Prediction of striatal D2 receptor binding by DRD2/ANKK1 TaqIA allele status

Abstract

In humans, the A1 (T) allele of the dopamine (DA) D2 receptor/ankyrin repeat and kinase domain containing 1 (DRD2/ANKK1) TaqIA (rs1800497) single nucleotide polymorphism has been associated with reduced striatal DA D2/D3 receptor (D2/D3R) availability. However, radioligands used to estimate D2/D3R are displaceable by endogenous DA and are non-selective for D2R, leaving the relationship between TaqIA genotype and D2R specific binding uncertain. Using the positron emission tomography (PET) radioligand, (N-[(11) C]methyl)benperidol ([(11) C]NMB), which is highly selective for D2R over D3R and is not displaceable by endogenous DA, the current study examined whether DRD2/ANKK1 TaqIA genotype predicts D2R specific binding in 2 independent samples. Sample 1 (n = 39) was composed of obese and non-obese adults; sample 2 (n = 18) was composed of healthy controls, unmedicated individuals with schizophrenia, and siblings of individuals with schizophrenia. Across both samples, A1 allele carriers (A1+) had 5-12% less striatal D2R specific binding relative to individuals homozygous for the A2 allele (A1-), regardless of body mass index or diagnostic group. This reduction is comparable to previous PET studies of D2/D3R availability (10-14%). The pooled effect size for the difference in total striatal D2R binding between A1+ and A1- was large (0.84). In summary, in line with studies using displaceable D2/D3R radioligands, our results indicate that DRD2/ANKK1 TaqIA allele status predicts striatal D2R specific binding as measured by D2R-selective [(11) C]NMB. These findings support the hypothesis that DRD2/ANKK1 TaqIA allele status may modify D2R, perhaps conferring risk for certain disease states. This article is protected by copyright. All rights reserved.

Full text

Prediction of striatal D2 receptor binding by DRD2/ANKK1 TaqIA
allele status
Sarah A. Eisenstein, Ph.D.1,2,*, Ryan Bogdan, Ph.D.3, Latisha Love-Gregory, Ph.D.4, Nadia
S. Corral-Frías, Ph.D.1, Jonathan M. Koller, B.S.1, Kevin J. Black, M.D.1,2,5,6, Stephen M.
Moerlein, Ph.D.2,7, Joel S. Perlmutter, M.D.2,5,8, Deanna M. Barch, Ph.D.1,2,3, and Tamara
Hershey, Ph,D1,2,3,5
1Psychiatry Department, Washington University in St. Louis, St. Louis, MO, USA 63110
2Radiology Department, Washington University in St. Louis, St. Louis, MO, USA 63110
3Psychological & Brain Sciences Department, Washington University in St. Louis, St. Louis, MO,
USA 63130
4Department of Medicine, Washington University in St. Louis, St. Louis, MO, USA 63110
5Neurology Department, Washington University in St. Louis, St. Louis, MO, USA 63110
6Anatomy and Neurobiology Department, Washington University in St. Louis, St. Louis, MO, USA
63110
7 Biochemistry Department, Washington University in St. Louis, St. Louis, MO, USA 63110
8Programs in Physical Therapy and Occupational Therapy, Washington University in St. Louis, St.
Louis, MO, USA 63110
Abstract
In humans, the A1 (T) allele of the dopamine (DA) D2 receptor/ankyrin repeat and kinase domain
containing 1 (
DRD2/ANKK1
) TaqIA (rs1800497) single nucleotide polymorphism has been
associated with reduced striatal DA D2/D3 receptor (D2/D3R) availability. However, radioligands
used to estimate D2/D3R are displaceable by endogenous DA and are non-selective for D2R,
leaving the relationship between TaqIA genotype and D2R
specific
binding uncertain. Using the
positron emission tomography (PET) radioligand, (
N
‐[11C]methyl)benperidol ([11C]NMB), which
is highly selective for D2R over D3R and is not displaceable by endogenous DA, the current study
examined whether
DRD2/ANKK1
TaqIA genotype predicts D2R specific binding in 2
*Corresponding author Sarah A. Eisenstein, Psychiatry Department, Campus Box 8225, Washington University in St. Louis, St. Louis,
MO 63110, Phone: (314) 362-7107, Fax: (314) 362-0168, eisensteins@npg.wustl.edu (SAE).
Author roles
SAE, RB, and TH wrote the manuscript. SAE, RB, LLG, NSCF, JMK, KJB, SMM, JSP, DMB, and TH contributed to study design
and methods. All authors reviewed and edited the manuscript.
Conflict of Interest
KJB: ACADIA Pharmaceuticals (advisory board, speakers bureau, research funding), Auspex Pharmaceuticals (consultant), Psyadon,
Inc. (research funding), Neurocrine Biosciences, Inc. (research funding), and U. S. patent # 8,463,552 and patent application #
13/890,198.
JMK: U. S. patent # 8,463,552 and patent application # 13/890,198.
DMB: Roche (consultant), Takeda Pharmaceuticals U.S.A., Inc. (consultant), Pfizer (consultant), Amgen (consultant).
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independent samples. Sample 1 (
n
= 39) was composed of obese and non-obese adults; sample 2
(
n
= 18) was composed of healthy controls, unmedicated individuals with schizophrenia, and
siblings of individuals with schizophrenia. Across both samples, A1 allele carriers (A1+) had
5-12% less striatal D2R specific binding relative to individuals homozygous for the A2 allele
(A1−), regardless of body mass index or diagnostic group. This reduction is comparable to
previous PET studies of D2/D3R availability (10-14%). The pooled effect size for the difference in
total striatal D2R binding between A1+ and A1− was large (0.84). In summary, in line with studies
using displaceable D2/D3R radioligands, our results indicate that
DRD2/ANKK1
TaqIA allele
status predicts striatal D2R specific binding as measured by D2R-selective [11C]NMB. These
findings support the hypothesis that
DRD2/ANKK1
TaqIA allele status may modify D2R, perhaps
conferring risk for certain disease states.
GRAPHICAL ABSTRACT
We investigated the difference in striatal dopamine D2 receptor binding, as measured by PET with
(
N
-[11C]methyl)benperidol ([11C]NMB), between A1 allele carriers (A1+) and individuals
homozygous for the A2 allele (A1−) of the
DRD2/ANKK1
TaqIA single nucleotide
polymorphism. In Study 1, A1+ had 5-12% less striatal [11C]NMB binding than A1−.
