Linkage but lack of association for blood pressure and the alpha-adducin locus in normotensive twins

Linkage but lack of association for blood pressure and the á- Andreas Busjahn, Atakan Aydin, Nina von Treuenfels, Hans-Dieter Faulhaber, Hans-RuÈdiger Gohlke, Hans Knoblauch, Herbert Schuster and Background á-adducin is a cytoskeletal protein involved Conclusions The á-adducin gene locus is relevant to with sodium-pump activity in the renal tubule. The á- blood pressure regulation in normal subjects. Failure to adducin gene locus has been linked to hypertension and a ®nd an association between higher blood pressures and polymorphism identi®ed which is associated with the 460 Trp mutation suggests that this mutation may hypertension; however, the role of the á-adducin gene become important only when hypertension is triggered, or locus in normal blood pressure regulation is not de®ned.
that other variations in á-adducin are present which have We performed a combined linkage and association study in not yet been discovered. J Hypertens 1999, 17:1437±1441 normotensive monozygotic (MZ) and dizygotic (DZ) twins & Lippincott Williams & Wilkins.
and their parents to address this issue.
Methods We studied 126 MZ and 70 DZ twin pairs and Journal of Hypertension 1999, 17:1437±1441 parents of DZ twins. Blood pressure values and responsesto a cold pressor test were obtained. Cardiac dimensions Keywords: adducin, association, blood pressure, genes, genetics, were measured echocardiographically. Three microsatellites adjacent to the á-adducin gene were Franz Volhard Clinic and Max DelbruÈck Center for Molecular Medicine, Medical studied as well as the 460 Trp mutation in the á-adducin Faculty of the ChariteÂ, Humboldt University of Berlin, Germany.
Sponsorship: These studies were supported by a grant-in-aid `German Human Genome Project', Bundesministerium fuÈr Bildung und Forschung, Bonn, Results We obtained strong evidence for linkage (P < 0.001) between the á-adducin gene locus and systolic Correspondence and requests for reprints to Friedrich C. Luft, Franz Volhard Clinic, Wiltberg Strasse 50, 13122 Berlin, Germany.
blood pressure. However, we were not able to associate Tel: ‡49 30 9417 2202; fax: ‡49 30 9417 2206; e-mail: [email protected] the 460 Trp mutation with higher blood pressures, cold Received 27 April 1999 Revised 7 June 1999 pressor responses or cardiac dimensions.
tension. Persons heterozygous for the mutant Gly/Trp Adducin is an á/â heterodimeric cytoskeletal protein allele showed a greater decrease in blood pressure with present in many tissues, including the kidney The acute volume contraction than subjects homozygous for protein participates in the regulation of cell-signal the Gly/Gly wild-type allele. Furthermore, Gly/Trp transduction through changes in the actin cytoskeleton individuals also displayed greater decreases in blood Alterations in adducin have been shown to in¯u- pressure with hydrochlorothiazide treatment, than wild- ence the surface expression and maximum velocity of type homozygous hypertensive patients. These data the sodium±potassium pump Known point muta- implicate mutations in á-adducin as responsible for tions, one each in the á- and â-adducin subunits, salt-sensitive hypertension in man. Hypertension af- account for up to 50% of the difference in blood fects 20% of the general population. Furthermore, over pressure between the Milan hypertensive and normo- half of individuals reaching 70 years of age will develop tensive rat strains An association between allelic hypertension Thus, identi®cation of quantitative markers close to the á-adducin gene locus and hyper- trait loci (QTL) for blood pressure regulation in normal tension has been shown in man Recently, Cusi et subjects is of interest. There is precedence for such al. studied sibling pairs affected by hypertension.
loci: a recent study has shown the Liddle hypertension They were able to show that the á-adducin gene locus gene locus to be an important QTL for blood pressure is linked to hypertension in these subjects. They then in normotensive persons We used a combined performed salt-sensitivity testing in hypertensive indi- linkage and association approach to test the relevance viduals with or without the 460 Trp á-adducin allele.
of the á-adducin gene locus and its variation to blood The 460 Trp mutation was associated with hyper- pressure regulation in normal subjects.
