微滴数字PCR定量检测葡萄膜黑色素瘤患者<i>GNAQ</i>/<italic/>11突变基因

赵清; 陈莹莹; 侯宸; 曾少雪; 张宇辉; 张军军

doi:10.12182/20200760503

微滴数字PCR定量检测葡萄膜黑色素瘤患者GNAQ/11突变基因

Detection of GNAQ/11 Mutations in Uveal Melanoma Patients’ FFPE DNA with Droplet Digital PCR

摘要

摘要:
目的探讨基于微滴数字PCR定量检测葡萄膜黑色素瘤（uveal melanoma, UM）患者肿瘤组织GNAQ/11突变的可行性。
方法肿瘤标本取自2009−2015年间在四川大学华西医院确诊并行眼球摘除术的78例UM患者的甲醛固定石蜡包埋（FFPE）肿瘤组织，取标本前所有患者均未进行放疗或化疗。采用回顾性研究，以微滴数字PCR技术检测葡萄膜黑色素瘤GNAQ/11的突变情况，同时Sanger测序对目标基因进行DNA测序，比较两种检测方式结果的一致性。
结果 74例UM患者肿瘤组织GNAQ/11突变频率为91.9%。对Sanger测序与微滴数字PCR技术两种方式检测74例葡萄膜黑色素瘤患者GNAQ/11突变的结果进行一致性检验，Kappa系数=0.436，P=0.001。检测GNAQ/11突变基因的两种方法差异最常见的是Sanger测序没有检测出GNAQ/11突变。Sanger测序结果的出错率在异质突变组高于同质突变组（12/37 vs. 3/16，P=0.53），但差异无统计学意义。
结论微滴数字PCR与Sanger测序结果具有较好的一致性，葡萄膜黑色素瘤中GNAQ/11的突变率差异较大。数字PCR检测葡萄膜黑色素瘤患者GNAQ/11突变频率接近报道水平。肿瘤组织DNA来源于FFPE的样本更推荐使用灵敏度高的微滴数字PCR检测基因突变，Sanger测序易出现假阴性结果。

Abstract:
Objective To quantitatively detect GNAQ/11 mutations in uveal melanoma (UM) by droplet digital PCR (ddPCR).
Methods Formaldehyde-fixed paraffin-embedded (FFPE) tumor samples were taken from 78 UM patients with enucleation in West China Hospital between 2009 and 2015. None of the patients received radiotherapy or chemotherapy before enucleation. A retrospective study was conducted to detect GNAQ/11 mutation in UM by ddPCR. To compare the consistency of the results of the two detection methods, DNA sequencing was performed on the target gene by Sanger sequencing. 78 patients with UM were studied retrospectively. GNAQ/11 mutations in uveal melanoma was detected by ddPCR. The consistency of the results of the two detection methods was analyzed.
Results GNAQ/11 mutations frequency was 91.9%. The consistency test between Sanger sequencing and ddPCR of GNAQ/11 mutations in 74 patients with UM was conducted. Kappa coefficient=0.436, P=0.001. The error rate of Sanger sequencing results was significantly higher in the heterogeneous group than in the homogeneous group (12/37 vs. 3/16, P=0.53), but the difference was not statistically significant.
Conclusion The results of ddPCR and Sanger sequencing showed good consistency, and the mutation ratio of GNAQ/11 in UM was significantly different. GNAQ/11 mutation frequency in UM patients detected by ddPCR was close to the reported frequency. It is more recommended to use ddPCR with high sensitivity to detect gene mutations in samples of tumor tissue DNA derived from FFPE. Sanger sequencing is prone to false negative results.

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