RT-PCR法快速筛查视网膜母细胞瘤RB1基因突变的研究

发布时间：2018-06-13 18:53

本文选题：视网膜母细胞瘤 + RB1基因　；参考：《浙江大学》2011年硕士论文

【摘要】：研究背景视网膜母细胞瘤(retinoblastoma, RB)是婴幼儿最常见的眼科肿瘤,约占儿童恶性肿瘤的4%,也是一类严重的致死性肿瘤(占婴幼儿死亡率的第一位)。大部分RB患儿在5岁之前发病。开始发病时较常见的症状为白瞳症、斜视及视力下降,多因此就诊。病情进一步发展则相继出现青光眼、葡萄膜炎、玻璃体出血,甚至肿瘤转移、死亡等。 RB具有明显的家系遗传性,大多数呈常染色体显性遗传方式,外显率高达90%。位于13q14.1-q14.2上的RB1基因(OMIM:180200)已被确定为唯一的RB致病基因。RB1基因全长183 kb,包含27个外显子,属于较长的基因。RB1基因是人类发现的第一个抑癌基因,其编码的pRB蛋白广泛存在于各种细胞中。正常的RB1基因表达对抑制RB的发生是必不可少的,RB1基因的缺失或变异失活都会导致RB的发生。 RB在临床上可表现为单眼发病和双眼发病两种类型。双眼患者发病时间早,多在1岁以内,90%以上的病例都是遗传性的；单眼患者发病较晚,约15%为遗传性的。92.7%的双眼患者和14.6%的单眼患者均携带高外显的RB1种系基因突变。因此,RB家系患者和散发患者的RB1基因的变异组学研究,对其早期诊断、治疗、预后、遗传咨询、产前诊断、植入前遗传学诊断以及基因治疗等都至关重要。研究目的对1个浙江地区视网膜母细胞瘤家系中所有成员的外周血表达的RBI cDNA序列进行测序分析,以期快速获得基因组水平RB1基因的突变信息,探讨RB1基因突变与RB发病的相关性。研究对象一个居于浙江省杭州地区的RB家系(包含2例患者),以及10例正常健康对照。研究方法 (1)抽取所研究家系的所有成员和正常对照个体的外周血(抗凝),用Ambion RiboPure blood kit提纯外周血总RNA,用苯酚-氯仿法纯化基因组DNA； (2)取所有RNA样本,反转录-聚合酶链式反应(RT-PCR)扩增其RBI cDNA全序列并测序； (3)取所有DNA样本,PCR扩增RB1基因外显子并测序； (4) DNAssist软件及Chromas 2软件进行测序数据分析,并与Ensemb1、NCBI等人类遗传学权威数据库进行比对分析； (5)针对所发现的突变位点,利用碱基错配引物的等位基因特异性-聚合酶链式反应(AS-PCR法),验证所发现的突变,并且筛查家系中的嵌合体患者。研究结果 (1)所研究RB家系的2例RB患者(先证者：双眼；先证者之父：单眼)的外周血RB1基因cDNA均存在第14外显子的c.1363CT(p.R455X)杂合突变。 (2)RB患者的基因组均存在RB1基因g.76460CT杂合突变。 (3)家系中的2例RB患者的AS-PCR都扩增得到特异条带,其他成员均无条带。结论 (1)RB1基因c.1363CT(p.R455X)突变为所研究RB家系患者的首要致病因素。 (2)先证者的杂合突变遗传自他的父亲。 (3)用RT-PCR/DNA测序相结合的方法筛查RB1基因突变,是目前RB变异组学研究中最方便、快捷、廉价的技术。
[Abstract]:Background retinoblastoma (RBB) is the most common ophthalmic tumor in infants and young children, accounting for about 4% of malignant tumors in children. Most children with RB develop before the age of 5. The more common symptoms of onset are mydriasis, strabismus and decreased visual acuity. There were glaucoma, uveitis, vitreous hemorrhage, tumor metastasis, death and so on. RB had obvious heredity in pedigree, most of them were autosomal dominant inheritance, and the rate of exsertion was as high as 90%. RB1 gene, located on 13q14.1-q14.2, has been identified as the only RB pathogenicity gene. RB1 gene contains 27 exons in 183kb. it is the first oncosuppressor gene found in human. Its encoded PRB protein is widely present in various cells. Normal RB1 gene expression is essential to inhibit the occurrence of RB. Deletion or inactivation of Rb1 gene can lead to RB occurrence. RB can be classified as monocular and binocular. The onset time of binocular patients was early, and 90% of the patients within 1 year of age were hereditary, and 15% of the patients with monocular disease were hereditary, and about 15% of binocular patients and 14.6% of monocular patients were carrying high explicit RB1 gene mutation. Therefore, the mutation study of RB1 gene in RB pedigree and sporadic patients is very important for its early diagnosis, treatment, prognosis, genetic counseling, prenatal diagnosis, preimplantation genetic diagnosis and gene therapy. Objective to sequence and analyze the RBI cDNA sequences expressed in peripheral blood of all members of a family of retinoblastoma in Zhejiang province, in order to obtain the mutation information of RB1 gene at genomic level. To investigate the relationship between RB1 gene mutation and RB. Participants A RB pedigree (including 2 patients and 10 normal controls) living in Hangzhou, Zhejiang Province, was enrolled in the study. Methods 1) the peripheral blood (anticoagulant) was extracted from all the members of the family and the normal individuals. The total RNAs of peripheral blood were purified by Ambion RiboPure blood kit, and the genomic DNAs were purified by phenol-chloroform method. The whole RBI cDNA sequence was amplified and sequenced by reverse transcription-polymerase chain reaction (RT-PCR), and the exon of RB1 gene was amplified and sequenced by PCR from all DNA samples. DNAssist software and Chromas 2 software were sequenced and compared with human genetics authoritative databases such as Ensemb1NCBI. The allele-specific polymerase chain reaction (AS-PCR) method of base mismatch primer was used to verify the mutation and to screen the chimeric patients in the pedigree. Results 1) two RB patients (proband: binocular) from RB pedigree were studied. The RB1 gene cDNA in peripheral blood of proband father: monocular) had heterozygous mutation of exon 14, c. 1363 CTP. R455X. RB1 gene g.76460CT heterozygous mutation was found in the genomes of both patients. Specific bands were obtained from AS-PCR amplification of RB patients. No other members have stripes. Conclusion the mutation of RB1 gene c.1363CTA p.R455X) is the leading pathogenic factor in the RB family studied. The heterozygosity of the proband was inherited from his father. Y3) was sequenced by RT-PCR / DNA sequencing. Methods RB1 gene mutation was screened. It is the most convenient, fast and cheap technique in RB mutation study.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R739.7

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