Genome-wide association study of theta band event-related oscillations identifies serotonin receptor gene HTR7 influencing risk of alcohol dependence.
ABSTRACT Event-related brain oscillations (EROs) represent highly heritable neuroelectrical correlates of human perception and cognitive performance that exhibit marked deficits in patients with various psychiatric disorders. We report the results of the first genome-wide association study (GWAS) of an ERO endophenotype-frontal theta ERO evoked by visual oddball targets during P300 response in 1,064 unrelated individuals drawn from a study of alcohol dependence. Forty-two SNPs of the Illumina HumanHap 1 M microarray were selected from the theta ERO GWAS for replication in family-based samples (N = 1,095), with four markers revealing nominally significant association. The most significant marker from the two-stage study is rs4907240 located within ARID protein 5A gene (ARID5A) on chromosome 2q11 (unadjusted, Fisher's combined P = 3.68 × 10⁻⁶). However, the most intriguing association to emerge is with rs7916403 in serotonin receptor gene HTR7 on chromosome 10q23 (combined P = 1.53 × 10⁻⁴), implicating the serotonergic system in the neurophysiological underpinnings of theta EROs. Moreover, promising SNPs were tested for association with diagnoses of alcohol dependence (DSM-IV), revealing a significant relationship with the HTR7 polymorphism among GWAS case-controls (P = 0.008). Significant recessive genetic effects were also detected for alcohol dependence in both case-control and family-based samples (P = 0.031 and 0.042, respectively), with the HTR7 risk allele corresponding to theta ERO reductions among homozygotes. These results suggest a role of the serotonergic system in the biological basis of alcohol dependence and underscore the utility of analyzing brain oscillations as a powerful approach to understanding complex genetic psychiatric disorders.
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ABSTRACT: Recent data collected at six identical electrophysiological laboratories from the large national multisite Collaborative Study on the Genetics of Alcoholism provide evidence for considering the P3 amplitude of the event-related potential as a phenotypic marker for the risk of alcoholism. The distribution of P3 amplitude to target stimuli at the Pz electrode in individuals 16 years of age and over from 163 randomly ascertained control families (n = 687) was compared with those from 219 densely affected alcoholic families (n = 1276) in which three directly interviewed first-degree relatives met both DSM-III-R and Feighner criteria at the definite level for alcohol dependence (stage II). The control sample did not exclude individuals with psychiatric illness or alcoholism to obtain incidence rates of psychiatric disorders similar to those of the general population. P3 amplitude data from control families was converted to Z-scores, and a P3 amplitude beyond 2 SD's below the mean was considered an "abnormal trait." When age- and sex-matched distributions of P3 amplitude were compared, members of densely affected stage II families were more likely to manifest low P3 amplitudes (2 SD below the mean) than members of control families, comparing affected and unaffected offspring, and all individuals; all comparisons of these distributions between groups were significant (p < 0.00001). P3 amplitude means were also significantly lower in stage II family members, compared with control family members for all comparisons, namely probands, affected and unaffected individuals (p < 0.0001), and offspring (p < 0.01). Furthermore, affected individuals from stage II families, but not control families, had significantly lower P3 amplitudes than unaffected individuals (p < 0.001). Affected males from stage II families had significantly lower P3 amplitudes than affected females (p < 0.001). Recent linkage analyses indicate that visual P3 amplitude provides a biological phenotypic marker that has genetic underpinnings.Alcoholism Clinical and Experimental Research 10/1998; 22(6):1317-23. · 3.42 Impact Factor
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ABSTRACT: This study investigates early evoked gamma band activity in male adolescent subjects at high risk for alcoholism (HR; n=68) and normal controls (LR; n=27) during a visual oddball task. A time-frequency representation method was applied to EEG data in order to obtain stimulus related early evoked (phase-locked) gamma band activity (29-45 Hz) and was analyzed within a 0-150 ms time window range. Significant reduction of the early evoked gamma band response in the frontal and parietal regions during target stimulus processing was observed in HR subjects compared to LR subjects. Additionally, the HR group showed less differentiation between target and non-target stimuli in both frontal and parietal regions compared to the LR group, indicating difficulty in early stimulus processing, probably due to a dysfunctional frontoparietal attentional network. The results indicate that the deficient early evoked gamma band response may precede the development of alcoholism and could be a potential endophenotypic marker of alcoholism risk.International Journal of Psychophysiology 12/2006; 62(2):262-71. · 2.04 Impact Factor
- Journal of Clinical Neurophysiology 01/1998; 15(1):44-57. · 1.45 Impact Factor
Genome-Wide Association Study of Theta Band
Event-Related Oscillations Identifies Serotonin
Receptor Gene HTR7 Influencing Risk of
Mark Zlojutro,1* Niklas Manz,2Madhavi Rangaswamy,2Xiaoling Xuei,3Leah Flury-Wetherill,3
Daniel Koller,3Laura J. Bierut,4Alison Goate,4Victor Hesselbrock,5Samuel Kuperman,6
John Nurnberger Jr.,3John P. Rice,4Marc A. Schuckit,7Tatiana Foroud,3Howard J. Edenberg,3
Bernice Porjesz,2and Laura Almasy1
1Department of Genetics, Southwest Foundation for Biomedical Research, San Antonio, Texas
2Department of Psychiatry, Henri Begleiter Neurodynamics Laboratory, SUNY Downstate Medical Center, Brooklyn, New York
3Indiana University School of Medicine, Indianapolis, Indiana
4Department of Psychiatry, Washington University School of Medicine, St. Louis, Missouri
5Department of Psychiatry, University of Connecticut Health Center, Farmington, Connecticut
