带SDHAF2基因突变；多重连接依赖式探针扩增法没有在患者中发现有SDH基因大片段缺失；单体型分析证实了一个中国患者中的多发突变SDHD c.3G>C为创始者突变。更多还原

【Abstract】 Paragangliomas are rare tumors occurring at head and neck area. Recently, it has been revealed that paragangliomas have a relationship with germline mutations of succinate dehydrogenase encoding genes. To evaluate the role and characteristics of SDH gene mutations in Chinese patients, we collected DNA samples of familiar and sporadic patients with head and neck paraganliomas and screened for germline mutations. We also analyzed the pathogenicity of the mutations and genotyped the haplotypes of the patients with same mutations.The study consists of 4 portions:Part 1:Collection of clinical data and DNA samples of HNPGL patientsWe recalled the case records on PGL and select the HNPGL cases confirmed by pathologic diagnosis. We contacted the patients and informed them about our study. With their consents, we registered their detailed clinical and familiar information and we collected blood samples from every patient and the formalin-fixed and paraffin-embedded (FFPE) tissue samples from the department of Pathology. In total,34 blood samples and 83 FFPE tissue samples (including the 34 patients with available blood samples) were collected. DNA was extracted from 34 blood samples (9 males and 25 females) and 30 FFPE tissue samples (13 males and 17 females). Part 2:Genetic assay of SDH genes for point mutations.We designed primers for all of the SDHB, SDHC and SDHD exons according to our requirements. Genetic assays were done for 64 HNPGL patients. Each exon of SDH genes was amplified and direct sequenced. By sequence BLASTing, we revealed that 48.5% of the 64 patients carry SDH mutations (10 SDHB mutations,3 SDHC mutations and 18 SDHD mutations) and 9 novel mutations were identified.Part 3:Large deletion detection for SDH genesFor the 33 patients without any mutation of SDH genes, we detect the large deleton of SDH genes by multiplex ligation-dependent probe amplification. None of these patient was revealed to carry a SDH gene copy number variant.Part 4:Haplotype analysis of patients with a same mutationThree chinese patients were reported to carry the SDHD c.3G>C mutation and another 2 patients with this mutation were identified in our study. We constructed the haplotype for 4 of the 5 families and compared the STR combinations between the families. We also calculated the probabilities of the STR combinations shared by these families in a control group of 75 blood donors and the result showed that the same mutation shared by these families are due to a founder effect.Conclusion:There are 48.5% of the 64 patients carrying SDH mutations, including 10 SDHB mutations,3 SDHC mutations and 18 SDHD mutations. Nine novel mutations were identified. None was found to carry a SDHAF2 gene mutation or copy number variant. SDHD c.3G>C mutation is due to a founder effect.更多还原