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(Hypertension. 2001;38:204.)
© 2001 American Heart Association, Inc.

Scientific Contributions

Interaction of ₁-Na,K-ATPase and Na,K,2Cl-Cotransporter Genes in Human Essential Hypertension

Nicola Glorioso; Fabiana Filigheddu; Chiara Troffa; Aldo Soro; Paolo Pinna Parpaglia; Aristides Tsikoudakis; Richard H. Myers; Victoria L.M. Herrera; Nelson Ruiz-Opazo

From Clinica Medica, Universita di Sassari (N.G., F.F., C.T., A.S., P.P.P.), Sassari, Italy; and Whitaker Cardiovascular Institute, Evans Department of Medicine (A.T., V.L.M.H., N.R.-O.) and Department of Neurology (R.H.M.), Boston University School of Medicine, Mass.

Correspondence to Nelson Ruiz-Opazo, PhD, Whitaker Cardiovascular Institute, Boston University School of Medicine, 700 Albany St, Boston, MA 02118, E-mail nruizo{at}bu.edu; and Nicola Glorioso, MD, Clinica Medica University of Sassari, Viale S Pietro 8, 07100, Sassari, Italy. E-mail glorioso@ssmain.uniss.it

	Abstract

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Abstract—— Essential hypertension is a common disease the genetic determinants of which have been difficult to unravel because of its clinical heterogeneity and complex, multifactorial, polygenic etiology. Based on our observations that ₁-Na,K-ATPase (ATP1A1) and renal-specific, bumetanide-sensitive Na,K,2Cl-cotransporter (NKCC2) genes interactively increase susceptibility to hypertension in the Dahl salt-sensitive hypertensive (Dahl S) rat model, we investigated whether parallel molecular genetic mechanisms might exist in human essential hypertension in a relatively genetic homogeneous cohort in northern Sardinia. Putative ATP1A1-NKCC2 gene interaction was tested by comparing hypertensive patients (blood pressure [BP] >165/95 mm Hg) with normotensive controls age >60 years with BP <140/85 mm Hg. Genotype analysis with microsatellite markers revealed conformation to Hardy-Weinberg proportions for 6 alleles of both ATP1A1 (D1S453) and NKCC2 (NKCGT7) markers, respectively. Two-by-six ² analysis of alleles identified overrepresentation of ATP1A1 No. 4 and NKCC2 No. 4 alleles, respectively, in hypertensives compared with controls. With a qualitative trait framework, single-gene analysis detected association of both the ATP1A1 No. 4 allele (P=0.004, ²=8.094, df=1) and the NKCC2 No. 4 allele (P=0.0002, ²=14.279, df=1) with moderate to severe hypertension. Digenic analysis revealed that ATP1A1 No. 4–NKCC2 No. 4 allele interaction increases susceptibility to hypertension (P<0.0001, ²=22.3, df=1) beyond levels obtained in single-gene analysis. Analysis was also performed in a quantitative trait framework with BP as the continuous trait parameter. Digenic analysis of ATP1A1 No. 4–NKCC2 No. 4 allele interaction revealed significant association with systolic (1-way ANOVA, P=0.000076) and diastolic (P=0.00099) BP. Interaction was corroborated by 2x2 factorial ANOVA for interaction (systolic BP interaction term, P<0.05, diastolic BP interaction term, P=0.035). The data are compelling that ATP1A1 and NKCC2 genes are candidate interacting hypertension-susceptibility loci in human essential hypertension and affirm gene interaction as an important genetic mechanism underlying hypertension susceptibility. Although corroboration in other cohorts and identification of functionally significant mutations are imperative next steps, the data provide a genotype-stratification scheme, with 4-fold predictive value (odds ratio, 4.28; 95% confidence interval, 2.29 to 8.0), which could help decipher the complex genetics of essential hypertension.