6Department of Psychiatry Research, University of Iowa, Iowa City, Iowa
7Department of Psychiatry, University of California, San Diego, California
Received 10 September 2009; Accepted 16 September 2010
Additional Supporting Information maybefoundin theonline version of
protein ACN9 homolog; ADHD, attention-deficit/hyperactivity disorder;
CDKN2B, cyclin-dependent kinase inhibitor 2B; CEU, HapMap European-
American population (Utah residents); CHB, HapMap Han Chinese
population (Beijing); CHRM2, muscarinic cholinergic receptor 2;
CIDR, Center for Inherited Disease Research; COGA, Collaborative
Study on the Genetics of Alcoholism; dbGaP, database of Genotypes
and Phenotypes; DNA, deoxyribonucleic acid; DSM-IIIR, Diagnostic
and Statistical Manual of Mental Disorders, Revised Third Edition; DSM-
IV, Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition;
guanine nucleotide-binding protein 1; GRM8, metabotropic glutamate
receptor type 8; GWAS, genome-wide association study; HMO, Health
Maintenance Organization; IBS, identical by state; ICD-10, International
Classification of Diseases, Tenth Version; JNK, jun N-terminal protein
kinase; JPT, HapMap Japanese population (Tokyo); LD, linkage
disequilibrium; LOD, logarithm of odds; MAF, minor allele frequency;
MAP, mitogen-activated protein; MAS, McCune–Albright syndrome;
MDS, multidimensional scaling analysis; MG, measured genotype;
MTAP, methylthioadenosine phosphorylase; NCBI, National Center for
Biotechnology Information; NESP55, neuroendocrine secretory protein
55; NIAAA, National Institute on Alcohol Abuse and Alcoholism; NIDA,
odds ratio; P300, positive voltage deflection at latency of approximately
300ms; PC, principal component; PHP, pseudohypoparathyroidism;
disequilibrium test; QTLD, quantitative trait linkage disequilibrium;
SFBR, Southwest Foundation for Biomedical Research; SNP, single
nucleotide polymorphism; SSAGA, Semi-Structured Assessment for the
UTR, untranslated region; YRI, HapMap Yoruba Population (Ibadan,
Grant sponsor: National Institute on Alcohol Abuse and Alcoholism
(NIAAA) and National Institute on Drug Abuse (NIDA); Grant
HHSN268200782096C; Grant sponsor: National Institutes of Mental
Health (NIMH); Grant number: R01 MH59490; Grant sponsor:
Indiana Genomics Initiative of Indiana University (INGEN) and The
Lilly Endowment, Inc.
Dr. Mark Zlojutro, Department of Genetics, Southwest Foundation for
Biomedical Research, 7620 N.W. Loop 410, San Antonio, TX 78227.
Published online 2 November 2010 in Wiley Online Library
? 2010 Wiley-Liss, Inc.
Event-related brain oscillations (EROs) represent highly
heritable neuroelectrical correlates of human perception and
cognitive performance that exhibit marked deficits in patients
with various psychiatric disorders. We report the results of the
first genome-wide association study (GWAS) of an ERO endo-
phenotype—frontal theta ERO evoked by visual oddball targets
a study of alcohol dependence. Forty-two SNPs of the Illumina
HumanHap 1M microarray were selected from the theta ERO
GWASforreplication infamily-basedsamples(N¼1,095), with
four markers revealing nominally significant association. The
most significant marker from the two-stage study is rs4907240
the most intriguing association to emerge is with rs7916403 in
P¼1.53?10?4), implicating the serotonergic system in the
neurophysiological underpinnings of theta EROs. Moreover,
promising SNPs were tested for association with diagnoses of
alcohol dependence (DSM-IV), revealing a significant relation-
shipwiththe HTR7 polymorphismamong GWAS case–controls
(P¼0.008). Significant recessive genetic effects were also
-based samples (P¼0.031 and 0.042, respectively), with the
HTR7 risk allele corresponding to theta ERO reductions among
homozygotes. These results suggest a role of the serotonergic
system in the biological basis of alcohol dependence and under-
score the utility of analyzing brain oscillations as a powerful
approach to understanding complex genetic psychiatric
disorders. ? 2010 Wiley-Liss, Inc.
Key words: serotonin receptor gene (HTR7); serotonin
receptor (5-HT7); event-related oscillation (ERO); alcohol
dependence; genome-wide association study (GWAS)
specific frequency rhythms correlated to sensory perception and
cognitive performance, including conscious awareness, memory,
and stimulus processing [Llinas and Ribary, 1993; Klimesch et al.,
are highly heritable [van Beijsterveldt and Boomsma, 1994; van
Beijsterveldt et al., 1996] and are modulated by neurotransmitters
and signal transduction factors, qualities that make them advanta-
geous as intermediate phenotypes or endophenotypes that are less
complex, quantitative, and more proximate to gene function than
traditionalcognitivemeasuresandclinical diagnosesthat aremore
influenced by individual and familial environmental factors
[Tsuang and Faraone, 2000; Begleiter and Porjesz, 2006].
ty using non-invasive scalp electrodes when the subject is either at
rest, producing ongoing state measurements of neuroelectrical
activity, or engaged in cognitive tasks, yielding event-related po-
tentials (ERPs) that are time-locked to specific stimuli, events, or
and 600ms after a stimulus (Fig. 1). This neuroelectrical compo-
Almasy et al., 1999], associated with brain structure integrity
[McCarley et al., 1993; Ford et al., 1994] and with various task-
of strong GABAergic, cholinergic, and glutamatergic interactions
that elicit excitatory and inhibitory post-synaptic potentials, with
ing effects [Frodl-Bauch et al., 1999; Polich, 2007]. Various neuro-
logical disorders display reductions in P300 amplitude and
prolonged latency, foremost being alcohol dependence [Porjesz
and Begleiter, 1998] and schizophrenia [Ford, 1999], as well as a
spectrum of externalizing disorders [Iacono et al., 2003].
Recent studies involving time–frequency domain analysis have
determined that ERPs may not be unitary entities but arise from
phase resetting or superposition of different frequency bands in
Gruber et al.,2005]. These underlying neural phenomena, referred
to as event-related oscillations (EROs), appear to influence the
timing of brain activity and synchrony in networks of active
neurons, and thus are likely to represent a basic mechanism of
1999, 2001a; Fries, 2005; Buzsaki, 2006].