Keywords
rs1800497; PET; dopamine
Introduction
The role of striatal dopamine (DA) signaling in substance abuse and psychiatric disorders
has yet to be fully characterized. Postiron emission tomography (PET) studies with
displaceable DA D2/D3 receptor (D2/D3R) radioligands show that low striatal D2/D3R
availability may be associated with impulsivity (Clark et al., 2012), addiction to alcohol
(Volkow et al., 1996; Martinez et al., 2005), substance abuse (Volkow et al., 1990; Volkow et
al., 2001; Martinez et al., 2004; Fehr et al., 2008), and obesity (Wang et al., 2001; Haltia et
al., 2007; de Weijer et al., 2011) whereas high D2/D3R availability has been associated with
risk for schizophrenia (Laruelle, 1998), although this finding has not been replicated (Howes
et al., 2012; Kambeitz et al., 2014). Similarly, the A1 (T) allele of the single nucleotide
polymorphism (SNP) TaqIA (rs1800497), located in the ankyrin repeat and kinase domain
containing 1 (
ANKK1
) 10 kb downstream from the DA D2 receptor (
DRD2
) gene (Grandy
et al., 1989), is associated with addictive behavior including gambling (Comings et al.,
1996), substance abuse (Noble et al., 1993; Persico et al., 1996; Lawford et al., 2000; Chen
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et al., 2004; Messas et al., 2005), and binge eating (Davis et al., 2012) and with obesity
(Noble et al., 1994; Spitz MR, 2000; Thomas et al., 2001; Stice et al., 2008; Duran-Gonzalez
et al., 2011) while the A2 (C) allele is associated with risk for schizophrenia (Parsons et al.,
2007; Dubertret et al., 2010; Arab and Elhawary, 2015).
Individual variability in striatal D2/D3R availability is significantly heritable, as detected by
a twin study (Borg et al., 2015). In addition, the similar pattern of associations between
DRD2/ANKK1
TaqIA variants and D2/D3R availability with psychiatric and drug abuse risk
has led to speculation that
DRD2
’s role in these disorders may be mediated by D2R. Indeed,
the A1 allele of the
DRD2/ANKK1
TaqIA A1 variant has been associated with lower striatal
D2/D3R availability relative to the A2 allele in several postmortem (Noble et al., 1991;
Thompson et al., 1997; Ritchie and Noble, 2003; Gluskin and Mickey, 2016) and
in vivo
PET studies (Pohjalainen et al., 1998; Jonsson et al., 1999; Hirvonen et al., 2009a; Savitz et
al., 2013; Gluskin and Mickey, 2016). However, a SPECT study (Laruelle et al., 1998) and
two PET studies (Brody et al., 2006; Wagner et al., 2014) did not find this association, likely
due to study of diseased populations (Gluskin and Mickey, 2016). To date, studies have used
PET radioligands (e.g. [11C]raclopride (Thompson et al., 1997; Pohjalainen et al., 1998;
Jonsson et al., 1999; Brody et al., 2006; Hirvonen et al., 2009a; Savitz et al., 2013; Wagner
et al., 2014), [3H]spiperone (Noble et al., 1991; Ritchie and Noble, 2003)) and the SPECT
radioligand [123I]IBZM (Laruelle et al., 1998), which do not discriminate between D2R and
D3R and whose binding is affected by synaptic dopamine concentrations, leaving the link
between
DRD2/ANKK1
TaqIA genotype and D2R
specific
binding unclear. The novel PET
radioligand (
N
-[11C]methyl)benperidol ([11C]NMB) specifically binds to D2R in a
reversible manner, does not undergo agonist-mediated internalization, is resistant to
displacement by endogenous DA, and is selective for D2R over D3R by 200-fold (Moerlein
et al., 1997; Karimi et al., 2011). Thus, [11C]NMB is an ideal radioligand to use for the
study of D2R binding under various conditions including disease states and genotype status.
The current studies examined whether
DRD2/ANKK1
TaqIA allele status is associated with
striatal D2R
specific
binding. We analyzed data from two independent studies that employed
PET with [11C]NMB and included human participants genotyped for the
DRD2/ANKK1
TaqIA variant. Based on previous evidence (Pohjalainen et al., 1998; Jonsson et al., 1999;
Savitz et al., 2013; Gluskin and Mickey, 2016), we hypothesized that A1 allele carriers
(A1+) would have lower striatal D2R binding than individuals homozygous for A2 (A1−).
We also performed meta-analyses to generate pooled effect sizes that reflect the ability of
DRD2/ANKK1
TaqIA allele status to predict D2R specific binding across striatal regions.
Materials and Methods
Participants
For Study 1, participants were recruited for a study of obesity and D2R from the St. Louis
region via a research volunteer database, flyers, and word of mouth. Data from these
individuals regarding the relationship between obesity and striatal D2R were previously
presented (Eisenstein et al., 2013; Eisenstein et al., 2015b; Eisenstein et al., 2015a). Obese
(
n
= 24) and non-obese (
n
= 20) individuals, aged 18-40 years, were eligible for the study
based on strict inclusion and exclusion criteria (Eisenstein et al., 2013). Exclusion criteria
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included a diagnosis of type 2 diabetes (based on oral glucose tolerance test), history of
psychiatric or neurological diagnoses, tobacco or illegal substance use, and dopaminergic
medication use. Handedness was obtained by self-report. A subset of participants (24 obese
and 16 non-obese) were genotyped for the
DRD2/ANKK1
TaqIA (rs1800497)
polymorphism and completed PET neuroimaging. Some participants were not genotyped
because we did not have biological specimens from which to extract DNA (
n
= 4).
For Study 2, healthy controls (HC;
n
= 10), siblings of individuals with schizophrenia (SIB;
n
= 10), and individuals with schizophrenia or schizoaffective disorder (SCZ;
n
= 3) were
recruited for a study of schizophrenia, reward behavior, and D2R. Participants (age range
18-50 years) were recruited by word of mouth, flyers, and during recruiting visits to clinics
and mental health centers in St. Louis, MO. A trained research assistant administered the
Structured Clinical Interview for the DSM-IV(First et al., 2002) to all participants to
determine the lifetime and current history of Axis I disorders. Exclusionary criteria included
DSM-IV (American Psychiatric Association, 2000) diagnosis of substance abuse or
dependence, either currently or within the last 6 months; neurological disorder; history of
concussion or head injury; pregnancy; claustrophobia; presence in body of non-removable
metallic objects or implanted medical electronic devices; mental retardation; positive drug
urine test; and positive alcohol breathalyzer reading. HC must not have had lifetime or
family history of psychotic disorders; current mood or anxiety disorder except for specific
phobia but may have had a past Axis I disorder except for a psychotic disorder. The
exclusionary criteria for SIB were identical to that of HC except that the participant must
have had a sibling with confirmed diagnosis of schizophrenia or schizoaffective disorder.