Key Words: complex polygenic disease • hypertension, essential • genes • epistasis • genetics

	Introduction

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Essential hypertension is a complex, multifactorial, polygenic disease that is a major risk factor for cardiovascular morbidity and mortality in developed countries.¹ With a multifactorial disease wherein environmental factors interact with genetic determinants, susceptibility genes that are necessary but not sufficient to cause the disease are to be expected, in contrast to monogenic forms of hypertension. Because of its polygenic quantitative trait, whereby several hypertension-susceptibility genes are presumed, as corroborated by genetic hypertension animal modeling studies (4 to 8 genes),² gene-gene interaction (epistasis) is expected to dominate in essential hypertension.³ Because it is a clinically heterogeneous disease, multiple genetic subtypes are predicted to exist in essential hypertension, with overlapping, as well as discrete, subsets of hypertension-susceptibility genes and interacting environmental factors.⁴

This implied conundrum of genetic subtypes of human essential hypertension with overlapping and discrete susceptibility genes, epistasis for some but not all susceptibility genes, and variability in environmental risk factor response and occurrence presents a formidable challenge.^4,5 Paradigms that assume a simplified model of hypertension, such as major gene effect, have not proved productive, which is to be expected given the genetics of complex traits.⁶

Insight into hypertension genetics could come through parallels drawn from hypertension genetic animal model studies in which genetic subtypes and environmental factors can be experimentally regulated and informative crosses can be designed. Paradigms identified from genetic studies of animal models could provide the "foot-in-the-door" insight that would facilitate the unraveling of the genetic conundrum of multifactorial subtypes of human essential hypertension. Although we acknowledge the inherent shortcomings of animal models of human disease, genetic rat models of polygenic (essential) hypertension do provide a critical contextual level not attained by alternative accepted approaches such as analysis of modifier genes of human monogenic hypertension.

Investigative robustness can be attained through the a priori inclusion of gene interaction paradigms into the investigation design, restriction of the hypertension cohort to a relatively genetically homogeneous population, and stringent cohort phenotype characterization. Two biological frameworks can be applied to the analysis of interacting hypertension-susceptibility gene pairs: (1) hypertension as a qualitative trait, that is, hypertension versus normotension; and (2) hypertension as a quantitative trait, that is, a continuous range of blood pressure (BP) levels.

We focused our studies on the ATP1A1 and NKCC2 genes as a putative hypertension-susceptibility interacting gene pair based on data suggesting that interaction of ₁-Na,K-ATPase (ATP1A1) and Na,K,2Cl-cotransporter (NKCC2) genes increases susceptibility to salt-sensitive hypertension in Dahl S rats.⁷ We hypothesized that a similar genetic paradigm might be operative in subtypes of human essential (polygenic) hypertension. To test this hypothesis, a microsatellite marker close to the ATP1A1 gene (D1S453, 160 kb away)⁸ and a marker within the NKCC2 gene (NKCGT7)⁹ were used to genotype a Sardinian (hypertensive and normotensive) cohort. Although D1S453 is estimated to be 160 kb away from the known ATP1A1 transcription unit, there are no other known candidate genes in the 160-kb genomic region, thus validating the use of this marker. This microsatellite marker is more informative than the diallelic restriction fragment length polymorphism in ATP1A1 intron 1^10,11 and provides a more robust genetic paradigm. The data reported here suggest that the ATP1A1/D1S453 and NKCC2 loci interactively increase susceptibility to hypertension in humans in a northern Sardinian population.

