Pharmacogenomics and Pharmacogenetics of Hypertension: Update and Perspectives—The Adducin Paradigm

Paolo Manunta and Giuseppe Bianchi Division of Nephrology, Dialysis and Hypertension, University “Vita-Salute” San Raffaele Hospital, Milan, Italy

There is a growing literature on the potential prospective use of genome information to enhance success in finding new medicines. An example of a prospective efficacy of pharmacogenetic and pharmacogenomics is the detection and impact of adducin polymorphism on hypertension. Adducin is a heterodimeric cytoskeleton protein, the three subunits of which are encoded by genes (ADD1, ADD2, and ADD3) that map to three different chromosomes. A long series of parallel studies in the Milan hypertensive rat strain model of hypertension and humans indicated that an altered adducin function might cause hypertension through an enhanced constitutive tubular sodium reabsorption. In particular, six linkage studies, 18 of 20 association studies, and four of five follow-up studies that measured organ damage in hypertensive patients support the clinical impact of adducing polymorphism. As many modulatory genes and environment affect the adducin activity, the context must be taken into account to measure the clinical effect size of adducins. Pharmacogenomics is giving an important contribution to this end. In particular, the selective advantages of diuretics in preventing myocardial infarction and stroke over other antihypertensive therapies that produce a similar BP reduction in carriers of the mutated adducin may support new strategies that aim to optimize the use of antihypertensive agents for the prevention of hypertension-associated organ damage. J Am Soc Nephrol 17: S30–S35, 2006. doi: 10.1681/ASN.2005121346

he enormous variation in the individual response to optimal goal levels seems to be more important than specific antihypertensive treatment has led to acceleration of drug selection. Indeed, the results of different meta-analyses T the interest in pharmacogenomic and pharmacogenetic (6,7) concluded that the treatment with any commonly used studies with the consequent technological developments (1). regimen (angiotensin-converting-enzyme inhibitors, calcium Publications on these fields have increased sharply in the past antagonists, angiotensin-receptor blockers, diuretics, and ␤ 5 yr with the emergence of molecular genetics and genotyping blockers) reduces the risk for total major cardiovascular events, technologies in clinical investigations. The terms pharmacog- and larger reductions in BP produce larger reductions in risk. enomics and pharmacogenetics have been used interchangeably in Although the percentage of patients who had hypertension and the literature; therefore, it is useful to define them here. Phar- received treatment increased from 31 to 59% in the period from macogenomics refers to the application of genome-wide ap- 1976 to 1980 and to 70% in 2000, the percentage of patients with proaches to understanding genetic influences on drug response high BP controlled to Ͻ140/90 mmHg is only approximately and to developing novel drugs. It has the potential to uncover 25% in Western industrialized countries, and this values has additive or even synergistic influences of multiple genes on been constant over the past 15 yr. Despite the great number of drug response. Historically, pharmacogenetic studies were de- antihypertensive drugs that are available the poor BP control, veloped initially to understand individual differences in drug adverse effects represent approximately 80% of the causes for pharmacokinetics and metabolism, often focused on single the discontinuity of the antihypertensive therapy (8). Pharma- gene polymorphisms (SNP), especially in drug metabolism cogenomics may reduce the interindividual variation to anti- genes (1,2). hypertensive therapy by tailoring therapy to individual genetic The Seventh Report of the Joint National Committee on make-up. Prevention, Detection, Evaluation, and Treatment of High However, a recent systematic review (9) demonstrated that Blood Pressure (3), the guideline for the management of hyper- there is a lack of consistency in the findings of different phar- tension of both the European Society of Hypertension (4) and macogenomic studies. Once again, this inconsistency is due the British Hypertension Society (5), delineates specific high- mainly to the failure to take into account the various factors that risk conditions that are compelling indications for the use an- modulate the genotype–drug response phenotype (1). The tihypertensive drug classes that will be required to achieve goal same picture concerns genetics of essential hypertension. BP (140/90 mmHg, or 130/80 mmHg for