视网膜母细胞瘤家系的基因突变检测和遗传咨询

【作者】 江伟；

【导师】 赵家良； 睢瑞芳；

【作者基本信息】 中国协和医科大学 ， 临床医学， 2008， 博士

【摘要】 【背景】视网膜母细胞瘤(Retinoblastoma,RB)是婴幼儿最常见的眼内恶性肿瘤。其致病基因RB1位于染色体13q14,是人类发现的第一个抑癌基因。遗传性RB与非遗传性RB在临床表现、突变特征和处理方面各不相同。分子遗传学研究能够更早发现并明确RB风险,从而为进一步诊断和治疗提供依据,最终实现改善预后的目的,因此具有重要意义。RB1基因突变谱极为广泛,给检测带来困难,但分子生物学的发展已经提供了许多有效工具,联合应用能够获得良好的检出率。【目的】收集视网膜母细胞瘤患者(家系),完成基因诊断,了解突变携带状态,并将结果应用于遗传咨询;丰富RB突变谱。【方法】提取患者外周血和肿瘤组织(如果有)的基因组DNA,设计引物扩增RB1基因各外显子及其侧翼序列和启动子,直接测序与参考序列比对,以检测碱基更替、小片段缺失/插入等小突变。【结果】在1例遗传性RB家系外周血中检出生殖细胞突变g.39470 G＞T,产生终止密码子,先证者肿瘤组织中检出体细胞突变g.2078 ins C,证实肿瘤发生的“twohit”模型。在1例散发双侧RB患者外周血中检出生殖细胞突变g.45844 G＞A,产生终止密码子。其中g.39470 G＞T和g.45844 G＞A均为首次报告。另外在2例散发单侧RB患者中未检出生殖细胞突变,对其中1例肿瘤组织的序列分析显示可能存在杂合性缺失现象。【结论】直接测序法应用于视网膜母细胞瘤的基因诊断是可行的,利用较低成本即可检出大部分突变,阴性者可继续选择其他手段予以检测。基因诊断可应用于指导遗传咨询,对携带致病突变者加强随访可尽早发现肿瘤以保存视力。更多还原

【Abstract】 Background:Retinoblastoma(RB) is the most common intraocular malignancy in childhood.The retinoblastoma gene RB1 is located on chromosome 13q14,and known as the first tumor suppressor gene identified.Hereditary form and non-hereditary form of retinoblastoma vary a lot in terms of clinical features,mutation type and management. For hereditary RB,molecular genetic study can identify the patients at risk as early as possible,thus enabling a better prognosis.An extraordinarily broad spectrum of mutations has been determined for RB1 gene,whereas development of molecular biology has provided abundant accesses to a satisfactory detection rate when used in combination.Objective:To establish genetic diagnosis for the RB patients and families collected;to translate the genetic results to the clinical setting for genetic counseling;to update the RB1 mutation spectrum.Method:Mutation analysis was carried out by extraction of genomic DNA from peripheral white blood cell and tumor tissue(if applicable),respectively,amplification of each exon with its flanking introns and promoter region,direct sequencing and comparison with reference sequences.Results:In one case of hereditary RB,a germline mutation of g.39470 G＞T and a somatic mutation of g.2078 ins C were identified,validating the "two hit model".In one case of sporadic bilateral RB,a germline mutation of g.45844 G＞A was identified.The two substitutions were both novel mutations.No germline mutation was identified in another 2 sporadic unilaterally affected cases,but loss of heterozygosity was highly suspected in one of the patients. Conclusion:Direct sequencing was feasible for genetic diagnosis of retinoblastoma,as most mutations could be identified with relatively low costs,and negative results could proceed to other genetic techniques.Genetic diagnosis could be applied for genetic counseling,and close follow-up of carriers might allow early detection of the tumor to preserve eyesight.更多还原