Generally, EROs have been analyzed using traditional EEG
frequency band limits: delta (1–3Hz); theta (4–7Hz); alpha
(8–12Hz); beta (13–28Hz); and gamma (>29Hz). EROs have
been increasingly used to study the neural components of brain
function [Klimesch et al., 1997b, 2001; Schurmann et al., 1997;
responses are attributed to particular cognitive processes. For
-making [Basar et al., 1999; Schurmann et al., 2001], whereas theta
rhythms appear to mediate conscious awareness, attention, recog-
nition memory, frontal inhibitory control, and episodic retrieval
[Klimesch et al., 1994, 2001; Doppelmayr et al., 1998; Gevins et al.,
1998; Karakas et al., 2000a,b; Basar et al., 2001b]. The P300
component is composed of a superposition of different frequency
frontal topography, while the delta maximum has a more parietal
How to Cite this Article:
Flury-Wetherill L, Koller D, Bierut LJ, Goate
Rice JP, Schuckit MA, Foroud T, Edenberg
Association Study of Theta Band Event-
Related Oscillations Identifies Serotonin
Receptor Gene HTR7 Influencing Risk of
Am J Med Genet Part B 156:44–58.
ZLOJUTRO ET AL.
These oscillatory responses have served as useful endopheno-
types for measuring and deconstructing neurocognitive dysfunc-
alcohol dependence [Jones et al., 2004, 2006b; Porjesz et al., 2005;
Padmanabhapillai et al., 2006a,b; Rangaswamy et al., 2007]. The
Collaborative Study on the Genetics of Alcoholism (COGA) has
investigated event-related oscillatory responses in families densely
affected with alcoholism, finding significant reductions in evoked
theta and delta ERO amplitudes among affected subjects while
processing target stimuli [Jones et al., 2006b]. Studies examining
high-risk children of alcoholics for the same paradigm have
compared to normal children (ages 14–17 years) [Rangaswamy
alcoholism and represent a strong trait marker. Family-based
linkage analysis and candidate gene association studies of ERO
responses have identified several chromosome regions and genetic
variants that underpin these neuroelectrical activities and contrib-
ute to the risk of alcohol dependence. Most notably, significant
genetic linkage was reported on chromosome 7q31–34, with asso-
2006a]. In addition, several single nucleotide polymorphisms
(SNPs) in GRM8 (metabotropic glutamate receptor type 8),
another candidate gene located under the same linkage peak, are
associated with theta EROs to target stimuli at frontal, central, and
parietal regions [Chen et al., 2009]. Both of these receptor genes
et al., 2004; Chen et al., 2009], underscoring the utility of EROs as
endophenotypes in relation to psychiatric pathologies.
In the present article, we report the results of the first
genome-wide association study (GWAS) of an ERO endopheno-
during processing of target visual signals in a visual oddball para-
digm. The analysis used a sample of unrelated individuals drawn
from a study of alcohol-dependence. High ranking SNPs from the
GWAS were selected for replication analysis in a family-based
sample ascertained for alcohol dependence, implicating new
regions not detected by previous linkage analyses, with a number
of significant markers in or near genes that play important roles in
SNPs were also tested for association with diagnoses of alcohol
dependence in both samples.
Sample Recruitment and Assessment
of the multi-site COGA, a national consortium designed to study
the genetic predisposition to develop alcoholism and related phe-
SUNY Downstate Medical Center at Brooklyn, New York; Univer-
sity of Connecticut Health Center; Washington University School
of Medicine in St. Louis; University of California at San Diego;
University of Iowa Carver College of Medicine; Indiana University
School of Medicine; and Howard University College of Medicine.
Institutional review boards at each site approved the research
protocols in the COGA study and written consent was obtained
from each individual prior to participation.
Recruitment and assessment procedures have been outlined
previously [Begleiter et al., 1995; Reich et al., 1998; Foroud et al.,
FIG. 1. Illustration of a standard P300 waveform and its time–frequency decomposition. Top panel: visual oddball ERP waveforms from 48 normal,
healthy control subjects. Bottom panel: time–frequency representation (TFR) plot for EEG responses to target stimuli. White box highlights the
time–frequency region of interest (TFROI) and the inset head plot exhibits the frontal preponderance of the peak theta band activity.
46 AMERICAN JOURNAL OF MEDICAL GENETICS PART B
2000; Nurnberger et al., 2004]. Alcoholic probands were recruited
from inpatient and outpatient treatment centers and control fami-
lies were selected from Health Maintenance Organizations
(HMOs), drivers’ license records, and dental clinics, with the
objective of obtaining representative samples of the communities
at each recruitment site. Prior to administration of a neurophysio-
logical battery and drawing of blood for DNA extraction, alcohol-
dependent subjects were required to have been detoxified in a
30-day treatment program and not exhibit withdrawal symptoms.
Subjects were excluded from assessment for any of the following:
(1) recent substance and alcohol abuse (i.e., positive breath-
analyzer test and/or urine screen); (2) hepatic encephalopathy/
cirrhosis of the liver; (3) significant history of head injuries,
seizures, or neurosurgical procedures; (4) uncorrected sensory
deficits; (5) subjects taking medication known to influence brain
functioning (e.g., any psychotropic drugs); and (6) history or
symptoms of psychoses.
administered a validated poly-diagnostic instrument, the Semi-
Structured Assessment for the Genetics of Alcoholism (SSAGA)
[Bucholz et al., 1994; Hesselbrock et al., 1999]. The SSAGA allows
assessment of alcohol dependence by several criteria, including
Feighner et al. , DSM-IIIR [American Psychiatric Associa-
tion, 1987], DSM-IV [American Psychiatric Association, 1994],
and ICD-10 [World Health Organization, 1993].