patients with diabetes Among thousands of association studies that have used poly- and chronic kidney disease). Moreover, lowering BP to the morphisms in candidate genes, few studies demonstrated consistent findings. This may be due to the variability of the current statistical genetic method to define causation in com- Address correspondence to: Dr. Paolo Manunta, Division of Nephrology, Dial- plex polygenic multifactorial disease such as primary hyper- ysis, and Hypertension, University “Vita-Salute” San Raffale, Via Olgettina 60, 20131 Milan, Italy. Phone: ϩ39-0226433006; Fax: ϩ39-0226432384; E-mail: tension (10). Most of the genetic and pharmacogenomic studies [email protected] have major weaknesses related to three major problems:

1. Selection criteria: The accuracy and the amount of detailed Molecular Mechanisms of a Candidate Gene: phenotypic data that can be compared in case-control stud- Adducin ies are crucial to the interpretation of the data. For example, Adducin is a heterodimeric cytoskeleton protein and consists in case-control studies, the appropriate control subjects of an ␣ subunit (103 kD) and either a ␤ (97 kD) or a ␥ subunit must be above the age of onset of “primary” hypertension. (90 kD). Three human genes (ADD1, ADD2, and ADD3) that Gender, body mass index, ethnic origin, and other biologic map to different chromosomes encode these subunits (17). Ad- variables must be taken into account. ducin is highly conserved through the different species, thus 2. Inadequate statistical power of small studies to address com- suggesting a role in basic cellular functions. Adducin is a ubiq- plex genetic questions (11,12): Indeed, in case-control stud- uitously expressed cytoskeleton protein that is involved in the ies, the methods of statistical genetics are based on the formation of actin-spectrin lattice, actin polymerization, and assumption that a genetic variant is involved in hyperten- cell signal transduction (18,19), including an effect on Na-K sion when a higher frequency of this variant is present in ATPase. Figure 1 shows ADD1, ADD2, and ADD3 gene struc- hypertensive individuals (12). However, the biologic com- ture and SNP identified in human and rat hypertension. In plexity of polygenic-multifactorial diseases challenges humans, two polymorphisms of the ADD1 gene lead to amino these assumptions. Differences in evolutionary history, acid substitutions: Gly460Trp and S586C (20). Other polymor- population stratification and admixture, epistatic interac- phisms occur in the human ADD2 and ADD3. The first linkage tion, context dependency, etc. all may weaken the scientific and case-control studies demonstrated an association of the validity of methods that are based on allelic frequency (10). ADD1 Trp allele with hypertension (20). This may explain why the case-control studies on candidate Cell culture and cell-free system experiments helped to elu- gene have provided inconsistent findings across popula- cidate the molecular mechanisms that make adducin mutation tions in polygenic-multifactorial disease (13,14). responsible for the abnormal cell sodium handling: 3. Pharmacogenomics analyses should be used to differentiate 1. In renal cells, transfection with the rat-mutated ␣-adducin phenotypic heterogeneity, to segment populations that increases the Na-K pump activity and causes a rearrange- seem to be responsive or unresponsive to a medicine, or to ment of the actin cytoskeleton (21). define accurately individuals who might be at higher per- 2. In a cell-free system, rat-mutated adducin accelerates actin sonal risk for an adverse event: To address this important polymerization (21), and rat-mutated and human-mutated point, it is crucial to enroll patients who have never treated (ADD1 Trp) adducins bind to and activate the Na-K pump before. For instance, during a chronic treatment with an with higher affinity than the respective normal proteins angiotensin-converting enzyme inhibitor or angiotensin II (22). antagonist, a great amount of plasma renin, angiotensino- 3. Studies on the dynamics of the endocytotic processes in gen, angiotensin I, or angiotensin II is available both in the transfected cells have provided an interpretation for the circulation and at the tissue level. After 3 or 4 wk from increased cellular expression and activity of the Na-K therapy withdrawal, BP eventually increases also because pump caused by the expression of the ␣-adducin mutants these substances are increased both in plasma and in the (23). Cells that are transfected with either the human or the tissues. This pressor mechanism certainly is different from rat hypertensive ␣-adducin compared with cells that are what is responsible for the gradual increase in BP with age