0263-6352 & 1999 Lippincott Williams & Wilkins 1438 Journal of Hypertension 1999, Vol 17 No 10 used probes speci®c for the wild-type and mutated á- We recruited 196 pairs of twins (126 monozygotic (MZ) adducin gene sequences. The wild-type probe (for Gly and 70 dizygotic (DZ)) and the parents of the DZ twins 460) and the mutated probe for Trp 460 have been by advertisement to participate in studies involving described previously The probes were labelled and blood pressure regulation and cardiovascular pheno- hybridization studies of PCR products from the geno- types The subjects were all German Caucasians.
mic DNA were performed to identify the point muta- They were recruited from various parts of Germany.
tions in the á-adducin gene. A novel 59 nuclease assay The protocol was approved by Humboldt University's committee on the protection of human subjects, and written informed consent was obtained from all partici- We assessed linkage for blood pressure as a continuous pants. Blood was obtained for the determination of trait rather than differentiating between normotensive zygosity and other molecular genetic studies from all and hypertensive subjects Sibling pair analysis to the twins and the parents of the DZ twins. Each determine linkage does not require the speci®cation of participant underwent a medical history and physical a genetic model. The underlying trait can follow either examination. None had hypertension or any other Mendelian or non-Mendelian modes of inheritance.
chronic medical illness. Blood pressure was measured Analysis was done by using a structural equation after 5 min (two measurements, 1 min apart) with a modelling approach as implemented in the MX standardized mercury sphygmomanometer in the sit- package From the four alleles harboured by the ting, standing and recumbent positions by a trained parents for a given locus, each child randomly inherits physician. The mean of the two measurements was two. Thus, a pair of siblings may have inherited either the same or different alleles. More speci®cally, they may share zero, one or two alleles identical by descent M-mode and two-dimensional echocardiograms were (IBD). If the locus under study is a QTL, phenotypic recorded with patients in the left-lateral decubitus similarity of siblings (measured by the covariance) position. M-mode tracings that were guided two-dimen- should increase with the number of alleles they share.
sionally were recorded from the short parasternal axis at Assuming no dominance effects, the total variance of the chordal level between the free edges of the mitral the trait is due to the genetic effect of the QTL lea¯ets at the tips of the papillary muscles. Only (Varqtl), remaining additive genetic effects (VaraddGen) tracings with optimal visualization of left ventricular interfaces were used. In our echocardiographic labora- tory, the range of variability of observations by a single reader is 0±1.5 mm for the left ventricular dimensions and 0±0.5 mm for the wall thickness. Interventricular Accordingly, the covariance of the three types of septal thickness (IVS) and posterior-wall (PW) thick- siblings as determined by their IBD status can be ness were measured in all patients, and left ventricular dimensions were calculated by the Penn formula according to the guidelines of the American Society of For the linkage study, the DZ pairs were used as ordinary sibling pairs, but with the advantage of perfect age matching and reduced environmental variation affecting the phenotype. By genotyping the parents, we For linkage analysis, a model is speci®ed estimating were able to perform an identity by descent (IBD) Varqtl, VaraddGen and Varenv so that the likelihood of the analysis. The power of the twin model in elucidation of empirical variance±covariance matrix of the sibs, complex genetic disease has recently been demon- weighted by the probability of sharing zero, one or two strated by Martin et al. The novelty of utilizing alleles identical by descent, is maximized. For each combined linkage and association sibling pair analyses sibling pair and each locus, the proportion of alleles for quantitative traits, as we employ here, has recently IBD, based on parental genotypes, is calculated using a been emphasized by Fulker et al. The MZ twins multi-point approach as implemented in MAPMAKER/ were used to estimate allele frequencies for the mar- SIBS To test for a QTL effect, the difference in kers tested. The zygosity was veri®ed with the use of model ®t for models with and without a QTL effect is ®ve polymerase chain reaction-ampli®ed microsatellite calculated as a ÷2 statistic. Since we used a candidate markers as described in detail elsewhere We gene approach, we accepted P , 0.01 to test for signi®- examined three microsatellite markers at the á-adducin cant linkage in accordance with the criteria de®ned by gene locus on chromosome 4 as described previously Lander and Kruglyak The high power of the namely D4S43, D4S95 and D4S228/E24. We then variance±covariance-based analysis, nearly twofold Adducin as QTL for blood pressure Busjahn et al. 1439 greater than the squared trait differences-based ap- Demographic data, phenotypic values (mean 6 SD), proach by the Haseman/Eston method, has been con- heredity estimates (á2) and correlations (r) for monozygotic (MZ) ®rmed in a recent simulation study Thus, while signi®cant linkage results obtained in smaller samples are still reliable, failure to detect linkage raises the issue of a lack of power and should not be interpreted Parameters of the quantitative genetic models were estimated by structural equation modelling using the MX program developed by Neale The variability of any given phenotype within a population can be broken down into genetic in¯uences (VaraddGen), envir- onmental in¯uences shared by the twins within a family (VarsharedEnv) and effects of random environment s the results of linkage analysis for the tested loci in the DZ twins using systolic and diastolic Var ˆ VaraddGen ‡ VarsharedEnv ‡ Varenv blood pressure in the sitting, standing and recumbent position as the phenotype. The blood pressure values For MZ and DZ, the covariance of their phenotype is are corrected for age and gender, although uncorrected values gave similar results. We found strong evidence for linkage of systolic blood pressure in the siting and standing positions to the á-adducin gene locus. For systolic blood pressure in the recumbent position, the results are suggestive for linkage. These results estab- lish the á-adducin gene locus as a QTL for systolic Heritability analysis in twin studies can estimate addi- blood pressure. We were not able to show such tive components of genetic variability as well as two evidence for linkage to diastolic blood pressure.