GWAS Case–Control Sample
All alcohol-dependent cases and controls in the GWAS sample
(N¼1,884) are genetically unrelated. Cases (N¼1,192) were
diagnosed for DSM-IV alcohol dependence at each clinical assess-
the GWAS if considered unaffected during their entire lifetimes
according to any of the four alcohol dependence criteria. To avoid
pleiotropic genetic components that contribute to multiple sub-
stance abuse phenotypes, controls for the GWAS also did not meet
diagnostic criteria for other illicit substance abuse or dependence.
Furthermore, controls were required to be 25 years or older and to
have consumed alcohol at some point in their lives to ensure that
their unaffected status was not due to lack of exposure to alcohol.
Based on the results of a multidimensional scaling analysis (MDS)
predominantly clustered among two subgroups characterized as
being of either European (74.3%; N¼1,399) or African ancestry
(25.7%; N¼485) (refer to the section on GWAS genotyping for
more detail). Twenty-one individuals did not cluster with either of
the two groups and were excluded from analysis. A subset of the
subjects (N¼1,064) had measurements for theta ERO energy
available for GWAS analysis (see Fig. 1 for overview of research
Family-Based Replication Sample
For the second stage, 42 promising SNPs from the GWAS were
tested for association with theta EROs in a set of 262 genetically
informative, multiplex alcohol dependence pedigrees (N¼1,095
individuals) (Fig. 2). The ethnic composition of the sample based
on self-reporting is predominantly European-American (78.5%;
N¼862) and African-American (13.9%; N¼153). Both linkage
al., 2004] and family-based association analyses [Edenberg et al.,
2004; Wang et al. 2004; Edenberg and Foroud, 2006; Jones et al.,
2006a; Dick et al., 2007] have been performed previously in these
families. Although some members of these families, primarily
probands, were included among the GWAS case–controls, over-
lapping individuals were removed from this family-based
sample using the program PEDSYS, and thus the results from each
association test are independent.
Electrophysiological Data Collection
and EEG acquisition hardware and software programs. EEG was
recorded using a fitted electrode cap (Electro-Cap International,
electrode.Continuous EEG wassampled asthesubjectsperformed
the standard visual oddball paradigm that was selected to elicit the
event-related potential containing the P300 component [Porjesz
et al., 1998]. The task involves the presentation of three types of
visual stimuli: 12.5% are target (X), 75% are non-target (squares)
and 12.5% are novel (non-repeating colored polygons). Subjects
responded to the appearance of target stimuli by pressing a button
with either the left or right index finger. The artifact control and
earlier articles on theta and delta EROs [Jones et al., 2006b;
Rangaswamy et al., 2007].
ERO Energy Estimation
Estimates of localized energy of non-stationary event-related
potential time series were obtained using a time–frequency repre-
sentation (TFR) method, the S-transform [Stockwell et al., 1996;
Visual oddball event-related electrophysiological data for the
targetconditions wereanalyzed.Mean values werecalculated from
the S-transform TFR for use as phenotypes within time–frequency
regions of interest (TFROIs) that are specified by frequency band
on the total amplitude of the theta (4.0–7.0Hz) frequency band in
latency window in the event-related waveforms measured at the
frontal midline channel (Fz electrode) (see Fig. 1), a brain region
ed for the P300 response [Karakas et al., 2000b]. This TFROI was
established by examination of target condition grand-mean TFRs
and selecting a time region that bound observed stimulus evoked
increases in theta band energy relating to a subcomponent of the
P300 ERP. Similar to the P300 ERP amplitude, the theta ERO
as a covariate in the genetic analyses.
were recorded (with ?4 SD outliers excluded from genome-wide
ZLOJUTRO ET AL.
association analysis), corresponding to 640 cases with alcohol
dependence and 424 controls, ranging in age from 17 to 69 years,
and consisting of 570 males and 494 females (see Table I). Of the
cases, 242 were selected from densely affected families that have
been studied in previous COGA family-based linkage and associa-
tion analyses, but none of these were included in the family-based
GWAS Genotyping and Quality Control
Genotyping was performed by the Center for Inherited Disease
Research (CIDR) at John Hopkins University using the Illumina
Infinium II assay protocol [Gunderson et al., 2006] for hybridiza-
tion toIllumina HumanHap1MBeadChips(Illumina,SanDiego,
CA). Details are reported by Edenberg et al.  and both
protocols and the data set are available on the National Center
for Biotechnology Information (NCBI) database dbGaP (available
at http://www.ncbi.nlm.nih.gov/sites/entrez?db¼gap; accession
number: phs000125.v1.p1). Blind duplicate reproducibility was
FIG. 2. Schematic of two-stage association study of the theta ERO phenotype.
Cases and Controls With Theta ERO Measurements
Mean age (SD)
Mean theta ERO (SD)
48 AMERICAN JOURNAL OF MEDICAL GENETICS PART B
Additional quality control measures were applied to the dbGaP
data for both the samples and the SNPs. Samples having genotypes
for at least 98% of the SNPs were considered for inclusion in
analyses. These samples were rigorously checked for cryptic relat-
edness, population stratification, consistency with reported sex,
and related quality control issues [Edenberg et al., 2010]. For the
theta ERO sample subset (N¼1,064) used in the GWAS analysis
(see below), 771 individuals were classified as European-American
and 293 as African-American. The inclusion thresholds for SNPs
were a minimum call rate of 98%, minor allele frequency (MAF)
>0.01, and Hardy–Weinberg P-values >0.001 for the European-
American and African-American groups. Based on these criteria,
90,394 SNPs were removed, reducing the number of SNP markers
in the final genotype data set to 951,071.
