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484 Original article

Serine/threonine kinase 39 is

a candidate gene for primaryhypertension especially in women: results from two cohortstudies in SwedesCristiano Favaa,b, Elisa Danesea,c,M, Martina Montagnanaa,c,M,Marketa Sjogrena, Peter Almgrena, Gunnar Engstroma, Peter Nilssona,Bo Hedblada, Gian C. Guidic, Pietro Minuzb and Olle Melandera

Background As recently pinpointed by a genome-wide

association study the serine/threonine kinase 39 (STK39) is

a candidate gene for hypertension. This kinase is strongly

implicated in sodium reabsorption by the kidney through its

modulating effect on furosemide-sensitive and thiazide-

sensitive channels. The aim of our study was to test the

effects of the STK39 rs35929607A>G polymorphism on

blood pressure (BP) levels and the prevalence and

incidence of hypertension in middle-aged Swedes

participating in two urban-based surveys in Malmo

(Sweden).

Methods The rs35929607A>G polymorphism was

genotyped in 5634 participants included in the

cardiovascular cohort of the ‘Malmo Diet and Cancer-

cardiovascular arm’ (MDC-CVA) study and successively in

17 894 participants of the ‘Malmo Preventive Project’ (MPP)

both at baseline and at reinvestigation after a mean of

23 years. The effect of the same single nucleotide

polymorphism on salt sensitivity was tested in 39

participants of the Salt Reduction to Avoid Hypertension

study.

Results Both before and after adjustment for covariates,

the functional rs35929607A>G polymorphism was

associated with higher SBP and DBP values in the

MDC-CVA, but not in the MPP. In both surveys, the

polymorphism was associated with hypertension

prevalence; after adjustment using the autosomal-

dominant model, the odds ratio for hypertension ranged

between 1.077 (MPP at baseline) and 1.151 (MDC-CVA)

with P-value less than 0.05. After stratification for sex,

the results remained statistically significant in women,

opyright © Lippincott Williams & Wilkins. Unautho

�E.D. and M.M. contributed equally to the writing of this article.

0263-6352 � 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins

but not in men. Carriers of the G-allele displayed an increase

in salt sensitivity.

Conclusion Our results from two large cohort studies

support previous evidence about the association of the

STK39 rs35929607A>G variant with hypertension, especially

in women. If further confirmed in successive studies, owing to

its pivotal role in sodium reabsorption at the renal tubule

level, STK39 might prove to be a suitable target for

antihypertensive therapy. The greater effect of the STK39

rs35929607A>G polymorphism in women with respect to

men deserves further investigation. J Hypertens 29:484–491

Q 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins.

Journal of Hypertension 2011, 29:484–491

Keywords: blood pressure, genetics, hypertension, Ste20-related proline–alanine-rich kinase, serine/threonine kinase 39

Abbreviations: ABP, ambulatory blood pressure; AHT, antihypertensivetreatment; BP, blood pressure; GWAS, genome-wide association study;MDC-CVA, Malmo Diet and Cancer-cardiovascular arm; MPP, MalmoPreventive Project; NCC, NaþClS channel; NKCC, NaþKþClS channel; SARAH,Salt Reduction to Avoid Hypertension; SPAK, Ste20-related proline–alanine-rich kinase; STK39, serine/threonine kinase 39; WNK, without lysine kinase

aDepartment of Clinical Sciences, Skane University Hospital, Lund University,Malmo, Sweden, bDepartment of Medicine and cDepartment of Life andReproduction Sciences, University Hospital of Verona, Verona, Italy

Correspondence to Cristiano Fava, MD, PhD, Division of Internal Medicine C,Department of Medicine, University Hospital of Verona, Piazzale LA Scuro 10,37134 Verona, ItalyTel: +39 045 8124414; fax: +39 045 8027465; e-mail: cristiano.fava@med.lu.se

Received 17 September 2010 Revised 27 October 2010Accepted 16 November 2010

See editorial comment on page 434

IntroductionA recent genome-wide association study (GWAS) in

the American Old Order Amish, an isolated population

descended from a small number of common founders

who emigrated from Switzerland in the eighteenth

century, identified serine/threonine kinase 39 (STK39) as

a possible candidate gene for hypertension [1].

Although other recent GWASs failed to find the same

association, a successive GWAS conducted in 1017

African–Americans found supportive evidence for associ-

ations between some single nucleotide polymorphisms

(SNPs) in the STK39 gene and SBP and DBP [2].

