Kiewiet Gea, Westra Dineke, de Boer Eddy N, van Berkel Emma, Hofste Tom G J, van Zweeden Martine, Derks Ronny C, Leijsten Nico F A, Ruiterkamp-Versteeg Martina H A, Charbon Bart, Johansson Lennart, Bos-Kruizinga Janneke, Veenstra Inge J, de Sain-van der Velden Monique G M, Voorhoeve Els, Heiner-Fokkema M Rebecca, van Spronsen Francjan, Sikkema-Raddatz Birgit, Nelen Marcel
Department of Genetics, University Medical Center Groningen, University of Groningen, 9713 GZ Groningen, The Netherlands.
Department of Human Genetics, Radboud University Medical Center, 6500 HB Nijmegen, The Netherlands.
Int J Neonatal Screen. 2024 Mar 7;10(1):20. doi: 10.3390/ijns10010020.
In this study, we compare next-generation sequencing (NGS) approaches (targeted panel (tNGS), whole exome sequencing (WES), and whole genome sequencing (WGS)) for application in newborn screening (NBS). DNA was extracted from dried blood spots (DBS) from 50 patients with genetically confirmed inherited metabolic disorders (IMDs) and 50 control samples. One hundred IMD-related genes were analyzed. Two data-filtering strategies were applied: one to detect only (likely) pathogenic ((L)P) variants, and one to detect (L)P variants in combination with variants of unknown significance (VUS). The variants were filtered and interpreted, defining true/false positives (TP/FP) and true/false negatives (TN/FN). The variant filtering strategies were assessed in a background cohort (BC) of 4833 individuals. Reliable results were obtained within 5 days. TP results (47 patient samples) for tNGS, WES, and WGS results were 33, 31, and 30, respectively, using the (L)P filtering, and 40, 40, and 38, respectively, when including VUS. FN results were 11, 13, and 14, respectively, excluding VUS, and 4, 4, and 6, when including VUS. The remaining FN were mainly samples with a homozygous VUS. All controls were TN. Three BC individuals showed a homozygous (L)P variant, all related to a variable, mild phenotype. The use of NGS-based workflows in NBS seems promising, although more knowledge of data handling, automated variant interpretation, and costs is needed before implementation.
在本研究中，我们比较了下一代测序（NGS）方法（靶向测序（tNGS）、全外显子组测序（WES）和全基因组测序（WGS））在新生儿筛查（NBS）中的应用。从50例经基因确诊的遗传性代谢疾病（IMD）患者的干血斑（DBS）和50份对照样本中提取DNA。分析了100个与IMD相关的基因。应用了两种数据过滤策略：一种仅检测（可能）致病（（L）P）变异，另一种检测（L）P变异以及意义未明的变异（VUS）。对变异进行过滤和解读，定义真/假阳性（TP/FP）和真/假阴性（TN/FN）。在4833名个体的背景队列（BC）中评估变异过滤策略。在5天内获得了可靠结果。使用（L）P过滤时，tNGS、WES和WGS的TP结果（47个患者样本）分别为33、31和30，纳入VUS时分别为40、40和38。排除VUS时，FN结果分别为11、13和14，纳入VUS时分别为4、4和6。其余的FN主要是纯合VUS样本。所有对照均为TN。3名BC个体显示纯合（L）P变异，均与可变的轻度表型相关。在NBS中使用基于NGS的工作流程似乎很有前景，不过在实施之前还需要更多关于数据处理、变异自动解读和成本方面的知识。