After reviewing the results from the primary genome-wide associ-
ation analysis, top-ranking SNPs were annotated for function (i.e.,
synonymous, non-synonymous, splice-site, intronic, etc.) and
aligned against the human genome assembly build 36.3 using the
program WGAViewer ver. 1.25 [Ge and Goldstein, 2007]. Forty-
two SNPs were chosen from the GWAS results and successfully
genotyped in the family-based sample (see Supplementary Online
Material). Of these, 30 were selected from the top 250 ranking
markers located within or near (<50kb) genes of interest based on
genic function and expression patterns, including regions exhibit-
ing high regulatory potential and interspecies conservation (e.g.,
evolutionary and sequence pattern extraction through reduced
representations—ESPERR score) [Taylor et al., 2006]. These were
supplemented by 12 other SNPs outside the top 250, but still
nominally significant (P¼0.05), identified as potentially relevant
(HTR2A and HTR7)]. Genotyping assays were designed for the
Sequenom MassArray system (Sequenom, San Diego, CA) using
MassArray Assay Design Software. Genotyping used iPLEX assays
(Sequenom), with alleles discriminated by mass spectrometry.
Assays were tested on two independent groups, 40 unrelated
European-American individuals and 40 unrelated African-
American individuals (samples obtained from Coriell Cell
in both groups were not genotyped in the COGA families. All
SNPs were tested for Mendelian inheritance using the program
PEDCHECK [O’Connell and Weeks, 1998]. Marker allele fre-
quencies were computed using the program USERM13 [Lange
et al., 1988].
Statistical Genetic Analyses
Genome-wide association tests were performed on 1,064 frontal
theta ERO measurements and corresponding genotypic data using
a standard quantitative trait measured genotype (MG) method
[Boerwinkle et al., 1986] implemented in the new GWAS software
package PLINK [Purcell et al., 2007a]. Under a simple fixed effects
additive MG model, SNP genotypes are coded 0 for heterozygotes,
?1 for one homozygote, and 1 for the other homozygote and then
the variation of the trait mean by genotype is assessed via a general
linearregression.Populationstratification, awell-knownsource of
confounding for case–control association studies, was corrected
ed significance values [Devlin and Roeder, 1999], which accom-
modates over-dispersion of the chi-square statistic due to
heterogeneity through the estimation of an inflation statistic; and
adjustment of genotypes and phenotypes for population ancestry
through linear regressions of principal component (PC) scores
score-based correction was assessed by examining the GC correc-
tion factor. Furthermore, neurophysiological endophenotypes are
known to vary between males and females and by age and thus age
model. Case/control status was not used as a covariate in the main
analysis, despite observed theta ERO deficits among the GWAS
cases, in order to detect genetic variants that may contribute to the
susceptibility of alcohol dependence through their effects on
electrophysiology. If a gene affects brain function, as indexed by
electrophysiological measures, and these differences in brain func-
tion also contribute to risk of alcohol dependence, then correcting
we are most interested in localizing. However, secondary analyses
producing negligible differences among the top-ranking SNPs as
compared to the main GWAS results. The full set of P-values that
emerged from the PLINK association analysis were loaded and
visualized in Haploview ver. 4.1 [Barrett et al., 2005] and WGA-
disequilibrium (LD) patterns and haplotype maps, generate quan-
latest available genomic databases.
For the family-based replication study, SNP association analysis
was conducted with the statistical computer software SOLAR
[Almasy and Blangero, 1998] using the quantitative trait linkage
disequilibrium(QTLD)procedure.Thisprocedure performs atest
for population stratification (based on the likelihood comparison
of between-family and within-family association parameters of
used association tests: MG test (see above) and the quantitative
transmission disequilibrium test (QTDT) [Abecasis et al., 2000].
AlthoughQTDTis a family-based associationtest that controls for
spurious associations due to population stratification by scoring
allelictransmissions basedonparental genotypes, intheabsenceof
stratification the classical MG approach uses more of the relative
association information and exhibits more power than the QTDT
[Havill et al., 2005] and thus is reported for the replication results
when this condition is met.
SNPs exhibiting significant or near significant association with
theta ERO measurements for both sample sets were further tested
for association with diagnoses of alcohol dependence (DSM-IV).
For the case–control data set, these analyses were performed in
sex, and the first and second PCs as covariates. For the family
sample, a liability threshold model with an ascertainment correc-
tion was implemented in SOLAR, also assuming an additive mode
of gene action. Markersthat producedsignificant results underthe
ZLOJUTRO ET AL.
additive model for either sample were additionally tested for
association under both recessive and dominant genetic models
(i.e., pairwise comparison of frequencies for either the minor or
major homozygotes against the other two genotype classes). Hap-
lotype-based tests were also carried out for the HTR7 region in the
H?1 degrees of freedom that jointly tests all H haplotypes by
comparing the alternate (each haplotype having a unique effect)
versus the null (no haplotypes having any different effect) [Purcell
et al., 2007b].
Stage I: GWAS in Unrelated Individuals
Estimates of theta ERO energy of the TFROI were calculated using
ing a mean score of 3.58?1.12mV and range of 0.86–8.05mV. For
means were 3.47?1.12mV and 3.76?1.11mV, respectively
(P¼3.1?10?5). After stringent data cleaning and quality control,
GWAS was performed on theta ERO data for 951,071 SNPs, with
age, sex, and PCs 1 and 2 included as covariates. The quantile–
in Figure 3. No strong deviation is observed from the expected
median chi-squaredstatisticis0.94),indicating nobiased inflation
of the test statistic or population stratification artifacts.
displaying P-values <1.0?10?5(N¼28) listed in Table II. The
top-ranking SNP (rs2784) has a P-value of 2.4?10?7, falling
short of the threshold for genome-wide significant association
SNPs listed in Table II, are located within the intronic regions of
TAOK3 on chromosome 12q24 (NCBI build 36.3bp 117,071,989–
involved in mitogen-activated protein (MAP) kinase (ERK and
JNK) signaling pathways [Zhang et al., 2000] that play important
roles in neuronal growth and differentiation [Rueda et al., 2002;
Harada et al., 2004; Kanzawa et al., 2006]. A 199kb ‘‘strong LD’’
block (bp 117,073,452–117,272,386) encompassing rs2784 and
neighboring top-ranking SNPs was identified by the D’ confidence
interval algorithm employed in Haploview ver. 4.1 [Gabriel et al.,
2002]. The pairwise r2scores for rs2784 with other top-ranking
TAOK3 markers (P<1.0?10?5) range from 0.75 (rs7314987) to
0.97 (rs17512142). Results from a haplotype-based association test
one of the haplotypes (frequency of 0.095) producing a highly
significant P-value of 6.5?10?7.