transfected with the wild-type variant show a higher Na-K that underlies the development of “primary” hypertension. pump activity and an impaired Na-K pump endocytosis in Conversely, a long (years) period of treatment with diuret- basal conditions (23) as well as in response to natriuretic ics may favor the development of an insulin-resistant status signals such as dopamine. Deficient endocytosis of the that, per se, may increase the risk for organ damage that Na-K pump therefore might be an important factor contrib- may appear in the subsequent years (15). Therefore, pa- uting to the increased renal tubular reabsorption observed tients who are already on antihypertensive therapy should in humans (24,25), carrying the mutated adducin variant. In be enrolled only for a particular type of study. Further- fact, an efficient endocytosis of the sodium transporting more, when BP is measured as office BP, it should be proteins is crucial for blunting the rise in systemic arterial recorded several times, or, alternatively, 24-h BP monitor- pressure when body sodium increases. ing should be used. Impact of Adducin Family Genes on Human Our research group approached the dissection of this com- Hypertension plexity throughout a series of studies on animal models and In support of these findings are the data collected in human patients at different levels of biologic organization: Whole hypertension: Among hypertensive patients, plasma renin is body, organ, cell, subcellular structures, protein, and genes (16). lower in carriers of the ADD1 Trp allele than in wild-type These studies led to the identification of adducin polymor- homozygotes (20,26). Patients with low renin hypertension phism as one of the genetic mechanism that, by modulating the have higher BP in the presence of mutated ␣-adducin, and constitutive capacity of tubular cell to reabsorb sodium, may ADD1 Trp/Trp homozygotes experience the largest increase in favor the development of hypertension with its organ compli- BP (26–28). Furthermore, carriers of ADD1 Trp allele, com- cations. pared with the Gly/Gly homozygotes, show an increased prox- S32 Journal of the American Society of Nephrology J Am Soc Nephrol 17: S30–S35, 2006

Figure 1. ADD1, ADD2, ADD3 gene structure and single-nucleotide polymorphism (SNP) identified in human and rat hypertension. Exon-intron structure of ADD genes is indicated by boxes and lines; filled and open boxes represent the exons for coding and noncoding regions of mRNA, respectively. Black filled boxes in ADD2 and ADD3 genes indicate alternative spliced exons in ADD2-␤4 and ADD3-␥2 isoforms, respectively. The position of human and rat (underline) SNP is indicated by the arrow. The SNP coordinate is based on the amino acid change (one-letter code, italics) for missense mutation and nucleotide change for silent or intronic mutation. dbSNP ID for human SNP is reported in brackets. The human and rat gene location is reported on the right.

imal tubular reabsorption measured by lithium clearance (25) converting enzyme D allele or salt intake. When DNA markers and a larger increase of BP after a saline infusion (24). that mapped at a much larger distance from the ADD1 locus In most tissues that are involved in cardiovascular homeosta- were used, negative results were found by four studies (35–38). sis (kidney, brain, heart, and vessels), adducin is expressed as a Within this large distance, many haplotype blocks were in- heterodimeric protein that consists of ␣ and ␥ subunits. There- cluded (39). Positive results were also obtained in 18 of 20 fore, this protein structure justifies the search for an interaction association studies that, in addition to BP, investigated vari- between ADD1 and ADD3. Recently (29), we described an ables that reflect body sodium or the renin-angiotensin system epistatic interaction between the adducin genes (ADD1 and (16,26). Four of five studies (40–44) showed an association ADD3) in a large cohort of never-treated hypertensive individ- between ␣-adducin polymorphism and organ damage in hy- uals (n ϭ 512), in which BP was determined with 24-h ambu- pertensive individuals. There were mixed results from case- latory BP monitoring. Patients who carried both the mutated control studies or studies in predominantly normotensive pop- ADD1 Trp allele and ADD3 G/G had the higher systolic BP and ulations that did not consider the above-mentioned variables diastolic BP values (approximately 8 mmHg; P ϭ 0.002). (see [16,26] for a detailed discussion of this issue). Furthermore, in a study (30) that was conducted in 642 participants who were randomly recruited from three Euro- pean populations, peripheral and central pulse pressure (PP), Pharmacogenomics Studies of Role of an index