Regulatory polymorphisms in human DBH affect peripheral gene expression and sympathetic activity

Abstract

Rationale: Dopamine β-hydroxylase (DBH) catalyzes the conversion of dopamine to norepinephrine in the central nervous system and peripherally. DBH variants are associated with large changes in circulating DBH and implicated in multiple disorders; yet causal relationships and tissue-specific effects remain unresolved.

Objective: To characterize regulatory variants in DBH, effect on mRNA expression, and role in modulating sympathetic tone and disease risk.

Methods and results: Analysis of DBH mRNA in human tissues confirmed high expression in the locus coeruleus and adrenal gland, but also in sympathetically innervated organs (liver>lung>heart). Allele-specific mRNA assays revealed pronounced allelic expression differences in the liver (2- to 11-fold) attributable to promoter rs1611115 and exon 2 rs1108580, but only small differences in locus coeruleus and adrenals. These alleles were also associated with significantly reduced mRNA expression in liver and lung. Although DBH protein is expressed in other sympathetically innervated organs, mRNA levels were too low for analysis. In mice, hepatic Dbh mRNA levels correlated with cardiovascular risk phenotypes. The minor alleles of rs1611115 and rs1108580 were associated with sympathetic phenotypes, including angina pectoris. Testing combined effects of these variants suggested protection against myocardial infarction in 3 separate clinical cohorts.

Conclusions: We demonstrate profound effects of DBH variants on expression in 2 sympathetically innervated organs, liver and lung, but not in adrenals and brain. Preliminary results demonstrate an association of these variants with clinical phenotypes responsive to peripheral sympathetic tone. We hypothesize that in addition to endocrine effects via circulating DBH and norepinephrine, the variants act in sympathetically innervated target organs.

Keywords: dopamine β-hydroxylase; gene expression/regulation; genetic association; genetic polymorphism; human; myocardial infarction.

Figure 1

A. Comparison of DBH mRNA expression across tissues including locus coeruleus (LC), adrenal (AD) and small bowel (SB) using a Taqman assay targeting the exon 11–12 boundary, standardized to GAPDH. Each group contains 6–8 individual samples of each tissue type. B. DBH mRNA expression targeting the exon 2–3 boundary, standardized with PGK1 using qRT-PCR for 16 LC, and 41 liver samples. In both cases, the qRT-PCR cycle thresholds are standardized to the house keeping gene; a higher Ct denotes lower expression.

Figure 2

Allelic DBH mRNA expression ratios measured at marker rs1108580 (black) and rs77905 (grey) in liver (A) and locus coeruleus and adrenal gland (B). The values are plotted as a ratio of major over minor allele for rs1108580; all AEI ratios for rs110580 were >1. As rs77905 is not in LD with rs1108580, AEI ratios ranged from >1 to <1 with near equal distribution in either direction; for comparison, these ratios are shown as absolute values in only one direction (>1). Livers heterozygous for rs1611115 are indicated with ↔. The horizontal line demarks the threshold for significant AEI (1.3). Each bar is the mean AEI measurement from one subject.

Figure 3. DBH mRNA levels grouped by genotype

The data represent the mean (n=2, average standard deviation of 0.27) of qRT-PCR cycle thresholds standardized to PGK1 mRNA as the house keeping gene. There was a significant difference between groups (p=0.03) and between AA and GG genotypes for rs1108580 (*p=0.005) in liver (A), and a significant effect of rs1611115 in liver (**p=0.0001) (B), and lung (p=0.02) (D). A one-way ANOVA showed an overall significant difference between the groups (p=0.01) when liver expression was grouped by number of minor alleles of rs1108580 and rs1611115 (C).