Macek M, Mercier B, Macková A, Miller P W, Hamosh A, Férec C, Cutting G R
Center for Medical Genetics, CMSC 1004, Johns Hopkins University School of Medicine, Baltimore, Maryland 21287-3914, USA.
Hum Mutat. 1997;9(2):136-47. doi: 10.1002/(SICI)1098-1004(1997)9:2<136::AID-HUMU6>3.0.CO;2-7.
More than 500 mutations have been identified in the CFTR gene, making it an excellent system for testing mutation scanning techniques. To assess the sensitivity of denaturing gradient gel electrophoresis (DGGE), we collected a representative group of 202 CFTR mutations. All mutations analyzed were detected by scanning methods other than the DGGE approach evaluated in this study. DGGE analysis was performed on 24 of the 27 exons and their flanking splice site sequences. After optimization, 201 of the 202 control samples produced an altered migration pattern in the region in which an alteration occurred. The remaining sample was sequenced and found not to have the reported mutation. The ability of DGGE to identify novel mutations was evaluated in three Asian CF patients with four unknown CF alleles. Three novel Asian mutations were detected-K166E, L568X, and 3121-2 A-->G (in homozygosity)-accounting for all CF alleles. These results indicate that an optimized DGGE scanning strategy is highly sensitive and specific and can detect 100% of mutations.
在囊性纤维化跨膜传导调节因子（CFTR）基因中已鉴定出500多种突变，这使其成为测试突变扫描技术的理想系统。为了评估变性梯度凝胶电泳（DGGE）的灵敏度，我们收集了一组具有代表性的202种CFTR突变。本研究中评估的DGGE方法以外的扫描方法检测到了所有分析的突变。对27个外显子中的24个及其侧翼剪接位点序列进行了DGGE分析。经过优化，202个对照样本中的201个在发生改变的区域产生了改变的迁移模式。对其余样本进行测序，发现没有报告的突变。在三名患有四个未知CF等位基因的亚洲CF患者中评估了DGGE识别新突变的能力。检测到三个新的亚洲突变——K166E、L568X和3121-2 A→G（纯合子）——占所有CF等位基因。这些结果表明，优化的DGGE扫描策略具有高度的敏感性和特异性，能够检测到100%的突变。
