Investigation of G72 (DAOA) expression in the human brain.
BACKGROUND
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Benzel
Isabel
I
Psychiatry, GlaxoSmithKline, New Frontiers Science Park, Harlow, Essex, CM19 5AW, UK. isabel.benzel@gmx.net
Kew
James N C
JN
Viknaraja
Ramya
R
Kelly
Fiona
F
de Belleroche
Jacqueline
J
Hirsch
Steven
S
Sanderson
Thirza H
TH
Maycox
Peter R
PR
D001714:Bipolar Disorder / Q000235:genetics*; D001921:Brain / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics*; D002460:Cell Line; D002874:Chromosome Mapping; D002882:Chromosomes, Human, Pair 13 / Q000235:genetics; D003605:D-Amino-Acid Oxidase / Q000235:genetics; D004789:Enzyme Activation / Q000235:genetics; D020022:Genetic Predisposition to Disease / Q000235:genetics; D018698:Glutamic Acid / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D015336:Molecular Probe Techniques; D011110:Polymorphism, Genetic / Q000235:genetics; D011401:Promoter Regions, Genetic; D054730:Protein Interaction Domains and Motifs / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics*; D012559:Schizophrenia / Q000235:genetics*
D002352:Carrier Proteins; C475071:DAOA protein, human; D047908:Intracellular Signaling Peptides and Proteins; D012333:RNA, Messenger; D018698:Glutamic Acid; C417983:DAO protein, human; D003605:D-Amino-Acid Oxidase