Expression of the Longest RGS4 Splice Variant in the Prefrontal Cortex Is Associated with Single Nucleotide Polymorphisms in Schizophrenia Patients

Abstract

The Regulator of G protein signaling 4 (RGS4) gene is a candidate susceptibility gene for schizophrenia (SCZ). Previous studies showed that the mRNA level of the longest splice variant RGS4-1 was decreased in the dorsolateral prefrontal cortex (DLPFC) of SCZ patients compared with healthy controls. In this pilot study, we examined the possible mechanisms of RGS4-1 mRNA reduction in SCZ. We genotyped SNP1 (rs10917670), rs2661347, SNP4 (rs951436), SNP7 (rs951439), SNP18 (rs2661319), and rs10799897 (SNP9897) and tested the methylation status of CpG islands of the RGS4 gene in the postmortem DLPFC samples obtained from subjects with SCZ and bipolar disorder as well as healthy controls. RGS4-1 mRNA level was associated with five SNPs (SNP1, rs2661347, SNP4, SNP7, and SNP18) and their haplotypes but not with SNP9897. In addition, this study revealed that RGS4-1 mRNA was low in subjects with specific genotypes of SNP1, rs2661347, SNP4, SNP7, and SNP18. Lower RGS4-1 mRNA expression in the DLPFC of SCZ is associated with SNPs in the 5' regulatory region of the RGS4 gene but not with the methylation status of its CpG islands.

Keywords: allele; bipolar disorder; genotype; postmortem; psychiatric; regulatory region.

Figure 1

Location of SNPs in the RGS4 gene. SNP1, SNP4, and SNP7 are located in the 5′ regulatory region of RGS4. SNP18 is in the second intron and SNP9897 is in the fourth intron of the RGS4 gene.

Figure 2

Effects of SNP1 and rs2661347 on RGS4-1 mRNA expression levels. RGS4-1 mRNA expression level in genotype TT of SNP1 was significantly lower than that of the genotype CC [F(3,77) = 4.20, overall p = 0.0083, SNP1 genotype p = 0.0263] (A). The genotype TT of rs2661347 had significantly lower expression level of RGS4-1 mRNA compared to the genotype AA [F(3,77) = 4.69, overall p = 0.0046, rs2661347 genotype p = 0.0141] (B).

Figure 3

Effects of SNP1 and rs2661347 genotypes on RGS4-1 mRNA expression among the groups. In the schizophrenia group (SCZ), the RGS4-1 mRNA expression level in the subjects with genotype CC of SNP1 were lower than the people in the normal control (NC) group with same genotype CC (adjusted p = 0.0324) of SNP1 (A). In the SCZ group, the patients with the genotype AA of rs2661347 (p = 0.0984) tended to have lower mRNA expression than those with the same genotype in the NC group (B). Boxes represent 25th and 75th percentile distribution. Bars outside the box represent the SD.

Figure 4

Association of RGS4-1 mRNA expression levels with the genotypes of SNP4 and SNP7. The genotype AA of SNP4 was associated with the lower expression level of RGS4-1, and CC was related with the higher expression of RGS4-1 [F(3,77) = 4.93, overall p = 0.0035, SNP4 genotype p = 0.0104] (A). The genotype TT of SNP7 was associated with the lower expression level, and CC with higher expression level of RGS4-1 [F(3,77) = 4.20, overall p = 0.0083, SNP7 genotype p = 0.0263] (B).

Figure 5

Effects of SNP4 and SNP7 genotypes on RGS4-1 mRNA expression among the groups. (A). In the schizophrenia (SCZ) group, the subjects with genotype CC of SNP4 tended to have lower mRNA expression level of RGS4-1 than the subjects with the same genotype CC of SNP4 in NC (p = 0.0984) (B). The mRNA of RGS4-1 in SCZ patients with genotype CC of SNP7 was lower than those of the individuals with the same genotype CC in NC (p = 0.0373).

Figure 6

Effects of SNP18 genotypes on RGS4-1mRNA expression. (A) Among all the groups, the individuals with the genotype AA of SNP18 had lower mRNA expression of RGS4-1 compared with the subjects with genotype GG of SNP18 [F (3,77) = 3.86 overall p = 0.0126; SNP18 genotype p = 0.0413]. (B) In SCZ, the subjects with genotype GG of SNP18 had lower mRNA expression level of RGS4-1 compared with the subjects with the same genotype GG of SNP18 in NC (p = 0.0393).

Figure 7

Effects of haplotypes on RGS4-1 mRNA expression. (A) Effect of haplotype 1(Hap1) on the mRNA expression of RGS4-1. The individuals with diplotype of two non-Hap1 have lower RGS4-1 mRNA expression level (p = 0.0413). (B) In the SCZ group, non-Hap4 carriers have lower RGS4-1 mRNA expression level compared to those with non-Hap4 in NC (p = 0.0192). Hap1 is CACCG, Hap4 is TTATA.

Figure 8

Schematic representation of linkage disequilibrium (LD) between RGS4 SNPs in the SCZ, BPD, and NC groups. The LD block (thick black outline) consisting of four SNPs spans <1 kb (hence the Haploview program shows it as 0 kb). The number shown in each box in the figure corresponds to r² which measures LD. r² Values were calculated from the genotype data from the entire samples by Haploview. 1 = rs10917670 (SNP1), 2 = rs2661347, 3 = rs951436(SNP4), 4 = rs951439(SNP7), 5 = rs2661319(SNP18), 6 = rs10799897.