A single nucleotide polymorphism in dopamine beta hydroxylase (rs6271(C>T)) is over-represented in inflammatory bowel disease patients and reduces circulating enzyme

Abstract

Inflammatory bowel diseases (IBD) are associated with altered neuronal regulation of the gastrointestinal (GI) tract and release of norepinephrine (NE). As sympathetic innervation of the GI tract modulates motility, blood flow, and immune function, changes in NE signaling may alter the risk of developing IBD. Dopamine beta-hydroxylase (DβH), the enzyme responsible for NE production, has been suggested to play a critical role in IBD, however the exact mechanism is unknown. We hypothesized that genetic variants of DβH could increase the risk of IBD. We performed genetic analysis on 45 IBD patients and 74 controls. IBD patients were screened by targeted exome sequencing and compared with NeuroX DβH single nucleotide polymorphism (SNP) genotyping data of the controls. Serum DβH protein levels for 15 IBD patients and 13 controls were evaluated using immunoblots and competitive ELISA. Seven SNPs were observed from DβH targeted exome sequencing in the 45 IBD patients. A single non-synonymous SNP, rs6271 (Arg549Cys), had a significant association with IBD patients; the odds ratio was a 5.6 times higher SNP frequency in IBD patients compared to controls (p = 0.002). We also examined the function and availability of the protein in both the IBD and control patients' sera bearing DβH Arg549Cys. Both control and IBD subjects bearing the heterozygote allele had statistically lower DβH protein levels while the intrinsic enzyme activity was higher. This is the first report of a noradrenergic genetic polymorphism (rs6271; Arg549Cys) associated with IBD. This polymorphism is associated with significantly lower levels of circulating DβH.

Fig 1. Map of DβH gene with all exonic SNPs and rs# observed in the targeted deep sequencing of IBD patients.

Fig 2. Expression results for DβH in sera from IBD subjects with different genotypes of Arg549Cys.

(A) The relative expression levels of DβH among wild-type, heterozygous and homozygous IBD sera. Numbers at the top refer to patient designators. (B) The relative expression levels of DβH controlled for transferrin among wild-type, heterozygous and homozygous IBD serums. Overall comparison of wild-type and heterozygous subject sera are shown as mean±SEM (p = 0.0091; as measured with unpaired t-test). C) The distribution of DβH quantity in both study cohorts as estimated from ELISA assays. Based on the ELISA, heterozygotes for Arg549Cys display lower circulating DβH compared to wildtype in both Control (p = 0.0056) and IBD (p = 0.0001).

Fig 3. The levels of conversion of tyramine to octopamine in serum samples from IBD and control subjects with different genotypes.

Relative activities after normalization to the DΒH expression levels measured by ELISA and represented in Fig 2. Overall comparison of wildtype and heterozygotes subject serum conversion of tyramine to octopamine (normalized to the amount of DΒH present; quantified as pmol/min/pg DβH, measured with an unpaired t-test).