FIG. 3. Quantile–quantileplotofgenome-wideassociationresults.
Observed P-values (y-axis) for theta ERO measurements are
plotted for 951,071 SNPs against expected P-values (x-axis)
under the null distribution for no association.
FIG. 4. Genome-wide association results. P-values (?log10) for 951,071 SNPs are plotted in order of chromosome position.
50AMERICAN JOURNAL OF MEDICAL GENETICS PART B
In addition to TAOK3, SNPs listed in Table II are located in
or nearby other genes, including MTA phosphorylase gene
(MTAP), ARID (A-T rich interaction domain) protein 5A
(ARID5A), cyclin-dependent kinase inhibitor 2B (CDKN2B),
guanine nucleotide-binding protein 1 (GNAS1), and subunit of
histone deacetylase-dependent SIN3A (SUDS3). None of these
genes have been previously associated with brain oscillation
Stage II: Replication in Family-Based Sample
From the GWAS results and SNP annotation performed with
WGAViewer ver. 1.25, SNPs were prioritized for follow-up geno-
Weinberg equilibrium, predicted functions, LD context, and gene
in the family-based replication sample (N¼1,095). The corre-
sponding theta ERO data set has a mean value of 3.99?1.18mV
and range of 1.05–8.56mV, with no evidence of kurtosis, and an
likelihood variation decomposition implemented in SOLAR. The
second stage association analysis produced six significant or near
significant SNPs for the MG test (no evidence of stratification or
Hardy–Weinberg disequilibrium was detected for these particular
and directions (relative to the minor allele) that are consistent
SNPs are presented in Table III, along with combined P-values for
the two stages using Fisher’s combined probability test [Fisher,
1925]. The lowest P-value observed for the family-based sample is
3.9?10?4(Bonferroni-corrected 1.6?10?2) for rs7916403 locat-
ed within intron 1 of the 5-hydroxytrptamine (serotonin) 7 recep-
P-value score of 1.5?10?4for the two stages. The association for
subset (P¼9.97?10?4), suggesting potential population-specific
ERO effects for this particular locus (see below for additional
analyses). The other three significant SNPs from the follow-up
association analysis are: rs4907240 downstream from ARID5A on
chromosome 2q11; rs13831 located in the 30-UTR of GNAS1 on
TABLE II. Association Results for Theta ERO Phenotype (P<1?10?5)
aBased on NCBI reference assembly build 36.3.
bA1 (minor):A2 (major) alleles.
cMinor allele frequency.
dMinor allele frequency of European-American sample.
eMinor allele frequency of African-American sample.
fDistance to nearest gene.
ZLOJUTRO ET AL.
chromosome 20q13; and non-synonymous rs2294015 of annexin
A13 (ANXA13) on chromosome 8q24.
markers from the GWAS, eight of which were genotyped but none
achieving significance. The directions of genetic effect for all eight
TAOK3 SNPs are the same as those obtained from the GWAS (i.e.,
minor alleles associated with theta ERO reductions); however, the
largest effect size is 0.092 (SE 0.078) for rs7314987, approximately
one-fourth of the regression coefficient estimated for the top
GWAS SNP rs2784. The MAF values in the family-based sample
(N¼1,095) are similar to those reported for the GWAS case–
controls (N¼1,064), ranging from 0.11 to 0.13, suggesting com-
parable power for detecting association with the ERO phenotype.
deviation from HWE. Thus, the observed discrepancy in associa-
tions across the two stages may simply represent a GWAS false
positive for this particular string of markers within the TAOK3 LD
block or stem from underlying differences in genetic substructure
between the data sets that relate to the high-density affected family
or case–control sample designs. If the smaller effect size estimate
from the family sample is the more accurate one, the detection of
TAOK3 in the GWAS sample was a low probability event, which
would be difficult to replicate without a large sample size.
Association With Alcohol Dependence
and high-risk subjects with limited alcohol exposure, it has been
development of alcoholism and may serve as an effective endo-
phenotype to the condition [Rangaswamy and Porjesz, 2008].
Thus, the four SNPs identified above as potential genetic determi-
nants of theta EROs were further examined for their association to
analysis for affected status (with PCs, age, and sex as covariates
in additive genetic effects model) produced one significant result
among the four SNPs—rs7916403 from serotonin receptor gene
allele is 1.24 [95% CI: 1.06–1.45], with an increased OR of 1.32
[95% CI: 1.02–1.71] when tested for a full recessive model
(P¼0.036), suggesting a significant recessive mode of genetic
influence on the development of alcoholism by the HTR7 risk
allele. Concordantly, the mean theta ERO value for the G/G
homozygotes is 3.51?1.11mV, revealing a near significant oscil-
latory deficit (P¼0.084) relative to the combined mean for the
other two genotype classes (3.62?1.12mV).
For the family-based samples, no significant evidence of associ-
ation was found for any of the four SNPs in an additive genetic
allele (frequency of 0.52), which produced an OR of 1.13 [95% CI:
0.94–1.35; P¼0.19] for alcohol dependence. However, consistent
with the results obtained for the case–control samples, significant
evidence for an increased risk with the number of G alleles was
G/G homozygotes is 3.85?1.06mV, again revealing an oscillatory
deficit (P¼0.013), relative to the combined mean of the other two
Population-Specific Effects of rs7916403
association analyses were conducted on the European-American
sample (N¼771), the association between theta EROs and
rs7916403 (P¼9.97?10?4) is stronger than what was observed
?0.29]in the direction of the G allele. For European-Americans in
the family-based replication sample (N¼862), the HTR7 risk
allele (G) was also found to be significantly associated with theta
ERO (P¼0.025), with a genetic effect of ?0.12 [95% CI: ?0.11
to ?0.22]. Additionally, diagnoses for alcohol dependence were
examined separately for European-Americans, revealing a signifi-
cant OR of 1.27 [95% CI: 1.06–1.52] for the G allele in the GWAS
of genetic effect, an increased OR of 1.39 [95% CI: 1.07–1.81] was
observed (P¼0.015). For the replication sample, no significant
evidence of association with alcohol dependence was found, al-
1.17 [95% CI: 0.95–1.15] for an additive genetic effects model
(P¼0.15); and OR of 1.36 [95% CI: 0.94–1.96] under a recessive
TABLE III. Significant Associations for Theta ERO From Case–Control GWAS and Family-Based Replication
Stage I: GWASStage II: replication
Effect size (b?SE)a
Effect size (b?SE)a
aGenetic effect in the direction of minor allele.
bUnadjusted P-values obtained from Fisher’s combined probability test [Fisher, 1925].
