CLINICAL AND FUNCTIONAL IMPLICATIONS OF THE ARG549CYS (rs6271) POLYMORPHISM IN DOPAMINE BETA-HYDROXYLASE
Open Access
- Author:
- Gonzalez-Lopez, Eugene
- Graduate Program:
- Neuroscience
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- June 28, 2019
- Committee Members:
- Kent Eugene Vrana, Dissertation Advisor/Co-Advisor
Gregory Steven Yochum, Committee Chair/Co-Chair
Kirsteen Nairn Browning, Committee Member
Andras Hajnal, Committee Member
Gregory Steven Yochum, Outside Member
Matthew D Coates, Special Member - Keywords:
- Parkinson’s Disease
Dopamine-β-Hydroxylase
Norepinephrine
Single Nucleotide Polymorphism
Inflammatory Bowel Disease
Human Genetics - Abstract:
- The role of genetics in understanding the pathogenesis and progression of disease remains an important area of investigation. In recent decades, several single nucleotide polymorphisms (SNPs) in the promoter and gene coding regions of dopamine beta-hydroxylase (DβH), the enzyme required for the synthesis of norepinephrine (NE), have emerged as a risk factor in the onset and progression of several neurological diseases and disorders (Chapter 1). Therefore, the primary goal of this dissertation was to examine the intersection of genetic variants in DβH with functional modification of the protein and individual susceptibilities to disease. In Chapter 2, utilizing next-generation sequencing, we examined the entire coding region of the DβH gene on a cohort of inflammatory bowel disease (IBD) patients using an unbiased approach. We identified a single highly significant association of a coding region DβH genetic polymorphism (Arg549Cys; rs6271) with IBD. This polymorphism is located in a highly conserved tetramerization domain that is thought to be essential for the maintenance of proper DβH function. Also, the presence of the amino acid change resulted in a dramatic, and significant, ten-fold reduction in circulating DβH protein in both IBD patients and control subjects bearing the heterozygous allele for Arg549Cys. Moreover, the intrinsic enzyme activity was higher. We did not observe an effect of disease on DβH protein levels. These findings are the first report of a noradrenergic genetic polymorphism significantly associated with IBD. In Chapter 3, we examined whether rs6271 was also enriched in Parkinson’s disease (PD). We also examined 33 other coding region SNPs believed to be involved in neurological conditions. The National Institute of Neurological Disorders and Stroke (NINDS) Parkinson’s Disease Biomarkers Program (PDBP) NeuroX SNP array provides a ready (and inexpensive) assessment of SNP profile of the DβH gene (among others). This study identified a significant association of a coding region DβH genetic polymorphism rs6271 with PD. Similar to Chapter 2 (IBD), we did not observe any other coding-region SNPs in DβH with a similar enrichment. However, unlike Chapter 2, we found that the disease reduces circulating DβH levels comparing wild-type PD patient to wild-type controls. We postulated that the Arg549Cys SNP affects protein assembly and alters the structural distribution of the DβH protein. To test this hypothesis, in Chapter 4, we utilized genotyped patients’ sequence data and serum resources provided by the Colorectal Disease Biobank (CDB) at Penn State College of Medicine to examine the structural distribution of soluble DβH protein. First we used size exclusion chromatography (SEC) to explore a complete wild-type DβH protein (where the entire coding region of the DβH gene had no missense polymorphisms) and observed that the majority (~95%) of DβH protein is in fractions that represent the monomer fraction (74 kDa); minimal amounts of dimer (152 kDa), tetramer (294 kDa) and large aggregates (650 kDa) were observed. We observed distinct differences in the protein assembly based on Arg549Cys genotype status. Once again, similar to both Chapters 2 & 3, we found a distinct molecular phenotype in the heterozygous patient sera samples. We observed 90% less monomer and a 10-fold increase in large aggregates in sera bearing a single allele of Arg549Cys compared to wild-type sera. Our finding from Chapters 2-4 shows that a genetic polymorphism in DβH may have effects outside of the traditional allele dosage observations where the most significant impact on protein availability and activity is in the heterozygous Arg549Cys in IBD, PD and control samples compared to the homozygous wild-type and single homozygous variant. There is a gap in the current knowledge of DβH variants, their possible functional implications, and their potential roles in disease. This work evaluated the functional consequences of the Arg549Cys variant in DβH and provided its first practical implication in PD and IBD. We hypothesize that the Arg549Cys polymorphism contributes to increased susceptibility by influencing NE levels and altering the structural integrity of the protein, thereby changing the enzyme activity and expression of the active quaternary structure. The approaches outlined in this dissertation provide new options for identifying the specific effectors (SNPs, mutations) that may be responsible for increased susceptibility to disease by influencing NE levels. Using protein quantification with size exclusion chromatography and genetic data can provide an additional tool for determining the functional role of potential DβH SNPs on protein structure and enzyme activity.