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 具有针对性的下一代新生儿综合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）利用高通量初级（第一层）筛查检测与转诊配对， 临床跟踪测试，以确定婴儿谁是某些遗传性疾病的影响。必要时， 在转诊进行后续检测之前进行二级检测，以减少假阳性的数量 （筛选确定未受影响的阳性样品）。假阳性新生儿筛查具有不希望的 对家庭和公共卫生实验室转诊系统的后果，包括： 额外的确证性检测，转诊中心的额外检测负担，父母焦虑，亲子关系 问题，并增加了额外的测试和抽血的婴儿的压力。强烈需要减少 通过在转诊前进行二级分子或生化检测来降低新生儿筛查的假阳性率。 这个第二阶段项目将是我们成功的第一阶段研究的延续，第一阶段研究产生了一个综合的 二级靶向下一代测序（tNGS）工作流程，能够识别点突变和 干血斑（DBS）标本的大缺失/重复事件。我们将继续关注庞培 疾病和粘多糖样沉积症I型（MPS I）-两种溶酶体储存疾病，最近 在美国推荐用于通用NBS，但是由于 假缺陷变异的发生率更高。目前，通过额外的生化分析或基因检测进行的第二级检测 已知致病性变体的测序用于鉴定假缺陷并减少假阳性测试 rates.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）开发生物信息学工具， 不确定显著性（VUS）截止值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）检测器;以及3）整合额外的酶测量 和人口统计学数据与tNGS评分。人口统计学数据先前已被证明与 由于生物学因素和DBS样品变异性，测定了酶活性。我们的算法将提供一个 更好的疾病状态呼叫和相关数据，以改善随访护理，为疾病提供关键预测 发病和治疗考虑。 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， 作为试剂盒提供给目前正在筛查或计划筛查庞贝氏症和MPS I的公共卫生实验室。 使用我们的测试确定的受影响的个人将更早地进行随访， 加快疾病确诊和治疗的进程。这些特征对庞培来说尤为重要 已知治疗干预延迟数天会对长期健康产生负面影响的疾病 结果。通过这项工作制定的方法有可能扩大到涵盖数十个 相同的原始干血斑样本中的酶缺乏。

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.