	Methods

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Study Population
The study cohort comprised 811 subjects, with 587 patients with essential hypertension (mild, moderate, and severe) and 224 normotensive controls enrolled at the Hypertension Center, Clinica Medica of the University of Sassari Medical School, Sassari, Italy. The study protocols were approved by the local ethics committee of Clinica Medica, University of Sassari Medical School. All patients and controls were white, unrelated, born in different domains of North Sardinia that have been demonstrated previously to have a high degree of genetic homogeneity,^12,13 ascertained to be Sardinian for at least 6 generations, and resided in Sardinia. Hypertensive patients with BP >160/95 mm Hg (n=318 of 587) age 21 to 72 years and with no secondary hypertension etiology were considered in the digenic analysis of hypertension as a discrete disease trait, that is (+) disease/hypertension versus (-) disease/normotension (Table 1). Older patients were included only if they had been diagnosed as hypertensive well before 55 years of age by our group and followed up since then. BP measurements were obtained with patients not taking any medications, as described previously.¹⁴ Patients with mild hypertension (n=269 of 587; average BP 160/95 mm Hg but systolic BP/diastolic BP >140/90 mm Hg) were excluded from the digenic analysis of hypertension as a discrete trait to increase robustness of the discrete trait cohort but were included in the expanded cohort analysis (n=811) for gene interaction analysis of hypertension as a quantitative trait (Tables 3 and 4). Secondary hypertension and major comorbid conditions were excluded as described previously.¹⁴ Family history of hypertension was investigated, and a complete pedigree was defined. To exclude erroneous control subjects with late-onset hypertension, normotensive controls (n=224) were limited to those older than 60 years who had not been previously diagnosed or treated as hypertensive, had no family history of hypertension and cardiovascular or cerebrovascular disease, and had BP values <135/85 mm Hg on at least 4 occasions as described previously.¹⁴ This selection process ascertains phenotype characterization of both hypertensive patients and normotensive controls, critical prerequisites for genetic analysis.

View this table: [in this window] [in a new window]   Table 1. Phenotype Characteristics and Single-Gene Analysis of Hypertension as a Discrete Trait

View this table: [in this window] [in a new window]   Table 3. Digenic Analysis of ATP1A1 and NKCC2 Loci Based on BP as a Quantitative Trait

View this table: [in this window] [in a new window]   Table 4. 2x2 Factorial ANOVA Determination of ATP1A1-NKCC2 Gene Interaction

Genotyping
Genotyping was performed blinded to phenotype characteristics. The D1S453 and NKCGT7 microsatellite markers were obtained from Research Genetics. The location of the D1S453 marker (3.77 centiRad; 160 kb) with respect to the ATP1A1 gene was obtained by radiation hybrid mapping with a radiation hybrid panel of human chromosome 1. Genotyping was performed as described previously with 20 ng of DNA.¹⁵ Alleles were identified by 6% nondenaturing polyacrylamide gel electrophoresis (1/2x tris-borate-EDTA, 750 V for 14 hours). Gels were exposed to autoradiograms, and results were ascertained on 2 independent, blinded genotype readings.

Gene Interaction Paradigms and Statistical Analysis
Analysis of ATP1A1 and NKCC2 allele frequencies in hypertension as a discrete trait, that is, hypertension versus normotension, was performed by ² analysis (SigmaStat). To objectively determine differential allele frequencies, if any, global comparative frequency analysis was first performed by 2x6 [2 (hypertension versus normotension)x6 (alleles 1 versus 2 versus 3 versus 4 versus 5 versus 6)] analysis. This identifies which alleles predominate in hypertensives (ie, hypertensive alleles) compared with normotensives. As the next step, these putative hypertension alleles (allele 4 for both ATP1A1 and NKCC2) were then tested further in 2x2 ² analysis [2 (hypertension versus normotension)x2 (allele 4 versus alleles 1/2/3/5/6)] analysis to validate observations obtained in the global 2x6 ² analysis.

Digenic analysis (analysis of 2 genes together) was then performed to investigate the putative interaction of allele 4 of both ATP1A1 and NKCC2 genes by 2x2 ² analysis [2 (hypertension versus normotension) x2 ([4¹/X¹+4^NKC/X^NKC] versus all other genotype permutations as a group {[X¹/4¹+N^NKC/N^NKC] or [N¹/N¹+X^NKC/4^NKC] or [N¹/N¹+N^NKC/N^NKC]} wherein 4¹ represents allele 4 of the ATP1A1 locus, 4^NKC is allele 4 of the NKCC2 locus, X¹ is any ATP1A1 allele, X^NKC is any NKCC2 allele, and N represents any allele except allele 4.

