SLC1A4, SLC1A5, SLC6A5 and SLC6A9 were located on chromosome 2p13-15, 19q13.3, 11p15.2-p15.1, and 1p33, respectively. Suggestive evidence for linkage of chromosome 2p14-p13, where SLC1A4 is located, with schizophrenia has been reported in schizophrenia families from Palau and Ireland [35, 36]. However, the subsequent mutation screening failed to find any sequence polymorphism segregated with the illness in the SLC1A4 region of the Palauan families . In addition, negative association of SLC1A4 with schizophrenia was reported in the German population . There has been no linkage with schizophrenia reported to the chromosome regions where SLC1A5, SLC6A5 or SLC6A9 are located . Moreover, exclusion of linkage between schizophrenia and SLC1A5 in 23 English and Icelandic schizophrenia families was reported . Recently, negative associations of schizophrenia with polymorphisms in SLC6A9 and SLC6A5 were reported in the Chinese and the German population, respectively [40, 41]. We investigated the association of SLC1A4, SLC1A5, SLC6A5 and SLC6A9 genes with schizophrenia in the Japanese population by analysing total 21 common SNPs.
Since the frequencies of genotyped SNPs are over 0.3, the expected detection powers of the four genes are over 0.80, assuming the genotype relative risk of 1.8. However, assuming lower genotype relative risk of 1.5 or 1.3, the expected detection powers for the four genes dropped to 0.50 – 0.53 or 0.24 – 0.25, respectively. Consequently, the negative finding for genes and SNPs excluded from the analyses using the Full-size Sample Set in this study may be due to type II error at lower relative risks, and they need to be investigated further in an enlarged sample size.
Out of the 21 SNPs analyzed, two within SLC1A4, (SNP4 and SNP6, 330 cases and 319 controls) and one within SLC6A5, (SNP5, 328 cases and 307 controls) have recently been reported to show no association with schizophrenia in the German population [38, 41]. We also observed no association of these SNPs with the disease in our Screening Sample Set. The SNP1 in SLC6A5 of which we observed a significant association with the disease, was not included in the report mentioned above.
In LD analysis of the initial screening of the 21 SNPs distributed in the four genes, modest LD (D' > 0.3) was observed in all combinations of adjacent SNPs in controls except for the combinations of SNP4-SNP5 of SLC1A4 and SNP2-SNP3 of SLC6A5, suggesting recombination hot spots in the two regions (6.6 kb and 7.6 kb, respectively) (Figure 2). We compared the LD structure to the publically open database, HapMap . The LD gap we observed in the SLC6A5 region was not observed in the HapMap LD structure from either Japanese or Chinese population data (D' = 0.817 and D' = 1, respectively). The other LD gap, which was observed in the SLC1A4 region, failed to be compared due to the absence of the novel SNP we found.
We observed significant single-marker associations in SNP2 and SNP3 of SLC1A4 in the Screening Sample Set. However, we failed to confirm these findings in the Full-size Sample Set. We attribute to type I error due to the small sample size used in the Screening Sample Set. On the other hand, the single-marker association of SNP1 (rs894747) in SLC6A5 region, although it does not show the significant association with the disease in the independent 300 case and 320 control samples (0.092), it does show the significant association in the Full-size Sample Set (P = 0.018). We consider that the nonsignificant result observed in the enlarged samples may be due to the small sample size. SNP1 is located in the intergenic region, 2,355-bp upstream from the transcription start site. In the negative association report of SLC6A5 in German population described above, four SNPs and one short-tandem-repeat distributed in intron 1~intron 11, but no SNP located in the upstream region were analysed . In our Full-size Sample Set, the G allele was more frequently observed in schizophrenics (44.4%) than in controls (38.6%). Therefore, the G allele may be in LD with a risk allele for schizophrenia (odds ratio, 1.27; 95% confidence interval, 1.04~1.55). We conclude that at least one susceptibility locus for schizophrenia is located within or nearby SLC6A5, whereas SLC1A4 SLC1A5 and SLC6A9 are unlikely to be major susceptibility genes for schizophrenia in the Japanese population. No potential regulatory elements were previously identified in the region where SNP1 is located . It is necessary to search for functional SNPs in the haplotype block where SNP1 is located. A copy number variation (CNV) has been reported in the European population on the chromosome 11p15.1, containing exon 15 of SLC6A5 . None of the 6 SNPs we genotyped is located within the CNV. Although the frequency of the CNV in the Japanese population is unknown, SNP1 may be associated with the variant devoid of exon 15, which is a strong candidate of the susceptible allele. Therefore, it is necessary to test the association of the CNV with schizophenia in Japanese sample sets. The positive association observed in SLC6A5 also needs to be validated in different ethnic populations.