A growing chain of evidence linking genetic variation in angiotensinogen with essential hypertension : focus on “ A haplotype of human angiotensinogen gene containing 217 A increases blood pressure in transgenic mice compared with 217

THE PREVALENCE OF HYPERTENSION across the globe continues to rise despite advances in treatment and an ever-growing battery of antihypertensive drugs. Untreated hypertension remains an important risk factor for cardiovascular diseases, including stroke. The renin-angiotensin system (RAS) has long been recognized as a critical mediator of blood pressure regulation, RAS blockers are particularly effective antihypertensive agents. Continued interest in clinically blocking the system is evidenced by the recent FDA approval of the renin inhibitor aliskiren as a first-line antihypertensive (16). Despite the impact of environmental and behavioral factors, it is now widely accepted that there exists a strong genetic component that influences the susceptibility to hypertension. Angiotensinogen (AGT) is the substrate of angiotensin II (ANG II) and the only known target of the aspartyl protease renin. Numerous studies have offered convincing evidence physiologically linking AGT with hypertension. These include a strong correlation between plasma AGT levels with blood pressure, decreased blood pressure after administration of antiAGT antibodies, increased blood pressure after injection of AGT, and increased blood pressure in animals with AGT transgenes (10, 20, 23, 27). Perhaps the seminal study mechanistically implicating aberrant AGT gene regulation as a cause for hypertension came from Smithies and colleagues (18, 26) who reported that increasing the number of copies of the mouse AGT gene resulted in increased AGT expression and elevated pressure. Other studies implicate tissue-specific dysregulation of AGT as a potential cause of hypertension (7, 19, 21). Genetic evidence implicating AGT in essential hypertension in humans was first reported in 1992 by Jeunemaitre et al. (17). AGT was also reported to be associated with preeclampsia (pregnancy-induced hypertension) (28). Like other complex diseases, linkage was confirmed in some populations (3, 4, 28) but was refuted in others (1). At first, the primary variant alleles of AGT investigated were those encoding amino acid changes at positions 174 (T174M, rs4762) and 235 (M235T, rs699) (17, 28). Since then, there have been hundreds of association studies trying to establish a functional link between these alleles of AGT and essential hypertension. Unfortunately, an exhaustive review of this extensive literature finds an approximately equal number of papers supporting and refuting their importance. This all suggests that M235T may derive its physiological importance, if any, from linkage disequilibrium with other functional polymorphisms (12). If this is true, where are they located? Nakajima et al. (22) identified nine single nucleotide polymorphisms (SNPs) in the AGT promoter that defined five common haplotypes. Based on sequence comparisons alone, many of these SNPs may effect the binding of transcription factors (8). We evaluated the transcriptional activity of eight naturally-occurring haplotypes of the AGT promoter containing polymorphisms at 14 positions (9). Our results confirmed studies by others suggesting that variants at 20 (rs5050) and 217 (rs5049) are potentially the most important physiologically (13, 14, 29). Accordingly, the data accumulated to date support a concept that polymorphisms in the AGT promoter may act cell specifically to differentially regulate AGT transcription in ANG-producing tissues. However, no matter how convincing the in vitro data are, it would take a giant “leap of faith” to conclude, based only on current evidence, that variation at 217 and 20 in the human AGT promoter is physiologically and genetically relevant in humans. Jain et al. (15) present experimental data functionally implicating one of these variants in hypertension. Jain et al. (15) used a method of gene targeting first described by Bronson et al. (2) and previously employed by us to examine allele-specific AGT expression (5, 6). The experimental strategy entails using gene targeting in mouse embryonic stem cells to insert transgenes differing only at selected polymorphic positions to a defined locus in the genome. The hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus is the one employed because it is 1) permissive for expression of many transgenes, 2) does not effect tissueand cell-specific expression of the transgene, and 3) importantly, homologous recombination at this locus is easily selected. That independent HPRT-targeted transgenes become inserted at a