Human dopamine beta-hydroxylase (DBH) regulatory polymorphism that influences enzymatic activity, autonomic function, and blood pressure

Abstract

Rationale: Dopamine beta-hydroxylase (DBH) plays an essential role in catecholamine synthesis by converting dopamine into norepinephrine. Here we systematically investigated DBH polymorphisms associated with enzymatic activity as well as autonomic and blood pressure (BP)/disease phenotypes in vivo.

Methods and results: Seventy genetic variants were discovered at the locus; across ethnicities, much of the promoter was spanned by a 5' haplotype block, with a larger block spanning the promoter in whites than blacks. DBH secretion was predicted by genetic variants in the DBH promoter, rather than the amino acid coding region. The C allele of common promoter variant C-970T increased plasma DBH activity, epinephrine excretion, the heritable change in BP during environmental stress in twin pairs, and also predicted higher basal BP in three independent populations. Mutagenesis and expression studies with isolated/transfected DBH promoter/luciferase reporters in chromaffin cells indicated that variant C-970T was functional. C-970T partially disrupted consensus transcriptional motifs for n-MYC and MEF-2, and this variant affected not only basal expression, but also the response to exogenous/co-transfected n-MYC or MEF-2; during chromatin immunoprecipitation, these two endogenous factors interacted with the motif.

Conclusions: These results suggest that common DBH promoter variant C-970T plays a role in the pathogenesis of human essential hypertension: common genetic variation in the DBH promoter region seems to initiate a cascade of biochemical and physiological changes eventuating in alterations of basal BP. These observations suggest new molecular strategies for probing the pathophysiology, risk, and rational treatment of systemic hypertension.

Figure 1. DBH polymorphism discovery

1A. Haplotype blocks at the DBH locus for American populations of European and African ancestry. Haplotype blocks in the DBH region for European American population (left) and African population (right). LD map and haplotype blocks were constructed by Haploview, using our resequencing data. The “solid spine” method was used to position haplotype block boundaries. The four-color scheme (from white, blue, pink, red) represents the increased value of LD with maximum D’ as one in bright red. Block-1 is longer in Whites than in Blacks, extending further into the coding region. 1B. Human DBH proximal promoter elements, common polymorphisms, and motifs altered by common genetic variants. Motifs altered by common variant C-970T were predicted by phylogenetic footprinting, as detailed in Methods. Elements within the core proximal promoter are assembled from several previous reports in the literature, rather than being identified in this report; of note, this very proximal region (-210/-1 bp) was invariant during our resequencing of n=88 subjects (2n=176 chromosomes). The short homeodomain 4-bp motif (ATTA) indicates potential homeodomain sites. Regions are not drawn to scale.

Figure 1. DBH polymorphism discovery

1A. Haplotype blocks at the DBH locus for American populations of European and African ancestry. Haplotype blocks in the DBH region for European American population (left) and African population (right). LD map and haplotype blocks were constructed by Haploview, using our resequencing data. The “solid spine” method was used to position haplotype block boundaries. The four-color scheme (from white, blue, pink, red) represents the increased value of LD with maximum D’ as one in bright red. Block-1 is longer in Whites than in Blacks, extending further into the coding region. 1B. Human DBH proximal promoter elements, common polymorphisms, and motifs altered by common genetic variants. Motifs altered by common variant C-970T were predicted by phylogenetic footprinting, as detailed in Methods. Elements within the core proximal promoter are assembled from several previous reports in the literature, rather than being identified in this report; of note, this very proximal region (-210/-1 bp) was invariant during our resequencing of n=88 subjects (2n=176 chromosomes). The short homeodomain 4-bp motif (ATTA) indicates potential homeodomain sites. Regions are not drawn to scale.

Figure 2. DBH promoter variants: Effects on BP and catecholamines in San Diego twin pairs

Mean values ± SEM are shown, calculated from all individuals as indicated in each bar (both members of each twin pairs, MZ and DZ) in that group, by GEE. Analyses were adjusted by age and sex. BP results were also adjusted for the effects of anthypertensive medications in the 8.8% treated for hypertension. 2A: Haplotype effects on basal/resting BP. 7-SNP haplotype-1 (suppl. Table 5B) is significantly associated with basal SBP. 2B: Stress-induced BP changes. Effects of C-970T genotype on both pre- and post-cold stress SBP in twins. 2C: Catecholamines. Joint effects of C-970T genotype on both urine epinephrine excretion and blood pressure.

Figure 2. DBH promoter variants: Effects on BP and catecholamines in San Diego twin pairs

Mean values ± SEM are shown, calculated from all individuals as indicated in each bar (both members of each twin pairs, MZ and DZ) in that group, by GEE. Analyses were adjusted by age and sex. BP results were also adjusted for the effects of anthypertensive medications in the 8.8% treated for hypertension. 2A: Haplotype effects on basal/resting BP. 7-SNP haplotype-1 (suppl. Table 5B) is significantly associated with basal SBP. 2B: Stress-induced BP changes. Effects of C-970T genotype on both pre- and post-cold stress SBP in twins. 2C: Catecholamines. Joint effects of C-970T genotype on both urine epinephrine excretion and blood pressure.

