Published online before print February 16, 2004, doi: 10.1161/01.HYP.0000120121.43524.cd
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
(Hypertension. 2004;43:766.)
© 2004 American Heart Association, Inc.
Scientific Contributions |
WNK4 Intron 10 Polymorphism Is Not Associated With Hypertension
From the Basic & Clinical Genomics Laboratory, School of Medical Sciences and Institute of Biomedical Research, University of Sydney, Australia.
Correspondence to Brian J. Morris, Basic & Clinical Genomics Laboratory, School of Medical Sciences and Institute of Biomedical Research, Building F13, University of Sydney, NSW 2006, Australia. E-mail brianm{at}physiol.usyd.edu.au
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
A polymorphism in intron 10 of the serine-threonine kinase with no lysine (K) 4 gene WNK4 (G
A, base 1156666 on chromosome 17) has recently been associated with essential hypertension in a white American population. We have attempted to replicate this finding in a well characterized cohort of 184 unrelated hypertensive Australians of British extraction in which biological power was enhanced by them each having 2 hypertensive parents. Controls were 219 normotensive ethnically matched subjects whose parents were both normotensive. Genotyping was performed using the homogeneous MassEXTEND Assay. This showed a frequency of 0.10 for the minor allele in each group (P=0.88). Moreover, blood pressure, body mass index, sex, and plasma lipid levels were similar across genotypes. In conclusion, our study provides no support for an association of the intron 10 variant of WNK4 with essential hypertension in the Anglo-Australian population studied.
Key Words: kinase • hypertension, essential • genetics • polymorphism • sodium transport
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Alocus for essential hypertension has been reported on chromosome 17 in several chromosomal microsatellite scans,1–3 as had been noted for the equivalent region in the genome of genetically hypertensive rats.4,5 Recently, interest has focused on WNK4 (chromosome 17q21.2), which encodes a serine-threonine kinase with no lysine (K), but instead has a cysteine at a key position in the active site. Such interest was sparked by the finding that mutations in WNK4 are responsible for a monogenic form of hypertension pseudohypoaldosteronism type II (PHAII; Online Mendelian Inheritance in Man no. 145260),6 an autosomal dominant disorder, which is characterized by severe hypertension with hyperkalemia and metabolic acidosis. WNK4 is expressed almost exclusively in the kidney, where it localizes to the distal convoluted tubule and the cortical collecting duct, more specifically to the intracellular junctions in the distal convoluted tubule and in both the cytoplasm and intercellular junctions in the cortical collecting duct.6 Although essential hypertension clearly differs from rare monogenetic hypertensive disorders, genes for the latter have previously been implicated in the former, eg, the positive association findings with essential hypertension for single nucleotide polymorphisms (SNPs) of the aldosterone synthase gene7,8 (a severe mutation of which was originally implicated in the rare hypertensive condition glucocorticoid-remediable aldosteronism9,10) and also of the epithelial sodium channel11–13 (severe mutations of which have been implicated in Liddle’s syndrome10,13,14).
A SNP in WNK4 (GA, intron 10; bp 1156666) was reported recently in Hypertension to be associated with essential hypertension in white Americans.15 A finding of such potential importance calls for urgent confirmation in other settings. We therefore tested this SNP in case-control groups with high biological power as a result of them having parents who both had the same blood pressure (BP) status as the subject. These cohorts have been studied extensively and have been shown to be capable of demonstrating genetic associations with hypertension, should these be present.8,16–18
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Subjects
There were 184 unrelated essential hypertension patients and 219 unrelated normotensive controls. All subjects were volunteers from Sydney and surrounding areas and were of British ancestry. Ascertainment details have been described previously.8,16–19 To be included, subjects had to be free of diabetes, heart disease, renal disease, or secondary causes of hypertension. Hypertension was defined as having a systolic/diastolic BP of
140/90 mm Hg on 3 separate occasions before treatment. Normotensive subjects had a BP <130/90 mm Hg. Only hypertensives who were the offspring of parents who both had hypertension and normotensives whose parents were both normotensive after age 50 were included. Hypertensives with 2 affected parents represent 10% of hypertensive patients,19 ie, the hypertensives selected had been drawn from a population of 1840 hypertensives. By using such selection criteria, we have increased the power of this type of study to reveal any genetic contribution to the trait and were able to simultaneously reduce the sample size required. For example, for a polymorphism with a minor allele frequency of 0.20 that contributes to hypertension with a disease odds ratio of 1.5, the n value required for 80% power to detect an existing association could be reduced by >75% (unpublished calculations). The study had ethical approval and all subjects gave informed consent.
