Linkage Disequilibrium Patterns and Functional Analysis of RGS4 Polymorphisms in Relation to Schizophrenia

Regulators of G-protein signaling (RGS) function as GTPase-activator proteins for heterotrimeric G-protein alpha (Gα) subunits and accelerate the hydrolysis of Gα-bound GTP.1 Thus, they shorten the duration of intracellular signaling of many G-protein–coupled receptors. To date, over 28 RGS proteins have been identified and most appear to have selective receptor targets.1,2 RGS proteins may therefore modulate intracellular effects of G-protein–coupled neurotransmitters. RGS proteins also appear to be responsive to stress, eg, regulator of G-protein signaling 4 (RGS4) expression is altered following chronic stress in rodents or following dexamethasone treatment of cells in vitro.3 Thus, a regulatory role for RGS4 in maintaining the milieu interior is plausible. RGS genes and their protein products are highly conserved, supporting critical roles in cellular function.4 The role of RGS proteins in several human diseases has been investigated. Drug addiction and brain injury may be associated with altered levels of some RGS mRNA species.5,6 Perhaps, the most extensive evidence relates to RGS4 and schizophrenia (SZ).7 It was initially suggested that RGS4 mRNA levels were significantly lower in postmortem samples from the dorsolateral prefrontal cortex of subjects with SZ, compared with matched controls.8 These studies suggest reduced expression of RGS4 in selected regions of the brain, and replicate studies have been published.9,10 In contrast, others did not detect significant case-control differences in postmortem brain samples.11 Because RGS4 maps to chromosome 1q23.3, a region implicated in prior SZ linkage studies,12 we conducted association studies using RGS4 genetic polymorphisms in family-based samples. These samples evaluate transmission of polymorphisms from heterozygous parents to affected offspring; significant transmission distortion is indicative of genetic association. Among case-parent “trio” families, we detected transmission distortions at 4 individual single-nucleotide polymorphisms (SNPs), and at haplotypes (combinations of these SNPs indicating chromosomal segments), in 2 independent samples of Caucasian ancestry.13 These samples were recruited separately by our group (Pittsburgh [PITT]) and by the National Institute of Mental Health (NIMH) Collaborative Genetics Initiative. The associated SNPs were denoted SNPs 1, 4, 7, and 18 (rs10917670, rs951436, rs951439, and rs2661319, respectively). However, the alleles associated in the PITT sample differed from those in the NIMH sample, leading to associations with 2 different haplotypes in these samples (haplotypes G-G-G-G, and A-T-A-A, respectively). The haplotypes together account for over 80% of the variation in this genomic region in US Caucasian samples. Suggestive transmission distortion with the A-T-A-A haplotype was also detected in an independent Indian sample gathered by our group, prompting us to propose RGS4 as a putative susceptibility gene for SZ.13 The associated region spans the 5′ upstream sequence and the first exon of the gene (6.09 kb). Since the initial report, 3 independent case-control comparisons and 2 family-based analysis have reported significant associations at the same SNPs, but the associated alleles have also differed between studies.14–18 Significant association with any of these SNPs was not detected in a Brazilian,19 a Chinese,20,21 a US sample,22 a UK sample,23,24 or a Japanese sample.25 In view of the discrepancies, we conducted meta-anlaysis of all available genotypes. Our published meta-analyses suggest risk due to at least 2 common haplotypes in the presence of heterogeneity.26 Two other meta-analyses that evaluated smaller subsets of these samples did not find significant association.27,28 There are several explanations for these divergent results, apart from stochastic variation. If an association exists, there could be one unidentified risk allele against the background of these 2 common haplotypes. Alternatively, 2 different (unidentified) risk variants could exist on these haplotypes. Risk may also be conferred by these haplotypes for different subgroups of SZ, and differences in ascertainment criteria could thus lead to divergent associations. Because of linkage disequilibrium (LD), polymorphisms in close proximity at a genomic segment can be correlated at the population level. Thus, the SNPs analyzed to date may indicate primary association with another polymorphism. Hence, the putative primary association, if present, could be in the intronic regions, the promoter sequences, or the upstream or downstream regions. To help discriminate among these possibilities, we identified all common RGS4 polymorphisms and evaluated patterns of LD. These SNPs were genotyped in our family-based and case-control samples. No exonic variations were detected in the initial study, including the postmortem samples used for the expression analyses (n = 36). Thus, the functional impacts of the associations are uncertain. Because some of the associated SNPs and haplotypes are located in the upstream region of RGS4, we evaluated the possibility that they may alter transcription. Using in vitro dual-luciferase reporter systems, the promoter activities of selected SNPs and haplotypes were analyzed.