Introduction
Genetic elements contribute to 30–50% of the blood pressure variability in human essential hypertension [1]. Studies aiming to identify contributing genes will allow us to recognize those vulnerable individuals, and to classify patients in subgroups with definite genetic and pathogenic mechanisms, to achieve better prevention and therapeutics. Genes involved in renal sodium handling are of particular interest because alterations in renal function and sodium reabsorption are associated with long-term increases in blood pressure. Good examples are the genes encoding for adducin α, β and γ subunits. Adducin is ubiquitously expressed and is involved in multiple functions, including cell motility and synaptic transmission [2,3]. The role of adducin includes stimulation of Na+-K+-ATPase activity, the key enzyme for tubular Na transport [4].
The hypothesis that adducin is important in hypertension originated with the development of an experimental genetic model, the hypertensive Milan rat strain [5]. The proposed molecular mechanism in the Milan rat strain involved adducin polymorphism, leading to increased Na+-K+ ATP-ase activity and enhanced renal tubular sodium reabsorption, facilitating the development of hypertension. An α-adducin allele was shown to co-segregate with blood pressure in the Milan rat strain [6].
Initial clinical studies associated the α-adducin polymorphism (consisting of tryptophan instead of glycine at amino acid number 460, Gly460Trp) with hypertension [7]. This was followed by a report of selective association with hypertension in Caucasian populations characterized by low plasma renin activity [5]. Hypertensives with at least one copy of the Gly460Trp allele and low plasma renin activity showed enhanced proximal tubular reabsorption and a more pronounced fall in blood pressure after chronic diuretic treatment or acute sodium depletion [8–10]. This suggested a genetic basis for selective anti-hypertensive therapy in groups of patients characterized by specific mechanisms, such as those involved in the renal handling of sodium.
In the Milan rat strain, the γ-adducin polymorphism was later associated with hypertension through non-additive gene–gene (epistatic) interaction with the α-subunit [5]. Two recent reports in the Journal of Hypertension[11,12] provide clinical correlates for the α- and γ-adducin epistatic interaction. In a population of never-treated hypertensive subjects, Gly460Trp polymorphism was associated with very small increases in blood pressure and lower plasma renin activity and ouabain, supporting the hypothesis that the α-adducin allele stimulates Na+ reabsorption, and of altered renal sodium retention in hypertension. In turn, the Ala386Gly γ-adducin allele was associated with higher blood pressures in the Gly460Trp population [11]. In a population-based study of three different European groups, epistatic interactions between α- and γ-adducin were associated with alterations in peripheral and central pulse pressure, and index of vascular stiffness as a consequence of chronically increased blood pressure [12]. In carriers of the Gly460Trp polymorphism, the peripheral and central pulse pressure increased with the presence of the γ-adducin Ala386Gly allele. In addition, α-adducin GlyGly homozygoty was associated with a lower urinary Na+/K+ ratio among α-adducin Trp allele carriers and with higher urinary aldosterone excretion among α-adducin GlyGly homozygotes. The proposed association between adducin alleles, low renin hypertension, renal sodium reabsorption and response to diuretics, now confirmed by family studies, appears of to be interest because of its promise of identifying a subgroup of essential hypertensive patients with specific genetic attributes and increased sensitivity to a particular class of anti-hypertensive medications.
The original adducin hypothesis, as formulated 10 years ago, generated numerous replication and expansion studies. Unfortunately, as is the case with most if not all initial studies on the genetics of hypertension, the results of replication studies and a further analysis of the genetic and environmental context are not encouraging.
Frequency of the α-adducin allele in human populations
The prevalence of adducin variants differs among populations, and is much higher in oriental (Chinese and Japanese) and lower in black South Africans than in Caucasians [5,8,13–15]. More importantly, the frequency of the Gly460Trp allele in a Scandinavian population was even lower in hypertensives than in normotensive controls [16].
