Mehling Lena-Maria, Spottke Annika, Heidbreder Anna, Young Peter, Madea Burkhard, Hess Cornelius, Courts Cornelius
Institute of Forensic Medicine, University Hospital Bonn, Stiftsplatz 12, 53111, Bonn, Germany.
Department of Neurology, University Hospital Bonn, Sigmund-Freud-Straße 25, 53127, Bonn, Germany.
Int J Legal Med. 2017 Sep;131(5):1261-1270. doi: 10.1007/s00414-017-1609-3. Epub 2017 May 22.
Gamma-hydroxybutyric acid (GHB) acts as an agonist of the GABA receptor, where GHB induces a depressant effect in the central nervous system. Besides its therapeutic application, GHB is also used as a date rape drug. However, the detection of GHB ingestion proves to be difficult due to its narrow detection window. The aim of this pilot study was to assess differential gene expressions after GHB intake to identify potential biomarkers for the detection of GHB intake. To this aim, alteration in gene expression of ALDH5A1, AKR7A2, EREG, and PEA15 was investigated via quantitative PCR (qPCR). Data normalization was based on a previously established and empirically derived normalization strategy. Blood samples of patients (n = 3) therapeutically taking sodium oxybate solution (GHB) and of donors without GHB intake (n = 49) were analyzed and compared. All qPCR procedures and results are reported according to the MIQE guidelines. Investigation of suitable reference genes using established algorithms suggested PPIB and FPGS as best-suited normalizers. Alterations in gene expression relating to GHB intake could not be confirmed to a forensically sufficient degree. However, significant differences in expression of EREG in the control group were observed, when time-point of sample collection was considered, indicating circadian rhythm. The study's main limitation is the small number of study subjects. Herein, we are first to present an empirically derived strategy for a robust normalization of qPCR data from the analysis of GHB-induced gene expression in human blood. We present results of the analysis of differential expression of ALDH5A1, AKR7A2, EREG, and PEA15 in the GHB-negative population. Finally, we report our findings on the effect of GHB intake on the expression of these genes and their presumable potential as GHB biomarkers.
γ-羟基丁酸（GHB）作为γ-氨基丁酸（GABA）受体的激动剂，在中枢神经系统中诱导抑制作用。除了其治疗用途外，GHB还被用作约会强奸药物。然而，由于其检测窗口期狭窄，检测GHB摄入情况很困难。这项初步研究的目的是评估GHB摄入后基因表达的差异，以确定检测GHB摄入的潜在生物标志物。为此，通过定量聚合酶链反应（qPCR）研究了乙醛脱氢酶5A1（ALDH5A1）、醛酮还原酶7A2（AKR7A2）、表皮调节素（EREG）和增殖相关蛋白15（PEA15）基因表达的变化。数据标准化基于先前建立并经实证推导的标准化策略。对接受γ-羟基丁酸钠溶液（GHB）治疗的患者（n = 3）和未摄入GHB的献血者（n = 49）的血样进行了分析和比较。所有qPCR程序和结果均按照《MIQE指南》报告。使用既定算法对合适的内参基因进行研究，结果表明肽基脯氨酰异构酶B（PPIB）和叶酸多聚谷氨酸合成酶（FPGS）是最适合的标准化基因。与GHB摄入相关的基因表达变化在法医层面上未能得到充分证实。然而，考虑样本采集时间点时，对照组中EREG的表达存在显著差异，表明存在昼夜节律。该研究的主要局限性是研究对象数量较少。在此，我们首次提出一种基于实证推导的策略，用于对分析人血中GHB诱导基因表达的qPCR数据进行稳健标准化。我们展示了GHB阴性人群中ALDH5A1、AKR7A2、EREG和PEA15差异表达分析的结果。最后，我们报告了GHB摄入对这些基因表达的影响及其作为GHB生物标志物的潜在可能性的研究发现。
