ORIGINAL ARTICLE

Association Testing of the Positional

and Functional Candidate Gene SLC1A1/EAAC1
in Early-Onset Obsessive-compulsive Disorder
Diane E. Dickel, BA; Jeremy Veenstra-VanderWeele, MD; Nancy J. Cox, PhD; Xiaolin Wu, MD, PhD;
Daniel J. Fischer, MSW; Michelle Van Etten-Lee, PhD; Joseph A. Himle, PhD;
Bennett L. Leventhal, MD; Edwin H. Cook, Jr, MD; Gregory L. Hanna, MD

Context: The first 2 independent linkage studies for
obsessive-compulsive disorder (OCD) identified a
region on 9p24 with suggestive evidence for linkage.
The glutamate transporter gene solute carrier family 1,
member 1 (SLC1A1) is a promising functional candidate
in this region because altered glutamatergic concentra-
tions have been found in the striatum and anterior cin-
gulate in neuroimaging studies of pediatric OCD.
Objective: To determine whether genotypes at poly-
morphisms in the SLC1A1 gene region are associated with
early-onset OCD.
Design: Family-based analysis of association using the
transmission disequilibrium test, confirmed using the fam-
ily-based association test.
Setting: Anxiety disorders program in an academic medi-
cal center.
Participants: Seventy-one probands with DSM-III-R or
DSM-IV OCD and their parents.
Methods: Nine single nucleotide polymorphisms spaced
throughout the SLC1A1 gene region were genotyped.
Results: Significant association was detected at
rs3780412 (P=.04) and rs301430 (P=.03), 2 common
adjacent single nucleotide polymorphisms in the 3!
region of SLC1A1. Analysis by sex revealed that associa-
tion at rs3780412 was limited to male probands
(P=.002). Significant association was also detected for
the T/C haplotype at rs301430-rs301979 (P = .03), the
only haplotype block identified among the 9 single
nucleotide polymorphisms. Analysis by sex also
revealed that the haplotype association was limited to
male probands (P=.003). A deletion in the 3! flanking
region of SLC1A1 was also detected that imperfectly
segregated with OCD in a large, multigenerational fam-
ily with multiple affected individuals.
Conclusions: The 3! region of SLC1A1 may contain a
susceptibility allele for early-onset OCD, with differen-
tial effects in males and females. The results also pro-
vide further support for the involvement of a glutama-
tergic dysfunction in the pathogenesis of early-onset
OCD.
Arch Gen Psychiatry. 2006;63:778-785

Author Affiliations:
Department of Human
Genetics, University of Chicago
(Ms Dickel and Drs Cox and
Wu), and Institute for Juvenile
Research, Department of
Psychiatry, University of Illinois
at Chicago (Drs Veenstra-
VanderWeele, Leventhal, and
Cook), Chicago, Ill; and
Department of Psychiatry,
University of Michigan, Ann
Arbor (Mr Fischer and
Drs Van Etten-Lee,
Himle, and Hanna). Ms Dickel
is now with the Department of
Genome Sciences, University of
Washington, Seattle. Dr
Veenstra-VanderWeele is now
with the Center for Molecular
Neuroscience, Vanderbilt
University Medical Center,
Nashville, Tenn.