SIB were unrelated to SCZ who completed this study. SCZ must have met DSM-IV criteria
for diagnosis of schizophrenia or schizoaffective disorder. Participants must have voluntarily
abstained from medications such as DA agonists and antagonists and other psychotropic
drugs for at least 4 weeks. During each study visit, evidence of alcohol use during the last 24
hr and recent use of drugs of abuse was obtained by breathalyzer and from urine sample
drug test, respectively. Handedness was obtained by self-reported preferred hand for writing.
A total of 8 HC, 8 SIB, and 2 SCZ were genotyped for the
DRD2/ANKK1
TaqIA
(rs1800497) polymorphism and completed PET neuroimaging. Some participants were not
genotyped because they participated in the study after genotyping was carried out (
n
= 2) or
we did not have biological specimens from which to extract DNA (
n
= 3).
Participants in both studies provided written informed consent prior to participation. The
study protocols were approved by the Washington University School of Medicine (WUSM)
Human Research Protection Office and the Radioactive Drug Research Committee, and
carried out in accordance with the principles expressed in the Declaration of Helsinki.
DNA Extraction and Genotyping
Blood (Study 1) and saliva (Study 2) were obtained from participants and DNA was
extracted. Participants were genotyped for the
DRD2/ANKK1
TaqIA (rs1800497)
polymorphism (A1/A2; T/C) by the Sequenom Technology Core at WUSM, the Molecular
Psychiatry Core, and the Adipocyte Biology and Molecular Nutrition Core at WUSM using
mass-spectrometry (Study 1), pyrosequencing (Study 1 and Study 2), and a pre-designed
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Taqman SNP genotyping assay (Study 1; Applied Biosystems; Waltham, MA), respectively.
Subjects were categorized as A1 allele carriers (A1+) or A2 allele homozygotes (A1−).
Magnetic resonance imaging and PET Imaging
For Study 1, the methods used to obtain magnetic resonance image (MRI) and PET scans
were reported (Eisenstein et al., 2013). Briefly, MRI scans were obtained on the Siemens
MAGNETOM Tim Trio 3T using a 3-D MP-RAGE sequence (sagittal orientation, TR =
2400 ms, TE = 3.16 ms, flip angle = 8 degrees, slab thickness = 176 mm, FOV = 256 × 256
mm, voxel dimensions = 1 ×1 × 1 mm) and PET scans were obtained on the Siemens CTI
ECAT/EXACT HR+. The radioligand [11C]NMB was prepared using an automated system
previously described (Moerlein et al., 2004; Moerlein et al., 2010). Radiochemical purity of
[11C]NMB was ≥ 96% and specific activity was ≥ 1000 Ci/mmol (39 TBq/mmol).
Participants received 6.4-18.1 mCi [11C]NMB intravenously.
For Study 2, structural magnetic resonance T1-weighted anatomical images were obtained
with the Siemens Biograph mMR PET/MR scanner using a 3-D MP-RAGE sequence
(sagittal orientation, TR=2400 ms, TE=2.67 ms, flip angle=7 degrees, slab thickness=192
mm, FOV=256×256 mm; voxel dimensions= 1×1×1 mm). PET images were acquired
simultaneously with the radioligand [11C]NMB. [11C]NMB was prepared as described for
Study 1. Radiochemical purity of [11C]NMB was ≥ 95% and specific activity was ≥ 1000
Ci/mmol (36 TBq/mmol). Participants received 5.9-18.8 mCi [11C]NMB intravenously.
MR and PET image processing has been previously described in detail (Eisenstein et al.,
2012; Eisenstein et al., 2013).
A priori
regions of interest (ROIs) including dorsal and
ventral areas of the striatum (putamen, caudate, and nucleus accumbens (NAc)) were
identified using FreeSurfer (Fischl et al., 2002) on the MP-RAGE MR images for each
participant. Dynamic PET images were co-registered to each other and to the MP-RAGE
image for each individual as previously described (Eisenstein et al., 2012). ROIs and the
cerebellar reference region were resampled in the same atlas space and decay-corrected
tissue activity curves were obtained from the dynamic PET data for every ROI. For both
studies,
a priori
regions of interest (ROIs) included putamen, caudate, nucleus accumbens
(NAc), dorsal striatum (putamen + caudate), and total striatum (putamen + caudate + NAc).
D2R non-displaceable binding potentials (BPNDs) were obtained for each ROI with the
Logan graphical method with whole cerebellum as a reference region (Antenor-Dorsey et
al., 2008). D2R BPNDs for each ROI were averaged across left and right hemispheres to
reduce the number of comparisons for primary analyses and because we did not have
a priori
hypotheses about laterality effects.
Meta-analyses
Cohen’s
d
effect sizes were estimated for Study 1 and Study 2. To obtain pooled effect sizes
for differences in D2R specific binding between A1+ and A1−, meta-analyses were
performed including Study 1 and Study 2 for each ROI.
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Primary Statistical Analyses
Data from each study was analyzed separately due to use of different PET scanners
Hierarchical linear regressions were used to determine whether
DRD2/ANKK1
TaqIA allele
status predicted D2R BPND for each ROI. Step 1 included covariates age, education level,
ethnicity (White vs. not), and gender and Step 2 included group (obese vs non-obese or HC
vs SIB vs SCZ). Step 3 included allele status (A1+ vs A1−) and Step 4 included the
interaction between group and allele status. We calculated BPND means for each ROI
adjusted for age, education level, ethnicity, gender and diagnostic group. We then calculated
the percent difference in D2R BPND between A1+ vs A1− groups. Cohen’s
d
effect sizes for
each study were calculated using means and standard deviations and number of individuals
in each allele group (A1+ and A1−). Meta-analyses were performed with Revman 5.3
software (Cochrane IMS, Oxford, UK). Weighted mean difference (MD) with the
corresponding 95% CI was reported as the pooled effect size.
I2
and
χ2
tests determined
heterogeneity and
p
< 0.10 was considered significant. Since heterogeneity did not exist
across studies (
I2
≤ 54%,
p
≥ 0.14), a fixed-effects model was used to calculate pooled effect
size. Forest plots were generated with
p
< 0.05 considered significant.
