应用多重实时荧光定量PCR技术联合筛查新生儿两种原发性免疫缺陷病及脊髓性肌萎缩症的研究  

Combined screening of two primary immunodeficiency diseases and spinal muscular atrophy in neonates by multiplex real-time fluorescence quantitative PCR

作　　者：张超 杨建滨[1] 尚世强[2] 陈迟[1] 毛华庆[1] 黄晓磊[1] 洪芳[1] 缪海霞 赵涵怡 杨茹莱[1] Zhang Chao;Yang Jianbin;Shang Shiqiang;Chen Chi;Mao Huaqing;Huang Xiaolei;Hong Fang;Miao Haixia;Zhao Hanyi;Yang Rulai(Department of Genetics and Metabolism,Children′s Hospital,Zhejiang University School of Medicine,National Clinical Research Center for Child Health,Hangzhou 310052,China;Department of Clinical Laboratory,Children′s Hospital,Zhejiang University School of Medicine,National Clinical Research Center for Child Health,Hangzhou 310052,China)

机构地区：[1]浙江大学医学院附属儿童医院遗传与代谢科、国家儿童健康与疾病临床医学研究中心,杭州310052 [2]浙江大学医学院附属儿童医院实验检验中心、国家儿童健康与疾病临床医学研究中心,杭州310052

出　　处：《中华检验医学杂志》2025年第2期249-257,共9页Chinese Journal of Laboratory Medicine

基　　金：国家重点研发项目(2022YFC2703401)。

摘　　要：目的探讨应用多重实时荧光定量PCR技术联合筛查新生儿重症联合免疫缺陷病(SCID)、X连锁无丙种球蛋白血症(XLA)及脊髓性肌萎缩症(SMA)的可行性,为患儿的早期筛查和诊治提供依据。方法横断面研究。收集2021年7月至2023年1月经冷链递送至浙江省新生儿疾病筛查中心的滤纸干血片103240份,采用多重实时荧光定量PCR方法联合检测干血片中T细胞受体切除环(TREC)、Kappa缺失重组切除环(KREC)浓度及运动神经元存活基因1(SMN1)外显子7缺失,以核糖核酸酶P/MRP 30000亚基(RPP30)作为内参基因。筛查阳性的新生儿进一步经实验室其他相关项目检测以辅助诊断,基因测序结果作为确诊金标准。同时收集本院已确诊1例SCID、3例XLA及2例SMA患儿样本用于阳性验证。分析新生儿基本信息与TREC/KREC检测浓度的相关性,并进行各因素组间差异性分析。结果应用多重实时荧光定量PCR技术筛查并确诊新生儿1例SCID、2例XLA、9例SMA以及7例其他遗传性疾病(4例DiGeorge综合征、1例21-三体综合征、1例努南综合征和1例超雄综合征);筛查新生儿SCID、XLA及SMA的阳性预测值分别为2.44%(1/41)、2.78%(2/72)和9/9。已临床确诊的1例SCID、3例XLA和2例SMA样本作为阳性验证样本,经该方法全部检出。TREC/KREC检测结果与新生儿采血时间、性别、体重、胎龄、分娩方式的r值分别为0.162/0.187、0.066/0.032、0.045/0.042、-0.015/-0.088、0.014/0.068(P均<0.05),有相关关系。结论浙江省依托现行的新生儿疾病筛查平台,应用多重实时荧光定量PCR技术开展新生儿2种原发性免疫缺陷病及脊髓性肌萎缩症的联合筛查具有可行性。Objective To explore the feasibility of joint screening of the two primary immunodeficiency diseases[severe combined immunodeficiency(SCID)and X-linked agammaglobulinemia(XLA)]and spinal muscular atrophy(SMA)in newborns by multiplex real-time quantitative PCR technology,and to provide evidence for early screening,diagnosis and treatment of children.MethodsCross-sectional study.From July 2021 to January 2023,a total of 103240 dry blood spots samples of newborns were collected which were delivered to Neonatal Disease Screening Center of Zhejiang by cold chain transportation.The concentrations of the T cell receptor excision ring(TREC),Kappa deletion of the recombinant excision loop(KREC),and exon 7 deletion of Survival Motor Neuron 1(SMN1)gene in dry blood spots were simultaneously detected by multiplex real-time fluorescence quantitative PCR,taken ribonuclease P/MRP 30000 subunits(RPP30)as an internal reference gene.The positive newborns were further diagnosed by other laboratory tests and gene sequencing was taken as gold standard.Children samples from 1 case of SCID,3 cases of XLA and 2 cases of SMA were used for positive verification.The correlation between detected concentration of TREC/KREC and basic information in newborns were analyzed.The differences among groups for each factor were analyzed.ResultsOne case of SCID,2 cases of XLA,9 cases of SMA and 7 cases of other genetic diseases(4 cases of DiGeorge syndrome,1 case of trisomy 21 syndrome,1 case of Noonan syndrome and 1 case of super male syndrome)were identified by multiplex real-time fluorescence quantitative PCR.The positive predictive values of screening neonatal SCID,XLA and SMA were 2.44%(1/41),2.78%(2/72)and 9/9 respectively.Taking the samples from clinically diagnosed 1 case of SCID,3 cases of XLA and 2 cases of SMA as positive validation samples,which were all identified.The detected results of TREC/KREC correlated with time of blood collection,sex,weight,gestational age and delivery mode of newborns,whose r values were 0.162/0.187,0.066/0.032,0

关 键 词：新生儿筛查 多重荧光定量PCR 重症联合免疫缺陷 X连锁无丙种球蛋白血症 脊髓性肌萎缩 儿童 

分 类 号：R722.1[医药卫生—儿科]