O
BSESSIVE-COMPULSIVE DIS-
order (OCD) is a severe
psychiatric disorder char-
acterized by repetitive
thoughts that cause anxi-
ety and ritualistic behaviors or mental ac-
tions aimed at relieving this anxiety. The Na-
tional Comorbidity Survey Replication
reported that OCD is the anxiety disorder
with the highest percentage (50.6%) of se-
rious cases.1 Estimates of its lifetime preva-
lence in adolescents and adults range from
1% to 3%.2-4 Obsessive-compulsive disor-
der is rare in young children, but its preva-
lence rises exponentially with increasing age
through adolescence.5 The average age at on-
set in epidemiologic studies1,3,6 of OCD is
in late adolescence or early adulthood. The
National Comorbidity Survey Replication
found a median age at onset of 19 years, with
21% of cases starting by age 10 years.2 Males
generally have earlier onset than females,
contributing to a preponderance of males
in most child and adolescent samples.6-8 In
contrast, there is a slight preponderance of
females in most adult samples.3,9
See also pages 717 and 769
Obsessive-compulsive disorder is
thought to be a complex genetic disorder
based on several lines of evidence. Mono-
zygotic twins show a higher concordance
rate of OC symptoms than do dizygotic twins
(80%-87% vs 47%-50%, respectively).10,11
Controlled family studies using adult pro-
bands demonstrate that the lifetime preva-
lence of OCD is significantly higher in case
relatives vs control relatives: Pauls et al,12
10.3% vs 1.9%, and Nestadt et al,13 11.7%
vs 2.7%. An early age at onset of OC symp-
toms in family studies with adult probands

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 is strongly associated with a more familial form of OCD.12-14
Recent controlled studies using pediatric probands found
that the lifetime prevalence of OCD was significantly higher
in case relatives vs control relatives: Hanna et al,15 22.5%
vs 2.6%, and do Rosario-Campos et al,16 22.7% vs 0.9%.
The only published genome scan of OCD used fami-
lies ascertained through pediatric probands to identify a
region on chromosome 9p24 with suggestive evidence
for linkage.17 A parametric linkage analysis using a
dominant Mendelian model of inheritance yielded a
logarithm of odds score of 2.25 that declined to 1.97
with additional participants and markers in that region.
In an effort to replicate this finding, Willour and col-
leagues18 genotyped 13 microsatellite markers from
chromosome 9p24 in 50 families. Their study identified
an overlapping region with suggestive evidence for link-
age centered only 0.5 centimorgan from the original
finding. Furthermore, Taylor and colleagues19 reported
a 9p monosomy in a patient with Tourette syndrome
and OCD. These findings indicate that 9p24 is a strong
candidate region for early-onset OCD. Association stud-
ies in this region have been mixed, however, with 1
study18 finding modest association at 2 microsatellite
markers flanking the solute carrier family 1, member 1
gene (SLC1A1), GATA62F03 (P = .02) and D9S288
(P=.05), and 1 study20 finding no evidence of associa-
tion at 2 single nucleotide polymorphisms (SNPs) in
SLC1A1 intron 3 (P=.42).
The SLC1A1 gene, which codes for the glutamate trans-
porter EAAC1 (EAAT3), is the most promising candi-
date gene in the region shared by the 9p24 linkage and
association findings and the reported 9p monosomy.
EAAC1 is a high-affinity glutamate transporter primar-
ily expressed in neurons, intestine, kidney, liver, and heart.
EAAC1 couples the transport of L-glutamate into cells
with the cotransport of sodium and hydrogen and the
countertransport of potassium, an important step in pro-
tecting neurons from glutamate excitotoxicity (for a re-
view see Kanai and Hediger21). The SLC1A1 knockout
mice show decreased spontaneous locomotor activity and
dicarboxylic aminoaciduria.22
The SLC1A1 gene is a plausible functional candidate
and a strong positional candidate. Pediatric patients with
OCD have been shown to have reduced glutamatergic con-
centrations in the anterior cingulate cortex23 and signifi-
cantly increased concentrations in the caudate com-
pared with unaffected controls.24 Caudate glutamatergic
concentrations and OC symptoms decrease concur-
rently with paroxetine treatment.24 Cerebrospinal fluid
glutamate levels were also found to be higher in adults
with OCD compared with adult control subjects.25
Although a previous study20 by our group found no
linkage disequilibrium (LD) between OCD and SLC1A1,
that study examined only 2 SNPs, both in intron 3 of the
gene. We expand this study to examine the association
between SLC1A1 and early-onset OCD by genotyping
more SNPs that more comprehensively cover the gene
and by using a larger patient sample. We use a family-
based approach to detect association, the transmission
disequilibrium test (TDT), which has less power than
methods using cases and hypernormal controls26; how-
ever, the TDT avoids the possibility of population strati-
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fication bias that may be present even in relatively ho-
mogeneous populations. 27-29 We report significant
association with 2 common adjacent SNPs in the 3! re-
gion of SLC1A1. We also describe a deletion in the 3!
flanking region of SLC1A1 in a large, multigenerational
family with multiple affected individuals.