52 AMERICAN JOURNAL OF MEDICAL GENETICS PART B
Haplotype-Based Association Analysis of
Serotonin Receptor Gene HTR7
According to NCBI Build 36.3, the HTR7 gene is positioned
between bp 92,490,555 and 92,607,651 on chromosome 10, with
its three splice variants encoding either 3 or 4 exon regions and
varying in the length of their carboxy terminal ends. A total of 35
SNPs encompassing HTR7 and its adjacent upstream and down-
stream regions (within 20kb) met the QC standards in the GWAS.
Of these, 22 SNPs capture all observed alleles at r2?0.8 as deter-
D’ confidence interval algorithm, five LD blocks were identified
within the HTR7 gene, with rs7916403 incorporated into a 5.8kb
block in intron 1 that includes SNPs rs11186309 and rs1935351.
Omnibus tests (H?1 degrees of freedom) were conducted on this
LD block, revealing significant joint-haplotype association with
alcohol dependence (P¼0.0074). When correcting for rs7916403,
the association with alcoholism largely disappears (P¼0.098).
Three alternate haplotypes were observed at this particular LD
0.51) producing a significant OR of 1.24 for alcohol dependence
(P¼0.0081). When controlling for the G-T-G haplotype, the
significant omnibus association with alcoholism once again
theta oscillations (Fz electrode) to visual targets in a visual oddball
been related to fronto-limbic interactions associated with a com-
plex set of cognitive functions, including conscious awareness,
attention, memory-related processes, and frontal inhibitory con-
trol, and has exhibited marked energy reductions in alcoholics and
their offspring [Jones et al., 2006b; Rangaswamy et al., 2007].
Significant linkage and association has been previously reported
for CHRM2 and GRM8 polymorphisms for the theta ERO endo-
phenotype and diagnoses (DSM-IV and ICD-10) of alcohol
dependence [Jones et al., 2004, 2006a; Chen et al., 2009], implicat-
ing both the cholinergic and glutamatergic neurotransmitter sys-
tems. None of the top GWAS hits in the present study come from
rs1362178 (P¼0.0034) from GRM8 and rs17168817 (P¼0.033)
from CHRM2. None of the most associated markers (P<0.01;
N¼10) identified in these earlier studies were captured by the
Illumina HumanHap 1M microarray and thus they could not be
evaluated in the GWAS. Of the 18 previously reported SNPs
only rs2299498 from GRM8 (Chen et al., 2009) exhibits nominal
significance (P¼0.028) in this study. Although these findings for
CHRM2 and GRM8 are modest, they are not unexpected. Associ-
primarily through linkage analysis, a method designed to detect a
different set of genetic effects than those of GWAS [Manolio et al.,
2009; Psychiatric GWAS Consortium Coordinating Committee,
from the joint effects of multiple rare or uncommon variants in
TABLE IV. Association With Diagnoses of Alcohol Dependence for Additive and Full Recessive Effects Models
OR (95% CI)a
OR (95% CI)b
OR (95% CI)a
OR (95% CI)b
aOdds ratio (OR) for the minor allele (unless noted otherwise) under a standard additive genetic effects model.
bOR for a full recessive genetic effects model for the G allele of rs7916403 (i.e., two-way test of G/G homozygotes versus G/T heterozygotes and T/T homozygotes).
cOR for G allele of rs7916403.
ZLOJUTRO ET AL.
these genes, we would not expect to detect these genes in GWAS
studies, which are designed to capture associations with common
The results from the GWAS and follow-up analyses implicate
several genes influencing theta EROs that were not detected by
previous linkage and association studies (Table III). Based on
combined P-values, the most significant marker to emerge is
rs4907240 located downstream of ARID5A (P¼3.7?10?6), with
effect sizes of0.25?0.06 and0.10?0.06mVforthe minorallelein
the GWAS and replication, respectively. The protein encoded by
ARID5A belongs to a family of diverse proteins with recognized
roles in development, tissue-specific gene expression, and cellular
growth regulation [Wilsker et al., 2002; Patsialou et al., 2005],
although with no reported role in brain development or neuro-
electrical activity. In contrast, the next top-ranked SNP, rs13831,
is within the 30-UTR of GNAS1 (estimated coefficients of
?0.21?0.05 and ?0.15?0.05mV), a locus with a complex, im-
printed expression pattern for multiple transcripts, including the
G-protein alpha subunit (GSa) involved in the cellular actions of
neurotransmitters, autocrine/paracrine factors, and hormones
[Bastepe, 2007; Plagge et al., 2008], as well as neuroendocrine
secretory protein 55 (NESP55) that is transcribed exclusively from
the maternal allele [Weiss et al., 2000]. Behavioral studies of mice
with a knockout of the NESP55 transcript have revealed increased
reactivity to novel environments, indicating a role of maternally
expressed NESP55 in controlling exploratory and risk-taking be-
possibility that other types of behavior are under its influence
[Plagge et al., 2005].
