Multiplex Analysis of Inborn Errors of Metabolism

描述(由申请人提供)：该项目的总体目标是开发串联质谱仪，用于对已有或正在开发治疗方案的溶酶体储存疾病的新生儿子集进行筛查。新生儿筛查卡上干血斑的冲床被用作溶酶体酶的来源，并与合适缓冲液中的一组底物孵育，以形成产物。后者在一套内标的帮助下，通过串联质谱仪进行定量。我们建立了六种溶酶体酶的串联质谱仪多重分析方法。这项6-plex检测将在西澳州新生儿筛查实验室进行试点，随机抽取100,000名新生儿的干血点。下一阶段是开发高灵敏度的试剂来检测与另外6种溶酶体储存疾病相关的6种硫酸酶。我们还将开发串联质谱仪分析三肽基蛋白酶I(缺乏症导致神经元蜡样脂褐素沉积症2)、溶酶体酸性脂肪酶(缺乏症导致Wolman病和胆固醇酯储存疾病)。然后，我们将开发一种多重方法，使用最少的干血斑点冲头和检测缓冲液对所有13种酶进行新生儿筛查。一旦这种多重分析被开发出来，我们将在佤邦新生儿筛查实验室对n=100,000个随机新生儿的干血点进行第二次试点研究。我们还将使用下一代测序技术对酶活性低于截止值的干血斑点进行外显子DNA测序。这些数据将使我们能够确定筛查试验的阳性预测值和假阳性率。这些初步研究将探索新生儿筛查可治疗的溶酶体储存疾病的可行性。

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.