Genetic and physiological data implicating the new human gene G72 and the gene for D-amino acid oxidase in schizophrenia

Abstract

A map of 191 single-nucleotide polymorphism (SNPs) was built across a 5-Mb segment from chromosome 13q34 that has been genetically linked to schizophrenia. DNA from 213 schizophrenic patients and 241 normal individuals from Canada were genotyped with this marker set. Two 1,400- and 65-kb regions contained markers associated with the disease. Two markers from the 65-kb region were also found to be associated to schizophrenia in a Russian sample. Two overlapping genes G72 and G30 transcribed in brain were experimentally annotated in this 65-kb region. Transfection experiments point to the existence of a 153-aa protein coded by the G72 gene. This protein is rapidly evolving in primates, is localized to endoplasmic reticulum/Golgi in transfected cells, is able to form multimers and specifically binds to carbohydrates. Yeast two-hybrid experiments with the G72 protein identified the enzyme d-amino acid oxidase (DAAO) as an interacting partner. DAAO is expressed in human brain where it oxidizes d-serine, a potent activator of N-methyl-D-aspartate type glutamate receptor. The interaction between G72 and DAAO was confirmed in vitro and resulted in activation of DAAO. Four SNP markers from DAAO were found to be associated with schizophrenia in the Canadian samples. Logistic regression revealed genetic interaction between associated SNPs in vicinity of two genes. The association of both DAAO and a new gene G72 from 13q34 with schizophrenia together with activation of DAAO activity by a G72 protein product points to the involvement of this N-methyl-d-aspartate receptor regulation pathway in schizophrenia.

Figure 1

Strategy used to identify potential schizophrenia genes in chromosome 13q. (a) Schizophrenia linked region (50 cM) derived from several studies (–15). The position of the 5-Mb region selected for association studies is marked. (b) Allele frequencies association tests performed by Armitage trend test are shown as –Log (P value) for 191 markers. Thresholds for P values of 5E-02 (straight line) and 1E-02 (dotted line) are indicated. Statistically significant markers (P value lower than 5E-02) obtained in this study are shown: 99-13064/328 (M-1), 99-62753/178 (M-2), 99-62654/305 (M-3), 99-26234/336 (M-4), 99-26189/164 (M-5) in Bin B and 99-16105/152 (M-12), 99-5919/215 (M-22) in Bin A. These last two markers present P values lower than 1E-02. (c) Location of genes found in the 5-Mb analyzed region. Predicted genes found in the proximal 2-Mb portion of the region are shaded red (1, CanIon; 2, IT6BL1; 3, FHF4; 4, TPP2; 5, PHSP8; 6, XPG; 7, SLC10A2); unpredicted genes found in the distal 3 Mb described in this study are shaded blue (8, G90; 9, G72; 10, G30).

Figure 2

Structure of LG72 protein and evolution of its putative orthologues in primates. DNA from four different primates (chimpanzee, gorilla, gibbon, and rhesus monkey) were amplified by using specific primers from the human G72 gene. Amplicons were sequenced, and exons were defined by homology to the corresponding human exons. Alignment of theoretical polypeptide sequences corresponding to pLG72 in human and these different primate species is shown. pLG72 translation in chimpanzee and gorilla is altered by a 4-nt insertion causing a frameshift and a premature stop. In rhesus monkey, the insertion of one T at the end of Exon M causes a frameshift and a premature stop.

Figure 3

In vitro and ex vivo translation of the G72 candidate gene. (a) Coupled in vitro transcription/translation assays in the presence of [³⁵S]-Met for both LG72 (lane 1) and LG30 (lane 2) cDNAs, unprimed lysates (lane 3), and a luciferase cDNA control (lane 4). (b) Expression vectors harboring the nontagged LG72 cDNA either in the antisense orientation (−) (lane 2) or in the sense orientation (+) (lane 3) were transfected into COS-7 cells and the expressed proteins analyzed with a purified anti-pLG72 serum. A His-Xpress-pLG72 recombinant protein (5 ng) purified from bacteria was run as a control (time exposure for this lane is shorter than for the two others). The His-Xpress tag is 33 amino acids long.

Figure 4

Oxidation of d-serine by DAAO in the presence of LG72 protein. Activity is expressed as amount H₂O₂ generated as a result of oxidation of d-serine. The concentration of DAAO was constant (45 ng/μl), the concentrations of pLG72 varied from 0 to 680 ng/μl, and BSA was added to maintain the equal total protein concentration. The curves represent the mean of the three measures for each incubation point. Confidence interval (“error bars”) = mean (three measures) plus or less 1.96. *, Standard deviation (three measures).