environmental in¯uences, shared and non-shared envir- onmental in¯uences These values estimate the We tested the hypothesis that systolic blood pressure relative amount of the variable's in¯uence on inter- and diastolic blood pressure may be in¯uenced by individual differences up to a sum of 1. Genetic as well different genes. The extent to which both systolic and as environmental effects were estimated by the best diastolic blood pressure are in¯uenced by the same ®tting model as selected by the ÷2 value. Statistical genes was measured by the genetic correlation (rg). For analysis was conducted using the SPSS program (SPSS blood pressure measured in the sitting position, the Inc., Chicago, Illinois, USA). Adjustment of blood value was rg ˆ 0.53. To test the signi®cance of the pressure values for sex and age was done by multiple genetic correlation, submodels of the bivariate analysis linear regression with the unstandardized residuals as were calculated setting either the shared or speci®c genetic in¯uences to zero. Both submodels resulted in a signi®cant loss of ®t (P , 0.01), con®rming the Finally, we performed a bivariate analysis to determine importance of both the shared as well as the speci®c the degree to which systolic and diastolic blood pres- sure are in¯uenced by different genes or different sets of genes This analysis relies on maximum like- Results of linkage analysis. The difference between lihood estimates of genetic in¯uences.
structural equation modelling with/without quantitative trait loci (QTL) effect and corresponding P values for a QTL effect are given Demographic data, blood pressure values and heredity estimates of blood pressure and echocardiographic vari- ables in 252 MZ and 140 DZ twins are given in There were no signi®cant differences between MZ and DZ twins for any of the variables examined.
Systolic and diastolic blood pressure were heritable.
The heritability estimates for systolic blood pressure Diastolic BP increase (cold pressor test) were about double those for diastolic blood pressure.
The echocardiographic parameters also demonstrated BP, blood pressure; NS, not signi®cant.
1440 Journal of Hypertension 1999, Vol 17 No 10 the results of the association analysis.
in¯uences shared by the traits and speci®c genetic For this analysis, the DZ twins and one of the MZ in¯uences are estimated. The extent to which both twins of each pair were genotyped, 266 subjects in all.
systolic and diastolic blood pressure are in¯uenced by We found no association between wild-type Gly/Gly the same genes can then be measured by the genetic homozygosity, Gly/Trp heterozygosity or Trp/Trp correlation. Our results indicates that about 50% of the homozygosity on blood pressure or heart size. Hardy± genetic in¯uences on blood pressure affect both systolic and diastolic blood pressure, an example of pleiotropy.
The remaining genetic in¯uences are speci®c to either systolic blood pressure or diastolic blood pressure. To The important ®nding in this study is that the á- test the signi®cance of the genetic correlation, submo- adducin gene locus was linked to systolic blood pres- dels of the bivariate analysis are calculated, setting sure in young healthy individuals. To our knowledge, either the shared or speci®c genetic in¯uences to zero.
this is the ®rst observation to document the importance Both submodels con®rmed the importance of both the of á-adducin to blood pressure regulation prior to the shared and the speci®c genetic in¯uences in our development of hypertension. The result implies that variability in the á-adducin gene must exist, which in¯uences blood pressure. Interestingly, we could not We believe our approach of identifying linkage in show that the 460 Trp polymorphism contributed to normotensive DZ twin subjects and their parents is a this variability in normal individuals. Our ®ndings are uniquely powerful method to establish the relevance of con®ned to systolic blood pressure. We found no genes regulating blood pressure in normal people. In an linkage to diastolic blood pressure. There are several earlier study, we found strong evidence for linkage of possible explanations. The genes for regulatory systems the IGF-1 and renin gene loci to blood pressure in¯uencing systolic and diastolic blood pressure may The subsequent step is to identify all the genetic not invariably be the same, a hypothesis we tested.
variants in these candidate genes so that these variants Systolic blood pressure may be more accurately meas- can be retested in terms of an association study in the ured. Finally, the robustness of genetic variance on same subjects. There is precedence for this approach.
blood pressure in our study was almost twice as great We recently employed multiplex sequencing to identi- for systolic as for diastolic blood pressure.