METHODS
PARTICIPANT ASCERTAINMENT
Participants were ascertained in 2 separate groups for earlier
genome scan and candidate gene studies. The first group was
ascertained as described previously.15,17,20 Briefly, this group con-
sisted of 21 parent-child trios from singleplex families and 19
parent-child trios from 7 multiplex families. All the probands
were directly interviewed and met the DSM-III-R30 criteria for
OCD. (The DSM-IV had not yet been published when subject
ascertainment began.) Probands ranged in age from 6 to 17 years
(mean±SD, 14.4±3.8 years), and all had onset of OC symp-
toms at 3 to 14 years of age (mean±SD, 8.8±3.9 years). The
exclusion criteria were (1) a diagnosis of a chronic neurologic
disorder other than tic disorder; (2) a lifetime DSM-III-R diag-
nosis of mental retardation, autistic disorder, bipolar disor-
der, or schizophrenia; (3) currently living away from both bio-
logical parents; and (4) adoption.
The second group of participants was recruited for the pur-
pose of an expanded genome scan and candidate gene studies.
The probands in the second group were recruited from clinics
in the University of Michigan Health System and from local chap-
ters of the Obsessive Compulsive Foundation. All the probands
were directly interviewed to determine whether they met the DSM-
IV31 criteria for a lifetime diagnosis of OCD. The second group
of probands consisted of 11 males and 15 females ranging in age
from 7 to 64 years (mean±SD, 28.4±17.5 years). Age at onset
of OC symptoms in these probands ranged from 3 to 18 years
(mean±SD, 8.0±3.7 years). The exclusion criteria for the sec-
ond group of probands were (1) the onset of OC symptoms af-
ter age 18 years; (2) a lifetime DSM-IV diagnosis of autistic dis-
order, schizophrenia, or bipolar I disorder; (3) adoption; (4) if
younger than 18 years, currently living away from both biologi-
cal parents; and (5) a first-degree relative with a lifetime DSM-IV
diagnosis of autistic disorder, schizophrenia, or bipolar I disor-
der. The study was approved by the institutional review board
of the University of Michigan Medical School.
After providing informed consent and assent, probands and
relatives younger than 18 years were interviewed using the
Schedule for Affective Disorder and Schizophrenia for School-
Age Children—Epidemiologic Version–5.32 This interview was
completed independently with a parent of the participant and
with the participant. Probands and relatives 18 years and older
were interviewed using the Structured Clinical Interview for
DSM-IV Axis I Disorders.33 Both interviews were supple-
mented with sections on OCD and tic disorders derived from
the Schedule for Tourette and Other Behavioral Syndromes.34
The section on OCD included a series of questions modified
to cover all the criteria for a lifetime DSM-IV diagnosis of OCD31
and a checklist from the Yale-Brown Obsessive Compulsive
Scale35,36 modified to obtain information about the lifetime oc-
currence of specific obsessions and compulsions.
All interviews were audiotaped and coded on paper to
assess reliability, maintain quality control, and achieve diag-
nostic consensus. All the interviewers had at least a master’s
degree and clinical training in either child or adult psychopa-
thology. They were trained to at least 90% diagnostic agree-
ment with the individual instruments. After completion of all
WWW.ARCHGENPSYCHIATRY.COM

rs301443
rs10814987 Position 4 584 919
Position 4 473 303 rs301979
Position 4 566 851
rs1980943 rs3780412
Position 4 504 246 Position 4 562 480
rs10115600 rs10758629
Position 4 509 369 Position 4 518 816 rs301434
Position 4 572 082
rs10739062 rs301430
Position 4 492 848 Position 4 566 680
Figure 1. Solute carrier family 1, member 1 gene (SLC1A1) organization and
single nucleotide polymorphism (SNP) locations. The horizontal line
represents the genomic sequence; vertical bars, exons, with tall bars
representing translated regions and short bars representing untranslated
regions. The positions are from the University of California at Santa Cruz
Genome Browser, May 2004 Assembly, and are measured in base pairs from
the p-terminus of chromosome 9. The rs10758629 SNP was not included in
the statistical analysis.
interviews for an individual, all available materials (personal
interview data, family history data, and clinical records) were
collated.