Genotyping
DNA was extracted from whole blood using a QIAamp DNA Blood Mini Kit (Qiagen). The genomic sequence for WNK4 was obtained from the Ensembl Human Genome browser (http://www.emsembl. org/Homo_Sapiens). The GA polymorphism in intron 10 of the WNK4 gene15 was detected using the homogeneous MassEXTEND Assay (Sequenom, http://www.sequenom.com). The MassEXTEND assay is based on primer extension whereby allele-specific polymerase chain reaction products are produced. Each primer extension product has a unique molecular mass, which allows genotyping by MALDI-TOF mass spectrometry. Genotyping was performed by the Australian Genome Research Facility.
Statistical Analysis
Total observed alleles on all chromosomes were calculated from genotype data. 
2 Analysis was used to test for differences in allele and genotype frequencies between hypertensive patients and normotensive controls. Comparison of demographic parameters across genotypes was by one-way ANOVA. Each was conducted using StatView (Abacus Concepts). Linkage disequilibrium was tested in the largest group, the normotensives, by the method of Hill.20
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
The characteristics of normotensive control subjects and hypertensive patients are shown in Table 1. Genotype frequencies did not deviate from Hardy-Weinberg equilibrium. Genotype and allele frequencies in the hypertensive group were similar to those in the normotensive group (Table 2) (for alleles, OR=1.0; 95% CI: 0.6 to 1.5). For a relative risk of hypertension of 1.6,21 our study had 95% power to detect a significant association (P<0.05) of the WNK4 variant with hypertension. Comparison across genotypes for the parameters in Table 1 also showed no significant differences. In the case of pretreatment systolic BP, values were 172±25, 178±26, and 191±30 mm Hg (n=120, 19, and 2, respectively; P=0.37), and for diastolic BP were 106±15, 108±14, and 110 mm Hg (n=118, 19, and 1, respectively; P=0.84).
|
|
The intron 10 variant was not in linkage disequilibrium with another polymorphism in intron 1013 (D'=0.037; P 0.05), and haplotype analysis of these 2 variants between the hypertensive and normotensive groups failed to show any association with hypertension (data not shown).
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Our data presented here argue strongly against the hypertension association for WNK4 reported recently by Erlich et al,15 applying to our hypertensive population, and raises the question that, at best, the previous association finding may not be generally applicable. The present finding adds, moreover, to our previous negative finding for another variant in intron 10 of WNK4.22 Erlich et al noted the association with hypertension in white, but not in black, hypertensives.15 Whereas we found that the frequency of the minor allele was 0.10 in each group, they found frequencies of 0.13 in 165 hypertensives and 0.07 in 91 normotensives. The significance of this difference was marginal (P=0.04). Moreover, they tested 8 polymorphisms, and given such multiple comparisons, their finding could represent a false-positive. Furthermore, their white study groups appeared to contain greater genetic heterogeneity than ours, which were restricted to white subjects of Anglo-Celtic extraction. In addition, the selection of our hypertensive population on the basis of a strong family history of hypertension meant that our cohort of patients, because of inherent biological power, had an increased likelihood of demonstrating a genetic contribution to hypertension. Thus, if an association of the WNK4 variant with hypertension existed in our population, our study should have been able to detect it.