Results
Participants
In Study 1, 40 individuals were genotyped for the
DRD2/ANKK1
TaqIA (rs1800497)
polymorphism. For unknown reasons, one obese, A1/A2 participant’s D2R binding values
were greater than 2.5 (2.55-3.14 across all ROIs) standard deviations above the A1+ group
mean and was thus excluded from analyses. Only one individual was homozygous for the
A1 allele. Therefore, this participant’s data was pooled with that from A1/A2 individuals for
comparisons to individuals homozygous for A2. The final dataset included 14 obese and 7
non-obese A1+ and 9 obese and 9 non-obese A1−. The distributions of obese and non-obese
were not different between A1+ and A1− (
χ2
= 1.11,
p
= 0.29). Handedness distribution did
not differ between A1+ and A1− (
χ2
= 1.81,
p
= 0.18). Participant demographics are
presented in Table 1.
In Study 2, 8 HC, 8 SIB, and 2 unmedicated SCZ were genotyped for the
DRD2/ANKK1
TaqIA (rs1800497) polymorphism and had PET D2R binding data. At the PET imaging
visit, SCZ had not taken antipsychotic medications for ≥ 9 months and did not display overt
signs of psychopathology. There were no individuals homozygous for the A1 allele. 6 HC
and 3 SIB were A1+ and 2 HC, 5 SIB, and 2 SCZ were A1−. Group distribution was not
different between A1+ and A1− (
χ2
= 4.50,
p
= 0.11). Handedness distribution did not differ
between A1+ and A1− (
χ2
= 0,
p
= 1). Participant demographics are presented in Table 1.
Genotyping Quality Control
Across both studies
DRD2/ANKK1
TaqIA genotype did not deviate from Hardy-Weinburg
Equilibrium in either sample (Study 1:
χ2
= 2.8,
p
= 0.10; Study 2:
χ2
= 2.0,
p
= 0.16) or
within the African American (AA) and Caucasian (C) subsamples (AA, Study 1:
χ2
= 3.0,
p
= 0.08; Study 2:
χ2
= 1.3,
p
= 0.25; C, Study 1:
χ2
= 0.9,
p
= 0.34; Study 2:
χ2
= 0.7,
p
=
0.39). Distribution of allele frequencies did not differ between AA and- C in either study
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(Study 1:
χ2
= 0.71,
p
= 0.40; Study 2:
χ2
= 0.22,
p
= 0.64) or among groups (obese/non-
obese or HC/SIB/SCZ) across either study (Study 1:
χ2
= 1.1,
p
= 0.29; Study 2:
χ2
= 4.5,
p
= 0.11).
Prediction of Striatal D2R Binding by DRD2/ANKK1 TaqIA Allele Status
In Study 1,
DRD2/ANKK1
TaqIA allele status predicted D2R binding in total striatum and
nucleus accumbens (both
β
≥ 0.29,
p
≤ 0.05; both Δ
R2
≥ 0.07; Fig 1a-b,Table 2) and in
dorsal striatum at trend-level significance (
p
= 0.09, Table 2).
DRD2/ANKK1
TaqIA allele
status and D2R binding in putamen and caudate were not significantly related (both
β
≤
0.23,
p
= 0.11, Table 2). Across ROIs, D2R binding was lower in A1+ relative to A1− by
5-8%). Neither a main effect of obesity group (obese/non-obese) nor an interactive effect
with
DRD2/ANKK1
TaqIA allele status was observed for D2R binding in any ROI (all
p
≥
0.13, Table 2). D2R BPND means (S.D.s), percent difference in binding, and estimated
Cohen’s
d
effect sizes are presented in Table 3.
In Study 2,
DRD2/ANKK1
TaqIA allele status predicted D2R binding in total striatum,
dorsal striatum, and putamen (all
β
≥ 0.41,
p
≤ 0.05; all
R2
≥ 0.11; Fig 1c-d,Table 4).
DRD2/ANKK1
TaqIA allele status did not significantly predict caudate or NAc D2R
binding (both
p
≥ 0.09,Table 4). Across all ROIs, D2R binding was lower in A1+ relative to
A1− by 8-12%. Diagnostic group (HC, SCZ, SIB) did not significantly predict D2R binding
in any ROI (all
p
≥ 0.13) but the relationship was near significant for caudate (
p
= 0.07), in
which SIB (mean BPND (S.D.) = 3.8 (0.6)) and SCZ (mean BPND (S.D.) = 3.9 (0.1)) tended
to have greater D2R binding relative to HC (mean BPND (S.D.) = 3.4 (0.6)). It should be
noted, however, that SCZ had a small sample size (
n
= 2). Group and
DRD2/ANKK1
TaqIA
allele status did not interact to affect D2R binding in any ROI (all
p
≥ 0.44). D2R BPND
means (S.D.s), percent difference in binding, and estimated Cohen’s
d
effect sizes are
presented in Table 3.
Meta-Analyses
The pooled analyses of Study 1 and Study 2 revealed that D2R specific binding was
significantly lower in A1+ relative to A1− across all striatal ROIs (Figs 2, 3).
Discussion
We show in two independent studies that
DRD2/ANKK1
TaqIA (rs1800497) allele status
predicts striatal D2R
specific
binding in humans, such that A1+ had lower D2R binding
relative to A1−. This relationship did not depend on group membership (i.e. non-obese vs
obese or controls versus psychosis). Our findings extend previous reports linking
DRD2/
ANKK1
TaqIA allele status to D2/D3R availability in postmortem striatal tissue (Noble et
al., 1991; Thompson et al., 1997; Ritchie and Noble, 2003; Gluskin and Mickey, 2016) and
in vivo
molecular imaging data in humans (Pohjalainen et al., 1998; Jonsson et al., 1999;
Hirvonen et al., 2009a; Savitz et al., 2013; Gluskin and Mickey, 2016). Pooled effect sizes,
or weighted mean differences, for total and dorsal striatal differences in D2R specific
binding between A1+ and A1− were larger (0.84, 0.69) than that calculated for
in vivo
D2/D3R availability studies (0.57; Gluskin and Mickey, 2016). However, the 95%
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confidence intervals for total striatum (0.30, 1.37) in our D2R study overlaps with that of
D2/D3R availability studies (0.27, 0.87; Gluskin and Mickey, 2016), indicating that our
effect size estimates are consistent with those of previous D2/D3R studies. Of note,
DRD2/
ANKK1
TaqIA status accounted for 5-14% of the variance in striatal D2R binding (Tables
2, 4), comparable to 7% of striatal D2/D3R availability in previous
in vivo
PET studies
(Gluskin and Mickey, 2016). Differences in participant characteristics, image analysis
methods such as use of arterial input function, outcome measures (
B
max versus BPND), and
scanner sensitivity may contribute to variability in strength of the relationship between
DRD2/ANKK1
TaqIA allele status and D2R binding or D2/D3R availability.