Association of α-adducin polymorphism and hypertension
The initial positive studies reported an association of adducin polymorphism and hypertension in an Italian population [7]. Follow-up studies of different populations did not replicate the initial report, with the exception of one study of a Black population born and living in Africa [14]. No association between the Gly460Trp allele and hypertension occurred in Asian (Chinese or Japanese) [15,17], Scottish [18], Scandinavian [16] or American populations [19,20]. The conclusion, from a series of studies supported by the Family Blood Pressure Program, was that the α-adducin gene did not have a major impact on the occurrence of hypertension [21]. Yet another report found an association in Whites, but not in Blacks, where the adducin polymorphism appeared to be protective against hypertension [22].
Association of α-adducin polymorphism with low renin hypertension and salt sensitivity
Attempts to link the Gly460Trp in specific subgroups of patients have also been inconsistent. The report of an association of Gly460Trp polymorphism with salt sensitivity and lower renin activity [8,23] was confirmed in Japanese populations [13,24–26] but the findings did not hold for Scottish [27] or Hispanic [28] populations. In the Hispanic population, the Gly460Trp allele associated, in a subset of hypertensives with salt sensitivity, only with greater salt-dependent modulation of NO excretion [28].
Context-dependent effects
Age and gender condition the adducin–hypertension association. The association of Gly460Trp polymorphism with low renin hypertension holds only for young subjects [24]. A report of a large cohort of a Japanese population concluded that the Gly460Trp polymorphism associates with hypertension only in females [29].
Pharmacogenetic studies and association with end organ damage
The initial pharmacogenetic studies that focused on blood pressure responses to thiazide diuretics have not been confirmed [30]. In a population-based, case–control study of a single nucleotide polymorphism in survivors of myocardial infarct or stroke, diuretics were associated with a lower risk of myocardial infarction and stroke but not with mean levels of blood pressure [31]. This suggests that in salt-sensitive hypertensives, diuretic therapy decreases the incidence of cardiovascular events through mechanisms other than the direct lowering of blood pressure, and that blood pressure differences may not predict the effects of drugs on cardiovascular end points. In an Irish population, the Gly460Trp variant did not associate with nephropathy or hypertension in type 1 diabetic patients [32]. Moreover, the α-adducin polymorphism has been recently associated with a significant protective effect on myocardial infarct [33].
In conclusion, follow-up studies have not replicated or confirmed the initial hypothesis of a major role of adducin polymorphisms in essential hypertension, and the role of adducin genes in hypertension remains elusive. Failure to confirm the role of candidate genes is not restricted to adducin, and has been recently reported for the ACEI/D and the AGT M235T polymorphisms [34,35]. Problems with studies of genetic associations in complex heterogeneous disorders are not limited to studies on essential hypertension. The frustration was so high that the question was raised as to whether or not association studies should be published at all in high-impact Journals [36]. Experts in the field are reaching an emerging consensus on these problems, and on the solutions to avoid confusion and further disappointments. Below, some of the problems and the proposed solutions are identified.
The problems
Complexity of the disease: influence of the environment and genetic context
A complex combination of processes, metabolic systems and intermediate traits, involving a redundancy of balancing pressor and depressor roles, controls blood pressure. Hypertension is probably a polygenic disease with complex interactions of networked genes with environmental stimuli, and a few alleles in a handful of genes are not likely to explain increased blood pressure. The pathogenic mechanisms are very complex and include elements of the metabolic syndrome, obesity and cardiovascular risk factors. Because of this, the influence of any genetic factor such as adducin is likely to be small, and dependent on the genetic context and environmental factors. This is why apparently there is a decreased support for genetic linkage with increasing sample size.
Population heterogeneity
The prevalence, age of onset, severity and complications of hypertension are not similar among ethnic groups, indicating the contribution of different sets of genes. The effect of only one gene is likely to be small and inconsistent across different genetic and environmental backgrounds. A genetic variation may only have a significant effect in particular subgroups of the population defined by some other genetic or environmental context, with minimal or no association in other subgroups of the population.
The expectations that initial findings will hold in different populations, indicating a universal association between the gene and the phenotype, usually fail. For the reasons stated above, differences among populations are not surprising but rather expected [37].