Comparative analysis has found the entire coding sequence to be identical for WNK4 in stroke-prone spontaneously hypertensive and normotensive Wistar-Kyoto rats, and thus failed to support a role of WNK4 in polygenic hypertension.23 Moreover, no differences in WNK4 gene expression were observed in the distal nephron of kidneys from each strain and a secondary effect of BP on transcriptional regulation was excluded.23
Although a linkage peak for hypertension in the WNK4 region has been reported in several studies,1–3 many genome scans have failed to see either a hypertension or a BP linkage peak in this region.24,25 It has been pointed out recently25 that the greatest statistical support for the locus on chromosome 17 was in a study by Julier et al1 of groups recruited on the basis of diabetes. In the study by Baima et al, the rapid decay in linkage seen over a small chromosomal area of <1 cM2 is consistent with inaccurate marker placement and hence inter-marker distance or, less likely, to allele mis-specification.25 The study having the greatest power and best study design is that of Levy et al,3 and those studies that failed to find a chromosome 17 linkage peak might have been underpowered or comprised subjects with phenotypic differences, such as obesity, meaning that their hypertension may have a different genetic basis.24,25 Although the largest genome scan performed to date yielded a negative result for chromosome 17,26 the possibility remains of a minor contributory locus here or one that manifests only in, for example, diabetic hypertension.25
In conclusion, the present investigation offers no support for a previous association of an intron 10 polymorphism of WNK4 and essential hypertension. Our finding further lessens, but does not completely eliminate, the possibility that WNK4 could play a part in the cause of essential hypertension. Because it is unclear what functional significance this intron polymorphism would have, other polymorphisms in the coding or regulatory regions of WNK4 should perhaps be tested before ruling out completely the involvement of WNK4 in the cause of essential hypertension.
Perspectives
Mutations in WNK4 have been identified as causing the rare monogenic form of hypertension PHAII. The conjecture that it is possible that mutations in WNK4, which cause more subtle changes than those observed in PHAII, may contribute to polygenic hypertension is attractive, particularly because WNK4 is located in a region that demonstrates a quantitative trait locus for hypertension in humans and rats. The results of our association studies fail to support a role for WNK4 in essential hypertension in our Anglo-Australian population. Future work should be directed at other genes under the chromosome 17 linkage peak, but only in populations in which there is convincing evidence that a linkage peak truly exists in this region. The possibility that a true contributory locus to hypertension might only exist in diabetic subjects, whose blood pressure elevation might have a different cause than classic essential hypertension, should be borne in mind. Whether this could involve WNK4, or whether a completely different gene is involved, requires extensive further investigation. More work is also needed to see whether involvement of WNK4 could be restricted to a particular population, ethnic, or racial group.
|
Acknowledgments |
|---|
This study was supported by a grant from National Health and Medical Research Council of Australia. We thank Judith O’Neill for help in the collection of patient samples and Adam V. Benjafield for assistance with some of the statistical analyses.
Received September 30, 2003; first decision October 22, 2003; accepted January 20, 2004.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Julier C, Delepine M, Keavney B, Terwilliger J, Davis S, Weeks DE, Bui T, Jeunemaitre X, Velho G, Froguel P, Ratcliffe P, Corvol P, Soubrier F, Lathrop GM. Genetic susceptibility for human familial essential hypertension in a region of homology with blood pressure linkage on rat chromosome 10. Hum Mol Genet. 1997; 6: 2077–2086.
2. Baima J, Nicolaou M, Schwartz F, DeStefano AL, Manolis A, Gavras I, Laffer C, Elijovich F, Farrer L, Baldwin CT, Gavras H. Evidence for linkage between essential hypertension and a putative locus on human chromosome 17. Hypertension. 1999; 34: 4–7.
