Patterns of linkage disequilibrium (LD) across the CHGB locus
To visualize association patterns, 16 SNPs (each in Hardy Weinberg equilibrium) were scored and plotted by Haploview as pair-wise LD parameter r2
across the ~14 kbp locus. The proximal promoter (including common variants A-296C and A-261T) was maintained within a single block in both white and black subjects. The allele and genotype frequencies differed between white and black populations (on-line supplementary Table 2
). Although pair-wise r2
values were generally higher in white than black subjects, just 2 LD blocks spanned the locus in each group (). The original resequencing strategy and SNP discovery have been described (43
Human CHGB promoter genetic variation
Domains and motifs in the CHGB promoter
diagrams known motifs in the CHGB promoter, and superimposes common variants. Functional domains in the core/proximal promoter (such as the TATA box, cAMP response element, and G/C-rich regions) were invariant in ~180 people (~360 chromosomes) subjected to systematic polymorphism discovery by resequencing.
Two very common SNPs (MAF >30%) occur in the proximal promoter: A-296C and A-261T, whose allele, diploid genotype, and haplotype frequencies differed by ethnicity (on-line/supplementary Table 2
). The A-296C variant lies in a c-FOS transcriptional control motif (-298/-291), while A-261T lies in recognition motifs for both YY1 (-264/-259) and SRY (-265/-260).
CHGB promoter haplotype/reporter activity assays
To probe the functional significance of the two common promoter variants for transcriptional efficiency, we inserted each of the 4 haplotypes of the 2 promoter SNPs (A-296C and A-261A) into the luciferase reporter plasmid pGL3-Basic (supplementary Figure 1
). After transfection into rat chromaffin (PC12) cells, these 4 haplotypes yielded substantially different luciferase reporter activity (). Site-specific effect analysis of reporter activity showed that both A-296 and A-261 have context-dependent actions. The strengths of combinations of A-296C and A-261T were: AT>CT>AA>CT (p=8.46E-07, ). By 2-way ANOVA: overall F=107.2, p=8.46E-07; A-296C, p=2.09E-05; A-261T, p=0.064; 296-by-261 interaction, p=3.07E-07.
CHGB promoter variants A-296C and A-261T: Context-dependent allelic effects on luciferase reporter activity
We evaluated responses of CHGB promoter haplotypes to agents simulating natural secretory stimuli (). PACAP increased promoter activity by ~4.1-7.7-fold (p=4.86E-14), while nicotine increased activity by ~0.6-1.5-fold (p=1.34E-8). There were differences among the 4 haplotypes in response to PACAP (p=1.9E-5), though nicotine responses were similar (p=0.31).
CHGB promoter variant A-296C
The A-296C variant lies in an evolutionarily conserved region among humans, non-human primates, and other mammals (supplementary Figure 2
). During EMSA, a labeled oligonucleotide representing the C allele was shifted by PC12 nuclear proteins; specificity was suggested by displacement with the same (C) allele when unlabeled (supplementary Figure 3
A-296C occurred in a potential recognition site for transcription factor c-FOS, with a 7/8 base consensus match for the A allele, declining to 6/8 for the C allele (). When interrogated by ChIP, endogenous c-FOS binding to the motif was detected in all four haplotypes, though unexpectedly more intense for the -296C than the A-296 allele ().
Functional characterization of CHGB promoter common variant A-296C
When a plasmid expressing c-FOS was co-transfected into PC12 cells with CHGB promoter/reporters, all 4 CHGB haplotypes responded (p=2.36E-9), though unequally (p=7.23E-5). On a haplotypic background of the A-261 allele, c-FOS stimulated A-296 and -296C similarly, though on a background of the -261T allele, the transcriptional response of A-296 was far greater than -296C (). Thus, the A-296C response to exogenous c-FOS seemed to be context-dependent.
CHGB promoter variant A-261T
A-261T variant also occurs in an evolutionarily conserved region (supplementary Figure 4
). During EMSA the T allele was more effectively shifted by PC12 nuclear proteins than the A allele (supplementary Figure 5
). During EMSA, an oligonucleotide spanning the A allele was shifted by PC12 nuclear proteins, while the T allele was shifted to a lesser degree; specificity was suggested, especially for the A allele, by displacement with the same allele when unlabeled (supplementary Figure 5
A-261T occurred in potential recognition motifs for the transcription factors SRY and YY1: the A allele displayed a superior match to both SRY (5/6 bases) and YY1 (6/6 bases) motifs, as compared to the T allele (). Involvement of endogenous SRY and YY1 was probed by ChIP (): for both SRY and YY1, the A allele was more effectively bound than the T allele, on either A-296C haplotypic background.
Functional characterization of CHGB promoter common SNP A-261T
When a plasmid expressing SRY was co-transfected into PC12 cells with CHGB promoter/reporters, all four haplotypes showed decreased reporter activity (p=1.15E-10, ), though the degree of inhibition depended on A-296C background (p=4.29E-8).
When co-transfected with a YY1 expression plasmid, CHGB promoter reporter activity increased for each haplotype (p=7.6E-10), and the effect was more prominent for the A-261 allele than the -261T allele, regardless of A-296C context (p=1.64E-4, ).