Our results contrast with those of Laruelle et al. (1998), in which D2/D3R availability did
not differ between A1+ and A1−. However, that study used SPECT, which has lower
resolution than PET. In addition, a large proportion of the sample in Laruelle et al. (1998)
were patients with schizophrenia, some of whom may have been taking neuroleptics that
increase D2/D3R availability in schizophrenia (Silvestri et al., 2000). Two other previous
studies did not find differences in baseline D2/D3R availability between A1+ and A1−
(Brody et al., 2006; Wagner et al., 2014) but these included diseased populations including
smokers (Brody et al., 2006) and traumatic brain injured individuals (Wagner et al., 2014).
Variability in D2/D3R availability measurement due to small sample sizes may have
contributed to null findings in these studies. In addition, recent meta-analysis of these
D2/D3R studies revealed that the difference in D2/D3R availability between A1+ and A1− is
robust in healthy individuals but not in diseased individuals (Gluskin and Mickey, 2016),
suggesting that disease may modify this association. Intriguingly,
extra
striatal D2/D3R
availability, as measured by PET with [11C]FLB457, was
elevated
in A1+ relative to A1−
(Hirvonen et al., 2009b), indicating that there may be differential regulation of D2/D3R
across brain regions by
DRD2/ANKK1
TaqIA allele status. Therefore future studies may
investigate the effects of disease on the relationship between TaqIA allele status and D2R
specific binding as well as differences in
extra
striatal D2R specific binding as measured by
PET with [11C]NMB between A1+ and A1−.
There was no significant effect of group on D2R binding in either study. Nor did group
interact with TaqIA allele status to predict striatal D2R binding. We have not previously
found there to be differences in striatal D2R between an overlapping sample of non-obese
and obese individuals (Eisenstein et al., 2013). In the case of study 2, the SCZ group was not
large enough to fairly compare striatal D2R binding to HC and SIB. Neither study is truly
large enough to investigate the interaction between group and allele status on striatal D2R
binding. Therefore, caution should be used in interpreting the null results of these studies.
Rather, we emphasize our main finding that when group (and other covariates) was
controlled for, striatal D2R binding was lower in A1+ individuals compared to A1−
individuals in 2 independent studies.
The mechanism by which the TaqIA variant, which resides in a noncoding region 10 kb
downstream from the
DRD2
gene (Grandy et al., 1989), influences D2R binding remains
unknown. Strong linkage disequilibrium with one or more functional variants, including the
DRD2
intronic SNPs rs2283265 and rs1076560 (Zhang et al., 2007) and the
ANKK1
missense SNP rs7118900 (Hoenicka et al., 2010), may influence receptor expression
Eisenstein et al. Page 8
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(Comings et al., 1991; O'Hara et al., 1993). A likely candidate is the C957T variant (rs6277),
which disrupts mRNA stability and synthesis of D2R (Duan et al., 2003) and is in linkage
disequilibrium with the TaqIA SNP (Duan et al., 2003). The C957T variant relates to
decreased D2/D3R availability as measured
in vivo
with [11C]raclopride (Hirvonen et al.,
2004). However, the C957T variant appears to affect striatal D2/D3R availability by
changing receptor affinity while the TaqIA A1 polymorphism contributes to variability in
D2/D3R availability by changing
B
max (Hirvonen et al., 2009a). The TaqA1 A1 allele may
be instead be in linkage disequilibrium with a functional variant that affects presynaptic DA
signaling such as decreased inhibition of striatal DA synthesis (Duan et al., 2003; Laakso et
al., 2005), which may displace D2/D3R radioligand binding. However, since endogenous
DA does not displace [11C]NMB and we observed lower D2R binding in A1+, it is more
likely that the TaqIA allele is in linkage disequilibrium with a functional variant in the
DRD2
or
ANKK1
gene that directly affects D2R binding.
Limitations of the currently described studies include small sample sizes and heterogeneity
of sample composition. Therefore, our power to detect group differences in D2R binding or
allele status and interactions between group and allele status was low. Differences in PET
scanner characteristics precluded combining data from the two studies for analyses, which
would have provided more power. Nonetheless, we still detected relationships between
DRD2/ANKK1
allele status and striatal D2R specific binding in the predicted direction and
with small to large effect sizes, independent of group membership and PET scanner used.
Finally, none of the studies described, including ours, had enough data from healthy A1+
homozygotes to actually test the hypothesis that D2/D3R availability or D2R specific
binding is lower in these individuals relative to A1+/A1− and A1−/A1−. To test this
hypothesis, given the rare occurrence of homozygosity for A1+, future studies must
intentionally select enough healthy participants with A1/A1 to have enough power to detect
differences in striatal D2/D3R availability or D2R specific binding between A1/A1, A1/A2,
and A2/A2.
In summary, the two independent studies described here showed that
DRD2/ANKK1
TaqIA
allele status relates to individual differences in striatal D2R
specific
binding, such that A1+
individuals had greater binding relative to A1−. The use of the novel D2R specific
radioligand [11C]NMB with insensitivity to displacement by endogenous DA facilitated
measurement of D2R specific binding, in contrast to D2/D3R radioligands such as
[11C]raclopride which lacks the same specificity and may be displaced by varying levels of
endogenous DA (Moerlein et al., 1997; Karimi et al., 2011). Therefore these studies
replicate and extend previous findings from postmortem (Noble et al., 1991; Thompson et
al., 1997; Ritchie and Noble, 2003) and PET studies (Pohjalainen et al., 1998; Jonsson et al.,
1999; Savitz et al., 2013) performed with D2/D3R radioligands. Our findings also support
the hypothesis that the A1 allele (or linked functional variant) may influence risk for
substance abuse and psychiatric disorders via D2R, which can be formally tested with
mediation analyses.
