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目的 验证成纤维细胞生长因子受体 3(fibroblastgrowth factor receptor3,FGFR3)跨膜区1138位核苷酸为先天性软骨发育不全突变热点及用变性梯度电泳方法筛查的效果。方法 对 17例临床诊断为先天性软骨发育不全患者进行基因组 DNA聚合酶链反应 -限制性酶切片段长度多态性 (polymerasechain reaction- restriction fragment length polymorphism,PCR- RFL P)分析 ,并用变性梯度凝胶电泳(denaturing gradient gel electrophoresis,DGGE)进行其它突变位点的筛查。结果 17例患者中 14例RFL P检测显示能被 Sfc 酶切 ,提示存在 1138位核苷酸 G→ A的转换 ,且均为杂合子。 17例用 Msp 酶切均阴性 ,提示无 G→C的颠换。DGGE方法的筛查中 ,酶切阳性的 14例标本均显示存在杂合型突变。3例PCR- RFL P检测阴性的标本未显示突变位点 ,提示在该扩增区域确实不存在突变位点。结论 FGFR3跨膜区 1138核苷酸 G→A的突变是软骨发育不全的主要发病机理 ,DGGE可作为筛查某一区域基因突变的敏感而可靠的检测手段 ,尤其是对杂合子的检测。
Objective To verify the nucleotide sequence of 1138 nucleotide in the transmembrane region of fibroblast growth factor receptor 3 (FGFR3) as a hot spot in congenital achondroplasia and screening by denaturing gradient electrophoresis. Methods Genomic DNA polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was performed in 17 patients with congenital achondroplasia and was analyzed by denaturing gradient gel electrophoresis Other mutations were screened by denaturing gradient gel electrophoresis (DGGE). Results Of the 17 patients, RFLP analysis of 14 cases showed that it was cleaved by Sfc, suggesting the presence of 1138 nucleotide G → A transitions, all of which were heterozygous. 17 cases were negative with Msp digestion, suggesting no transversion of G → C. In the screening of the DGGE method, the 14 positive specimens showed heterozygous mutations. Three specimens negative for PCR-RFL P did not show the mutation sites, suggesting that there are indeed no mutation sites in the amplification area. Conclusion The mutation of G → A at nucleotide 1138 in the transmembrane region of FGFR3 is the main pathogenesis of achondroplasia. DGGE can be used as a sensitive and reliable method to screen genetic mutations in a certain region, especially for the detection of heterozygotes.