Interaction analysis was also performed to investigate the ATP1A1-NKCC2 interaction contribution to BP as a quantitative continuous trait (actual numerical values of BP instead of qualitative disease state; ie, hypertension versus normotension) in 2 ways. One-way ANOVA (SigmaStat) was performed to compare the following genotype groups: N¹/N¹+N^NKC/N^NKC, 4¹/X¹+N^NKC/N^NKC, N¹/N¹+4^NKC/X^NKC, and 4¹/X¹+4^NKC/X^NKC. Interaction analysis of ATP1A1-NKCC2 genes with BP as a quantitative trait (Table 4) was performed by 2x2 factorial ANOVA (2-way ANOVA) with ATP1A1 and NKCC2 genotype as the independent variables and BP as the dependent variable to assess whether both genes interactively increase BP. Interaction terms (F) and corresponding significance probability values are presented.

	Results

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To increase robustness of the cohort under analysis, we ascertained the following. First, we limited genetic diversity by restricting the cohort under study to a relatively isolated genetic population of northern Sardinia.^12,13 Second, we minimized subtype heterogeneity by defining the hypertension phenotype using stringent, clinically pertinent criteria to distinguish hypertensive patients and normotensive controls.

To define gene interaction, we tested the hypothesis using a multistep framework of analysis. First, selection of the candidate hypertension-susceptibility interacting gene pair should be based on biological precedence for interaction. As an interacting gene pair, ATP1A1 and NKCC2 genes fulfill this criterion based on biochemical data documenting the functional linkage of the 2 ion transporters through a common substrate, Na⁺, and based on genetic studies in animal models implicating ATP1A1-NKCC2 interaction.⁷ Second, digenic (2-gene interaction) analysis based on a valid phenotype and genotype stratification scheme should detect increased correlation with hypertension when the 2 putative interacting genes are analyzed as an interacting pair compared with respective single-gene analysis, as shown for diabetes.¹⁶ To execute this step, we first determined which allele of the ATP1A1 and NKCC2 genes exhibited increased frequency in hypertensives in contrast to normotensives. These alleles would then make up the interacting pair subsequently tested for association with hypertension as a discrete disease phenotype.

The phenotype stratification scheme compares ascertained hypertensive patients (n=318) with group means for systolic BP (SBP) of 175.4±16.8 mm Hg and diastolic BP (DBP) of 110.1±10.1 against control normotensives with group means for SBP of 127.8±11.7 and DBP of 77.7±7.3 (Table 1). Both groups contained equivalent representation of both genders (Table 1). The genotype stratification scheme compares the ATP1A1 and NKCC2 alleles with greatest frequency in hypertensives and normotensives. To pinpoint these hypertension-associated alleles, genotyping was performed with D1S453 (ATP1A1) and NKCGT7 (NKCC2) markers, respectively (Figure). This genotyping screen identified 6 ATP1A1 and 6 NKCC2 alleles, arbitrarily numbered 1 to 6. Genotypes for both loci were tested for Hardy-Weinberg proportions in the study population, which showed that ATP1A1 genotypes (²=6.851; df=10; P=0.739) and NKCC2 genotypes (²=11.082; df=13; P=0.604) do not deviate from Hardy-Weinberg predicted frequencies. Global comparative analysis of allele frequencies (alleles 1 to 6) between normotensives and hypertensives (Table 1) in a 2x6 ² analysis detected significant differences between hypertensives and normotensives (Table 1) at the ATP1A1 (P=0.014) and NKCC2 (P<0.0001) loci. The increased representation of the fourth ATP1A1 allele (4¹) and fourth NKCC2 allele (4^NKC) in hypertensives and their respective significant unit contribution to the overall ² of the 2x6 tables (for ATP1A1 No. 4 allele, 17.41% in normotensives and 24.84% in hypertensives; for NKCC2 No. 4 allele, 15.85% in normotensives and 25.76% in hypertensives; Table 1) suggests that these alleles might represent putative molecular variants whose dysfunction contributes to hypertension pathogenesis in this cohort. To test this notion, association analysis of allele 4 frequencies compared with non-4 alleles as a group (alleles 1 to 3, 5, and 6) was performed between normotensives and hypertensives in a 2x2 ² analysis. Differences with significant probability values were detected for ATP1A1 allele 4 (P=0.004) and NKCC2 allele 4 (P=0.0002; Table 1).

