Multiplex Analysis of Inborn Errors of Metabolism

先天性代谢缺陷的多重分析

• 批准号: 8696104
• 负责人: Michael H Gelb
• 金额: $ 64.22万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-08-01 至 2018-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8696104
• 关键词: Affect; Agreement; Area; Biological Assay; Blood; Buffers; Cholesterol Ester Storage Disease; Clinical Trials; Collection; DNA; DNA Sequence; Data; Development; Disease; Early treatment; Enzymes; Genes; Genotype; Goals; Grant; Inborn Errors of Metabolism; Incubated; Individual; Infusion procedures; Laboratories; Life; Lysosomal Storage Diseases; Methods; Monitor; Mucopolysaccharidosis I; Neonatal Screening; Neuronal Ceroid-Lipofuscinosis; Newborn Infant; Outcome; Phase; Pilot Projects; Predictive Value; Protocols documentation; Reagent; Research; Sample Size; Sampling; Source; Spottings; Staging; Sulfatases; Technology; Testing; Treatment outcome; Wolman Disease; Work; assay development; base; disease-causing mutation; enzyme activity; exome; exome sequencing; interest; mass spectrometer; multiple reaction monitoring; next generation; next generation sequencing; public health relevance; screening; tandem mass spectrometry; therapy development; tripeptidyl-peptidase I

DESCRIPTION (provided by applicant): The overall goal of this project is develop tandem mass spectrometry for newborn screening of the subset of lysosomal storage diseases for which treatment options exist or are being developed. A punch from a dried blood spot on a newborn screening card is used as a source of lysosomal enzymes, and this is incubated with a collection of substrates in a suitable buffer to allow formation of products. The latter are quantified by tandem mass spectrometry with the aid of a set of internal standards. We have developed a tandem mass spectrometry multiplex assay of six lysosomal enzymes. This 6-plex assay will be piloted in the WA state newborn screening laboratory on n=100,000 dried blood spots from random newborns. The next phase is to develop highly sensitive reagents to assay 6 sulfatase enzymes relevant to 6 additional lysosomal storage diseases. We will also develop tandem mass spectrometry assays for tripeptidyl protease I (deficiency causes neuronal ceroid lipofuscinosis 2), lysosomal acid lipase (deficiency causes Wolman disease and cholesterol-ester storage disease). We will then develop a multiplex method for newborn screening of all 13 enzymes using the minimum number of dried blood spot punches and assay buffers. Once this multiplex assay has been developed, we will carry out a second pilot study in the WA state newborn screening lab on n=100,000 dried blood spots from random newborns. We will also carry out exome DNA sequencing using next-generation sequencing on dried blood spots that give an enzyme activity below the cut-off value. This data will allow us to determine the positive predictive values and false positive rates for the screening assay. These pilot studies will explore the feasibility of newborn screening of treatable lysosomal storage diseases.

描述（由申请人提供）：本项目的总体目标是开发串联质谱法，用于新生儿筛查溶酶体贮积病亚组，治疗方案已存在或正在开发中。来自新生儿筛查卡上的干血斑的穿孔被用作溶酶体酶的来源，并且将其与适当缓冲液中的底物集合一起孵育以允许形成产物。后者通过串联质谱法在一组内标物的帮助下进行定量。我们开发了一种对六种溶酶体酶进行串联质谱多重检测的方法。该6重测定将在WA州新生儿筛查实验室中对来自随机新生儿的n= 100，000个干血斑进行试点。下一阶段是开发高灵敏度的试剂来检测与另外6种溶酶体贮积病相关的6种硫酸酯酶。我们还将开发三肽基蛋白酶I（缺乏导致神经元蜡样脂褐质沉积症2），溶酶体酸性脂肪酶（缺乏导致沃尔曼病和胆固醇酯储存病）的串联质谱测定。然后，我们将开发一种多重方法，使用最少数量的干血斑穿孔和测定缓冲液对所有13种酶进行新生儿筛查。一旦开发出这种多重检测方法，我们将在WA州新生儿筛查实验室对随机新生儿的n= 100，000个干血斑进行第二次试点研究。我们还将使用下一代测序技术对干血斑进行外显子组DNA测序，这些血斑的酶活性低于截止值。这些数据将使我们能够确定筛选试验的阳性预测值和假阳性率。这些试点研究将探索新生儿筛查可治疗的溶酶体贮积病的可行性。