Identification of glycosaminoglycans for newborn screening and therapeutic monitoring of mucopolysaccharidoses

摘要 用于新生儿筛查和疗效监测的糖胺多糖的鉴定 粘多糖症 粘多糖病(MPS)是一组11种罕见的遗传性代谢性疾病，每种疾病都是由一种缺陷引起的 在一种特定的酶中，这种酶是分解复杂糖所必需的，称为糖胺多聚糖(GAG)。这个 在各种组织中堆积的GAG会导致一系列的健康问题，包括心脏、骨骼、关节和 神经系统并发症，逐渐恶化，导致行动受限和过早死亡。 因此，通过新生儿筛查(NBS)在出生时识别受影响的新生儿至关重要。然而， 美国目前只有MPS I和MPS II的检测方法可用，并不是所有的州目前都在筛查 MPS疾病。一种可以筛查MPS障碍并通过亚型识别的方法将是 对国家统计局社区具有巨大价值；使用这样一个平台进行纵向研究的未来试点数据将 提供必要的证据，以推荐统一的NBS治疗经批准的治疗方法治疗MPS障碍。大多数人 目前可用的治疗的结果是减少了GAG的积聚，从而监测了GAG 水平是MPS治疗制度的重要组成部分。目前，用于总GAG测量的标准测试 使用串联质谱仪(MS/MS)或二甲基亚甲基蓝对尿样进行分析 一些限制包括：灵敏度差、样本量大、周转时间长(&gt；3天)。 为了克服国家统计局和MPS治疗监测方面的这些挑战，我们建议开发一种 新型高通量数字微流体(DMF)平台，用于识别可以 用于筛查疾病和监测疾病治疗。我们将使用新颖(专利)的组合 待定)酶调节分析以测量1)总的GAG水平(以确定在 NBS)和2)特定的GAG类别(以确定在NBS期间哪个特定的GAG升高)。应用程序 靶向下一代测序(TNGs)作为第三个测试将确定存在哪些特定的MPS/亚型。 GAG调制分析将在我们的自动化DMF试剂盒和平台上进行，该平台可以执行 GAG对干血点(筛查)或全血样本(治疗性监测)进行分析。这个 TNGs分析将在我们内部CLIA认证的实验室进行。 我们先前证明了GAG微量平板检测肝素、皮肤素和角质形成蛋白的可行性。 硫酸盐。我们将开发总GAG和硫酸软骨素的检测方法，并将所有GAG检测转换为 DMF平台。TNGs基因面板也将被开发出来，供国家统计局使用。初步分析 将评估化验小组的性能，并计划与黄金标准进行方法比较 验证平台等价性的测试。我们的创新和突破性解决方案用于识别 GAG将极大地提高NBS在州公共卫生实验室对更多MPSS的摄取率。这个 我们的平台的多功能性还可以监测治疗期间的个人GAG水平，这将增加显著的价值 商业化过程中的命题。

ABSTRACT Identification of glycosaminoglycans for newborn screening and therapeutic monitoring of mucopolysaccharidoses Mucopolysaccharidoses (MPS) are a group of 11 rare inherited metabolic diseases, each caused by a deficiency in a specific enzyme necessary for the breakdown of complex sugars termed glycosaminoglycans (GAGs). The accumulation of GAGs in various tissues causes a spectrum of health problems, including heart, bone, joint, and nervous system complications, which progressively worsen and lead to restricted mobility and premature death. Therefore, it is critical that affected newborns are identified at birth through newborn screening (NBS). However, only assays for MPS I and II are currently available in the U.S., and not all states are presently screening for MPS diseases. A methodology that can screen for MPS disorders, and identify by subtype, would be of tremendous value to the NBS community; future pilot data from a longitudinal study with such a platform would provide evidence needed to recommend uniform NBS for MPS disorders with approved therapies. The majority of currently available treatments result in a reduction in GAG accumulation and therefore monitoring of GAG levels is a crucial component of MPS treatment regimes. Standard tests for total GAG measurement are currently performed on urine samples using tandem mass spectrometry (MS/MS) or dimethyl methylene blue and have several limitations including: poor sensitivity, large sample volumes, and long turnaround times (>3 days). To overcome these challenges in NBS and therapeutic monitoring for MPS, we propose to develop a novel high-throughput digital microfluidic (DMF) platform for identification of GAGs that can be leveraged to screen for disease AND monitor disease treatment. We will use a combination of novel (patent pending) enzyme modulation assays to measure 1) total GAG levels (to identify if MPS disease is present during NBS) and 2) specific GAG categories (to determine which specific GAG is elevated during NBS). Application of targeted next generation sequencing (tNGS) as a 3rd test will determine which specific MPS/subtype is present. The GAG modulation assays will be performed on our automated DMF cartridge and platform, which can perform the GAG analyses either on dried blood spot (screening) or whole blood sample (therapeutic monitoring). The tNGS analysis will be developed in our in-house CLIA-certified laboratory. We previously demonstrated feasibility of GAG microtiter plate assays for heparan, dermatan, and keratan sulfates. We will develop assays for total GAGs and chondroitin sulfate, and translate all GAG assays to the DMF platform. The tNGS gene panel will also be developed for use during NBS. Preliminary analytical performance of the assay panel will be assessed, and a method comparison is planned against the gold standard assays to demonstrate platform equivalence. Our innovative and groundbreaking solution for identification of GAGs will dramatically increase the rate of uptake of NBS for more MPSs in state public health laboratories. The versatility of our platform to also monitor individual GAG levels during treatment will add significant value proposition during commercialization.