View larger version (15K): [in this window] [in a new window]   Schematic of human chromosomes 1 and 15 spanning the ATP1A1 and NKCC2 loci, respectively. The positions of the microsatellite markers are denoted in centimorgans.

To determine whether ATP1A1 and NKCC2 loci interact to increase hypertension-susceptibility in this Sardinian cohort, the frequency of the combined genotype carrying at least 1 No. 4 allele for both ATP1A1 and NKCC2 (4¹/X¹+4^NKC/X^NKC) was compared between normotensives and hypertensives in the study population in a 2x2 ² analysis (Table 2). Because multiple comparisons were not performed to select allele 4 of both genes as an interacting gene pair, the need for correction was not necessary. If both loci epistatically influence BP, greater statistical association with hypertension will be found in a digenic analysis in contrast to respective single-locus analyses, as reported for salt-sensitive hypertension in Dahl S rats⁷ and in diabetes.¹⁶ As shown in Table 2, the combined 4¹/X¹+4^NKC/X^NKC genotype was significantly overrepresented in hypertensives compared with normotensives (P<0.0001). The increase in association with hypertension for ATP1A1 allele 4 and NKCC2 allele 4 when analyzed as interacting loci (²=22.3, df=1; Table 2) compared with association as individual alleles in the 2x2 ² analysis (ATP1A1 ²=8.094, df=1 and NKCC2 ²=14.279, df=1; Table 1) suggests that ATP1A1 and NKCC2 genes constitute a hypertension-susceptibility gene pair. The odds ratio for the combined effect on BP was 4.28 (95% confidence interval [CI] 2.29 to 8.0).

View this table: [in this window] [in a new window]   Table 2. Gene Interaction Analysis Based on Genotype Frequencies

As a third step in the framework, the role of ATP1A1 and NKCC2 gene interaction was further tested by determining its contribution to the quantitative variation of BP by 1-way ANOVA with a valid genotype stratification scheme, as well as without a stratification scheme in a 2-way factorial ANOVA. An expanded cohort spanning the full spectrum of BP was used for this analysis (n=811), now including mildly hypertensive patients with SBP between 140 and 160 mm Hg in addition to the normotensives and hypertensives analyzed above in the digenic analysis of hypertension as a discrete trait (that is, hypertension versus normotension). As shown in Table 3, ANOVA revealed that there were no significant differences in BP in subjects carrying only 1 No. 4 allele—either an ATP1A1 No. 4 allele [X¹/4¹+N^NKC/N^NKC] or an NKCC2 No. 4 allele [N¹/N¹+X^NKC/4^NKC]—or in subjects who were noncarriers for allele No. 4 of both genes [N¹/N¹+N^NKC/N^NKC]. In contrast, patients digenic for ATP1A1 No. 4 and NKCC2 No. 4 alleles—[4¹/X¹+4^NKC/X^NKC] genotypes—showed significantly higher SBP (P=0.000076) and DBP (P=0.00099) than noncarriers [N¹/N¹+N^NKC/N^NKC]. This result is concordant with the notion that the ATP1A1 and NKCC2 genes interactively increase susceptibility to high BP in this Sardinian cohort.

To further validate the potential gene interaction detected by digenic analysis, we performed a 2x2 factorial ANOVA of the entire cohort (n=811; no prior stratification) using SBP and DBP as dependent variables. As shown in Table 4, the interaction term (ATP1A1xNKCC2) probability values for SBP (P<0.05) and DBP (P=0.035) support the notion that ATP1A1 and NKCC2 genes interactively influence BP in this human cohort.

