INTRODUCTION
Some variability in drug response can be explained by genetic
variability in drug targets. Single nucleotide polymorphisms (SNP) in
receptor encoding genes, may result in altered function, substrate
binding affinity, expression, and both up- and downregulation of
receptors.1
In the cardiac β-adrenoceptor (AR) pathway β-ARs themselves and Gs
protein α-subunit
have been found to date to display some genetic variation in humans that
might result in different response to inotropic therapy.2
The
β1-AR
gene (ADRB1 ; RefSeq NM_000684.2) has two commonly variable
sites, at amino acid positions 49 and 389. The most studied AR gene
polymorphism is the Arg389Gly human β1-AR gene polymorphism. It is
associated with significantly elevated adenylate cyclase (AC)/protein
kinase A activities and hence β1-AR- stimulated cardiac contractility in
the Arg389 variant carriers compared with Gly389 carriers.3
Arg389 β1-AR is more common than Gly389 β1AR in individuals of European
descent.
A second human β1-AR polymorphism is the p.Ser49Gly variation,
associated with increased long-term agonist-promoted receptor
downregulation for the Gly49 over the Ser49 variant.4,5 The beneficial effect of Gly49 variant on survival
of heart-failure patients, is supporting the concept that β1-AR
desensitization is protective in heart failure. 6
Three nonsynonymous polymorphisms have been shown to exist in the coding
region of the β2-AR gene (ADRB2, RefSeq NM_000024.5), at amino
acid positions 16, 27, and 164. 7 Gly16Arg and
Gln27Glu variations are in linkage disequilibrium and thus constitute a
haplotype, which affects receptor downregulation. And a third
polymorphism, Thr164Ile, a rare polymorphism (Ile164, heterozygous
frequency ~0.03) that confers impaired receptor G
protein coupling and reduced acetylcholine mediated signalling,
resulting in severely depressed or absent agonist-promoted cardiac
contractility. 8–10
The β-AR-Gs protein system is essential for the activation of AC in
cardiac smooth muscle, which regulates cardiac output and peripheral
vascular resistance. 11,12 Retrospective studies
suggest that the c.393C>T polymorphism in the human gene
encoding the α-subunit of Gs protein (GNAS, RefSeq NM_000516.4)
has an effect on response to β-blockade and heart rate after exercise
stress test. 13,14 Data from a prospective study
suggest that the c.393C>T polymorphism of GNAS is
functionally relevant in vivo . The results of a modified
dobutamine
stress echocardiography protocol show that individuals with homozygous
or heterozygous C393 have an increased cardiovascular agonistic response
to dobutamine.15
According to PharmGKB database, β1-AR gene Arg389Gly and GNAS gene
c.393C>T polymorphisms are associated with haemodynamic
response to dobutamine in vivo . 16 However,
there are no data on the effect of the genetic variability of β-ARs or
Gs protein α-subunit on haemodynamic response to agonist stimulation in
early neonatal life.
Our study aimed to find out whether the known SNPs in the β1-, β2-AR and
GNAS genes play a role in the variability of cardiovascular response to
dobutamine treatment in critically ill neonates.
METHODS
The study was conducted as a sub study of dobutamine
pharmacokinetic-pharmacodynamic (PKPD) study, approved by the Ethics
Committee of the University of Tartu and registered at the EU Clinical
Trials Register under number 2015–004836-36. A prospective 2-centre
study was performed from April 2016 to December 2017 in Tallinn
Children’s Hospital, Tallinn, Estonia and Tartu University Hospital,
Tartu, Estonia. Full details of the study can be found elsewhere.17
Briefly, neonates hospitalised to NICU within the first 72 hours of life
and needing inotropic therapy were studied. Written informed consent for
genetic analysis was signed by parents or guardians separately from the
main dobutamine PKPD study consent before study inclusion.
Whole blood samples of 0.3 ml were collected from indwelling arterial or
venous catheter within the study period. The samples were transported
immediately to the Department of Clinical Genetics, United Laboratories,
Tartu University Hospital, Tartu, Estonia. DNA was extracted from blood
lymphocytes by Nucleospin Tissue kit (Macherey-Nagel). Sanger sequencing
was performed for ADRB1 gene exon 1, ADRB2 gene exon 1 and
GNAS gene exon 5, accordingly including the following variants of
interest: β1-AR p.Gly389Arg, β1-AR p.Ser49Gly, β2-AR Gly16Arg, β2-AR
Gln27Glu, GNAS c.393C>T. PCR primers used are provided in
Supplementary Table 1. Sanger sequencing was performed with same primers
as for PCR amplification and products were sequenced bidirectionally
using ABI GeneScan 3130XL instrument.
