NEWBORN SCREENING FOR SEVERE COMBINED IMMUNODEFICIENCIES USING TRECS AND KRECS: SECOND PILOT STUDY IN BRAZIL

ABSTRACT Objective: To validate the quantification of T-cell receptor excision circles (TRECs) and kappa-deleting recombination excision circles (KRECs) by real-time polymerase chain reaction (qRT-PCR) for newborn screening of primary immunodeficiencies with defects in T and/or B cells in Brazil. Methods: Blood samples from newborns and controls were collected on filter paper. DNA was extracted and TRECs, and KRECs were quantified by a duplex real-time PCR. The cutoff values were determined by receiver operating characteristic curve analysis using SPSS software (IBM®, Armonk, NY, USA). Results: Around 6,881 samples from newborns were collected and TRECs and KRECs were quantified. The TRECs values ranged between 1 and 1,006 TRECs/µL, with mean and median of 160 and 139 TRECs/µL, respectively. Three samples from patients with severe combined immunodeficiency (SCID) showed TRECs below 4/µL and a patient with DiGeorge syndrome showed undetectable TRECs. KRECs values ranged from 10 to 1,097 KRECs/µL, with mean and median of 130 and 108 KRECs/µL. Four patients with agammaglobulinemia had results below 4 KRECs/µL. The cutoff values were 15 TRECs/µL and 14 KRECs/µL and were established according to the receiver operating characteristic curve analysis, with 100% sensitivity for SCID and agammaglobulinemia detection, respectively. Conclusions: Quantification of TRECs and KRECs was able to diagnose children with T- and/or B-cell lymphopenia in our study, which validated the technique in Brazil and enabled us to implement the newborn screening program for SCID and agammaglobulinemia.摘要 研究目的：本研究旨在验证通过实时聚合酶链反应（qRT-PCR）定量检测T细胞受体切除环（T-cell receptor excision circles, TRECs）与κ删除重组切除环（kappa-deleting recombination excision circles, KRECs）的方法，用于巴西地区T细胞和/或B细胞缺陷型原发性免疫缺陷病的新生儿筛查。 研究方法：采用滤纸片采集新生儿及对照人群的血液样本。提取基因组DNA后，通过双重实时PCR对TRECs与KRECs进行定量分析。临界值通过受试者工作特征曲线（receiver operating characteristic curve, ROC曲线）分析确定，所用软件为SPSS（IBM®，美国纽约州阿蒙克市）。 研究结果：共收集约6881份新生儿血液样本并完成TRECs与KRECs定量检测。TRECs浓度范围为1~1006拷贝/微升，均值与中位数分别为160拷贝/微升与139拷贝/微升。3份来自重症联合免疫缺陷（severe combined immunodeficiency, SCID）患者的样本TRECs浓度低于4拷贝/微升，1例迪格奥尔格综合征（DiGeorge syndrome）患者的TRECs未检出。KRECs浓度范围为10~1097拷贝/微升，均值与中位数分别为130拷贝/微升与108拷贝/微升。4例无丙种球蛋白血症（agammaglobulinemia）患者的KRECs检测结果低于4拷贝/微升。经受试者工作特征曲线分析确定的临界值为：TRECs 15拷贝/微升、KRECs 14拷贝/微升，二者对重症联合免疫缺陷与无丙种球蛋白血症的检测灵敏度均为100%。 研究结论：本研究结果显示，TRECs与KRECs定量检测可用于诊断T细胞和/或B细胞淋巴细胞减少症患者，验证了该技术在巴西的适用性，为开展重症联合免疫缺陷与无丙种球蛋白血症的新生儿筛查项目奠定了技术基础。