STK39 encodes a serine/threonine kinase known as a

Ste20-related proline–alanine-rich kinase (SPAK), and

it belongs to the STE20-family protein kinases [1].

In renal tubules, these protein kinases act as bridges

between without lysine kinase-1 (WNK1) and WNK4

and their ion cotransporters, such as the NaþKþCl�

channel (NKCC) and the NaþCl� channel (NCC) [3].

In fact, it has been demonstrated that WNK1 and WNK4

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DOI:10.1097/HJH.0b013e328342b2c1

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Serine/threonine kinase 39 gene and hypertension Fava et al. 485

phosphorylate SPAK at two conserved residues, namely a

T-loop Thr residue and a Ser residue in the S-motif [4],

and that this activates the kinase. In turn, SPAK can

directly phosphorylate human NCC at three sites, and it

may also regulate NKCC2, as demonstrated in a yeast

two-hybrid screen [5] and in vitro [6].

These ion cotransporters are the targets of well known

antihypertensive drugs: NKCC2 is antagonized by fur-

osemide and NCC is sensitive to thiazide diuretics.

Mutations in the genes encoding these ion channels have

been implicated in the Mendelian forms of hypotension

(Bartter and Gitelman’s syndromes [7]), whereas

mutations in the WNK1 and WNK4 genes are causative

for pseudohypoaldosteronism type 2 featuring hyperten-

sion and hyperkalemia [8].

Thus, the WNK-SPAK-NKCC2/NCC pathway of salt

renal reabsorption is strongly implicated in blood pres-

sure (BP) homeostasis and hypertension development;

the fact that several genetic variants in STK39 were

associated with BP phenotypes in the GWAS support

this hypothesis. However, owing to the possibility of false

positive results, these findings need to be replicated in

independent cohorts.

The STK39 gene maps to chromosome 2q24.3, spans

approximately 300 kb and contains 18 exons; the SNPs

associated with BP by GWAS are located within introns

1–8 with no coding or splice variants identified by

sequencing [1]. Between the associated polymorphisms,

rs35929607 is located in a conserved intronic element

which, based on a multispecies sequence alignment, is

equally or more conserved evolutionarily compared with

the coding exons. The minor G-allele, tested with

luciferase reporter gene constructs, enhances transcrip-

tion in vitro and is predicted to upregulate the expres-

sion of STK39. Thus, this functional variant has been

indicated by the authors of the GWAS as responsible

for their findings of augmented BP in carriers of the

G-allele.

The aim of the present study was to test if the STK39rs35929607 polymorphism was implicated in hyperten-

sion prevalence and development in two large urban-

based cohort studies ongoing in the Malmo urban area:

the Malmo Diet and Cancer-cardiovascular arm (MDC-

CVA) and the Malmo Preventive Project (MPP), which

together include more than 20 000 people.

Participants and methodsAn extended version of the methods with further

details about the study populations and definitions of

hypertension and genotyping are reported in the

‘Supplementary methods and results’ section, http://

links.lww.com/HJH/A65.

All study participants provided written informed consent.

The procedures were in accordance with the institutional

opyright © Lippincott Williams & Wilkins. Unauth

guidelines. The Ethics Committee of the Medical

Faculty of Lund University approved the study.

Participants

Malmo Diet and Cancer-cardiovascular arm and Malmo

Preventive Project

The MDC study is an urban-based prospective study

ongoing in Malmo, southern Sweden. In all, 28 449

participated out of an eligible population of 74 000

between 1991 and 1996 [9]. BP, along with other cardio-

vascular risk factors, was measured in a random sub-

sample referred to as the MDC-CVA (n¼ 6103). Success-

fully extracted genomic DNA was available from 5763

MDC-CVA participants.

The MPP is an urban-based prospective study that

screened 33 346 Swedish participants from the city of

Malmo during 1974–1992 (attendance rate 71%) [10]. Of

the individuals participating in the initial screening, 4931

have died and 551 were lost after follow-up for other

reasons. Twenty-five thousand of the eligible individuals

were invited for a rescreening visit from 2002 to 2006,

including a physical examination with BP measurement.

DNA was obtained from 18 240 individuals participating

in the rescreening.