Best-estimate lifetime diagnoses were made independently
by at least 2 investigators (D.J.F., M.V.E.-L., J.A.H., and G.L.H.,
with G.L.H. reviewing the diagnostic information for all par-
ticipants) using DSM-IV criteria.31 Definite OCD was diag-
nosed if an individual met all the diagnostic criteria. To avoid
forcing closure on inadequate diagnostic information, partici-
pants were interviewed again if necessary to clarify incom-
plete or contradictory information. When major disagree-
ments occurred between 2 diagnosticians, consensus diagnoses
were reached with the assistance of a third diagnostician fol-
lowing procedures developed for the diagnosis of other psy-
chiatric disorders.37 The interrater reliability of this diagnostic
process was studied in a sample of 108 individuals. There was
good diagnostic agreement, as evidenced by a "=0.91 for OCD,
a "=0.91 for tic disorder, and an intraclass correlation coeffi-
cient of 0.94 for age at onset of OC symptoms.
Blood samples were obtained from all the probands and di-
rectly interviewed relatives and from 10 relatives without di-
rect interviews. All 31 complete parent-child trios from among
the 20 families containing at least 1 complete trio in the sec-
ond group of participants were used for the association stud-
ies described herein.
DNA EXTRACTION
Peripheral whole blood samples were obtained by venipunc-
ture from each consenting individual and were frozen at either
–20°C or –70°C until DNA extraction. DNA was extracted from
300 µL of blood using a DNA purification kit (Puregene; Gen-
tra Systems, Minneapolis, Minn).
GENOTYPING
Single nucleotide polymorphisms, selected for complete cov-
erage of the SLC1A1 gene, were chosen from SNP genotyping
assays (TaqMan; Applied Biosystems, Foster City, Calif ). Any
SNPs that had minor allele frequencies less than 0.2 in white
populations were excluded. Two pairs of SNPs were expected
to be in strong LD based on near-identical allele frequency,
and 1 SNP from each pair was excluded. Ten SNPs remained
for typing, and a tagging SNP search using a software tool
(SNPbrowser version 2.0; Applied Biosystems) showed that these
SNPs provide adequate coverage of the SLC1A1 gene and the
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immediate flanking region (haplotype R2#85%). The average
spacing between SNPs across the gene was 12.4 kilobases (kb).
Figure 1 shows the locations of the SNPs used.
Polymerase chain reactions (PCRs) for the SNP genotyp-
ing assays contained 10 ng of dry DNA, 2.5 µL of 2$ TaqMan
Universal Master Mix (Applied Biosystems), 0.25 µL of 20$
SNP Genotyping Assay Mix, and 2.25 µL of water, for a total
volume of 5 µL. All the reactions were performed using a
PerkinElmer 9700 Thermocycler (Applied Biosystems) under
the following conditions: 1 AmpErase step at 50.0°C for 2 min-
utes, 1 enzyme activation step at 95.0°C for 10 minutes, and
40 alternating cycles of denaturation at 92.0°C and reanneal-
ing and extension at 58.0°C for 1 minute. The fluorescence in-
tensity of the final reaction product was measured using an LJL
Analyst AD fluorescence microplate reader and LJL Criterion
Host Software (LJL Biosystems, Sunnyvale, Calif ).