Eisenstein et al. Page 9
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Acknowledgments
We thank Emily Bihun, Samantha Ranck, Melissa Cornejo, Arthur Schaffer, and Danielle Kelly for their help with
recruiting participants. We thank Heather Lugar and Jerrel Rutlin for their help with processing MR scans. The
studies presented in this work were conducted using the scanning and special services in the MIR Center for
Clinical Imaging Research located at the Washington University Medical Center.
Funding
This work was supported by a National Alliance for Research on Schizophrenia and Affective Disorders Young
Investigator Award to SAE, the National Institutes of Health (R01 DK085575 to TH, R01 MH066031 to DMB,
UL1 TR000448, R01 NS41509 to JSP; R01 NS075321 to JSP; R01 NS058714 to JSP; T32 MH014677 to NSCF;
P30 DK056341 to the Washington University Nutrition Obesity Research Center; P30 DK020579 to the
Washington University Diabetes Research Center), Barnes Jewish Hospital Foundation (Eliot Stein Family Fund
and Parkinson Disease Research Fund) to JSP; the American Parkinson Disease Association (APDA) Advanced
Research Center at Washington University to JSP, the Greater St. Louis Chapter of the APDA to JSP, the
McDonnell Center for Systems Neuroscience New Resource Proposal Award to SAE, and the Gregory B. Couch
Award to DMB. RB received additional funding from the Klingenstein Third Generation Foundation and NIH (R01
AG045231).
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Figure 1.
In Study 1,
DRD2/ANKK1
TaqIA (rs1800497) allele status predicted D2R specific binding
in
a
) striatum (putamen + caudate + nucleus accumbens) and
b
) nucleus accumbens. The
data from the A1/A1 individual was pooled with data from A1/A2 for statistical analysis. In
Study 2,
DRD2/ANKK1
TaqIA (rs1800497) allele status predicted D2R specific binding in
c
) striatum and
d
) putamen. D2R BPND, dopamine D2 receptor non-displaceable binding
potential; NAc, nucleus accumbens; HC, healthy control; SIB, sibling of individual with
schizophrenia; SCZ, individual with schizophrenia or schizoaffective disorder. *, **,
p
≤
0.05, = 0.01
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Figure 2.
Forest plots of the pooled effect sizes for reduced
a
) striatal and
b
) dorsal D2R specific
binding in A1+ relative to A1− individuals according to study. In both cases, the pooled
effect sizes were significant. Size of square is proportional to weight of mean. CI,
confidence interval; df, degrees of freedom; IV, inverse variance (statistical method).
Eisenstein et al. Page 16
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Figure 3.
Forest plots of the pooled effect sizes for reduced D2R specific binding, as measured by
[11C]NMB, in A1+ relative to A1− individuals in Study 1 and Study 2 in
a
) putamen,
b
)
caudate, and
c
) nucleus accumbens. Pooled effect sizes were significant for each ROI. Size
of square is proportional to weight of mean. CI, confidence interval; df, degrees of freedom;
IV, inverse variance (statistical method).
Eisenstein et al. Page 17
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Author Manuscript Author Manuscript Author Manuscript Author Manuscript
Eisenstein et al. Page 18
Table 1
Participant demographics for Studies 1 and 2. Mean (S.D.) shown.
Study 1
Non-obese
(
n
= 16) Obese
(
n
= 23)
Age (years) 28.8 (5.6) 32.3 (6.2)
Education
(years) 16.2 (1.4) 15.0 (1.9)
BMI (kg/m2)22.2 (2.1) 40.2 (4.9)
Gender 11 F/5 M 19 F/ 4 M
Ethnicity 13 Caucasian, 1 African
American, 1 Hispanic, 1 Other 12 Caucasian, 11
African American
Handedness 14 right, 2 non-right 23 right
Allele
distribution 7 A1/A2, 9 A2/A2 1 A1/A1, 13 A1/A2, 9
A2/A2
Study 2
Healthy Control
(
n
= 8) Sibling
(
n
= 8) Schizophrenia
(
n
= 2)
Age (years) 35.5 (10.3) 33.3 (7.6) 36 (5.7)
Education
(years) 12.6 (1.5) 14.9 (1.6) 12.5 (0.7)
Gender 3 F/5 M 5 F/3 M 1 F/1 M
Ethnicity 4 Caucasian, 4 African American 5 Caucasian, 3 African
American 2 African
American
Handedness 6 right, 2 non-right 7 right, 1 non-right 1 right, 1 non-
right
Allele
distribution 6 A1/A2; 2 A2/A2 3 A1/A2, 5 A2/A2 2 A2/A2
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Table 2
Summary of hierarchical multiple linear regression analyses for prediction of striatal dopamine D2 receptor specific binding by
DRD2/ANKK1
Taq1A
(rs1800497) allele status in Study 1.