Blood pressure

We treated BP as a continuous variable both in the MDC-

CVA and the MPP before and after adjustment of the

measured BP values (see below) and as a dichotomized

trait (hypertension vs. normotension). Hypertension in

both cohorts was defined as being on antihypertensive

treatment or having a SBP/DBP equal or greater than 140/

90 mmHg (according to current diagnostic criteria) and

normotension as having a SBP/DBP less than 140/

90 mmHg.

In both studies, BP was measured by specially trained

nurses on the right brachial artery using a mercury

sphygmomanometer. The SBP was defined by ‘phase

I’ and the DBP was defined by ‘phase V’ Korotkoff

sounds.

Differences between the studies in the modality of BP

measurement were as follows:

In the MDC-CVA study, BP was measured after 10 min

of rest in the supine position. In the MPP, the first BP

reading was taken after 1 min of rest in the supine

position. Then, the participants were asked to stand

up and a second BP measurement was taken in the

upright standing position after 1 min. This procedure

was then repeated following an initial 10 min rest in

the supine position. The average BPs of all the partici-

pants with at least three valid measurements were used in

the present study. At reinvestigation in the MPP, BP was

measured twice in the supine position and all of the

measurements were recorded. The average BP of all

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486 Journal of Hypertension 2011, Vol 29 No 3

the participants with at least two valid measurements was

used in the present study.

Blood pressure adjustment

To overcome the possibility that a biased selection might

result from selecting only individuals who were free of

antihypertensive treatments, we conducted an analysis

adjusting the SBP and DBP of hypertensive individuals

that were taking antihypertensive drugs at the time of

investigation by two methods recently reviewed by Cui

et al. [11].

Fixed addition

On the basis of the known average treatment effects,

fixed increments of 10 mmHg SBP and 5 mmHg DBP

were added to the pressures of treated participants.

Stepped addition

To account for the number of drugs, stepped

increments of 8/4, 14/10, 20/16 and 26/22 mmHg

were added to the measured SBP/DBP of treated

individuals taking one, two, three and four drug classes,

respectively.

Data about antihypertensive medication, menopause

status and about hormone replacement therapy were

recorded from the standardized questionnaire and the

‘7 days book menu’, which individuals participating in

the two surveys were asked to fill out. Women who

confirmed that their menstruation had ceased or who

used HRT because of menopausal problems were classi-

fied as postmenopausal.

Salt sensitivity study (Salt Reduction to AvoidHypertension)Thirty-nine healthy participants (53.6� 11 years of age,

BMI 26.3� 3.1 kg/m2, number of men/women 20/19)

with no history of hypertension, diabetes or kidney dis-

ease completed a salt sensitivity study (Salt Reduction to

Avoid Hypertension, SARAH), published in 2007[12].

Their characteristics are presented in Supplementary

Table S6, http://links.lww.com/HJH/A65.

During a period of 8 weeks, the participants were given

all of their meals and drinks from a metabolic ward with a

constant daily energy intake. The diet contained

50 mmol of salt (NaCl) daily. In addition to this basal

intake, the study participants were given either 100 mmol

of salt in capsules (150 mmol of salt daily) for 4 weeks or a

corresponding number of placebo capsules (50 mmol of

salt daily) for 4 weeks in a random-order, double-blind

crossover design [12].

Ambulatory blood pressure (ABP) and 24-h urinary

excretion of sodium and potassium were measured at

baseline and after 4 weeks on the high salt intake diet

(150 mmol daily) or the low salt intake diet (50 mmol

daily).

opyright © Lippincott Williams & Wilkins. Unautho

Salt sensitivity was defined as the difference between

24-h systolic ABP (ABPM 90207 device; Spacelabs

Medical Inc., Redmond, Washington, USA) after 4 weeks

on the high salt diet and 24-h systolic ABP after 4 weeks

on the low salt diet. In addition, salt-induced changes in

diastolic ABP (diastolic salt sensitivity) were recorded,

and systolic and diastolic salt sensitivity were further

divided into daytime and night-time periods [12].

GenotypingThe STK39 rs35929607A>G polymorphism (dbSNP

accession number rs35929607) was determined by end-

point fluorescent measurements [13].

StatisticsContinuous variables are presented as the mean�SD. All

data, except for the power analysis, were analyzed with

SPSS statistical software (version 18.0; SPSS Inc., Chi-

cago, Illinois, USA). Power calculation was performed

using the Power and Sample Size calculator version 2.1.31

(Vanderbilt University Medical Center, Nashville,

Tennessee, USA).