STATISTICAL ANALYSIS
Departures from Hardy-Weinberg equilibrium were assessed
using the HWE program from the LINKUTIL package pro-
vided by Dr Jurg Ott (ftp://linkage.rockefeller.edu/software
/utilities/). PedCheck38 was used to identify genotype incom-
patibilities. The HaploView program (http://www.broad.mit
.edu/mpg/haploview/)39 was used to identify potential haplotype
blocks and to calculate intermarker LD, including LD with the
2 SNPs from the previous study of SLC1A1, rs10974625 and
rs1805311, that had been genotyped in a subset of the present
sample.20 HaploView was also used to calculate family-based
association using the TDT for individual markers and for the
only identified haplotype block (rs301430-rs301979), where
the rs301979G allele is present only on chromosomes that con-
tain the rs301430C allele. Because nonindependent trios were
present in multiple extended families in our sample and link-
age was previously reported in the 9p24 region, we also used
the empirical correction for linkage implemented in the family-
based association test (FBAT) to evaluate the possibility that
linkage alone accounted for significant findings using the
TDT.40-42 The “FBAT-e” and “HBAT-e” functions use a null hy-
pothesis of “no association in the presence of linkage” for geno-
types and haplotypes, respectively.42 Precise estimates of power
for this modification of the FBAT have not been published; how-
ever, the number of families informative for the statistic de-
creased to 0.74 to 0.77 of our complete families.
DELETION CHARACTERIZATION
Primers (sense: FAM-TTTTGGCCAGGGAGATAGAA-3! and an-
tisense: 5!-GTTTCTTCATGAATCACTGGACATGGTG-3!) were
designed to amplify a region surrounding rs301443 to confirm
the presence of the deletion and to determine its size. A PCR kit
(AmpliTaq Gold; Applied Biosystems) was used to genotype the
deletion. Each PCR contained 1.0 µL of 10-ng/µL genomic DNA,
1.0 µL of 10$ PCR Buffer II, 1.0 µL of 10mM deoxynucleoside
triphosphate mix, 1.2 µL of 25mM magnesium chloride, 0.08
µL of 5-U/µL AmpliTaq Gold polymerase, 0.1 µL of each 10µM
primer, and 5.52 µL of water for a total reaction volume of 10
µL. All the reactions were performed using a PerkinElmer 9700
Thermocycler under the following conditions: 1 enzyme acti-
vation step at 95.0°C for 12 minutes, 36 cycles of 3 alternating
steps (denaturation at 94.0°C for 15 seconds, annealing at 55.0°C
for 15 seconds, and extension at 72.0°C for 30 seconds), and 1
final extension step at 72.0°C for 10 minutes. The final PCR prod-
ucts were analyzed by capillary electrophoresis using an ABI 3730
(Applied Biosystems), and genotypes were called using Gen-
eMapper v3.5 (Applied Biosystems). All the participants were
screened for this deletion.
WWW.ARCHGENPSYCHIATRY.COM
 Sequencing was performed to determine the exact location
of the deletion. Three participants, 2 heterozygous for the de- I 1 2 3
letion and 1 homozygous without the deletion, were chosen 2/2
for sequencing. These individuals (IV:1, IV:7, and III:2, respec-
tively) are shown in Figure 2. A region surrounding rs301443 II 1 2 3 4 5 6
1/2 1/1 1/1
was amplified using the PCR conditions, and the reverse primer
is listed in the preceding paragraph. The same forward primer
III 1 2 3 4 5 6 7 8 9
was also used but without the FAM label. After PCR amplifi- 2/2 1/2 1/1 2/2 2/2 2/2 2/2
cation, 0.5 µL of 1-U/µL shrimp alkaline phosphatase (Roche,
Indianapolis, Ind), 0.5 µL of 10$ shrimp alkaline phospha- IV 1 2 3 4 5 6 7 8 9
tase reaction buffer, and 0.25 µL of exonuclease I (USB Corp, 1/1 1/2 2/2 2/2 1/1 2/2 2/2
Cleveland, Ohio) were added to each 10-µL PCR product to
degrade unincorporated deoxynucleoside triphosphates and
primers. The shrimp alkaline phosphatase reactions were per-
formed using a PerkinElmer 9700 Thermocycler under the fol- Male Affected Wild Type Proband
Female Unaffected Deletion
lowing conditions: 1 cycle at 37.0°C for 45 minutes and 1 cycle
at 95.0°C for 15 minutes. The previously mentioned PCR prim-
ers were diluted and used for sequencing, which was per- Figure 2. Pedigree for the family with incompatibilities for marker rs301443.