fy all variants in the â-2 adrenergic receptor gene in the normotensive offspring of hypertensive parents from Systolic and diastolic blood pressure may well be in¯u- the Bergen Blood Pressure Study Four mutations enced by different genes. For instance, systolic blood causing amino acid substitutions were found, which pressure may be more dependent on aortic elasticity were in linkage disequilibrium with each other. The than diastolic blood pressure, which in turn might be complexity of the resulting haplotype analysis is a more dependent on peripheral vascular resistance. To harbinger of the future dif®culties geneticists will test the hypothesis that systolic and diastolic blood encounter when considering the effects of multiple pressure are affected by different sets of genes, as well genes and their haplotypes simultaneously.
as pleiotropic genes, we performed a bivariate herit- ability analysis according to the methods of Neale and Our failure to ®nd an association between variances at Cardon Based on the variance±covariance matrix the 460 Trp site and blood pressure in these normal of the two traits in MZ and DZ twins, both genetic subjects by no means distracts from the potential importance of this polymorphism. Some of our subjects may develop hypertension at a later date, at which time such association may become apparent. The association Effects of the á-adducin polymorphism. The dizygotic twins and one of the monozygotic twins from each pair were was particularly prominent in terms of responses to volume loading and contraction, and in terms of blood pressure decreases with diuretic therapy in the studies of Cusi et al. and subsequent observations by Manunta et al. We did not examine salt sensitivity or resistance in this study. Conceivably, had we performed salt sensitivity testing in these normal sub- jects, we might have identi®ed allelic association. We did, however, examine the blood pressure responses to a stressor impulse, the cold pressor test. In an earlier Diastolic BP increase (cold pressor test) study we showed that this response is also in¯u- enced by genetic variance, but that different genes were likely to be responsible. Thus, our failure to ®nd There were no signi®cant differences between groups. BP, blood pressure.
an association between the á-adducin gene locus and Adducin as QTL for blood pressure Busjahn et al. 1441 this response may very well be predicated on the fact function in assembly of spectrin±actin complexes. J Biol Chem 1995; that different genes are involved. Kamitani et al. searched for association between the 460 Trp mutation 2 Tripodi G, Valtorta F, Torielli L, Chieregatti E, Salardi S, Trusolino L, et al.
Hypertension-associated point mutations in the adducin alpha and beta and high or low blood pressure from a Scottish cohort.
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Association of the alphá-adducin locus with essential hypertension. Hyper- 5 Cusi D, Barlassina C, Azzani T, Casari G, Citterio L, Devoto M, et al.
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hypertension. In the Milan strain, hypertension can be transplanted with the kidney, suggesting that á-adducin 8 Busjahn A, Faulhaber H-D, Viken RJ, Rose RJ, Luft FC. Genetic in¯uences expression in that organ is of primary importance, as on blood pressure with the cold pressor test: a twin study. J Hypertens opposed to á-adducin expression elsewhere Cross- 9 Busjahn A, Knoblauch J, Knoblauch M, Bohlender J, Menz M, Faulhaber immunization of cytoskeleton proteins between the H-D, et al. Angiotensin converting enzyme and angiotensinogen gene polymorphisms, plasma levels, and left ventricular size: a twin study. Hyper- Milan hypertensive and normotensive rat strains showed immunochemical differences in rat adducin 10 Sahn DJ, DeMaria A, Kisslo J, Weymen A. Recommendations regarding Such studies have not been performed with hu- quantitation in M-mode echocardiography: results of a survey of echocardio- graphic measurements. Circulation 1978; 58:1072±1083.
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Automated zygosity determination with microsatellites. J Reprod Med 1997; will have to be sequenced in a population such as ours, so that all genetic variants are known, as was done in 14 Livak KJ. Allelic discrimination using ¯uorogenic probes and the 59 nuclease the subjects of the Bergen Blood Pressure Study [23].
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higher blood pressure in these normal subjects. In 21 Neale MC, Cardon LR. Methodology for Genetic Studies of Twins and conclusion, our data support the notion that the á- Families. Dordrecht, The Netherlands: Kluwer Academic Publishers; 1992, adducin gene locus is important to blood pressure 22 Nagy Z, Busjahn A, BaÈhring S, Faulhaber H-D, Gohlke H-R, Knoblauch H, regulation in normal man. Sequencing the entire gene et al. Quantitative trait loci exist near the IGF-1, the Liddle syndrome, the in this subject cohort and in others will elucidate all á- angiontensin II-receptor gene and the renin loci in man. J Am Soc Nephrol adducin polymorphisms in¯uencing blood pressure.
23 Timmermann B, Mo R, Luft FC, Gerdts E, Busjahn A, Omvik P, et al. â-2 Such an effort in this and other genes will be necessary adrenoceptor genetic variation is associated with genetic propensity to to further elucidate the molecular genetics of blood essential hypertension: the Bergen Blood Pressure Study. Kidney Int 1998; pressure regulation in normal persons and essential 24 Manunta P, Cusi D, Barlassina C, Righetti M, Lanzani C, D'Amico M, et al.
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