Huang Q, Dryja T P, Yandell D W
Department of Ophthalnology, The First Affiliated Hospital, West China University of Medical Sciences, Chengdu 610041 P.R.China.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi. 1998 Apr 10;15(2):65-8.
To develop a diagnostic test for direct identification of disease-causing mutation in the patients with retinoblastoma and correct prediction of carrier- status in unaffected adults and newborns in the RB kindred.
Southern blot hybridized by Rb cDNA and other intragenic probes were used to detect big deletions or rearrangements at Rb gene locus. SSCP analysis and direct sequencing of primer-directed enzymatic amplification to identify point mutations as small as a single nucleotide change. RFLPs and VNTRs within the Rb gene were used as genetic markers for haplotype analysis.
The probands from 79 RB kindreds were identified to have Rb gene mutation, including 25 somatic mutations and 54 germline mutations (36 new germline mutations, 15 inherited mutations and 3 mosaicisms). The WBC DNAs from their family members were also analyzed for determining origin and carrier of mutation.
The direct identification of causing- cancer mutations by combining SSCP analysis and direct DNA sequencing showed many advantages than other indirect methods such as haplotype analysis. It can distinguish hereditary RB from nonhereditary RB and identify the unaffected carriers without family history and informes affected family member. This method is helpful in gene diagnosis and genetic counselling.
开发一种诊断测试，用于直接鉴定视网膜母细胞瘤患者中致病突变，并准确预测RB家族中未受影响的成年人和新生儿的携带者状态。
使用Rb cDNA和其他基因内探针进行Southern印迹杂交，以检测Rb基因位点的大片段缺失或重排。采用单链构象多态性分析（SSCP）和引物定向酶促扩增直接测序，以鉴定小至单个核苷酸变化的点突变。Rb基因内的限制性片段长度多态性（RFLPs）和可变数目串联重复序列（VNTRs）用作单倍型分析的遗传标记。
在79个RB家族的先证者中鉴定出Rb基因突变，包括25个体细胞突变和54个种系突变（36个新的种系突变、15个遗传突变和3个嵌合体）。还对其家庭成员的白细胞DNA进行了分析，以确定突变的起源和携带者。
与单倍型分析等其他间接方法相比，结合SSCP分析和直接DNA测序直接鉴定致癌突变具有许多优势。它可以区分遗传性RB和非遗传性RB，识别无家族史的未受影响携带者，并为受影响的家庭成员提供信息。该方法有助于基因诊断和遗传咨询。