CHGB promoter common variants A-296C and A-261T: Implications for hypertension in the population
Here we studied BP trait-extreme individuals of European ancestry, in order to enhance statistical power (29
). Chromosome-based haplotype analysis on subjects dichotomized into two groups (higher versus lower BP) indicated that individuals with the less common AT or CA haplotypes had a strong tendency to be hypertensive (odds ratio=4.898 for AT haplotype, p=3.19E-11; odds ratio=3.84 for CA haplotype, p=3.64E-10). People with the more common haplotype CT had a strong tendency to be normotensive (odds ratio=0.637, 95% CI=0.524-0.773, p=4.64E-6). The most common haplotype AA had no effect on blood pressure status. The overall effect of CHGB
haplotypes on BP status was substantial ( and ), whether analyzed by chromosome/haplotype (global χ2
=93.9, p=3.16E-20); or by diploid haplotype pairs (global χ2
CHGB promoter haplotypes and blood pressure in a population sample with extreme trait values
CHGB common promoter variant effects on blood pressure in a European ancestry population sample with extreme BP values
Single SNP-based allele tests showed far less power to detect BP associations (): A-296C (though not A-261T) had an effect on blood pressure status (p=0.038), with the A-296 allele tending towards hypertension. Of note, when subjects were stratified by sex, A-261T displayed significant (p<0.001/p=0.011) effects on SBP/DBP in males, though not females.
We also pursued association of the quantitative traits (SBP and DBP in mmHg) with CHGB
haplotypes (), and here we found substantial predictions for both SBP and DBP. With increasing copy number (0,1,2) of haplotype AT, SBP increased by ~29 mmHg (p=0.0002), while DBP increased by ~21 mmHg (p=1.59E-5), each in additive fashion. With increasing copy number of haplotype CA, SBP increased by ~13 mmHg (p=0.005) while DBP increased by ~12 mmHg (p=8.49E-6). With increasing copy number of haplotype CT, SBP decreased
by ~11 mmHg (p=0.0045), with a parallel decrease
in DBP by ~7 mmHg (p=0.011). When we adjusted BP values for the effects of antihypertensive medications in treated hypertensives (38
), the haplotype effects on SBP/DBP persisted or increased: AT, p=4.1E-5/p=6.83E-6; CA, p=0.008/p=2.04E-5; CT, p=0.002/p=0.008. Haplotypes AA and AT displayed more prominent effects on BP in females, while CT had a greater effect in males.
Promoter polymorphisms A-296C/A-261T interacted
non-additively to influence SBP and DBP (p=1.15E-6/p=1.07E-10, ). On a background of A-296C major allele homozygosity (A/A), the A-261T major (A) allele lowered
SBP/DBP by ~27/~22 mmHg, while on a background of A-296C minor allele homozygosity (C/C), the A-261T major (A) allele elevated
SBP/DBP by ~21/~18 mmHg. When treatment-adjusted (38
) BPs were analyzed, the interactions persisted or increased: SBP/DBP, p=4.41E-7/p=1.08E-10. The A-296C-by-A-261T (SNP-by-SNP) interaction was confirmed on analyses of the dichotomous BP trait (higher versus lower): p=3.71E-15.
SBP/DBP values predicted by CHGB
promoter haplotypes were, in rank order (, left): CT<AA
CA<AT (SBP/DBP, p=6.16E-8/p=5.18E-12). On ANOVA, CHGB
promoter genetic variation accounted for ~2.3% of SBP variance and ~3.4% of DBP variance in this primary care population.
Parallel effects of human CHGB promoter hapotypes in vivo and in cella
Of note, the 4 promoter haplotypes display the same rank order for effects on BP in vivo and luciferase reporter activity in chromaffin cells in cella (, right).
Extension to a second population: sub-Saharan African hypertension
An association of CHGB
promoter haplotypes and hypertension was also found in a Nigerian population selected for extreme BP values (top and bottom 25th
%iles)(p=0.007 for BP status, on-line Table 3
). Here haplotype -296C/A-261 increased SBP by ~34 mmHg (p=0.002) and DBP by ~22 mmHg (p=3.52E-4, , left). The rank order of overall haplotypic variation on blood pressure (, right) was: AA, AT<CT
CA (SBP/DBP, p=0.007/p=0.002). While haplotype CA was associated with higher BP in both black and white subjects, haplotype AT predicted higher BP only in whites. However, CHGB
promoter allele and haplotype frequencies differed substantially between black and white populations (on-line supplementary Table 2
), perhaps contributing to different haplotype effects on BP; for example haplotype AT is relatively unusual in whites but far more common in blacks ( and on-line Table 2
promoter haplotypes did not clearly differ in effects on BP between sexes.
“Intermediate” trait: Association of heritable BP response to environmental stress with CHGB promoter variants A-296C and A-261T
In predominantly (~90%) normotensive white twin pairs (n=163 pairs), the DBP response to environmental (cold) stress was substantially heritable (h2
promoter individual genotypes A-296C (A>C, p=0.0237) and A-261T (A>T, p=0.037) predicted ΔDBP in the cold stress test. Because of the modest sample size (2n=326 chromosomes), we observed only 5 examples of CA haplotypes and one AT haplotype, so analyses could only be performed for haplotypes AA and CT. Consistent with basal BP in white BP extremes (), the CT haplotype decreased
the stress ΔDBP, while the AA haplotype increased
ΔDBP (); thus, the rank order of effects of the haplotypes on BP was preserved in twins (AA>CT), though the effects of the more prominent BP-increasing haplotypes (CA, AT) could not be quantified in this predominantly normotensive sample.
CHGB promoter common polymorphisms A-296C/A-261T: Haplotype effects on ΔDBP in the environmental (cold) stress test in twin pairs