Technology development for rapid detection and diagnosis of metabolic disorders

快速检测和诊断代谢紊乱的技术开发

• 批准号: 8767347
• 负责人: Curt Scharfe
• 金额: $ 8.42万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-07-10 至 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8767347
• 关键词: Amino Acids; Archives; Biochemical; Biochemical Genetics; Biological Assay; Birth; Blood; Blood specimen; CTSD gene; California; Candidate Disease Gene; Carbamoyl Transferases; Carbamoyl-Phosphate Synthase (Ammonia); Child; Citric Acid Cycle; Clinic; Clinical; Clinical Management; Code; Collaborations; Copy Number Polymorphism; Creatine; DNA; Data; Data Analyses; Defect; Detection; Diagnosis; Diagnostic; Dideoxy Chain Termination DNA Sequencing; Disease; Early Diagnosis; Early treatment; Energy Metabolism; Enzymes; Family; Gene Mutation; Genes; Genetic; Genetic Markers; Genomic DNA; Genotype; Health; Healthcare; Heterogeneity; Homeostasis; Inborn Errors of Metabolism; Individual; Inherited; Laboratories; Libraries; Liquid Chromatography; Long-Chain-Acyl-CoA Dehydrogenase; Measures; Metabolic; Metabolic Diseases; Metabolic Marker; Metabolism; Methods; Mitochondria; Mitochondrial Proteins; Mutation; Neonatal Screening; Newborn Infant; Nucleotides; Ornithine; Outcome; Parents; Pathway Analysis; Pathway interactions; Patients; Performance; Phenotype; Plasma; Play; Protocols documentation; Research; Role; Sampling; Screening Result; Secondary to; Sequence Analysis; Specificity; Specimen; Spottings; System; Technology; Test Result; Testing; Time; Variant; Whole Blood; Work; acylcarnitine; base; cost; disease phenotype; disorder subtype; follow-up; genetic variant; glutaric acidemia; improved; methylmalonic aciduria; new technology; next generation sequencing; novel; organic acid; programs; protein protein interaction; public health relevance; rapid detection; rapid diagnosis; screening; small molecule; tandem mass spectrometry; technology development

DESCRIPTION: Inborn errors of metabolism are detected at birth through public newborn screening (NBS) programs using tandem mass spectrometry (MS/MS), with early and definitive diagnosis critical to patient outcomes. However, a number of obstacles contribute to the delayed diagnosis of many diseases, including high rates of false positive screening results, the inability to differentiate disease subtypes, and biochemical heterogeneity leading to discrepancies in test results. Additionally, some disorders are not included in current NBS panels. Here we propose to investigate whether the combination of genetic and metabolite technologies established in our laboratories - based on targeted next-generation sequencing (NGS) and liquid chromatography tandem mass spectrometry (LC-MS/MS) - can shorten the time to diagnosis following an abnormal NBS result, as well as facilitate detection of additional disorders. We will focus on five disorders, three that exemplify the diagnostic challenges described above (VLCADD - very long-chain acyl-CoA dehydrogenase deficiency, GA1 - glutaric acidemia type I, and MMA - methylmalonic acidemia), and two that are not currently screened (OTCD - ornithine transcarbamoylase deficiency, and CPSD - carbamoylphosphate synthetase I deficiency). These disorders are all caused by mitochondrial enzyme defects that impact the homeostasis of a variety of pathways (e.g. amino acid and organic acid metabolism). Our genetic approach of the responsible disease genes utilizes complementary long padlock probes (cLPPs) for multiplex target capture and NGS, which will identify mutations and copy-number variations (CNVs) at clinical-grade accuracy and completeness and at very low cost. The metabolite approach involves a LC-MS/MS multiplex-marker panel combining both the standard NBS panel and additional markers (e.g. small molecule intermediates of energy metabolism). This strategy is based on the finding that metabolic changes detectable on the mitochondria-systems level, which are secondary to the primary enzyme defect identified in NBS, may play a role in the expression of the disease phenotype. Accordingly, our NGS panel of 524 genes not only contains the primary disease-causing genes but also functionally related candidate genes, which we have prioritized using a gene-network analysis of mitochondrial protein-protein interaction data. We will evaluate the performance of the two technologies using whole blood samples from children with confirmed disease and their parents, as well as archived dried blood spot (DBS) specimens from the patients collected at birth and obtained from the California Newborn Screening Program. Data analysis will utilize a new family-based, statistical sequence analysis to eliminate false-negative DNA variants, individual and combined metabolic marker analysis, and a systematic patient disease-phenotype analysis. In collaboration with the California NBS program, our results will contribute to the description and implementation of novel metabolic and genetic markers for screening of inherited metabolic conditions.

描述：先天性代谢缺陷是在出生时通过公共新生儿筛查 (NBS) 计划使用串联质谱 (MS/MS) 检测出来的，早期和明确的诊断对患者的预后至关重要。然而，许多障碍导致了许多疾病的延迟诊断，包括筛查结果假阳性率高、无法 区分疾病亚型和导致测试结果差异的生化异质性。此外，某些疾病未包含在当前的 NBS 小组中。在这里，我们建议研究我们实验室建立的基于靶向下一代测序 (NGS) 和液相色谱串联质谱 (LC-MS/MS) 的遗传和代谢技术的结合是否可以缩短 NBS 结果异常后的诊断时间，并促进其他疾病的检测。我们将重点关注五个方面 其中三种疾病例证了上述诊断挑战（VLCADD - 极长链酰基辅酶 A 脱氢酶缺乏症、GA1 - I 型戊二酸血症和 MMA - 甲基丙二酸血症），以及两种目前尚未筛查的疾病（OTCD - 鸟氨酸转氨甲酰酶缺乏症和 CPSD - 氨甲酰磷酸合成酶 I 不足）。这些疾病都是由线粒体酶缺陷引起的，影响多种途径的稳态（例如氨基酸和有机酸代谢）。我们对致病基因的遗传方法利用互补长挂锁探针 (cLPP) 进行多重目标捕获和 NGS，这将以临床级的准确性和完整性以及非常低的成本识别突变和拷贝数变异 (CNV)。代谢物方法涉及 LC-MS/MS 多重标记物组，结合了标准 NBS 组和其他标记物（例如能量代谢的小分子中间体）。该策略基于以下发现：在线粒体系统水平上可检测到的代谢变化（继发于 NBS 中确定的主要酶缺陷）可能在疾病表型的表达中发挥作用。因此，我们的 524 个基因的 NGS 面板不仅包含主要致病基因，还包含功能相关的候选基因，我们使用线粒体蛋白质-蛋白质相互作用数据的基因网络分析对这些基因进行了优先排序。我们将使用确诊疾病儿童及其父母的全血样本，以及从加州新生儿筛查计划获得的出生时收集的患者存档的干血斑 (DBS) 样本来评估这两种技术的性能。数据分析将利用新的基于家族的统计序列分析来消除假阴性 DNA 变异、个体和组合代谢标记物分析以及系统的患者疾病表型分析。与加州国家统计局项目合作，我们的结果将有助于描述和实施用于筛查遗传代谢状况的新型代谢和遗传标记。