The x2-test (Pearson’s) was used to compare group

frequencies and to test for deviations from Hardy–Wein-

berg equilibrium. Analysis of variance followed by

Tukey’s test and the t-test was used to compare group

means of continuous variables with the exception of the

salt sensitivity study, in which nonparametric tests were

applied. Multiple linear and logistic regression analyses

were used in the multivariate models with BP and hy-

pertension status as the dependent variables and geno-

type, age, sex, BMI, heart rate (when available), D-BMI,

follow-up years (when appropriate) and the interaction

variables (computed by multiplying the genotype

with the interacting variable; i.e. age, sex, etc.) as

independent variables.

All tests were two-sided and P-values less than 0.05 were

considered statistically significant.

ResultsThe clinical characteristics of the individuals included in

the study from both the MDC-CVA and the MPP (both at

baseline and at follow-up) are summarized in Table 1.

The power analysis is presented in Supplementary Table

S1, http://links.lww.com/HJH/A65.

The genotyping success rate was 97.8% (5634 of 5763

individuals) in MDC-CVA and 98.1% (17894 of 18240

individuals) in MPP. We found in MDC-CVA and MPP

69.5 and 68.5% rs35929607 GG-homozygotes, 27.5 and

28.5% GA-heterozygotes and 3 and 3% AA-homozygotes,

respectively.

Genotype distributions did not deviate from Hardy–

Weinberg equilibrium in any of the cohorts (predicted

heterozygosity 0.280, observed heterozygosity 0.275,

P¼ 0.24 in the MDC-CVA; predicted heterozygosity

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Serine/threonine kinase 39 gene and hypertension Fava et al. 487

Table 1 Anthropometric and metabolic features of the investigated participants in the Malmo Diet and Cancer-cardiovascular arm (baseline)and the Malmo Preventive Project (baseline and reinvestigation)

Variables Data available (n) MDC-CVA Data available (n) MPP at baseline Data available (n) MPP at follow-up

Sex (male) (%) 5634 41.7 17 894 63.3 17 894 63.3Age (year) 5634 57.5�5.9 17 894 45.2�7.4 17 874 68.2�5.8SBP (mmHg) 5634 141.2�19.0 17 565 126.8�14.1 17 693 144.9�20.0DBP (mmHg) 5634 86.9�9.4 17 560 85.3�8.7 17 690 83.6�10.6Heart rate (beats/min) 0 – 17 887 68.3�9.6 17 827 70.5�12.0BMI (kg/m2) 5627 25.9�4.0 17 887 24.3�3.4 17 790 27.2�4.1Obesity (%) 5627 13.5 17 887 5.6 17 790 21.7Hypertension (%) 5634 63.3 17 574 34.3 17 762 72.2Diabetes (%) 5090 8.7 17 780 3.2 17 641 13.2Current smoking (%) 5313 27.6 17 894 37.2 – –Menopause/perimenopause (% in women) 3121 78 3504 87.6 – –Antihypertensive therapy (%) 5634 16.1 17 864 4.4 17 891 38.3Hormone replacement therapy (% in women) 2811 19.9 3504 18.5 5752 12.3

MDC-CVA, Malmo Diet and Cancer-cardiovascular arm; MPP, Malmo Preventive Project.

0.285, observed heterozygosity 0.286, P¼ 0.58 in MPP;

and predicted heterozygosity 0.224, observed heterozyg-

osity 0.205, P¼ 0.97 in the SARAH study).

On the basis of the preliminary analyses on the data, we

focused our attention especially on the autosomal-domi-

nant genetic model, which is presented in the main

manuscript; data about autosomal-recessive and additive

genetic model are presented in the Supplementary

material, http://links.lww.com/HJH/A65.

Blood pressureCrude BP data and hypertension prevalence according to

genotype in the MDC-CVA and MPP (both at baseline

and reinvestigation) are presented in Supplementary

Table S2, http://links.lww.com/HJH/A65.

