Benzel Isabel, Kew James N C, Viknaraja Ramya, Kelly Fiona, de Belleroche Jacqueline, Hirsch Steven, Sanderson Thirza H, Maycox Peter R
Psychiatry, GlaxoSmithKline, New Frontiers Science Park, Harlow, Essex, CM19 5AW, UK.
BMC Psychiatry. 2008 Dec 11;8:94. doi: 10.1186/1471-244X-8-94.
Polymorphisms at the G72/G30 locus on chromosome 13q have been associated with schizophrenia or bipolar disorder in more than ten independent studies. Even though the genetic findings are very robust, the physiological role of the predicted G72 protein has thus far not been resolved. Initial reports suggested G72 as an activator of D-amino acid oxidase (DAO), supporting the glutamate dysfunction hypothesis of schizophrenia. However, these findings have subsequently not been reproduced and reports of endogenous human G72 mRNA and protein expression are extremely limited. In order to better understand the function of this putative schizophrenia susceptibility gene, we attempted to demonstrate G72 mRNA and protein expression in relevant human brain regions.
The expression of G72 mRNA was studied by northern blotting and semi-quantitative SYBR-Green and Taqman RT-PCR. Protein expression in human tissue lysates was investigated by western blotting using two custom-made specific anti-G72 peptide antibodies. An in-depth in silico analysis of the G72/G30 locus was performed in order to try and identify motifs or regulatory elements that provide insight to G72 mRNA expression and transcript stability.
Despite using highly sensitive techniques, we failed to identify significant levels of G72 mRNA in a variety of human tissues (e.g. adult brain, amygdala, caudate nucleus, fetal brain, spinal cord and testis) human cell lines or schizophrenia/control post mortem BA10 samples. Furthermore, using western blotting in combination with sensitive detection methods, we were also unable to detect G72 protein in a number of human brain regions (including cerebellum and amygdala), spinal cord or testis. A detailed in silico analysis provides several lines of evidence that support the apparent low or absent expression of G72.
Our results suggest that native G72 protein is not normally present in the tissues that we analysed in this study. We also conclude that the lack of demonstrable G72 expression in relevant brain regions does not support a role for G72 in modulation of DAO activity and the pathology of schizophrenia via a DAO-mediated mechanism. In silico analysis suggests that G72 is not robustly expressed and that the transcript is potentially labile. Further studies are required to understand the significance of the G72/30 locus to schizophrenia.
超过十项独立研究表明,13号染色体q臂上G72/G30基因座的多态性与精神分裂症或双相情感障碍有关。尽管遗传研究结果非常可靠,但预测的G72蛋白的生理作用至今尚未明确。最初的报告表明G72是D-氨基酸氧化酶(DAO)的激活剂,支持精神分裂症的谷氨酸功能障碍假说。然而,这些发现随后未能得到重复验证,而且关于内源性人类G72 mRNA和蛋白表达的报告极为有限。为了更好地理解这个假定的精神分裂症易感基因的功能,我们试图在相关人类脑区证明G72 mRNA和蛋白的表达。
通过Northern印迹法、半定量SYBR-绿和Taqman RT-PCR研究G72 mRNA的表达。使用两种定制的特异性抗G72肽抗体,通过蛋白质印迹法研究人组织裂解物中的蛋白表达。对G72/G30基因座进行深入的计算机分析,以尝试识别能够深入了解G72 mRNA表达和转录本稳定性的基序或调控元件。
尽管使用了高灵敏度技术,我们仍未能在多种人类组织(如成人大脑、杏仁核、尾状核、胎儿大脑、脊髓和睾丸)、人类细胞系或精神分裂症/对照尸检BA10样本中检测到显著水平的G72 mRNA。此外,通过蛋白质印迹法结合灵敏的检测方法,我们也未能在多个人类脑区(包括小脑和杏仁核)、脊髓或睾丸中检测到G72蛋白。详细的计算机分析提供了多条证据,支持G72明显低表达或不表达的情况。
我们的结果表明,在本研究分析的组织中通常不存在天然G72蛋白。我们还得出结论,在相关脑区缺乏可证明的G72表达不支持G72通过DAO介导的机制调节DAO活性和精神分裂症病理的作用。计算机分析表明G72表达不强烈,且转录本可能不稳定。需要进一步研究以了解G72/30基因座对精神分裂症的意义。