Dobutamine was administered at escalating doses of 5-20 μg
kg−1 min−1. Monitoring and recording
of heart rate (HR) and mean arterial pressure (MAP) were started before
dobutamine infusion and continued throughout the treatment period, left
ventricular ejection fraction (LVEF), left and right ventricular cardiac
output (LVO, RVO) were measured before and repeatedly during the
dobutamine treatment. 17
The effect of SNPs on haemodynamic parameters (HR, MAP, LVEF, LVO and
RVO) was analysed with linear mixed−effects models separately for each
SNP. The model building process was started with full model including
gestational age (GA), Score for Neonatal Acute Physiology Perinatal
Extension (SNAPPE-II) 18, age at recruitment,
antenatal glucocorticoid administration (only if GA <34
weeks), exposure to dobutamine measured as the area under the
time-plasma concentration curve (AUC) calculated according to the
published dobutamine population pharmacokinetic model using empirical
Bayes estimates of pharmacokinetic parameters in each neonate, and
interaction between AUC and SNP as fixed effects and random intercept
and random slope of AUC for each neonate. 17 Stepwise
backward elimination was used to retain only statistically significant
effects into model. Autocorrelation structure of order 1 with time after
dose (in minutes) as covariate was used in models for HR and MAP.
Key protein targets and ligands in this article are hyperlinked to
corresponding entries in http://www.guidetopharmacology.org, the
common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY.
RESULTS
Overall, 26 of the 28 PKPD study participants were consented to the
pharmacogenomic study. The median (range) gestational age was 30.9 (22.7
– 41.0) weeks, birth weight 1668 (465 – 4380) g, age at recruitment
6.3 (2.4 – 27.5) h, SNAPPE-II scores 14.5 (3.0 – 64.0), 5 min APGAR
score 7 (1 – 8), baseline LVEF 62.5 (51.0 – 79.0) %, RVO 126 (75 –
306) mL kg-1 min-1, LVO 128 (71 –
232) mL kg-1 min-1.
The incidences of investigated alleles were comparable to those of
general population
of Estonia in all observed SNPs. (Table 1)
We found that β1-AR Arg389Gly polymorphism influences HR increase during
dobutamine treatment and GNAS c.393C>T polymorphism affects
cardiac output response to dobutamine. The other studied SNPs did not
show statistically significant effect on dobutamine induced changes in
HR, MAP, cardiac output or LVEF. The linear mixed-effect models, where
the effect of SNPs on hemodynamic parameters was statistically
significant are presented in Table 2. HR adjusted to GA is dependent on
β1- AR Arg389Gly polymorphism so that in G/G (Gly) homozygotes and G/C
heterozygotes dobutamine increases HR more steeply than in C/C (Arg)
homozygotes (p=0.0008). (Figure 1) LVO adjusted to antenatal
glucocorticoid administration and SNAPPE-II is dependent on GNAS
c.393C>T polymorphism so that in T/T homozygotes and C/T
heterozygotes but not in C/C homozygotes LVO increases during dobutamine
treatment (p=0.0095). (Figure 2) RVO adjusted to SNAPPE-II and GA is
dependent on GNAS c.393C>T polymorphism so that in T/T
homozygotes and C/T heterozygotes but not in C/C homozygotes RVO
increases with dobutamine treatment (p=0.0025). (Figure 3)
Individual measured haemodynamic parameter values, population (marginal)
and individual (conditional) predictions from the final linear mixed
effects models plotted against dobutamine AUC are presented in the
supplementary figures S1-S3.
DISCUSSION
To the best of our knowledge this is the first study demonstrating the
role of polymorphisms in the β-AR pathway related genes in the
variability of response to inotropic therapy in critically ill neonates.
The main findings of the study were associations between Arg389Gly
polymorphism in β1-AR gene and HR as well as GNAS c.393C>T
polymorphism and cardiac output (RVO and LVO).
There is only sparse clinical data about the influence of studied
variants and cardiovascular response to inotropes. In our study, β1-AR
Arg389 homozygotes had lower heart rate in response to dobutamine
compared to heterozygotes and Gly389 homozygotes, with no effect on RVO
or LVO. In contrast, previous studies in adults found that human hearts
with Arg389 β1-AR variant, from both healthy subjects and patients with
heart failure, had substantially greater agonist-induced inotropy
compared with hearts from Gly389 carriers, in an ex vivoexperiment. 20 Healthy male volunteers homozygous for
the Arg389 β1-AR showed significantly higher increase in fractional
shortening upon cumulative doses of dobutamine as compared to subjects
carrying one or two copies of the Gly389 allele.21 In
a study of 10 and 8 male subjects homozygous Arg389 β1-AR and Gly389
β1-AR, respectively (to avoid influences of codon 49 polymorphism, all
were homozygous Ser49 β1-AR), dobutamine increased plasma-renin
activity, heart rate and contractility and decreased diastolic blood
pressure more potently in Arg389 β1-AR versus Gly389 β1-AR
subjects.22. In healthy individuals, HR and renin
responses to dobutamine in incremental doses were more than three-fold
greater among β1-AR Arg389 compared with Gly389 homozygotes.23 In the same study population, no significant
difference was found in haemodynamic response to dobutamine constant
infusion between β1-AR 389 genotypes. 24 The
difference in the effect on chronotropy in neonates, compared to adults,
may be explained by developmental physiology as well as differences in
β-AR signalling in the immature heart. As in neonates, especially in the
premature and critically ill with cardiac failure, HR is already high
before treatment, the lower HR in response to dobutamine treatment in
Arg389 β1-AR subjects may refer to better response to inotropic
treatment, with sequence variant specific difference in the change in
contractility not captured by our measurement methods.