Figure 3. DBH promoter activity: Role of common variant C-970T

3A. Site-directed mutagenesis at C-970T. On 4 common natural haplotype backgrounds, C-970 or -970T were point-mutated to -970T or C-970, creating 4 non-natural/artificial haplotypes. Results are compared by ANOVA followed by post-hoc T-tests. These basal (unstimulated) activity results are presented as firefly luciferase activity normalized to Renilla activity in the same cell lysate. As a negative control, the ratio of firefly/renilla luciferase activities for the promoterless luciferase reporter vector (empty vector pGL3-Basic) co-transfected with transfection efficiency control plasmid Renilla plasmid (pTK-RL) was 0.008±0.001. The absolute firefly luminescence for haplotype-1→firefly luciferase transfection was 1334.7±198.2 RLU/sec; 50 μl from each 500 μl cell lysate were used for luciferase assay. Each experiment was performed in triplicate, and such experiments were repeated at least three times. 3B. Trans-activation of C-970T by n-MYC. The n-MYC motif in the sequence is shown. Results are presented as fold-stimulation by exogenous/co-transfected n-MYC for each haplotype. Mock: Co-transfected n-MYC empty expression vector. The RLU/protein for haplotype-1→firefly luciferase transfection (without co-transfected n-MYC) was 9.98±0.31 RLU/μg, with an absolute firefly RLU/sec of 346±19.2. 50 μl of each 500 μl cell lysate was used for luciferase assay. 3C, Trans-activation of C-970T by MEF-2. The MEF-2 motif in the sequence is shown. Results are presented as fold over basal activity for each haplotype. Mock: Co-transfected MEF-2 empty expression vector. Basal RLU values were as reported in Figure 3A. 3D. ChIP (Chromatin ImmunoPrecipitation) at C-970T. Results of nucleosomal immunoprecipitations from PC12 cells. “Input” DNA derives from the nucleosomal preparation prior to immunoprecipitation. Antibodies directed against MYC or MEF-2 (or pre-immune serum) were used to precipitate nucleosomes derived from PC12 cells transfected with either the C-970 or -970T alleles, on promoter/reporter plasmids. The nucleosomal immunoprecipitates were interrogated with PCR primers spanning a 142 bp amplicon centered upon C-970T.

Figure 3. DBH promoter activity: Role of common variant C-970T

3A. Site-directed mutagenesis at C-970T. On 4 common natural haplotype backgrounds, C-970 or -970T were point-mutated to -970T or C-970, creating 4 non-natural/artificial haplotypes. Results are compared by ANOVA followed by post-hoc T-tests. These basal (unstimulated) activity results are presented as firefly luciferase activity normalized to Renilla activity in the same cell lysate. As a negative control, the ratio of firefly/renilla luciferase activities for the promoterless luciferase reporter vector (empty vector pGL3-Basic) co-transfected with transfection efficiency control plasmid Renilla plasmid (pTK-RL) was 0.008±0.001. The absolute firefly luminescence for haplotype-1→firefly luciferase transfection was 1334.7±198.2 RLU/sec; 50 μl from each 500 μl cell lysate were used for luciferase assay. Each experiment was performed in triplicate, and such experiments were repeated at least three times. 3B. Trans-activation of C-970T by n-MYC. The n-MYC motif in the sequence is shown. Results are presented as fold-stimulation by exogenous/co-transfected n-MYC for each haplotype. Mock: Co-transfected n-MYC empty expression vector. The RLU/protein for haplotype-1→firefly luciferase transfection (without co-transfected n-MYC) was 9.98±0.31 RLU/μg, with an absolute firefly RLU/sec of 346±19.2. 50 μl of each 500 μl cell lysate was used for luciferase assay. 3C, Trans-activation of C-970T by MEF-2. The MEF-2 motif in the sequence is shown. Results are presented as fold over basal activity for each haplotype. Mock: Co-transfected MEF-2 empty expression vector. Basal RLU values were as reported in Figure 3A. 3D. ChIP (Chromatin ImmunoPrecipitation) at C-970T. Results of nucleosomal immunoprecipitations from PC12 cells. “Input” DNA derives from the nucleosomal preparation prior to immunoprecipitation. Antibodies directed against MYC or MEF-2 (or pre-immune serum) were used to precipitate nucleosomes derived from PC12 cells transfected with either the C-970 or -970T alleles, on promoter/reporter plasmids. The nucleosomal immunoprecipitates were interrogated with PCR primers spanning a 142 bp amplicon centered upon C-970T.