	Discussion

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Altogether, the association data indicate that interaction of the ATP1A1 and NKCC2 genes increases susceptibility to essential (polygenic) hypertension in this northern Sardinian population. The concordance of results from multiple analytical paradigms (² analysis, 1-way ANOVA, and 2-way interaction ANOVA investigating hypertension as both a discrete disease trait and as a quantitative continuous trait) and concordance with observations obtained from linkage analysis of ATP1A1-NKCC2 interaction in the Dahl S genetic rat model⁷ provide compelling evidence for the hypothesis that ATP1A1 and NKCC2 genes constitute a hypertension-susceptibility interacting gene pair. This concordance is reassuring given that association of susceptibility loci in human hypertension can be expected not to be corroborated by linkage analysis despite an odds ratio (/ß, probability of having the disease with the associated allele/allele pair, , divided by the probability of having the disease without the allele, ß) equal to 9.99.¹⁷ However, the odds ratio for an ATP1A1-NKCC2 interaction association with hypertension of 4.28 (95% CI 2.29 to 8) is greater than the estimated Sardinian population odds ratio (<3) for hypertension given a positive family history of hypertension (unpublished data, N. Glorioso), thus validating the hypothesis that ATP1A1/D1S453-NKCC2/NKCGT7 digenic genotype predicts susceptibility for essential hypertension. It is possible that the mode of selection of normotensive controls (>60 years of age) could overestimate the odds ratio of the observed association in the general population. However, this is unlikely given that true normotensive controls should remain normotensive beyond the age of 60 years. Ascertainment of said bona fide controls in old-age-onset diseases (such as hypertension and Alzheimer’s disease) can be performed with greater confidence (>60 years), thus eliminating pseudonormotensive controls, that is, normotensives in the fourth and fifth decades of life but hypertensives in the sixth decade of life.

It is not surprising that this digenic combination increases susceptibility to hypertension given the physiological interaction of apical bumetanide-sensitive Na,K,2Cl-cotransporter and basolateral ₁-Na,K-ATPase as a functionally linked Na-transport system in renal thick ascending loop of Henle tubular cells.¹⁸ A Q276L ATP1A1 molecular variant showing increased Na/K coupling ratio^15,19,20 and intron-3 mutations affecting NKCC2 alternatively spliced exons 4B, 4A, and 4F have been implicated in Dahl salt-sensitive hypertensive rats.⁷ Whether similar alterations are associated with the 4¹ and 4^NKC alleles remains to be determined. Additionally, the possibility that another gene in linkage disequilibrium with the markers used are the susceptibility genes has not been ruled out, although this is unlikely given the ATP1A1-NKCC2 interaction precedence in the Dahl S rat model studies.⁷ Although this analysis is limited to association studies, we reiterate that association analysis is predicted to be more robust in identifying susceptibility loci of complex traits compared with linkage analysis,¹⁷ thus adding theoretical confidence to the results obtained.

The demonstration that ATP1A1 and NKCC2 genes are implicated in human hypertension pathogenesis, parallel to observations in rats,^7,15,19 suggests that animal model studies can provide critical turnkey insights into the molecular genetic mechanisms operative in human essential hypertension. Altogether, the data underscore gene interaction as a genetic paradigm of polygenic (essential) hypertension. This notion is supported by analysis indicating that gene interaction (epistasis) seems to be the rule rather than the exception for genes involved in complex traits.²¹ Gene interaction has also been reported for putative hypertension intermediate phenotypes, pressure response to saline loading, and plasma renin activity²²; however, 2-way interaction ANOVA was not significant, thus highlighting the importance of the findings for the ATP1A1-NKCC2 gene interaction reported here.

Although these results are tempered with the remaining tasks to be performed (testing in other hypertension cohorts to corroborate and determine the scope of the ATP1A1-NKCC2 interaction gene pair in different hypertension subtypes and delineation of the molecular basis of said interaction), they nevertheless substantiate a paradigm for future investigations of human essential hypertension-susceptibility loci, which hopefully will prove to be that elusive "foot in the door."

	Acknowledgments

 
This study was supported by NIH grant HL-58136. We thank Armand MacMurray for helpful discussions.

Received September 12, 2000; first decision November 10, 2000; accepted January 23, 2001.

	References

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