应用变性高效液相色谱技术检测肥大细胞病c-kit基因突变

本研究检测7例肥大细胞病患者c—kit基因激酶编码区突变情况，以探讨该基因变异在肥大细胞病诊断及治疗中的意义。应用PCR测定、变性高效液相色谱技术及DNA测序方法对患者进行c—kit基因外显子17～外显子19突变检测。结果表明：1例患者外显子17扩增产物在DHPLC中出现异常色谱峰，测序结果显示在外显子17出现A→T突变，即D816V；另2例患者外显子18／19经DHPLC检测，结果显示1个额外的色谱峰，测序结果证实在外显子18出现G→C改变，为同义突变L862L。结论：变性高效液相色谱技术是一种高效、可靠的突变检测方法，c—kit基因突变检测对肥大细胞病临床治疗有指导意义。

Application of Denaturing High Performance Liquid Chromatography to Mutation Detection of the c-kit Gene in Mastocytosis

The aim of study was to detect mutation of tyrosine domain of c-kit gene in mastocytosis using denaturing high performance liquid chromatography technique, and to investigate the significance of this gene mutation in diagnosis and therapy of mastocytosis. Genomic DNA was obtained from bone marrow or peripheral blood leukocytes using the phenol/chloroform method from 7 mastocytosis patients. PCR was performed with AmpliTaq Gold DNA polymerase and 100 ng genomic DNA to amplify the entire coding sequence and exon-intron boundaries of c-kit exon 17 approximately exon 19. Denaturing high-performance liquid chromatography (DHPLC) analysis was performed on a WAVE DNA Fragment Analysis System. Each PCR product was mixed with an equal quantity of amplified human placental DNA (served as normal control) and was denatured at 95 degrees C for 5 minutes, followed by slowly cooling down to room temperature by 1.5 degrees C per minute to allow heteroduplexes formation. All the conditions for the DHPLC analysis, including melting temperature and buffer gradients were determined using the Transgenomic software Navigator. Samples with extra peaks or with different peak form on DHPLC were directly sequenced using the BigDye Terminator Cycle Sequencing Reaction kit and ABI 3100 Genetics Analyser. The results showed that DHPLC revealed an aberrant peak in one patient in exon 17 and the D816V mutation was identified by direct sequencing. The other two patients had an extra peak for exon 18/19 and direct sequencing revealed a conservative sequence change (L862L) within exon 18. It is concluded that denaturing high performance liquid chromatography is a high efficiency and reliable technique for mutation detection of c-kit gene and the detection results would be helpful for the selection of therapy in mastocytosis.