Allele specific analysis of the ADRBK2 gene in lymphoblastoid cells from bipolar disorder patients

Abstract

G-protein coupled receptor kinase-3 (GRK3), translated from the gene, ADRBK2 has been implicated as a candidate molecule for bipolar disorder through multiple, converging lines of evidence. In some individuals, the ADRBK2 gene harbors the A-haplotype, a collection of single nucleotide polymorphisms (SNPs) previously associated with an increased risk for bipolar disorder. Because the A-haplotype encompasses the ADRBK2 promoter, we hypothesized that it may alter the regulation of gene expression. Using histone H3 acetylation to infer promoter activity in lymphoblastoid cells from patients with bipolar disorder, we examined the A-haplotype within its genomic context and determined that at least four of its SNPs are present in transcriptionally active portions of the promoter. However, using chromatin immunoprecipitation followed by allele-specific PCR in samples heterozygous for the A-haplotype, we found no evidence of altered levels of acetylated histone H3 at the affected allele compared to the common allele. Similarly, using a transcribed SNP to discriminate expressed ADRBK2 mRNA strands by allele of origin; we found that the A-haplotype did not confer an allelic-expression imbalance. Our data suggest that while the A-haplotype is situated in active regulatory sequence, the risk-associated SNPs do not appear to affect ADRBK2 gene regulation at the level of histone H3 acetylation nor do they confer measurable changes in transcription in lymphoblastoid cells. However, tissue-specific mechanisms by which the A-haplotype could affect ADRBK2 in the central nervous system cannot be excluded.

Figure 1

(A) 50kb segment of the ADRBK2 promoter. Box indicates Exon 1. Dots indicate the position of five A-haplotype SNPs from left to right: 19, 23, 28, 29 and 32. (B) Enrichment of ac-H3 across the ADRBK2 locus in lymphoblastoid cells. Horizontal axis indicates Chr 22 base pair coordinate and vertical axis is fold enrichment compared to ALB baseline. Enrichment of ac-H3 did not differ by diagnosis and for purposes of locus tiling, both control and A-haplotype data were averaged together to generate the ac-H3 enrichment profile (N=5) (C) Enrichment of ac-H3 in lymphoblastoid cells at two sites in the GAPDH promoter (positive controls) (D) Lack of ac-H3 enrichment at two regions of ADRBK2 promoter duplication (N=3). Coordinates in A, B and D correspond to chr 22 location, aligned by sequence homology. Vertical lines indicate locations of each SNP (B) or homologous flanking sequence (D) relative to ac-H3 state. (E) Enrichment of ac-H3 across the ADRBK2 locus in fetal brain tissue. (F) Enrichment of ac-H3 across the ADRBK2 locus in Y79 retinoblastoma cells. (G) SNP position relative to species conservation at the ac-H3 enriched 10kb section of the ADRBK2 promoter.

Figure 2

Allele-specific chromatin recovery was verified using anti-ac-H3 antibodies and allele-specific PCR primers directed at rs220030 from the imprinted gene, SNRPN. In the four heterozygotes identified, the paternal allele was more strongly associated with ac-H3, leading to marked imbalances in allelic association with the precipitated chromatin and subsequent recovery efficiency.

Figure 3

No evidence of allele-specific chromatin recovery at either (A) SNP 28 or (B) SNP 32 in heterozygotes bearing the A-haplotype. Both common and rare alleles were detected by allele-specific PCR in equal amounts after ChIP selection using anti ac-H3 antibodies to precipitate transcriptionally active chromatin. For SNP 28 N=11, for SNP 32 N=8. Three A-haplotype bearing subjects do not carry a rare SNP 32 allele. The differences in averages between alleles were not statistically significant (p>0.30) in either case as measured by a paired two-tailed T-test.

Figure 4

Allele-specific ADRBK2 expression was measured in lymphoblastoid cells from four A-haplotype heterozygous subjects using allele-specific PCR probes directed at the transcribed T/C SNP rs133839. The common T allele is cis to the A-haplotype. RNA was converted to cDNA and normalized against genomic DNA for each subject. Error bars represent SEM. There was no significant difference in allelic gene expression in any of the subjects. Differences between subjects are due to variability in the content of genomic DNA in the samples, whereas cDNA content between samples was similar.