In the MDC-CVA, after adjusting for age, sex and BMI,

with and without adjusting for antihypertensive treat-

ment, carriers of the G-allele showed significantly higher

SBP and DBP (Table 2 and Complementary Table C2,

http://links.lww.com/HJH/A65). In the MPP, although

the sign of the association remained positive, the associ-

ation was not statistically significant (neither at baseline

nor at reinvestigation; see Supplementary Table S3,

opyright © Lippincott Williams & Wilkins. Unauth

Table 2 b-Coefficient and standard error (in parentheses) for theserine/threonine kinase 39 rs35929607 AG polymorphism tested bylinear regression according to the autosomal-dominant geneticmodel and after different types of blood pressure adjustment in theMalmo Diet and Cancer-cardiovascular arm

Type of blood pressure adjustmentfor antihypertensive therapya Autosomal dominant P-value

Unadjusted for antihypertensive therapya

SBP (mmHg) 0.956 (0.508) 0.06DBP (mmHg) 0.510 (0.260) 0.04

Fixed additiona

SBP (mmHg) 1.155 (0.537) 0.03DBP (mmHg) 0.610 (0.274) 0.03

Stepped additiona

SBP (mmHg) 1.115 (0.538) 0.04DBP (mmHg) 0.594 (0.281) 0.03

Adjustments for age, sex and BMI. a Details about the kind of antihypertensiveadjustment can be found in the ‘Participants and methods’ section.

http://links.lww.com/HJH/A65). When hypertension

was analyzed, carriers of the G-allele displayed a higher

prevalence of hypertension in both the MDC-CVA and

the MPP (at baseline and reinvestigation; see Table 3 and

Complementary Table C3, http://links.lww.com/HJH/

A65). When 2398 participants (13.4%) who also partici-

pated in the MDC-CVA were excluded from the MPP,

the higher hypertension prevalence in STK39 rs35929607

G-carriers was no longer statistically significant at base-

line [odds ratio (OR) 1.057, 95% confidence interval (CI)

0.979–1.142; P¼ 0.16] or at reinvestigation (OR 1.061,

95% CI 0.980–1.150; P¼ 0.14).

Interaction with demographic variables and stratifiedanalysisMalmo Diet and Cancer-cardiovascular arm

In linear regression analysis, no interaction of the

rs35929607A>G polymorphism with either sex, age or

BMI was evident (P> 0.05 for all the tested interaction

variables), but after stratification for sex, female but not

male carriers of the G-allele showed higher SBP and DBP

(Supplementary table S4, http://links.lww.com/HJH/

A65) and higher hypertension prevalence with respect

to AA-homozygotes (Table 3).

Malmo Preventive Project

Neither evidence of interaction nor significant results

after stratifying for sex were observed at baseline or at

reinvestigation for SBP or DBP. However, after stratifica-

tion for sex, similarly to findings in the MDC-CVA,

female carriers of the G-allele had a higher hypertension

prevalence (both at baseline and follow-up; see Table 3)

and incidence with respect to AA-homozygotes, whereas

no significant differences were evident in men.

When 1113 women (17%) who also participated in the

MDC-CVA were excluded from the MPP, the higher

hypertension prevalence in STK39 RS35929607 G-

carriers was still present, but it was not statistically

significant at baseline (OR 1.095, 95% CI 0.957–1.254;

P¼ 0.16). However, it remained statistically significant at

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488 Journal of Hypertension 2011, Vol 29 No 3

Table 3 Odds ratio and 95% confidence interval for hypertension conferred by the serine/threonine kinase 39 rs35929607 AG polymorphismtested by logistic regression according to the autosomal-dominant genetic model in the Malmo Diet and Cancer-cardiovascular arm and theMalmo Preventive Project (both at baseline and at follow-up) and after stratification for sex

All Women Men

OR (95% CI) P-value OR (95% CI) P-value OR (95% CI) P-value

Hypertension prevalence (MDC-CVA) 1.151 (1.016–1.305) 0.03 1.209 (1.030–1.420) 0.02 1.058 (0.865–1.294) 0.58Hypertension prevalence (MPP at baseline) 1.077 (1.002–1.157) 0.04 1.127 (0.997–1.274) 0.06 1.050 (0.960–1.147) 0.28Hypertension prevalence (MPP at follow-up) 1.092 (1.014–1.177) 0.02 1.209 (1.074–1.361) 0.002 1.019 (0.926–1.122) 0.70Hypertension incidence (MPP at follow-up) 1.064 (0.976–1.159) 0.16 1.230 (1.072–1.411) 0.003 0.968 (0.867–1.081) 0.56

CI, confidence interval; MDC-CVA, Malmo Diet and Cancer-cardiovascular arm; MPP, Malmo Preventive Project; OR, odds ratio.

reinvestigation (OR 1.163, 95% CI 1.021–1.325;

P¼ 0.02), as did hypertension incidence at reinvestiga-

tion (OR 1.178,95% CI 1.012–1.370; P¼ 0.03)

Exploratory analysis of interactions of the serine/threonine kinase 39 rs35929607A>G polymorphismwith menopause status and HRT

Menopause

Because the effect of the polymorphism was especially

evident in women, to further investigate the potential

effects of menopause on this issue, we conducted

additional exploratory analyses.

