摘要目的 拟建立适合临床应用的新生儿筛查技术,结合Sanger测序技术,明确重症联合免疫缺陷(SCJD)相关基因突变,为患儿的早期筛查与诊治提供依据.方法 前瞻性研究.利用Taqman实时荧光PCR技术,建立定量检测滤纸干血斑中T细胞受体重排删除环(TRECs)的方法,进行方法学评价;收集2013年1月至2014年6月到重庆医科大学附属儿童医院就诊的SCID疑似患儿30例,检测干血斑TRECs拷贝数和外周血基因组DNA中IL2RG核酸序列;结合患儿临床诊断,分析两种方法的临床符合率.结果 自建荧光定量PCR法的最低检测量为103拷贝/ml,批内及批间变异系数分别为＜4.7％和＜9.1％.30例SCID疑似患儿中,17例TRECs含量低于参考区间,根据临床表现,最终均确诊为SCID,自建方法的临床符合率为17/17.17例SCID确诊患儿均为男孩,其中16例存在IL2RG基因突变(7例移码突变,6例错义突变,2例无义突变,1例剪切突变),1例为RAG1基因复合杂合错义突变.结论 建立以TRECs为基础的新生儿筛查技术,联合应用IL2RG基因分析技术,将有助于SCID患儿的早期发现、早期诊断及治疗方案的正确选择.

abstractsObjective To establish a newborn screening method suitable for clinical application,combined with Sanger sequencing assay for related gene mutation,and thereby to provide early screening and diagnosis basis for severe combined immunodeficiency (SCID).Methods Using Taqman real-time PCR technology,the quantitative detection method for T-cell receptor rearrangement excision circles (TRECs) from the dried blood spots was developed and the methodology evaluation was performed.From January 2013 to June 2014,a total of 30 suspected SCID cases in the Children's Hospital of Chongqing Medical University were enrolled.TRECs copies were measured in DNA isolated from the dried blood spots.IL2RG gene mutations were also tested in DNA from peripheral blood mononuclear cells by sequencing.Compared with the confirmed clinical diagnosis,the clinical coincidence rate of the above two methods was evaluated,respectively.Results The detection limit of Taqman real-time PCR technique was 103 copies/ml for TRECs from the dried blood spots.The intra-and inter-coefficients of variations were less than 4.7％ and less than 9.1％ respectively.Among 30 suspected SCID cases,17 cases,which were confirmed as SCID based on the clinical presentation,were observed that TRECs copies were below normal reference interval.The coincidence rate of Taqman real-time PCR technique and the confirmed diagnosis was 17/17.Among 17confirmed SCID cases,all were boys.IL2RG gene mutations were identified in 16 cases,including 7 frameshift mutations,6 missense mutations,2 silent mutations and 1 splice mutation.Additionally,a compound heterozygosity mutation in RAG1 gene was found in a confirmed SCID case without the IL2RG gene mutation.Conclusion TRECs-based newborn screening,and combination with IL2RG gene analysis technology,may provide technical support for early identification,diagnosis,and selection of treatment for SCID.