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

摘要 一种面向下一代的新生儿综合筛查算法的可行性和有效性 测序(TNGS)技术是Pompe和MPS I第二级测试的一部分 新生儿筛查(NBS)利用高通量的初级(1级)筛查分析与转介和 临床随访测试，以确定哪些婴儿受到某些遗传性疾病的影响。如有需要， 在转诊进行后续测试之前执行第二级测试，以减少假阳性的数量 (筛选确定不受影响的阳性样本)。假阳性新生儿筛查有不受欢迎的 对家庭和公共卫生实验室转诊系统的影响，包括：与 额外的验证性测试、转介中心的额外测试负担、父母焦虑、亲子关系 问题，并通过额外的测试和抽血增加了婴儿的压力。有强烈的需求来减少 通过在转诊前实施二级分子或生化测试来筛查假阳性新生儿的比率。 这个第二阶段的项目将是我们成功的第一阶段研究的继续，第一阶段的研究产生了一个 第二层目标下一代测序(TNG)工作流能够识别点突变和 干血斑(DBS)样本中的大量缺失/复制事件。我们将继续专注于庞贝 疾病和粘多糖病I型(MPS I)--最近发现的两种溶酶体储存障碍 推荐用于美国的通用NBS，但由于 假性缺陷变异的比率很高。目前，通过额外的生化分析或基因检测进行的二级检测 已知致病变异的测序用于识别假缺陷和减少假阳性检测 费率。我们将通过以下方式扩展我们的新型TNG第二层工作流：1)为TNG的变体开发生物信息学工具 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可变性(CNV)呼叫者；以及3)整合额外的酶测量 以及人口统计数据和TNGs分数。人口统计数据此前已被证明与 由于生物因素和DBS样本变异性而测得的酶活性。我们的算法将提供一个 更好的疾病状态呼叫和相关数据，用于改进后续护理，提供对疾病的关键预测 发病和治疗方面的考虑。 通过这项工作开发的第二级测试最初将作为诊断发送服务出售，最终 作为试剂盒提供给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。 使用我们的测试确定的受影响个人将被转介到更早的后续行动，并将有 加快了疾病确认和治疗的途径。这些特征对庞贝来说尤其重要 疾病，已知仅延迟几天的治疗干预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 同一原始干燥血斑样本的酶缺乏症。

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.