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 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 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.
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expression in that organ is of primary importance, as
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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-
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graphic measurements. Circulation 1978; 58:1072±1083.
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12 Fulker DW, Cherny SS, Sham PC, Hewitt JK. Combined linkage and
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linkage disequilibrium with some other as yet undiscov-
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13 Becker A, Busjahn A, Faulhaber H-D, BaÈhring S, Schuster H, Luft FC.
Automated zygosity determination with microsatellites. J Reprod Med 1997;
will have to be sequenced in a population such as ours,
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14 Livak KJ. Allelic discrimination using ¯uorogenic probes and the 59 nuclease
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In the study by Cusi et al. large numbers of mutant
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21 Neale MC, Cardon LR. Methodology for Genetic Studies of Twins and
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adducin gene locus is important to blood pressure
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VIII MULTIDISCIPLINARY SYMPOSIUM ON DESIGN AND EVALUATION OF DIGITAL CONTENT FOR EDUCATION VIII SIMPOSIO PLURIDISCIPLINAR SOBRE DISEÑO Y EVALUACIÓN DE CONTENIDOS DIGITALES PARA LA EDUCACIÓN PROGRAMA Versión del 3 de Junio de 2011 . Las presentaciones podrán hacerse en Español o en Inglés. Para cada Comunicación, se dispondrá de 15 minutos de presentación y discusión. Ciudad Real
Kanton St.Gallen Gesundheitsdepartement Fachkommission Infektion und Hygiene Empfehlungen Schutzimpfungen beim Medizinalpersonal Einführung Das Medizinalpersonal1 hat ein erhöhtes Risiko bestimmte Infektionskrankheiten zu erwerben. Einer- seits ergibt sich ein Infektionsrisiko durch den direkten Kontakt mit Patientinnen und Patienten, ande- rerseits können Medizinalpersonen