Step 1 Step 2 Step 3 Step 4
Striatum (N = 39)
Variable
B SE B β B SE B β B SE B β B SE B β
Education .14 .10 .21 .16 .11 .24 .16 .10 .25 .16 .10 .25
Age −.07 .03 −.37 −.08 .03 −.39 −.08 .03 −.42 −.08 .03 −.42
Gender .59 .39 .21 .54 .40 .19 .27 .40 .10 .27 .42 .10
White or not .44 .35 .18 .49 .36 .20 .46 .34 .19 .46 .36 .19
Group −.25 .37 −.10 −.42 .36 −.18 −.44 1.1 −.19
Allele status .68 .33 .29*.66 1.0 .28
Group × allele status .01 .69 .01
R2
.38 .39 .46 .46
F
for change in
R2
5.2,
p
< 0.01 0.45,
p
= 0.51 4.3,
p
= 0.05 0,
p
= 0.98
Dorsal Striatum (N = 39)
Variable
B SE B β B SE B β B SE B β B SE B β
Education .10 .09 .18 .12 .09 .21 .12 .09 .22 .12 .09 .22
Age −.06 .03 −.35 −.06 .03 −.38 −.07 .03 −.40 −.07 .03 −.41
Gender .42 .34 .18 .36 .35 .15 .16 .36 .07 .13 .37 .06
White or not .37 .31 .18 .42 .31 .21 .40 .31 .19 .44 .32 ..21
Group −.27 .32 −.13 −.40 .32 −.20 −.81 1.0 −.40
Allele status .51 .29 .26†.15 .90 .08
Group × allele status .26 .61 .30
R2
.32 .33 .39 .39
F
for change in
R2
4.0,
p
= 0.01 0.67,
p
= 0.42 3.1,
p
= 0.09 0.18,
p
= 0.67
Putamen (N = 39)
Variable
B SE B β B SE B β B SE B β B SE B β
Education .03 .04 .10 .04 .04 .15 .04 .04 .16 .04 .04 .16
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Step 1 Step 2 Step 3 Step 4
Age −.04 .01 −.47 −.04 .01 −.50 −.04 .01 −.52 −.04 .01 −.52
Gender .27 .16 .24 .23 .16 .20 .14 .17 .13 .13 .17 .11
White or not .11 .14 .11 .15 .14 .15 .14 .14 .14 .15 .15 .15
Group −.18 .15 −.18 −.23 .15 −.24 −.43 .46 −.44
Allele status .22 .14 .23 .05 .41 .05
Group × allele status .12 .28 .29
R2
.38 .40 .45 .46
F
for change in
R2
5.2,
p
< 0.01 1.4,
p
= 0.25 2.8,
p
= 0.11 0.20,
p
= 0.66
Caudate (N = 39)
Variable
B SE B β B SE B β B SE B β B SE B β
Education .07 .06 .22 .08 .06 .24 .08 .06 .25 .08 .26 .25
Age −.02 .02 −.23 −.02 .02 −.24 −.03 .02 −.27 −.03 .02 −.27
Gender .15 .21 .11 .13 .21 .10 .02 .22 .01 0 .23 0
White or not .26 .18 .22 .28 .19 .24 .26 .19 .22 .28 .20 .24
Group −.09 .20 −.08 −.17 .20 −.15 −.38 .62 −.33
Allele status .29 .18 .26 .10 .55 .09
Group × allele status .14 .37 .28
R2
.24 .25 .31 .31
F
for change in
R2
2.7,
p
= 0.04 0.22,
p
= 0.64 2.6,
p
=.11 0.14,
p
= .71
Nucleus Accumbens (N = 39)
Variable
B SE B β B SE B β B SE B β B SE B β
Education .04 .03 .27 .04 .03 .26 .04 .03 .28 .04 .02 .27
Age −.01 .01 −.30 −.01 .01 −.29 −.02 .01 −.32 −.02 .01 −.31
Gender .17 .09 .26 .18 .10 .27 .11 .10 .17 .14 .10 .21
White or not .07 .08 .13 .07 .09 .12 .06 .08 .11 .03 .08 .05
Group .02 .09 .03 −.02 .09 −.04 .37 .26 .64
Allele status .17 .08 .30*.51 .23 .91
Group × allele status −.25 .16 −1.0
R2
.37 .37 .45 .49
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Step 1 Step 2 Step 3 Step 4
F
for change in
R2
5.0,
p
< 0.01 0.1,
p
= 0.83 4.5,
p
= 0.04 2.4,
p
= 0.13
*
,
p
≤ 0.05;
†
,
p
= 0.09
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Table 3
Mean (S.D.) striatal D2 receptor specific binding (BPND) by
DRD2/ANKK1
TaqIA allele status.
NBPND
Unadjusted BPND
Adjusted
Percent difference in
Adjusted BPND between
A1+ and A1−Estimated Effect
Size (Cohen’s d)
Study 1
Total Striatum
Total Sample
A1+ 21 9.74 (0.80) 9.75 (0.99) 6.5% 0.69
A1− 18 10.45 (1.46) 10.43 (0.99)
Non-obese
A1+ 7 9.92 (0.86)
A1− 9 10.47 (1.53)
Obese
A1+ 14 9.65 (0.79)
A1− 9 10.43 (1.48)
Dorsal Striatum
Total Sample
A1+ 21 7.81 (0.70) 7.81 (0.88) 6.1% 0.58
A1− 18 8.32 (1.23) 8.32 (0.89)
Non-obese
A1+ 7 7.86 (0.69)
A1− 9 8.36 (1.28)
Obese
A1+ 14 7.78 (0.72)
A1− 9 8.28 (1.26)
Putamen
Total Sample
A1+ 21 4.02 (0.35) 4.02 (0.40) 5.2% 0.54
A1− 18 4.24 (0.59) 4.24 (0.41)
Non-obese
A1+ 7 3.98 (0.28)
A1− 9 4.25 (0.68)
Obese
A1+ 14 4.03 (0.39)
A1− 9 4.23 (0.54)
Caudate
Total Sample
A1+ 21 3.79 (0.41) 3.79 (0.54) 7.1% 0.54
A1− 18 4.08 (0.70) 4.08 (0.54)
Non-obese
A1+ 7 3.87 (0.43)
A1− 9 4.12 (0.65)
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NBPND
Unadjusted BPND
Adjusted
Percent difference in
Adjusted BPND between
A1+ and A1−Estimated Effect
Size (Cohen’s d)
Obese
A1+ 14 3.75 (0.40)
A1− 9 4.05 (0.78)
Nucleus Accumbens
Total Sample
A1+ 21 1.93 (0.21) 1.94 (0.24) 8.1% 0.71
A1− 18 2.13 (0.32) 2.11 (0.24)
Non-obese
A1+ 7 1.86 (0.07)
A1− 9 2.15 (0.09)
Obese
A1+ 14 1.86 (0.20)
A1− 9 2.14 (0.39)
Study 2
Total Striatum
Total Sample
A1+ 9 10.66 (1.70) 10.48 (1.1) 10.7% 1.14
A1− 9 11.54 (1.40) 11.73 (1.1)
Healthy Control
A1+ 6 10.87 (1.74)
A1− 2 10.04 (1.51)
Sibling
A1+ 3 10.26 (1.81)
A1− 5 12.24 (1.18)
Schizophrenia
A1+ 0 N/A
A1− 2 11.31 (0.66)
Dorsal Striatum
Total Sample
A1+ 9 7.62 (1.2) 7.48 (0.79) 11.7% 1.25
A1− 9 8.33 (0.95) 8.47 (0.79)
Healthy Control
A1+ 6 7.75 (1.27)
A1− 2 7.37 (1.11)
Sibling
A1+ 3 7.35 (1.4)
A1− 5 8.77 (0.85)
Schizophrenia
A1+ 0 N/A
A1− 2 8.20 (0.55)
Putamen
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NBPND
Unadjusted BPND
Adjusted
Percent difference in
Adjusted BPND between
A1+ and A1−Estimated Effect
Size (Cohen’s d)
Total
Sample
A1+ 9 4.19 (0.57) 4.07 (0.33) 11.3% 1.56
A1− 9 4.47 (0.5) 4.59 (0.33)
Healthy Control
A1+ 6 4.31 (0.58)
A1− 2 4.09 (0.61)
Sibling
A1+ 3 3.94 (0.58)
A1− 5 4.67 (0.47)
Schizophrenia
A1+ 0 N/A
A1− 2 4.33 (0.43)
Caudate
Total
Sample
A1+ 9 3.43 (0.70) 3.41 (0.5) 12.1% 0.94
A1− 9 3.87 (0.48) 3.88 (0.5)
Healthy Control
A1+ 6 3.44 (0.72)
A1− 2 3.29 (0.5)
Sibling
A1+ 3 3.41 (0.84)
A1− 5 4.10 (0.4)
Schizophrenia
A1+ 0 N/A
A1− 2 3.87 (0.12)
Nucleus Accumbens
Total Sample
A1+ 9 3.05 (0.45) 3.0 (0.32) 8.0% 0.81
A1− 9 3.21 (0.45) 3.26 (0.32)
Healthy Control
A1+ 6 3.11 (0.48)
A1− 2 2.67 (0.39)
Sibling
A1+ 3 2.91 (0.44)
A1− 5 3.47 (0.35)
Schizophrenia
A1+ 0 N/A
A1− 2 3.10 (0.11)
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BPND, non-displaceable binding potential; A1+,
DRD2/ANKK1
TaqIA (rs1800497) A1 allele carrier; A1−,
DRD2/ANKK1
TaqIA (rs1800497)
A2 allele homozygote; N/A, not applicable.
