Many previous studies have provided evidence that common SNPs at the AGT
locus influence transcription rates, plasma AGT levels, or blood pressure [9
]. Variants −1074T, −532T, −217A, −20C, and −6A within the promoter have been implicated in essential hypertension, individually or in combination, and replicated in a number of recent functional assays or association studies [13
]. Consistent with these studies, our analysis detects significant associations between these AGT
alleles and increased plasma AGT levels and essential hypertension.
Our analysis identifies a complete AGT
haplotype, H4, which contains the SNP alleles individually associated with essential hypertension and elevated plasma AGT. This is the fourth most common haplotype in the analysis cohort and has a frequency of 6–13% in European populations. Individuals with H4, primarily heterozygotes (e.g., H4/H1), account for a large proportion of the association signal in the data even though most copies of the −6A and 4072C are found on other haplotypes. Of the common Eurasian AGT
haplotypes, H4 uniquely contains promoter alleles −1178G, −1074T, −532T and −217A. Consistent with these results, Brand-Herrmann et al.
showed that the −532T/-6A AGT
haplotype is associated with elevated blood pressure (systolic, diastolic, ambulatory) in untreated hypertensive subjects [13
]. In addition, Jain et al.
] have recently shown that a human promoter haplotype containing −217A confers an allele-dose-specific increase in AGT
transcription in liver and kidney tissues compared to the −217G-containing promoter haplotype in transgenic mice.
A key factor affecting the strength of the association between AGT
genotype and plasma AGT levels was sodium intake. Associations were more numerous and more significant under physiological conditions of sodium depletion. It is well-established that sodium loading has an inhibitory effect on the RAAS system and renin expression in humans and model organisms [36
]. Our results suggest that differences in plasma AGT levels associated with AGT
SNP variation are detectable under low-sodium conditions, but that RAAS down-regulation under sodium loading reduces these allelic differences and associations. Thus, these results suggest that future AGT
-blood pressure association studies should account for physiological sodium status.
Several previous studies have found an influence of AGT
genetic variation on renal traits. Individuals homozygous for the −6A allele are over-represented among hypertensive patients with low renal plasma flow on high-salt diets [21
]. Younger hypertensive 235T homozygotes are characterized by a prematurely blunted renal vascular response to increased salt intake [40
]. The non-modulator hypertensive phenotype, characterized by blunted renal response to angiotensin II infusion under a high-sodium diet, is correlated with genotype 235TT
, but this effect is enhanced by adding other correlated genotypes from ACE
In our study, baseline associations AGT variation and renal plasma flow (RPF) were stronger under low-salt than high-salt conditions. More significant associations, however, occurred following angiotensin II infusion under high-salt conditions. While several interpretations are possible, we suggest that allelic variants found on the H4 haplotype background lead to increased responsiveness of AGT expression, and that H4 identifies a subset of the −6A–bearing individuals with an enhanced angiotensin II response and lower RPF. Conditions that activate RAAS systemically, such as sodium depletion and angiotensin II infusion, may have a greater effect on AGT H4 than on other haplotypes, thus producing detectable differences among haplotype classes. This model permits H4 to maintain higher angiotensinogen levels under RAAS-activating conditions, possibly leading to a higher set point for pressure natriuresis and blood pressure over time.
Increasing molecular evidence now suggests that several common diseases involving metabolism, sodium homeostasis, and hypertension may be the result of evolutionarily adapted alleles that are deleterious in modern environments [3
]. The ancestral AGT
−6A allele frequency decreases with latitude and, along with −217A, may confer positive heat adaptation [45
]. The derived −6G allele appears to be under positive selection in some non-African populations [31
]. Additionally, the frequency of the AGT
4072C allele (aa. 235T) is correlated with the unlinked CYP3A5*3 allele, which is also associated with salt and water retention [46
]. At these loci, alleles favoring salt retention, vasoconstriction, and a predisposition to hypertension are more common in African populations [44
A comparison of the common Eurasian AGT haplotypes in this study shows that H4 (and H5) are more divergent from the ancestral form of AGT than are the H3 and H6 haplotypes, even though the latter also contain the ancestral −6A promoter allele (). AGT haplotype H4 contains several human-specific alleles associated with elevated AGT levels (-1178G, −217A, 1164A, 6066A, 9597C). As humans underwent range expansion into more arid environments within Africa, specific mutations and recombination events may have produced AGT haplotype configurations (i.e., H4) that favored vasoconstriction, sodium retention, and limited volume depletion via RAAS activation and the effect of this activation in the kidney.
Figure 4 A neighbor-joining network for the six common Eurasian AGT haplotypes. The network is rooted by a hypothetical ancestral AGT haplotype based on the nucleotide present at the orthologous position in non-human primates for each of the 24 human AGT SNPs. (more ...)
Haplotype H4, along with a closely related haplotype that differs from H4 at position −532T only, accounts for 13% of our sample of sub-Saharan haplotypes. These two haplotypes are common in Africans, relative to other haplotypes. An independent assessment of 57 Nigerians found that one of seven common AGT
haplotypes was significantly associated with elevated plasma AGT levels [47
]. For nine shared markers located between −1074 and 4072, all alleles reported for this Nigerian haplotype are identical to haplotype H4. Consistent with the potential adaptive features described above, the frequency of haplotype H4 is high (23%) in tropical South Indians.
We suggest that haplotype H4 may be a predisposing factor for essential hypertension via elevated AGT levels. In this study, activation of the RAAS system, either through sodium depletion or angiotensin II infusion, produced stronger associations between haplotype variation and hypertension-associated phenotypes. Replication of these results in larger cohorts is feasible and necessary. In particular, the relatively low frequency of the H4 haplotype limited the present study to those effects seen in heterozygotes. Larger studies can assess whether haplotype H4 has significant additive effects in homozygotes. Additional analyses of the molecular mechanisms influencing transcriptional regulation of the human AGT gene by SNP and haplotype class are also needed. The distributions of hypertensive trait values for H4 and non-H4 groups overlap extensively, so caution is warranted in using this haplotype to predict plasma AGT, RPF, or hypertensive predisposition of a single individual. Nevertheless, identification of hypertensive patients with AGT haplotypes that increase plasma AGT levels could help to optimize pharmacological approaches to hypertension management.