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目的建立结节性硬化症TSC1/TSC2基因高通量测序技术以快速准确检测结节性硬化的TSC基因突变。方法对10例结节性硬化症患者及10例正常对照进行研究,利用长链PCR方法扩增TSC1及TSC2基因所有外显子区域,运用Ion PGMTM平台进行二代测序并运用常规测序验证。结果设计引物对TSC1及TSC2基因特异性扩增出7个长片段产物,产物长度介于13 kb~15 kb间;运用二代测序技术快速鉴定出10个致病突变,其中,7个突变位于TSC2基因,3个突变位于TSC1基因;所有突变经常规Sanger测序验证,结果一致。结论高通量二代测序技术可快速可靠诊断结节性硬化症TSC1、TSC2基因突变。
Objective To establish a TSC1 / TSC2 gene high-throughput sequencing technique for rapid and accurate detection of TSC mutations in tuberous sclerosis. Methods Ten patients with tuberous sclerosis and 10 normal controls were studied. All exon regions of TSC1 and TSC2 genes were amplified by long-chain PCR and sequenced by using Ion PGMTM platform and verified by routine sequencing. Results The primers were designed to amplify seven long fragments of TSC1 and TSC2 genes. The lengths of the products ranged from 13 kb to 15 kb. Ten disease-causing mutations were rapidly identified by the second-generation sequencing. Among them, seven mutations were located in TSC2 gene, three mutations in the TSC1 gene; all mutations by conventional Sanger sequencing validation, the results are consistent. Conclusion High-throughput second-generation sequencing can rapidly and reliably diagnose TSC1 and TSC2 gene mutations in tuberous sclerosis.