of vascular stiffness, was measured. Among carriers of Adducin the ADD1 Trp allele, peripheral and central PP were 5.8 and 4.7 The association between the ADD1 polymorphism and the mmHg higher in ADD3 GG homozygotes than in their AA response to diuretics has been evaluated in five studies counterparts, as a result of an increase in systolic BP. Moreover, (20,41,45–47). The rationale for these studies was that in the in 162 nuclear families and 70 unrelated individuals, a test presence of constitutive enhanced tubular sodium reabsorp- based on the transmission of ADD3 G allele across families tion, drugs such as diuretics should trigger less counterregula- confirmed the interaction between the ADD1 and ADD3 genes. tory mechanisms, thus yielding a more beneficial therapeutic These epistatic interactions, described in two separate studies, effect. Indeed, three studies (20,45,46) that were designed to test are biochemically consistent with the heterodimeric structure of this hypothesis and conducted in never-treated hypertensive the cytoskeleton protein adducin. patients demonstrated a greater BP response to hydrochlorothi- According to a PubMed literature search, after our initial azide (HCTZ) among carriers of the 460Trp allele. report (16,26), at least 67 articles addressed the association A negative study (47) involved 291 unrelated non-Hispanic between human hypertension and the ADD1 Trp allele. Six black and 294 unrelated non-Hispanic white adults aged 30 to human (20,30–34) linkage studies showed positive results 59.9 yr. The BP value recorded after at least 4 wk from discon- when a DNA marker that mapped to 30 kb from the ADD1 tinuation of the previous therapy was used as baseline to locus or SNP of one of the three adducin genes was considered evaluate the BP response to 25 mg of HCTZ given for 1 mo. either alone or in combination with each other or angiotensin- Certainly, this study was large enough to conclude that, in that J Am Soc Nephrol 17: S30–S35, 2006 Pharmacogenomics of Hypertension S33 context, no relationship exists between adducin genotypes and known side effects of diuretics are developed. However, the the BP response to the diuretics. However, the important dif- major problem to overcome in the exploitation of the full po- ference with the other similar but positive studies (20,45,46) is tential of pharmacogenomics is to abandon the “old,” “tradi- that the latter were performed in newly discovered and never- tional” paradigms regarding the definition of causation of a treated hypertensive individuals. After at least 1 mo of run-in given gene variant in a complex multifactorial disease such as and three measurements of BP on three different occasions, hypertension (16,26). Almost all of the reviews concluded that HCTZ was given for 2 mo, and BP was measured after 1 and 2 the available data on genetics or pharmacogenomics of “pri- mo of treatment. In these patients, the genotype–BP relation- mary” hypertension are conflicting and do not discuss the ship was not influenced by a previous therapy, different phases implications of the complexity of these diseases that arise from of hypertension (because they all were in a relatively early the genetic, environmental, or biologic interactions. hypertensive phase), and the variety of counterregulatory mechanisms that come into play with sudden therapy with- Acknowledgments drawal. In fact, even after 1 mo from therapy withdrawal, the This work was supported in part by grants from Ministero Universita` renin response to a standard dose of diuretic still is different e Ricerca Scientifica of Italy (FIRB grant RBNE01724C_001 to G.B. and from that observed in the never-treated hypertensive status (48). PRIN grant 2004069314_01 to G.B.), from Ministero della Salute (ICS In accordance with our findings are the results of an obser- 110.4/RF02353), and from EURNETGEN, EC funded research (grant vational study (41) on 1038 hypertensive patients who were QLG1-2000-01137). followed for approximately 10 yr and treated with a variety of antihypertensive drugs. These results show that in carriers of References the 460Trp ADD1 allele (38% of the population), the adminis- 1. Bianchi G, Staessen JA, Ferrari P: Pharmacogenomics of tration of diuretics halves the incidence of myocardial infarc- primary hypertension: The lesson from the past to look tion and stroke when compared with other antihypertensive toward the future. Pharmacogenomics 4: 279–296, 2003 treatments that produce a similar reduction of BP. The selective 2. Roses AD: Pharmacogenetics and drug development: The beneficial effect of diuretics over the other drugs was not path to safer and more effective drugs. 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