We also found the effect of GNAS c.393C>T polymorphism on
cardiac output during dobutamine treatment, T allele carriers increasing
RVO and LVO in response to dobutamine compared to no change or decrease
in C/C homozygotes. (Figures S2, S3) The only in vivo prospective study
in 18 healthy Chinese adults showed that individuals with homozygous or
heterozygous C393 had an increased cardiovascular agonistic response to
dobutamine. 15 This controversy remains difficult to
explain, as the exact molecular mechanism of the effect of the GNAS
c.393C>T polymorphism has not been identified.25 Further studies are urgently needed, as our very
first data in the field suggest potential untoward effect of decreasing
LVO and RVO with increasing dobutamine exposure in C/C homozygous
neonates.
Three of the studied polymorphisms (β1-AR Ser49Gly and β2-AR Gly16Arg
and Gln27Glu) have been associated with agonist promoted receptor
desensitisation and downregulation. 4,5,8–10 The lack
of association between these SNPs and cardiovascular response to
dobutamine in our study population may be explained by ontogeny of
β-ARs. Results of juvenile animal studies indicate that β-ARs in the
neonatal heart do not desensitize in response to agonist stimulation.26–29 Furthermore, the maintenance of cardiac β-AR
signalling in the face of intense stimulation is likely to play an
important role in the physiologic adaptation during perinatal
transition. 30 One study on preterm foetal sheep
demonstrates the downregulation of β-AR with prolonged dobutamine
treatment in preterm heart exposed to hypoxia-ischemia.31 The lack of the clinical evidence of receptor
downregulation in our study may have been influenced by the relatively
short observational period.
In addition to the unique situation of transitional circulatory response
in neonates several perinatal factors/ interventions, like prematurity,
glucocorticoid exposure or hypoxia-ischemia may contribute to the
effects of sequence variants on dobutamine response.31,32 Hence GA, age at recruitment, antenatal
glucocorticoid administration status and SNAPPE-II score as a marker for
perinatal illness severity were chosen to be included to the
multivariable linear models describing the effect of genetic variability
to the haemodynamic response to dobutamine.
Developmental changes in the expression of G protein subunits may
account for different response to β-adrenergic stimulation in adult and
neonatal populations. 33
Enhanced role of β2-AR and β3-AR, as compared to β1-AR, in chronotropic
effects of β-adrenergic agonists has been suggested in both immature rat
and large mammal heart. Possible underlying mechanism include immature
coupling between β1-AR and Gs in the early postnatal period.34
In our relatively small study group, we could not detect the role of the
studied genetic polymorphisms in predisposition to haemodynamic
instability and need for inotropic treatment during the transitional
period. The allele incidences of all studied SNPs were comparable to the
general population. It has been shown that in term neonates β1-AR Gly49
homozygosity may predispose to transient tachypnoea of the neonate.35 The possible role of AR related genetic variations
in perinatal cardiac failure needs to be addressed in larger association
studies.
The low number of participants is a major limitation of our study.
Assessment of cardiac function by ultrasound is operator dependent with
large inter-occasion and -observer variability. To limit this effect all
cardiac ultrasound studies were performed by 5 cardiologists in our
study and in each patient all examinations were performed by the same
specialist throughout the study period. Relatively short haemodynamic
data collection period limited the assessment of time-consuming
processes like receptor downregulation.
Conclusions. Our results suggest, that the large interindividual
variability of the cardiovascular effects of dobutamine in early
postnatal life may partly be explained by AR and GNAS sequence variants.
Larger pharmacogenomic studies are urgently needed for better targeted
dobutamine therapy in neonates. Results from adult studies cannot be
readily extrapolated to younger age groups due to developmental
differences in beta-adrenergic signalling and the physiology of early
transitional circulation.
ACKNOWLEDGEMENTS
The study was funded by the Estonian Research Council (PUT1197). MH
received support from the University of Tartu Foundation (Professor
Lembit Allikmets’ and Heino Kruse’s foundations).
The authors have no conflicts of interest to declare.
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Table 1. The incidence of studied
SNP alleles in the study population and Estonian population.