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法，用于对现有或正在开发治疗方案的溶酶体贮积病子集进行新生儿筛查。使用新生儿筛查卡上干燥血点的打孔作为溶酶体酶的来源，并将其与适当缓冲液中的底物集合一起孵育，以形成产物。后者通过串联质谱借助一组内标进行量化。我们开发了六种溶酶体酶的串联质谱多重测定。这种 6 重检测将在西澳州新生儿筛查实验室对随机新生儿的 n=100,000 个干血点进行试点。下一阶段是开发高灵敏度试剂来检测与另外 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着症 2）、溶酶体酸性脂肪酶（缺乏导致沃尔曼病和胆固醇酯贮积病）的串联质谱分析。然后，我们将开发一种多重方法，使用最少数量的干血点冲孔器和检测缓冲液对所有 13 种酶进行新生儿筛查。一旦开发出这种多重检测方法，我们将在西澳州新生儿筛查实验室对随机新生儿的 n=100,000 个干血点进行第二项试点研究。我们还将使用新一代测序技术对酶活性低于截止值的干血点进行外显子组 DNA 测序。这些数据将使我们能够确定筛查测定的阳性预测值和假阳性率。这些试点研究将探讨新生儿筛查可治疗的溶酶体贮积病的可行性。

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.