Feasibility and validation of an integrated newborn screening algorithm with targeted Next Generation Sequencing (tNGS) technology as part of a 2nd-tier test for Pompe and MPS I

作为 Pompe 和 MPS I 二级测试的一部分，采用靶向下一代测序 (tNGS) 技术的集成新生儿筛查算法的可行性和验证

• 批准号: 9909076
• 负责人: Arindam Bhattacharjee
• 金额: $ 77.87万
• 依托单位: BAEBIES, INC.
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-16 至 2021-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9909076
• 关键词: Affect; Age; Algorithms; Alleles; Anxiety; Biochemical; Bioinformatics; Biological Assay; Biological Factors; Blinded; Blood; Caring; Classification; Clinical; Copy Number Polymorphism; Cost Savings; Creatine Kinase; Data; Databases; Diagnostic; Disease; Ensure; Enzymes; Event; Family; Funding; Galactosidase; Gender; Genes; Gestational Age; Glycogen storage disease type II; Goals; Health; Hereditary Disease; Immunologics; Individual; Injury; Lead; Manuals; Measurement; Measures; Methodology; Methods; Modeling; Molecular; Mucopolysaccharidosis I; Muscle; Mutation; Neonatal Screening; Newborn Infant; Onset of illness; Outcome; Parents; Pathogenicity; Performance; Phase; Phenotype; Point Mutation; Population; Probability; Public Health; Published Database; Reporting; Research; Resort; Sampling; Services; Specimen; Speed; Spottings; Stress; System; Technology; Testing; Therapeutic Intervention; Time; United States Public Health Service; Validation; Variant; Work; base; bioinformatics tool; biomedical referral center; causal variant; clinical Diagnosis; cost; cost effective; cross reactivity; enzyme activity; enzyme deficiency; follow-up; glucosylceramidase; heuristics; improved; infancy; insight; interest; next generation sequencing; novel; sample collection; screening; tool; variant of unknown significance

ABSTRACT Feasibility and validation of an integrated newborn screening algorithm with targeted Next Generation Sequencing (tNGS) technology as part of a 2nd-tier test for Pompe and MPS I Newborn screening (NBS) utilizes high throughput primary (1st-tier) screening assays paired with referral and clinical follow-up testing to identify babies who are affected with certain inherited disorders. Where necessary, 2nd-tier tests are performed prior to referral for follow-up testing in order to reduce the number of false positives (screen positive samples that are determined to be unaffected). False-positive newborn screens have undesired consequences for both families and the public health lab referral system, including: high cost associated with additional confirmatory testing, extra testing burden on referral centers, parental anxiety, parent-baby bonding issues, and added stress to the baby with additional tests and blood draws. There exists a strong need to reduce the rate of false positive newborn screens by implementing 2nd-tier molecular or biochemical tests prior to referral. This Phase II project will be a continuation of our successful Phase I research, which generated an integrated 2nd-tier targeted next generation sequencing (tNGS) workflow capable of identifying both point mutations and large deletions/duplication events from dried blood spot (DBS) specimens. We will continue to focus on Pompe disease and Mucopolysaccharidosis Type I (MPS I) -- two lysosomal storage disorders that were recently recommended for universal NBS in the U.S., but have been challenging to implement as NBS tests due to the high rates of pseudodeficient variants. Currently, 2nd-tier testing via either additional biochemical analysis or gene sequencing for known pathogenic variants are used to identify pseudodeficiency and reduce false positive test rates. We will expand our novel tNGS 2nd-tier workflow by: 1) developing bioinformatic tools for variants of uncertain significance (VUS) cut-off and cross-reactive immunological material (CRIM) status prediction; 2) improving our existing copy number variability (CNV) caller; and 3) integrating additional enzyme measurements and demographic data with the tNGS score. Demographic data has previously been shown to correlate to measured enzyme activities due to biological factors and DBS sample variability. Our algorithm will provide a better disease state call and associated data for improved follow-up care, provide critical predictions for disease onset and treatment considerations. The 2nd-tier tests developed through this work will be sold initially as a diagnostic send-out service and eventually as kits to public health labs that are currently screening, or planning to screen for Pompe disease and MPS I. Affected individuals who are identified using our tests will be referred to follow-up earlier and will have an accelerated path to disease confirmation and treatment. These features are especially important for Pompe disease, where a delay in therapeutic intervention of just days is known to negatively impact long term health outcomes. The approaches developed through this work have the potential to be expanded to cover dozens of enzyme deficiencies from the same primary dried blood spot sample.

抽象的 具有目标下一代的集成新生儿筛查算法的可行性和验证 测序 (tNGS) 技术作为 Pompe 和 MPS I 二级测试的一部分 新生儿筛查 (NBS) 采用高通量初级（第一级）筛查测定法，并结合转诊和 临床后续测试，以确定患有某些遗传性疾病的婴儿。如有需要， 在转介进行后续测试之前进行第二层测试，以减少误报数量 （筛选确定不受影响的阳性样本）。新生儿筛查假阳性有不良影响 对家庭和公共卫生实验室转诊系统造成的后果，包括： 额外的确认性测试、转诊中心的额外测试负担、父母焦虑、亲子关系 问题，并通过额外的检查和抽血给婴儿增加压力。迫切需要减少 通过在转诊前实施二级分子或生化测试来降低新生儿筛查假阳性率。 这个第二阶段项目将是我们成功的第一阶段研究的延续，该研究产生了一个综合的 第二层靶向下一代测序 (tNGS) 工作流程能够识别点突变和 干血斑 (DBS) 样本中的大量缺失/重复事件。我们将继续关注庞贝 病和 I 型粘多糖贮积症 (MPS I)——最近发现的两种溶酶体贮积症 建议在美国进行通用 NBS，但由于 假缺陷变异的发生率很高。目前，通过额外的生化分析或基因进行第​​二层测试 对已知致病变异的测序用于识别假缺陷并减少假阳性测试 费率。我们将通过以下方式扩展我们新颖的 tNGS 第二层工作流程：1）开发用于变体的生物信息学工具 不确定意义（VUS）截止和交叉反应免疫物质（CRIM）状态预测； 2） 改进我们现有的拷贝数变异性 (CNV) 调用程序； 3) 整合额外的酶测量 以及人口统计数据和 tNGS 评分。人口统计数据之前已被证明与 测量由于生物因素和 DBS 样品变异性而产生的酶活性。我们的算法将提供 更好的疾病状态呼叫和相关数据以改善后续护理，为疾病提供关键预测 发病和治疗注意事项。 通过这项工作开发的第二层测试最初将作为诊断发送服务出售，最终将 作为目前正在筛查或计划筛查庞贝病和 MPS I 的公共卫生实验室的试剂盒。 使用我们的测试确定的受影响个人将被提早转介至后续行动，并将有一个 加速疾病确认和治疗的途径。这些功能对于 Pompe 来说尤其重要 已知治疗干预仅延迟几天就会对长期健康产生负面影响 结果。通过这项工作开发的方法有可能扩展到涵盖数十个 来自同一原代干血斑样本的酶缺乏症。