Neither the interaction between menopause status and

the polymorphism nor a stratified analysis according to

menopause gave significant results for BP or hyperten-

sion prevalence when tested in linear and logistic

regression analysis both in the MDC-CVA and the

MPP at baseline.

Exploratory analysis of the effects of HRTSuccessively, we looked at HRT and found no interaction

between the STK39 rs35929607 G-allele and HRT

regarding BP values and hypertension prevalence both

in the MDC-CVA and the MPP at baseline.

A borderline significant positive interaction of HRT with

the G-allele was evident for hypertension in the MPP at

reinvestigation (OR 1.39, 95% CI 0.99–1.95, P¼ 0.06 for

prevalent hypertension; OR 1.47, 95% CI 0.99–2.18,

P¼ 0.055 for incident hypertension), suggesting that

carriers of the G-allele could be at increased risk of being

or becoming hypertensive if treated with HRT.

In the analysis stratified according to HRT therapy, in

the MPP at reinvestigation, the magnitude of the risk

conferred by the G-allele was greater in women taking

HRT (OR 1.47, 95% CI 1.06–2.04, P¼ 0.02 for preva-

lent hypertension; OR 1.53, 95% CI 1.04–2.24, P¼ 0.03

for incident hypertension) compared with women not

taking HRT (OR 1.17, 95% CI 1.03–1.33, P¼ 0.01 for

prevalent hypertension; OR 1.19, 95% CI 1.03–1.38,

P¼ 0.02 for incident hypertension), further supporting

the potentially positive interaction between HRT and

hypertension.

opyright © Lippincott Williams & Wilkins. Unautho

Salt sensitivity studyIn the SARAH study, carriers of the STK39 AG and GG

genotypes showed higher D-systolic and D-diastolic ABP

values than did AA-homozygotes (statistically significant

for 24-h systolic ABP and nighttime diastolic ABP; see

Fig. 1).

DiscussionRecent GWASs have identified multiple loci and candi-

date genes for BP-related traits [1,14,15]. We found the

study recently published by Wang et al., in which several

intronic polymorphisms in the STK39 gene were associ-

ated with hypertension, to be of particular relevance. The

important renal salt reabsorption pathway this kinase

underlies made this result particularly meaningful and

prompted us to replicate it in our population.

We, thus, provide evidence that the STK39rs35929607A>G polymorphism is associated with hyper-

tension, at least in women, in two very large urban-based

surveys conducted in southern Sweden. Our analysis is

the largest ad-hoc replication of the finding. Despite the

fact that other GWASs have not pinpointed the same

locus [14–16], other investigators have recently focused

their attention on this interesting gene with conflicting

results. In the context of a GWAS in African–Americans,

Adeyemo et al. [2] were the first to replicate the positive

association between several SNPs in STK39 and hyper-

tension even if the result cannot be considered significant

at the genome-wide level. In contrast, a British study [17]

with a family design also using ABP monitoring failed to

find an association with BP, despite the fact that at least

three genetic variants (including the rs35929607 investi-

gated in the present survey) were associated with expres-

sion differences in peripheral blood cells [17].

The differences between these studies could be due to

differences in design, study population, genetic hetero-

geneity and/or statistical power. Our study has much

greater statistical power than the British family study,

and the effect of the polymorphism is quite modest,

especially when analyzed in the entire population

(a nearly 15% increase in hypertension prevalence in

the MDC-CVA and 8–9% in the MPP at baseline/rein-

vestigation); it is larger when only women are considered

(a nearly 21% increase in hypertension prevalence in

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Serine/threonine kinase 39 gene and hypertension Fava et al. 489

Fig. 1

Differences in blood pressure as measured by ambulatory blood pressure monitoring in carriers of the ‘deleterious’ STK39 rs35929607 G-allelecompared with AA-homozygotes after a 4-week high salt diet in the Salt Reduction to Avoid Hypertension study (see the text for details about theSalt Reduction to Avoid Hypertension salt sensitivity study). BP, blood pressure.

the MDC-CVA and 13–20% in the MPP at baseline/

reinvestigation).