3. Levy D, DeStefano AL, Larson MG, O’Donnell CJ, Lifton RP, Gavras H, Cupples LA, Myers RH. Evidence for a gene influencing blood pressure on chromosome 17. Genome scan linkage results for longitudinal blood pressure phenotypes in subjects from the Framingham Heart Study. Hypertension. 2000; 36: 477–483.
4. Hilbert P, Lindpainter K, Beckmann JS, Serikawa T, Soubrier F, Dubay C, Cartwright P, De Gouyen B, Julier C, Takahashi S, Vincent M, D. Ganten D, Georges M, Lathrop GM. Chromosomal mapping of two genetic loci associated with blood-pressure regulation in hereditary hypertensive rats. Nature. 1991; 353: 521–529.[CrossRef][Medline] [Order article via Infotrieve]
5. Jacob HJ, Lindpainter K, Lincoln SE, Kusumi K, Bunker RK, Mao Y-P, Ganten D, Dzau VJ, Lander ES. Genetic mapping of a gene causing hypertension in the stroke-prone spontaneously hypertensive rat. Cell. 1991; 67: 213–224.[CrossRef][Medline] [Order article via Infotrieve]
6. Wilson FH, Disse-Nicodeme S, Choate KA, Ishikawa K, Nelson-Williams C, Desitter I, Gunel M, Milford DV, Lipkin GW, Achard J-M, Feely MP, Dussol B, Berland Y, Unwin RJ, Mayan H, Simon DB, Farfel Z, Jeunemaitre X, Lifton RP. Human hypertension caused by mutations in WNK kinases. Science. 2001; 293: 1107–1112.
7. Zhu H, Sagnella GA, Dong Y, Miller MA, Onipinla A, Markandu ND, MacGregor GA. Contrasting associations between aldosterone synthase gene polymorphisms and essential hypertension in blacks and in whites. J Hypertens. 2003; 21: 87–95.[CrossRef][Medline] [Order article via Infotrieve]
8. Kumar N, Benjafield AV, Lin RCY, Wang WYS, Stowasser M, Morris BJ. Haplotype analysis of aldosterone synthase gene (CYP11B2) polymorphisms shows association with essential hypertension. J Hypertens. 2003; 21: 1331–1337.[CrossRef][Medline] [Order article via Infotrieve]
9. Lifton RP, Dluhy RG, Powers M, Rich GM, Cook S, Ulick S, Lalouel J-M. A chimeric 11ß-hydroxylase/aldosterone synthase gene causes glucocorticoid-remediable aldosteronism and human hypertension. Nature. 1992; 355: 262–265.[CrossRef][Medline] [Order article via Infotrieve]
10. Morris BJ. Molecular genetics in childhood hypertension (review). Prog Pediat Cardiol. 1999; 9: 203–212.
11. Baker EH, Dong YB, Sagnella GA, Roghwell M, Onnipinla AK, Markandu ND, Cappuccio FP, Cook DG, Persu A, Corvol P. Association of hypertension with T594M mutation in ß subunit of epithelial sodium channels in black people resident in London. Lancet. 1998; 351: 1388–1392.[CrossRef][Medline] [Order article via Infotrieve]
12. Persu A, Coscoy S, Houot AM, Corvol P, Barbry P, Jeunemaitre X. Polymorphism of the gamma subunit of the epithelial Na+ channel in essential hypertension. J Hypertens. 1999; 17: 639–645.[CrossRef][Medline] [Order article via Infotrieve]
13. Persu A. The epithelial Na+ channel: progressing from Liddle’s syndrome to essential hypertension (Editorial comment). J Hypertens. 2003; 21: 855–857.[Medline] [Order article via Infotrieve]
14. Hansson JH, Nelson-Williams C, Suzuki H, Schild L, Shimkets R, Lu Y, Canessa C, Iwasaki T, Rossier B, Lifton RP. Hypertension caused by a truncated epithelial sodium channel 
subunit: genetic heterogeneity of Liddle syndrome. Nat Genet. 1995; 11: 76–82.[CrossRef][Medline]
[Order article via Infotrieve]
15. Erlich PM, Cui J, Chazaro I, Farrer, L. A., Baldwin CT, Gavras H, DeStefano AL. Genetic variants of WNK4 in whiles and African Americans with hypertension. Hypertension. 2003; 41: 1191–1195.
