基于二步化学衍生专利技术筛查孤独症谱系障碍临床各亚型尿液代谢标志物

Autism spectrum disorder (ASD) represents a neurodevelopmental disorder with high incidence of disability. The global incidence of ASD increases greatly in recent years. However, ASD is still diagnosed exclusively through observation and standardized behavioral scales that may involve a great variability, due to a lack of laboratory biomarkers, which could greatly aid clinicians in providing earlier and more reliable diagnoses. Metabolomics studies based on gas chromatography-mass spectrometry (GC-MS) have found a number of metabolites correlated with ASD, however, some results reported among these studies were not consistent with each other because of a lack of stratifying ASD into homogeneous subgroups, as well as methodological limitations including lack in standardization for pretreatment and separation methods. Our group has developed a patented two-step chemical derivatization method for pretreatment of urine sample with higher efficiency under mild conditions compared with traditional methods, which protects thermal instability metabolites from decomposition, enabling simultaneous analysis of hundreds of metabolites including amino acids, organic acids and fatty acids and so on. Therefore, the current project intends to develop a standardized pretreatment and GC-MS method for comprehensive metabolomics analysis of urine samples between health controls and several ASD subtypes stratified by their phenotypes, to further obtain several specific metabolites, which could be usable as biomarkers for earlier and more reliable diagnoses in asymptomatic conditions.

孤独症谱系障碍（ASD）是一种社会致残率较高的神经发育障碍，且患病率呈急剧上升趋势。然而ASD临床诊断主要以观察、行为量表评估等主观指标为主，可变性大，缺乏客观的实验依据。近年来，基于气相色谱-质谱（GC-MS）的代谢组学研究报道了多个可能与ASD相关的代谢产物。但是，这些研究没有对ASD进行分型分类，且前处理条件苛刻，得到的结果彼此间差异较大。课题组前期研究获得了尿液二步衍生法前处理专利技术，与传统方法相比，该方法条件温和，效率高，保护了易分解成分，能同步分析氨基酸、有机酸、脂肪酸等上百个小分子代谢产物，扩展了代谢物筛查范围。为了获得稳定可靠的ASD代谢标志物，本项目拟从ASD临床表型分型入手，将ASD分为具有相同或者相近临床表型的各类ASD临床亚型，然后，基于课题组的专利技术，建立标准化的GC-MS分析方法，筛查ASD各类临床亚型代谢标志物，为ASD的早期筛查、诊断提供实验依据。

孤独症谱系障碍(autism spectrum disorder,ASD)是一种社会致残率较高的广泛性神经发育障碍，患病率呈逐年上升趋势，其临床诊断主要以行为量表评估等主观指标为主，缺乏客观的实验室诊断指标。大量代谢组学研究发现ASD同时伴有多条代谢路径紊乱，但由于ASD个体差异大，至今尚未获得特异性强的代谢产物可单独作为筛查诊断ASD的标志物，甚至还出现争议的研究结果。因此，需要对ASD进行分型分类研究。.课题组通过性别分组病例对照研究发现，女性ASD患儿中，肌酸、肌酐等代谢物的波动情况较女性正常儿有显著差异，而这种情况在男性患儿与男性对照组间没有差异。肌酸合成与转运过程中，雄激素能够促进肌酸转运蛋白将肌酸从血液转运到细胞，而雌激素抑制这种转运。当有芳香酶抑制剂的作用下，雄激素向雌激素转化受阻，造成雄激素累积，雌激素下降，从而大大促进了肌酸的转运。在女性患儿尿液中，我们发现一个与睾内酯（芳香酶抑制剂）结构相似的代谢物7alpha-hydroxytestololactone显著升高，推测该物质可能具有类似睾内酯的作用，能够抑制雄激素向雌激素转化，促进血液肌酸转运至细胞，进一步合成肌酐，从而在尿液中的肌酸显著降低，肌酐显著升高。相关的分子机制可能是众多导致ASD性别差异的原因之一。用肌酐/肌酸比值做ROC曲线分析，发现曲线下面积达到0.913，表明肌酐/肌酸比值可作为早期筛查ASD的一个客观指标。.基于这些代谢指标，与苏州博源医疗科技有限公司合作开发均相酶免疫检测试剂盒。通过靶向检测和绝对定量分析、验证，与代谢组学研究结果一致。目前试剂盒开发与注册都已接近完成，正在通过检测正常儿童样本建立参考范围。同时课题组计划开展更大样本量的验证工作，以保证结果的准确性，争取早日用于ASD的早期筛查预警。