A series of experiments in different animal models support

the theory that the WNK-SPAK-NCC system has a pivotal

role in the homeostasis of salt control. For example, in

mice, the mutation Wnk4 D561A was knocked-in, resulting

in increased phosphorylation of SPAK and the NCC. The

phosphorylation states of NCC and SPAK are enhanced by

low salt diets and reduced by high salt diets, and this

regulation was completely lost in the knock-in mice. The

increased phosphorylation can be reversed by spironolac-

tone, whereas the decreased phosphorylation can be

reversed by administration of exogenous aldosterone

[18]. More recently, knock-in mice in which SPAK cannot

be activated by WNKs showed significantly reduced BP

that was salt-dependent [19].

Furthermore, it has been shown that the regulation of

salt reabsorption through NCC and NKCC2, when

stimulated respectively by angiotensin II and intracellu-

lar chloride depletion, requires intact WNK4 and

WNK3–SPAK axes because the disruption of the SPAK

binding motifs abolishes this effect [20,21]. As already

emphasized, the genes encoding WNK4, NCC and

NKCC2 are also involved in the monogenic form of

hypertension in humans [7,8].

Thus, a very strong biological basis exists for the idea that

SPAK and the STK39 gene are involved in salt homeo-

stasis and, consequently, BP control in humans.

opyright © Lippincott Williams & Wilkins. Unauth

Our study further supports this association and indicates

that STK39 could be implicated in the pathophysiology of

hypertension in humans. If other studies support these

findings, the entire pathway could become a suitable

pharmacological target for antihypertensive therapy.

We focused our attention on the rs35929607A>G poly-

morphism due to its functionality because it has been

demonstrated, using two different approaches, that it can

enhance transcription in vitro and upregulate the expres-

sion of STK39 [1,17].

The clearer results in women were unexpected and have

not been previously investigated, but they are in accord-

ance with the demonstration that SPAK expression is

under both androgen and estrogen influences when

measured in LNCaP human prostate cancer cells. In fact,

it has been shown that SPAK expression can be upregu-

lated by androgens and inhibited by antiandrogen agents

and by phytoestrogen such as genistein [22,23]. Thus,

we speculate that sexual hormones could also influence

SPAK expression at the kidney level. Our exploratory

analysis of HRT, even if nonprespecified and made in a

sample not specifically collected for a pharmacogenetic

study, adds to this hypothesis by showing a potential

interaction between HRT treatment and the polymorph-

ism with respect to hypertension development. Further

studies in humans are needed to confirm the preferential

effect of this polymorphism in women, and animal

models could help clarify how this effect is mediated.

orized reproduction of this article is prohibited.

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490 Journal of Hypertension 2011, Vol 29 No 3

In our exploratory analysis of salt sensitivity, we also

found a possible explanation of the mechanism through

which STK39 rs35929607A>G SNP could affect hyper-

tension development.

In fact, we found that carriers of the G-allele, the allele

associated with hypertension development at the popu-

lation level, are more salt sensitive than AA-homozygotes

on the basis of a significant increase in D-ABP level when

passing from a low salt to a high salt diet. This result

further supports the hypothesis that SPAK could have

primary roles in the regulation of renal sodium channels

and salt reabsorption in humans.

This latter part of our study, even if highly underpowered

with respect to the others, is in perfect agreement and

reinforces the findings at the population level.

Moreover, several specific aspects of the salt sensitivity

study should be emphasized: first, salt sensitivity is a

much less complex phenotype than BP because it is

influenced by fewer environmental factors. In addition,

the heritability of salt sensitivity is higher than the

heritability of BP, with fewer genes likely conferring

gene–environment interactions [24]. Second, the ABP

monitoring used in this study gives a more accurate

estimate of the individual’s ‘true’ BP than office BP.

