基因捕获技术在视网膜母细胞瘤患者RB1基因突变筛选的应用

Application of gene capture technology on mutation screening of RB1 gene in retinoblastoma patients

目的探讨基因捕获技术在视网膜母细胞瘤（RB）患者RB1基因突变筛选中的应用。方法实验研究。2010年6月至2014年6月采集北京大学人民医院经病理学检查确诊的17例RB患者的外周血。通过设计RB1基因外显子及侧翼序列的靶向捕获探针，对患者外周血DNA进行测序分析，并采用Sanger法对候选变异进行验证。同时收集患者父母血样进行比较。结果在17例RB患者中，男性12例，女性5例；年龄0．5～23．0岁，平均（3．2±5．2）岁；双眼患者6例，单眼患者11例。4例患者存在RB1基因突变，其中双眼受累2例，单眼受累2例。2例新发现基因突变，包括RB1基因第15外显子c．1408A〉T错义突变，造成异亮氨酸突变为苯丙氨酸和RB1第19外显子0．1960G〉C错义突变，造成缬氨酸突变为亮氨酸。该2例患儿父母均未检测到基因突变。另外2例存在已报道的基因突变位点。其中1例患者的家族史阳性，其父亲为单眼RB患者，患者及其父亲均在第10外显子发生c．1030C〉T无义突变。1例患者发生了第3外显子c．371．372delTA的缺失突变，该父母未检测到基因突变。结论应用基因捕获技术联合Sanger测序可有效筛选RB患者RBI基因突变，发现了2例RB患者新的RB1生殖细胞突变，补充了RB1基因突变谱。

Objectives To analyze RB1 gene mutation in retinoblastoma （RB） patients using gene capture technology. Methods Experimental research. The clinical data of 17 RB patients were collected at Department of Ophthalmology, Peking University People＇s Hospital from June 2010 to Jun 2014. Peripheral blood samples of seventeen RB patients and their parents were collected and genomic DNA were extracted. DNA library from RB patients was mixed with designed gene capture probe of RB1 exons and its flanking sequences. The data were analyzed using bioinformatics software. To avoid the false positive, the abnormal sites were verified using the Sanger sequencing method. Results Totally, there were 17 RB patients, including 12 males and 5 females, from 0.5 to 23 years old, average ages were （3.2±5.2）years old. Both eyes were involved in 6 patients. The other 11 cases were only one eye was attacked. Four RB patients were found to have germline mutations, among whom 2 had bilateral tumors and 2 had unilateral tumors. 2 novel missense mutations were identified, including 15° exon c.1408A〉T （p. Ile470Phe） and e.1960G〉C （p. Val654Leu） at 19° exon. No RB1 mutation was identified in any of their parents. We also identified 2 mutations reported previously. One is e. 1030C〉T termination mutation at 10^th exon in a bilateral RB patients and his father, who was diagnosed with unilateral RB. The other is e.371-372delTA frame shift mutation at 3^rd exon. No mutation was found in their parents. Conclusions Two novel germline RBI mutations were found using gene capture technology, which enriched RB1 mutations library.