16. Wang WYS, Zee RYL, Morris BJ. Association of angiotensin II type 1 receptor gene polymorphism with essential hypertension. Clin Genet. 1997; 51: 31–34.[Medline] [Order article via Infotrieve]
17. Chambers SM, Morris BJ. Glucagon receptor gene mutation in essential hypertension. Nature Genet. 1996; 12: 122.[CrossRef][Medline] [Order article via Infotrieve]
18. Benjafield AV, Jeyasingam CL, Nyholt DR, Griffiths LR, Morris BJ. G-protein ß3 subunit gene (GNB3) variant in causation of essential hypertension. Hypertension. 1998; 32: 1094–1097.
19. Morris BJ, Zee RYL, Schrader AP. Different frequencies of angiotensin-converting enzyme genotypes in older hypertensive individuals. J Clin Invest. 1994; 94: 1085–1089.[Medline] [Order article via Infotrieve]
20. Hill WG. Estimation of linkage disequilibrium in randomly mating populations. Heredity. 1974; 33: 229–239.[Medline] [Order article via Infotrieve]
21. Weeks DE, Lathrop M. Polygenic disease: methods for mapping complex disease traits. Trends Genet. 1995; 11: 513–519.[CrossRef][Medline] [Order article via Infotrieve]
22. Benjafield AV, Katyk K, Morris BJ. Association of EDNRA, but not WNK4 or FKBP1B, polymorphisms with essential hypertension. Clin Genet. 2003; 63.
23. Monti J, Zimdahl H, Schulz H, Plehm R, Ganten D, Hubner N. The role of Wnk4 in polygenic hypertension: a candidate gene analysis on rat chromosome 10. Hypertension. 2003; 41: 938–942.
24. Morris BJ, Benjafield AV, Lin RCY. Essential hypertension: Genes and dreams. Clin Chem Lab Med. 2003; 41: 834–844.[CrossRef][Medline] [Order article via Infotrieve]
25. Knight J, Munroe PB, Pembroke JC, Caulfield M. Human chromosome 17 in essential hypertension. Ann Hum Genet. 2003; 67: 193–206.[CrossRef][Medline] [Order article via Infotrieve]
26. Caulfield M, Munroe P, Samani N, Pembroke J, Dominiczak A, Brown M, Benjamin N, Webster J, Ratcliffe P, O’Shea S, Papp J, Taylor E, Dobson R, Knight J, Newhouse S, Hooper J, Lee W, Brain N, Clayton D, Lathrop M, Farrall M, Connell J. Genome-wide mapping for human loci for essential hypertension. Lancet. 2003; 361: 2118–2123.[CrossRef][Medline] [Order article via Infotrieve]
This article has been cited by other articles:
 |
|
 |
|
  H. Zhang and J. A. Staessen Association of Blood Pressure With Genetic Variation in WNK Kinases in a White European Population Circulation, November 29, 2005; 112(22): 3371 - 3372. [Full Text] [PDF]
|
|
|
|
|
|
M. D. Tobin, S. M. Raleigh, S. Newhouse, P. Braund, C. Bodycote, J. Ogleby, D. Cross, J. Gracey, S. Hayes, T. Smith, et al. Association of WNK1 Gene Polymorphisms and Haplotypes With Ambulatory Blood Pressure in the General Population Circulation, November 29, 2005; 112(22): 3423 - 3429. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Meneton, X. Jeunemaitre, H. E. de Wardener, and G. A. Macgregor Links Between Dietary Salt Intake, Renal Salt Handling, Blood Pressure, and Cardiovascular Diseases Physiol Rev, April 1, 2005; 85(2): 679 - 715. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||