gene is the only one for which association with EH has been consistently replicated in multiple populations.13
Several polymorphisms across the AGT
gene have been associated with both EH and plasma AGT levels. Increased plasma AGT levels have been postulated as a causal factor for increased risk to EH. However, the lack of replication across populations has prevented the translation of this association to clinical applications. Differences in the genomic structure between human populations, including that of the Mexican population,18
have been described in the last decade, and these differences might be the source for this lack of replication. Thus, analysis of the risk-associated alleles in multiple interethnic populations may become an important tool to determine the role of genetic predisposition in the development of the disease.
This study identified 4 alleles associated with EH risk and were found to be independent risk factors in a logistic regression model. We were able to replicate the 2 most important associations, A-6 and C4072 (T235), and we identified 2 new markers associated with higher risk, C6309 and G12775. The association signal at intron 2 of the AGT
(C6309) is particularly interesting because another polymorphism was found recently in intron 2 (C6233T) associated with a major effect on the risk to EH in whites.8
An important point in our study is that we did not find any association between these SNPs and plasma AGT levels in the Mexican population, which reproduces the negative association between C4072T (T235M) and plasma AGT reported previously in a Mexican-derived population.6,23
This finding suggests that mechanisms other than an increased level of plasma AGT contribute to EH risk in the Mexican-Mestizo population. Interethnic variability in the genetic control of plasma AGT levels has been reported for Japanese and African-American children. Sato et al14
found no association between C4072T (T235M) and plasma AGT levels, and the SNP G-1074T associated with AGT levels did not increase the EH risk. In another study, Bloem et al24
were also unable to find an association between plasma AGT levels and C4072T (T235M).
In the Mexican-Mestizo population, the A-20C and C3389T (T174M) were found to be associated with plasma AGT levels, and the C3389 (T174) allele showed the strongest association. However, these SNPs were not associated with increased risk for EH. Both SNPs have been associated with EH and plasma AGT levels in other populations. For example, A-20C located in the promoter region of AGT
is associated with both in vitro changes in AGT
transcription and plasma AGT levels in the Japanese population.25,26
In a meta-analysis including 11079 subjects, an association between C3389T (T174M) and EH was identified in Asian and multiethnic populations but not in a European population.15
The functional mechanism by which C3389T (T174M) increases BP among carriers is currently unknown, and several studies have failed to show association between this SNP and plasma AGT levels.3,27,28
In our sample, levels of plasma AGT by itself did not increase the risk of EH nor showed association with any BP-related traits in our covariate-controlled linear regression analysis. In addition, a recent study failed to associate plasma AGT levels and BP-related traits in a family cohort of white ancestry.29
These results differ from those in an initial report that found a positive correlation between plasma AGT levels and diastolic BP.30
The observations that plasma AGT levels are not associated with any BP-related traits and are not independent risk factors for EH support the notion that, at least in our sample, the increased EH risk from the AGT
locus involves mechanisms other than an increase in plasma AGT levels alone.
To summarize these results, individual SNP analysis identified polymorphisms that were associated with EH risk but not were associated with plasma AGT levels; we also identified SNPs associated with plasma AGT levels that were not associated with EH risk. Plasma AGT is not an independent risk factor for EH and does not impact the BP-related trait.
If we consider that each SNP in a genomic region is fixed to another SNP by evolutionary forces, it would be anticipated that this haplotype background could be more informative than individual SNPs alone. Several publications have described the higher informative value of haplotype analysis as compared with individual SNP analysis.8,31–33
Our haplotype analysis found that the H1 and H5 are associated with risk to EH, and the H2 and H4 are associated with protection against EH. The lack of association between haplotypes H2 and H5 with hypertension after a permutation test could be influenced by sample size and modest haplotype frequency differences between cases and controls. For this reason, a replication study with a larger sample size could be useful to strengthen this analysis and contribute to clarifying the effect of these haplotypes on the risk of EH.
The SNP composition of these haplotypes shows that H1 contains the EH risk alleles and plasma AGT levels. The second risk-associated haplotype H5 also contains the EH risk alleles but lacks the A-20 allele in the promoter, similar to one of the ancestral haplotypes. Interestingly, H3, which includes the EH risk alleles but lacks the plasma AGT levels, did not increase the risk for EH. The protective haplotypes, H2 and H4, include plasma AGT level–associated alleles but not those from the EH risk alleles. Our analysis suggests a more complex model than a single polymorphism effect that involves a combination of variants within the AGT
gene, which modulate the risk for EH and plasma AGT levels. There are multiple indications that several polymorphisms within a gene position interact to affect quantitative trait variation. Thus, multiple locus interactions create a major locus that has a large effect on the observed phenotype (superallele).34
Quantitative trait mapping in Drosophila melanogaster
has shown that major gene effects are not necessarily attributed to single site polymorphisms but are the result of the combined effects of multiple associated polymorphisms.35
This phenomenon has also been described in human traits and diseases; for example, the apolipoprotein gene (APOB
) affects plasma low-density lipoprotein and high-density lipoprotein cholesterol,36
gene influences intestinal lactase activity,37
and ADRB2 influences the actions of catecholamines on bronchodilation and risk to asthma.38
Similarly, the AGT
gene has a demonstrated additive effect on the risk to EH, acting as a superallele, when specific SNPs are present.39,40
Genetic distance analysis on these haplotypes showed that H5 and H1 have the shortest distances from the ancestral chimpanzee haplotype. Considering that haplotypes H1 and H5 contain the major alleles and have the shortest distances with respect to the ancestral haplotype H0, we named these as human ancestral. The difference between H1 and H5 haplotypes is the presence of the A-20 allele in H5 and H0. The H2 and H4 have the longest distances from H0, suggesting a recent expansion of haplotypes. The Zapotecs, an ancestral Amerindian population, contain a similar genetic distance pattern for H1 and H5, supporting the ancestral character of these haplotypes and also the important genetic contribution of the Amerindian population on the modern Mexican-Mestizo population. These observations are in agreement with the ancestral-susceptibility model for common diseases in which the ancestral alleles reflect risk in the modern lifestyle, whereas in human ancestors these same ancestral alleles provided adaptive advantages to both a low-salt intake and vegetable-based diets.41
In conclusion, our results show heterogeneity in the effects of AGT polymorphisms on EH risk and plasma AGT levels. Two haplotypes act as superalleles for the risk to EH, containing EH risk alleles and plasma AGT levels. Two SNPs were associated with plasma AGT levels, but no association was identified between plasma AGT variation and risk to EH and BP-related traits. These findings suggest that population genetic diversity plays an important role in the control of intermediate traits and helps to elucidate interethnic variability in plasma AGT levels and the role of the AGT locus in EH in the Mexican-Mestizo population.
Our results contribute to understanding the influence of the AGT locus to the risk to EH in a population with a unique genomic ancestry as the Mexican ancestry. Expression analysis of intron 2 and its interactions with functional SNPs across the AGT may be interesting to determine its role in disease. Furthermore, cohort follow-up of genotyped individuals in the general population would be valuable to understand the effect of haplotypes on BP continuous traits and also the role of haplotypes in the clinical setting.