A TRECs screening assay and its combination with IL2RG gene analysis for diagnosis of severe combined immunodeficiency

Abstract

Objective 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 17 confirmed 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. (Chin J Lab Med, 2015, 38: 21-26) Key words: Severe combined immunodeficiency; Neonatal screening; Interleukin receptor common gamma subunit; Gene rearrangement, T-lymphocyte