BPND adjusted means are adjusted for age, gender, ethnicity, education, and group membership.
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Table 4
Summary of hierarchical multiple linear regression analyses for prediction of striatal D2R specific binding by
DRD2/ANKK1
Taq1A (rs1800497) allele
status in Study 2.
Step 1 Step 2 Step 3 Step 4
Striatum (N = 18)
Variable
B SE B β B SE B β B SE B β B SE B β
Education −.20 .18 −.24 −.28 .18 −.33 −.37 .16 −.44 −.28 .21 −.34
Age −.06 .04 −.31 −.05 .04 −.27 −.06 .03 −.29 −.06 .03 −.34
Gender 1.8 .72 .58 1.8 .67 .61 2.2 .60 .72 1.9 .70 .64
White or not 1.7 .62 .55 1.9 .60 .62 1.7 .52 .57 1.7 .55 .55
Group .67 .41 .29 .20 .41 .09 .05 .48 .02
Allele status 2.5 1.1 .41*.15 3.8 .03
Group × allele status 1.4 2.2 .42
R2
.58 .66 .77 .78
F
for change in
R2
4.6 4.6,
p
= 0.02 2.7,
p
= 0.13 5.1,
p
= 0.05 0.42,
p
= 0.53
Dorsal Striatum (N = 18)
Variable
B SE B β B SE B β B SE B β B SE B β
Education −.13 .14 −.21 −.19 .14 −.31 −.26 .12 −.43 −.21 .16 −.34
Age −.05 .03 −.33 −.04 .03 −.29 −.04 .02 −.32 −.05 .03 −.36
Gender 1.2 .54 .53 1.2 .51 .55 1.5 .45 .67 1.3 .53 .61
White or not 1.2 .47 .52 1.3 .45 .59 1.2 .39 .54 1.1 .41 .52
Group .50 .31 .30 .13 .30 .08 .03 .36 .02
Allele status 2.0 .82 .45*.51 2.8 .12
Group × allele status .89 1.6 .36
R2
.55 .63 .76 .77
F
for change in
R2
4.0 ,
p
= 0.03 2.6,
p
= 0.13 5.8,
p
= 0.04 .30,
p
= 0.60
Putamen (N = 18)
Variable
B SE B β B SE B β B SE B
p
B SE B β
Education −.10 .06 −.33 −.11 .06 −.39 −.15 .05 −.52 −.12 .07 −.40
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Step 1 Step 2 Step 3 Step 4
Age −.02 .01 −.35 −.02 .01 −.33 −.02 .01 −.36 −.03 .01 −.41
Gender .59 .24 .56 .61 .24 .58 .74 .19 .71 .66 .22 .63
White or not .58 .20 .55 .62 .21 .59 .57 .16 .54 .53 .17 .51
Group .14 .14 .18 −.05 .13 −.06 −.11 .15 −.14
Allele status 1.0 .35 .49** .14 1.2 .07
Group × allele status .54 .68 .46
R2
.63 .66 .81 .82
F
for change in
R2
5.4,
p
= 0.01 1.0,
p
= 0.33 8.8,
p
= 0.01 0.64,
p
= 0.44
Caudate (N = 18)
Variable
B SE B β B SE B β B SE B β B SE B P
Education −.03 .08 −.10 −.08 .08 −.23 −.11 .07 −.33 −.14 .07 −.41
Age −.02 .02 −.30 −.02 .02 −.25 −.02 .01 −.27 −.01 .01 −.18
Gender .57 .33 .47 .61 .30 .50 .74 .28 .60 .90 .26 .74
White or not .58 .33 .47 .68 .26 .56 .63 .24 .52 .57 .22 .47
Group .36 .18 .39 .18 .19 .20 .22 .17 .25
Allele status .95 .52 .39†.63 .48 .26
Group × allele status .16 .08 .37
R2
.46 .59 .69 .78
F
for change in
R2
2.7,
p
= 0.07 3.9,
p
= 0.07 3.4,
p
= 0.09 4.1,
p
= 0.07
Nucleus Accumbens (N = 18)
Variable
B SE B β B SE B β B SE B β B SE B β
Education −.07 .05 −.29 −.09 .05 −.37 −.11 .05 −.45 −.12 .05 −.50
Age −.01 .01 −.23 −.01 .01 −.19 −.01 .01 −.21 −.01 .01 −.15
Gender .60 .20 .69 .62 .19 .72 .69 .18 .80 .77 .19 .89
White or not .52 .17 .60 .57 .17 .66 .54 .16 .63 .51 .15 .59
Group .17 .11 .26 .07 .12 .11 .09 .12 .11
Allele status .52 .34 .30 .36 .34 .21
Group × allele status .08 .06 .25
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Step 1 Step 2 Step 3 Step 4
R2
.62 .68 .74 .78
F
for change in
R2
5.4,
p
= 0.01 2.3,
p
= 0.16 2.4,
p
= 0.15 1.9,
p
= 0.20
*
,
p ≤
0.05,
**
,
p ≤
0.01;
†
,
p
= 0.09
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