Complexity of pharmacogenetics
Does the adducin variant alone identify a subset of hypertensive patients who are particularly likely to benefit from diuretic therapy? The results of follow-up studies have also been discouraging. Hypertension is characterized by large inter-patient variability in response to drugs, and there are multiple genetic variants influencing response to anti-hypertensive therapy, including inter-individual variability in rates of drug metabolism. For example, numerous polymorphisms related to gut, liver, blood and renal function have been implicated in the response to diuretics, indicating the complex interplay of many pathways [38].
The solutions
Address the aetiological heterogeneity of essential hypertension
A single locus model is not adequate for analysis of a complex phenotype.
When there is aetiological heterogeneity, analysis of subgroups can enhance gene finding or hidden genetic heterogeneity because different hypertension genes operate in different subsets of families. Gene–gene, gene–genetic background and gene–environment interactions may be at play.
In addition, adequate genetic analysis and phenotype definition will not be possible until basic mechanisms and interactions are understood. Future studies should analyse the contribution of adducin not only on the pathophysiology of sodium handling, but also focus on the functional characteristics related to sodium transport, such as sodium pump activity and blood pressure responses to changes in sodium balance. Many cells express adducin subunits and this protein could play additional roles in multiple functions, such as the regulation of neurotransmitter activity [39].
Improve study design
There is an urgent need for a limited and generally accepted set of methods that permit appropriate assessment and comparison of individual results. To validate a hypothesis, further tests are necessary and require independent replication. Because negative evidence in replication studies is expected from statistical theory [37], the size of the sample and the power of the statistical tests utilized become crucial for analysis and selection of competing hypothesis.
Replication should reproduce the original definition of cases and controls, and include a population of similar extraction and similar ethnic background, using the same inclusion and exclusion criteria. The phenotype should be carefully defined, in terms of age of onset and present age, gender, disease severity and body size.
Environmental exposure and lifestyle must be similar to that of the original report [37].
Mutual influences among the metabolic effects of genetic variations are the norm rather than the exception, and there is a need to study gene–gene interactions and multiple polymorphisms within large epidemiological samples. Will the study of haplotypes (chromosomal segments preserved intact over many generations that may account for the vast majority of diversity in populations) be powerful predictors of disease [33]?
Subjects on antihypertensive treatments should be included because familial components of blood pressure variance are important, and this information is lost, reducing the evidence of linkage, if treated patients are excluded [40,41]. To deal with treatment effects we need special analytical methods [41].
Association studies of high quality should include large sample sizes and high statistical power, report associations that make biological sense containing an initial study as well as an independent replication, and systematically investigate the interaction among genes and the environment [42].
Although associations in population-based studies are useful, family-based analysis is more powerful for detecting linkage and/or linkage disequilibrium [35,43].
Focus on pharmacogenetics of selected populations
Will the patient's genetic profile be important in the future to decide how to treat hypertension? Work in pharmacogenetics promises to improve the safety and efficacy profile of commonly used medications and help individualize treatment, selecting drug therapies that maximize effectiveness and safety. Many questions remain to be answered before this fantasy becomes reality. Drug treatment based on genomic traits must be scrutinized rigorously because therapeutic recommendations may be valid for selected populations only [34].
Develop a multidisciplinary approach
Boerwinkle [21] has called for a collaborative multidisciplinary approach, with the combination of information obtained from similar studies, the sharing and combining of results before publication and the combining of data resources being necessary. Such an approach should also eliminate any positive publication bias and the tendency to over interpret marginal results.
Conclusions
It is discouraging that in spite of intense efforts (9113 PubMed-listed publications for ‘human hypertension/genetics’; 131 publications for ‘human hypertension/genetics/adducin’) the field has not advanced beyond initial hopes that have been crushed by disappointments. It appears that increased funding and further molecular and statistical sophistication will not be sufficient to overcome the obstacles, and may very well add to the confusion. On the other hand, we cannot abandon genetic studies on hypertension and other complicated diseases. Recognition of the complexity of hypertension and the difficulties ahead, improved study design, data sharing and collaborative studies, coupled with stringent criteria for acceptance in high impact Journals, should slowly advance our understanding of a disease of such immense importance for clinical practice and public health.
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