Exon-by-exon Screening for RB Germline Mutations Using Heteroduplex-SSCP Analysis  

作　　者：Qingjiong Zhang, Kensei Minoda, Ruiping Zeng, Zhongyao Wu Xueshan Xiao, Shiqiang Li, Fengshen ZhangZhongshan Ophthalmic Center Department of Medica GeneticsSun Yat-sen University of Medical Sciences , Guangzhou 510060 , ChinaDepartment of Ophthalmology, Teikyo University Ichihara Hospital, Ichihara , Chiba 299 - 01, Japan. 

出　　处：《眼科学报》1997年第1期5-11,共7页Eye Science

基　　金：Guang Dong Provincial Natural Science Foundation (QZ:930354, 960149 );the Misnistry of Public Health(QZ);the Ministry of Health and Welfare of Japan(KM);the Mishima Fund for Eye Research and International Exchange(QZ);the Sasagawa Medical Re

摘　　要：Objective: To disclose the nature of RBI germline mutations in Chinese and to develop a practical and effective way for mutational screening. Methods: Leukocyte DNA was prepared from 8 Chinese patients with hereditary retinoblastoma. PCR combined with nonisotopic heteroduplex-SSCP analysis was used to screen leukocyte DNA for RBI germline mutations, exon-by-exon, without the use of restriction endonuclease digestion. The mutations were finally identified by sequencing. In order to testify the effectiveness of this method, the same method was used to detect other 17 samples which have been previously analyzed by other methods. Results: Heterozygous germline mutations were detected in the leukocyte DNA of 6 out of 8 Chinese patients;G del/codon 46,T del/codon 131 ,CAGAA del/condon 257 -258, GCAgta→GCAgca/donor of exon 16, C@T/codon 661, and C→T/codon 787. Heteroduplex-SSCP analysis may detect RBI germline mutations in 68% (177 25) unselected patients, which is more effective than SSCP(56%) orObjective: To disclose the nature of RBI germline mutations in Chinese and to develop a practical and effective way for mutational screening. Methods: Leukocyte DNA was prepared from 8 Chinese patients with hereditary retinoblastoma. PCR combined with nonisotopic heteroduplex-SSCP analysis was used to screen leukocyte DNA for RBI germline mutations, exon-by-exon, without the use of restriction endonuclease digestion. The mutations were finally identified by sequencing. In order to testify the effectiveness of this method, the same method was used to detect other 17 samples which have been previously analyzed by other methods. Results: Heterozygous germline mutations were detected in the leukocyte DNA of 6 out of 8 Chinese patients;G del/codon 46,T del/codon 131 ,CAGAA del/condon 257 -258, GCAgta→GCAgca/donor of exon 16, C@T/codon 661, and C→T/codon 787. Heteroduplex-SSCP analysis may detect RBI germline mutations in 68% (177 25) unselected patients, which is more effective than SSCP(56%) or heteroduplex analysis(64%) alone. The corrected RBI mutation detection rate may be 80% if the samples were previously analyzed by Southern blotting, which is much higher than those reported on literature. Conclusion: Mutations involving a few base pairs in RBI gene are common in Chinese. Heteroduplex-SSCP analysis is more useful and effective than SSCP or heteroduplex analysis alone for the rapid screening for unknown mutations. Eye Science 1997; 13 :5 - 11 .

关 键 词：成视网膜细胞瘤 突变 SSCP 遗传因素 非遗传因素 

分 类 号：R774.1[医药卫生—眼科] R739.7[医药卫生—临床医学]