The other two significant SNPs to emerge from the two-stage
association analysis are rs2294015, a non-synonymous variant of
ANXA13 that encodes a phospholipid-binding protein involved in
cellular growth and signal transduction (coefficients of 0.21?0.05
and 0.09?0.05mV), and intronic rs7916403 from serotonin re-
the T allele). All four implicated SNPs were tested for association
relationship to the disease under a recessive mode of genetic
influence, with estimated ORs of 1.32 (P¼0.036) and 1.37
(P¼0.042) in the two samples (similar OR scores were observed
for the European-American subsets). Haplotype-based testing of a
5.8kb LD block encompassing the HTR7 variant lends support to
this finding, revealing a significant joint-association to alcohol
dependence (P¼0.0074) that weakens when the effects of
rs7916403 are controlled for (P¼0.098).
indicate important genetic effects on both neuroelectrical activity
and the risk of alcohol dependence. Although the HTR7 marker
shows a stronger association with theta band EROs in the family
affected families but that the neuroelectrical effect at this locus is
more easily detected on a genetic background with a higherload of
AD risk alleles. The HTR7 gene encodes a G-protein-coupled
neurotransmitter receptor of the serotonergic system. Serotonin
(5-HT) is one of the most widely used neurotransmitters in the
brain, whose effects are produced through its interaction with 14
receptor 5-HT7, and has been implicated in the etiology of numer-
ous psychiatric disease states, including depression, social phobia,
schizophrenia, obsessive–compulsive, migraine, and eating disor-
2008]. The distribution of 5-HT7binding sites in discrete areas of
the brain, such as the limbic system and thalamocortical regions,
overlaps the areas of neuroelectrical activity of theta EROs and
suggests a possible role in pathophysiology of affective disorders.
and 5-HT7(d)], encoding proteins varying in the length of their
ences have been observed in their signal transduction or tissue
distribution [Jasper et al., 1997; Heidmann et al., 1998]. The
development of 5-HT7antagonists [Thomas et al., 2003] and the
generation of a Htr7 knockout mouse [Hedlund et al., 2003] have
revealed a role for the receptor in the control of circadian rhythms
et al., 2008; Eriksson et al., 2008]. Pharmacologically, 5-HT7
receptors have a high affinity for a number of antidepressants and
antipsychotics, such as clozapine and risperidone [Roth et al.,
1994], underscoring their potential role in the neurophysiology of
psychiatric disorders. However, to date, purported human disease
et al., 2006].
Hence, the association between HTR7 and alcohol dependence
represents a novel insight into the genetic underpinnings of the
disorder. Alcohol dependence is considered to be part of a wider
spectrum of disinhibitory disorders that include externalizing
behavior and substance abuse, with many neurochemical factors
and pathways contributing to co-occurring disinhibitory person-
ality traits. Presently, several common genetic determinants for
the GABAergic, cholinergic, and glutamatergic systems (GABRA2,
CHRM2, and GRM8, respectively). Based on the results of the
present study, as well as pharmacological research [Buhot et al.,
2000], the serotonergic system may serve as a key modulator of
these interactive neuroelectrical systems, involved in different
‘‘cognitive pathways,’’ such as memory and learning processes,
structures [Frodl-Bauch et al., 1999; Cifariello et al., 2008]. More-
over, administration of 5-HT7antagonists in adolescent rats has
been shown to enhance behavioral impulsivity, while agonist-
induced activation of endogenous 5-HT7significantly increased
neurite length in striatal neuron primary cultures, suggesting
remodeling of neuronal plasticity in brain reward circuits [Leo
et al., 2009]. Previous studies on human brain oscillation
measures and alcohol dependence have not found any evidence
of genetic associationinvolving serotonin receptors.However, in a
pair of recent genome-wide linkage studies, a significant linkage
peak (LOD¼3.3) was identified at chromosome 10q23.3–10q24.1
for alcohol dependence in African-Americans [Gelernter et al.,
10q25.1 for a quantitative measure of alcoholism and illicit drug
dependence [Agrawal et al., 2008], a region that encompasses the
54AMERICAN JOURNAL OF MEDICAL GENETICS PART B
In conclusion, this two-stage GWAS has identified four genes
that may contribute to the expression of event-related theta
oscillations measured in the frontal region of the brain and are
worthy of further investigation: ARID5A, GNAS1, ANXA13, and
HTR7. Of these four, the serotonin receptor gene HTR7 also
appears to play a role in the risk for alcohol dependence, with a
significant association emerging in the case–control samples for
of 1.24 for an additive MG model, although significant recessive
effects for diagnoses of alcohol dependence were detected for
both sample sets that notably parallel the theta ERO deficits
observed among corresponding T/T homozygotes. Haplotype-
based tests of HTR7 LD blocks lend support to these findings,
with important effects contributed by the rs7916403 variant.
Therefore, the results of the study are compelling, implicating
the serotonergic system in the genetic susceptibility of alcohol
the functional importance of 5-HT7in the generation of theta
EROs and its contribution to the biological basis of alcohol
The Collaborative Study on the Genetics of Alcoholism (COGA),
L. Bierut, includes 10 different centers: University of Connecticut
Jr., and T. Foroud); University of Iowa (S. Kuperman and J.
Kramer); SUNY Downstate (B. Porjesz); Washington University
of California at San Diego (M. Schuckit); Howard University (R.
Taylor); Rutgers University (J. Tischfield); Southwest Foundation
Parsian and M. Reilly are the NIAAA Staff Collaborators. We
continue to be inspired by our memories of Henri Begleiter and
Kai Li, currently a consultant with COGA, P. Michael Conneally,
This national collaborative study is supported by the NIH Grant
U10AA008401 from the National Institute on Alcohol Abuse and
Alcoholism (NIAAA) and the National Institute on Drug Abuse
(NIDA). Dr. Mark Zlojutro receives support from the Cowles
by R01 MH59490 from the National Institutes of Mental Health.
Funding support for GWAS genotyping, which was performed
at the Johns Hopkins University Center for Inherited Disease
and Alcoholism, the NIH GEI (U01HG004438), and the NIH
contract ‘‘High throughput genotyping for studying the genetic
Family-based genotyping was performed using the facilities of the
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