儿童干扰素刺激基因表达检测方法的建立与应用

Establishment and application of detection method for interferon stimulated genes expression in children

目的建立儿童干扰素刺激基因(ISG)表达检测方法, 确立参考值范围, 初步探讨其临床应用价值。方法纳入2017年11月至2021年9月就诊于北京协和医院儿科的Ⅰ型干扰素病患者作为疾病组, 同时纳入健康儿童作为对照组。疾病组共纳入18例患儿, 其中男性8例, 女性10例, 共采集血液样本25份, 首次检测的中位年龄为8.5岁。对照组共纳入28名健康儿童, 年龄1~18岁, 中位年龄10.5岁, 其中男性15名, 女性13名。分别提取疾病组和对照组外周血总RNA并逆转录为互补DNA(cDNA)。以β肌动蛋白基因(β-Actin)和鸟氨酸脱羧酶抗酶基因(OAZ)为内参, 采用实时荧光定量聚合酶链反应(qPCR)检测干扰素诱导的四肽重复蛋白1基因(IFIT1)、干扰素α诱导蛋白27基因(IFI27)、干扰素诱导蛋白44样基因(IFI44L)、干扰素刺激基因15(ISG15)、唾液酸结合的免疫球蛋白样凝集素1基因(SIGLEC1)、含有S-腺苷甲硫氨酸结构域2基因(RSAD2)的相对表达量, 以6个ISG的中值为干扰素评分(IS)。去除正常对照中表达明显异常的样本, 将其他样本的cDNA混合作为参照, 重新检测各样本并计算IS, 将大于对照xˉ+2s的IS结果判断为异常。用独立样本t检验或Mann-WhitneyU检验比较组内和组间IS的差异。结果对照组IS均值为1.046, 标准差0.755, IS截断值为2.556。18例Ⅰ型干扰素病患者组IS异常的有15例(15/18), IS均值为27.010。与对照组相比, 干扰素疾病患者组IS明显升高(t=4.247, P=0.000 1)。该检测方法的准确度为91.30%(42/46), 精密度为7.47%(0.084/1.124), 敏感度为15/18, 特异度为96.43%(27/28)。结论本研究通过ISG表达的检测并计算IS, 为临床筛查以及动态监测Ⅰ型干扰素疾病变化提供了新的可靠的手段。

Objective To establish the detection method for the interferon stimulated genes(ISGs),calculate the cut-off value and test it in clinical practice.Methods Patients with type I interferonopathies who were admitted to Peking Union Medical College Hospital from November 2017 to September 2021 were chosen as the disease group,and healthy children were included as the control group.A total of 18 children were in the disease group,including 8 males and 10 females,with a median age of 8.5 years for the first test.From them 25 blood specimens were collected.A total of 28 healthy children,aged 1 to 18 years,with a median age of 10.5 years,including 15 males and 13 females,were included in the control group.Blood samples of 34 controls and 18 interferonopathies patients were collected,then total RNA extraction and cDNA synthesis were performed.Real-time quantitative polymerase chain reaction assays were run in duplicate to measure the expression of six ISGs:interferon induced protein with tetratricopeptide repeats 1(IFIT1),interferonαinducible protein 27(IFI27),interferon induced protein 44 like(IFI44L),interferon stimulated genes 15(ISG15),sialic acid binding Ig like lectin 1(SIGLEC1),and radical S-adenosyl methionine domain containing 2(RSAD2).The relative abundances of each target transcript was normalized to the expression level ofβ-Actin and OAZ.The median fold change of the six ISGs was used to create an interferon score(IS)for each individual.Samples with abnormal expressions were removed and the cDNA mix of the remaining samples was used as a calibrator to calculate the IS.We define an abnormal IS as being greater than+2 standard deviations above the mean of controls.Differences in IS between groups were compared using t-test or Mann-Whitney U-test.Results The mean IS of controls was 1.046,standard 0.755,and the cut-off value was 2.556.A total of 25 samples from 18 interferonopathies patients were tested.The mean value was 27.010 with a 15/18 abnormality rate.Compared with the control group,IS in patients was significantly higher,t=4.247(P=0.0001).The accuracy,precision,sensitivity,and specificity were 91.30%(42/46),7.47%(0.084/1.124),15/18,and 96.43%(27/28),respectively.Conclusion This study provides a new and reliable method for clinical screening and dynamic monitoring of typeⅠinterferonopathies by detecting ISGs expression and creating an IS.