Figure 3. DBH promoter activity: Role of common variant C-970T

3A. Site-directed mutagenesis at C-970T. On 4 common natural haplotype backgrounds, C-970 or -970T were point-mutated to -970T or C-970, creating 4 non-natural/artificial haplotypes. Results are compared by ANOVA followed by post-hoc T-tests. These basal (unstimulated) activity results are presented as firefly luciferase activity normalized to Renilla activity in the same cell lysate. As a negative control, the ratio of firefly/renilla luciferase activities for the promoterless luciferase reporter vector (empty vector pGL3-Basic) co-transfected with transfection efficiency control plasmid Renilla plasmid (pTK-RL) was 0.008±0.001. The absolute firefly luminescence for haplotype-1→firefly luciferase transfection was 1334.7±198.2 RLU/sec; 50 μl from each 500 μl cell lysate were used for luciferase assay. Each experiment was performed in triplicate, and such experiments were repeated at least three times. 3B. Trans-activation of C-970T by n-MYC. The n-MYC motif in the sequence is shown. Results are presented as fold-stimulation by exogenous/co-transfected n-MYC for each haplotype. Mock: Co-transfected n-MYC empty expression vector. The RLU/protein for haplotype-1→firefly luciferase transfection (without co-transfected n-MYC) was 9.98±0.31 RLU/μg, with an absolute firefly RLU/sec of 346±19.2. 50 μl of each 500 μl cell lysate was used for luciferase assay. 3C, Trans-activation of C-970T by MEF-2. The MEF-2 motif in the sequence is shown. Results are presented as fold over basal activity for each haplotype. Mock: Co-transfected MEF-2 empty expression vector. Basal RLU values were as reported in Figure 3A. 3D. ChIP (Chromatin ImmunoPrecipitation) at C-970T. Results of nucleosomal immunoprecipitations from PC12 cells. “Input” DNA derives from the nucleosomal preparation prior to immunoprecipitation. Antibodies directed against MYC or MEF-2 (or pre-immune serum) were used to precipitate nucleosomes derived from PC12 cells transfected with either the C-970 or -970T alleles, on promoter/reporter plasmids. The nucleosomal immunoprecipitates were interrogated with PCR primers spanning a 142 bp amplicon centered upon C-970T.

Figure 3. DBH promoter activity: Role of common variant C-970T

3A. Site-directed mutagenesis at C-970T. On 4 common natural haplotype backgrounds, C-970 or -970T were point-mutated to -970T or C-970, creating 4 non-natural/artificial haplotypes. Results are compared by ANOVA followed by post-hoc T-tests. These basal (unstimulated) activity results are presented as firefly luciferase activity normalized to Renilla activity in the same cell lysate. As a negative control, the ratio of firefly/renilla luciferase activities for the promoterless luciferase reporter vector (empty vector pGL3-Basic) co-transfected with transfection efficiency control plasmid Renilla plasmid (pTK-RL) was 0.008±0.001. The absolute firefly luminescence for haplotype-1→firefly luciferase transfection was 1334.7±198.2 RLU/sec; 50 μl from each 500 μl cell lysate were used for luciferase assay. Each experiment was performed in triplicate, and such experiments were repeated at least three times. 3B. Trans-activation of C-970T by n-MYC. The n-MYC motif in the sequence is shown. Results are presented as fold-stimulation by exogenous/co-transfected n-MYC for each haplotype. Mock: Co-transfected n-MYC empty expression vector. The RLU/protein for haplotype-1→firefly luciferase transfection (without co-transfected n-MYC) was 9.98±0.31 RLU/μg, with an absolute firefly RLU/sec of 346±19.2. 50 μl of each 500 μl cell lysate was used for luciferase assay. 3C, Trans-activation of C-970T by MEF-2. The MEF-2 motif in the sequence is shown. Results are presented as fold over basal activity for each haplotype. Mock: Co-transfected MEF-2 empty expression vector. Basal RLU values were as reported in Figure 3A. 3D. ChIP (Chromatin ImmunoPrecipitation) at C-970T. Results of nucleosomal immunoprecipitations from PC12 cells. “Input” DNA derives from the nucleosomal preparation prior to immunoprecipitation. Antibodies directed against MYC or MEF-2 (or pre-immune serum) were used to precipitate nucleosomes derived from PC12 cells transfected with either the C-970 or -970T alleles, on promoter/reporter plasmids. The nucleosomal immunoprecipitates were interrogated with PCR primers spanning a 142 bp amplicon centered upon C-970T.

Figure 4. Human DBH genetic variation: Schema for effects on autonomic and disease traits Intermediate phenotype hypothesis for DBH

Framework for integration of the experimental results on DBH genetic variation, early/proximate autonomic traits, later disease traits (such as hypertension), and changes in DBH secretion.