We have to acknowledge some limitations of our popu-

lation studies. More than 2000 participants (nearly 15%)

of the MPP sample were previously investigated in the

MDC-CVA, thus including these participants in the final

analysis could result in a ‘duplication’ bias, whereas

deleting them could result in an ‘inappropriate sampling’

bias. We chose to present both the analyses, which

unfortunately gave different results in the whole popu-

lation (a significant association when including them in

the MPP analysis, no significant association when delet-

ing them). This difference could be due to a lack of

statistical power because women were less represented in

the MPP than in the MDC-CVA. In any case, conclusions

about the effect of this polymorphism in the entire

population should be made cautiously. Nevertheless,

we had similar results in the female sample (a significant

association both when including and excluding them in

the MPP analysis), which strengthens the results, at least

in women.

Our findings cannot be generalized to populations with

genetic backgrounds different from that of our popu-

lation. Our results could be influenced by population

stratification and cryptic relatedness, even if in Scandi-

navians the first problem seems less common than in

other populations [25]. For cryptic relatedness, large

sample size with low inbreeding probably minimizes

the problem [26]. Some differences also exist between

the two populations investigated in the present study: the

mean age of the investigated participants is lower in the

MPP at baseline than in the MDC-CVA, and both results

opyright © Lippincott Williams & Wilkins. Unautho

are lower with respect to MPP at reinvestigation. This

difference is clearly important when considering the

increase in BP that occurs naturally during age, but we

emphasize that in all our analyses we considered ages

between the covariates and adjusted the final analysis for

its effect. In addition, the sex proportion varies such that

women are the majority in the MDC-CVA and only a

minority in the MPP; thus, we lost some statistical power

in the analysis stratified for sex. Another consideration

with respect to the MPP sample is that we could only get

DNA from participants who survived from the first to the

final examination (nearly 23 years of follow-up). Thus,

people at greater risk for cardiovascular disease (i.e.

carriers of deleterious polymorphisms) could have died

at a higher frequency than individuals not carrying a

deleterious polymorphism. This could have led to the

lower magnitude of the result in the MPP with respect to

MDC-CVA and to the lack of statistically significant

results for SBP and DBP traits.

Our adjustment for antihypertensive medications is a

relatively simple and widely adopted way to use data

coming from treated patients, and it has proven to aug-

ment familial genetic and shared environmental signals

without increasing the noise from individual-specific

sources of variation [11]. The exclusion of such partici-

pants greatly limits the power of genetic studies, not only

due to the decreased sample size but also due to the

exclusion of participants who have the highest probability

of carrying ‘prohypertensive’ genes. On the other hand,

this method constitutes an oversimplification that can

lead to bias.

Finally, we chose the rs35929607A>G polymorphism for

its in-vitro-demonstrated functionality, but we cannot

exclude the possibility that other SNPs in linkage dis-

equilibrium are responsible for our findings or that a tag-

SNP strategy could have had a better chance of capturing

the association of STK39 with hypertension.

Confirmation of positive findings derived from GWAS in

population-based sample of adequate size is warranted

before pursuing new candidate genes and pathways.

Our study, analyzing a total sample of more than 20 000

participants from the general population, represents a

reliable replication of the STK39 rs35929607A>G poly-

morphism conferring an increased risk of hypertension,

especially in women.

Owing to its pivotal role in sodium reabsorption at the

renal tubule level, the SPAK kinase might prove to be a

suitable target for antihypertensive therapy. Further

mechanistic studies should address the role of this kinase

with respect to the other kinases and sodium channels

involved in the pathway controlling renal tubular sodium

reabsorption.

The stronger effect of this polymorphism in women and

the exploratory analysis of sex hormones–STK39 gene

rized reproduction of this article is prohibited.

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Serine/threonine kinase 39 gene and hypertension Fava et al. 491

interaction add to the puzzle of sex difference in the

homeostasis of BP. Further investigations with the aim of

unraveling the complexity of sex/sex hormone–gene

interrelation in renal sodium handling and the develop-

ment of hypertension are needed.

AcknowledgementsThis study was supported by grants from the Swedish

Medical Research Council, the Swedish Heart and Lung

Foundation, the Medical Faculty of Lund University,

Malmo University Hospital, the Albert Pahlsson

Research Foundation, the Crafoord Foundation, the

Ernhold Lundstroms Research Foundation, the Region

Skane, Hulda and Conrad Mossfelt Foundation,

King Gustaf V and Queen Victoria Foundation and the

Lennart Hansson Memorial Fund. The authors acknowl-

edge the Knut and Alice Wallenberg Foundation for its

economic support of the SWEGENE DNA extraction

facility.

There are no conflicts of interest.

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