formed in both directions. Sequencing reactions contained 1.2 Genotypes for rs301443 appear as numbers below each genotyped
µL of PCR template, 1.2 µL of 1.6pM primer, 2.0 µL of ready individual. Genotypes for the 11–base pair deletion are symbolized by
reaction mix (BigDye v3.1 Terminator; Applied Biosystems), colored circles below the rs301443 genotypes. This deletion was observed
and 2.0 µL of water, for a total reaction volume of 6.4 µL. Se- only in this family and was found predominantly in affected individuals.
quencing reactions were analyzed by capillary electrophoresis
using an ABI 3730 (Applied Biosystems), and the resulting se-
quences were read using Sequencher v4.2 (Gene Codes Corp, male probands (P =.66). Table 1 gives the TDT results
Ann Arbor). for the overall sample and for male and female probands
separately. Pairwise LD between rs3780412 and rs301430
was intermediate (r2 =0.15, D!=0.64). Intermarker LD for
RESULTS all the markers is given in Table 2. The 2 markers geno-
typed in the previous study of SLC1A1 in OCD,
rs10974625 and rs1805311, were in nearly complete but
Before statistical analysis, 1 SNP (rs10758629, desig-
not perfect LD with rs3780412, rs301430, and rs301979
nated as C___1459316_10 by Applied Biosystems) was
(r2 =0.116-0.696, D!=0.94-1.0) but in only modest LD
dropped owing to poorly defined genotype clusters. Geno-
with the other SNPs.
types for the 9 remaining markers used in this study
Only 1 haplotype block was identified, and it com-
showed no deviation from Hardy-Weinberg equilib-
prised 2 markers, rs301430 and rs301979. The T/C hap-
rium. PedCheck detected 6 incompatibilities in the geno-
lotype of these 2 markers was undertransmitted in the
typing data. However, 5 of the 6 incompatibilities were
overall sample (%2 =4.90; P=.03) (Table 3). The under-
found only in SNP rs301443 in 1 large, multigenera-
transmission of this haplotype was also detected in male
tional family in a pattern indicating the presence of a de-
probands (%2 = 8.91; P = .003) but not in female pro-
letion. Because of the deletion, this family was not in-
bands (%2 =0.00; P#.99). The HBAT-e results paralleled
cluded in the association analysis for rs301443 but was
the TDT results, with undertransmission of this haplo-
included for the remaining markers. An 11–base pair (bp)
type in the overall sample (P =.02) that strengthened in
deletion downstream of SLC1A1, in an intron of the un-
the male probands (P =.004) but that was not detected
characterized gene chromosome 9 open reading frame
in the female probands (P=.74).
68 (C9ORF68), was observed in 10 members of this fam-
ily. Figure 2 shows the pedigree and genotypes for
rs301443 and the deletion for this family. The deletion COMMENT
spans 4 584 839 to 4 584 849 bp on chromosome 9 (lo-
cations from the p-terminus of chromosome 9 using the Two independent linkage studies17,18 and a report of a 9p
University of California at Santa Cruz Genome Browser, monosomy in a patient with OCD and Tourette syn-
May 2004 Assembly) and was not observed in any other drome19 implicate 9p24 as a candidate region for OCD.
participants in the sample. Previous association studies in this region, however,
Two SNPs showed nominally significant association have been limited and have produced mixed results.
with early-onset OCD in the overall sample. The higher- One study18 found modest evidence of association at 2
frequency A allele of rs3780412 and the lower- microsatellite markers: GATA62F03 (P=.02), which lies
frequency C allele of rs301430 were overtransmitted 613 kb 3! to SLC1A1, and D9S288 (P=.05), which lies
(%2 =4.19; P=.04 and %2 =4.91; P=.03, respectively). The 539 kb 5! to SLC1A1. The only previous study 20 to
overtransmission of the A allele of rs3780412 was ob- examine association at markers in SLC1A1 found no evi-
served in male probands (%2 = 9.53; P = .002) but not in dence of association. Our finding of nominally signifi-
female probands (%2 = 0.03; P = .87). The FBAT-e results cant association at 2 SNPs, rs3780412 and rs301430,
paralleled the TDT results, with trends for overtransmis- and a haplotype including a third SNP, rs301979, in
sion of rs3780412A (P=.09) and rs301430 (P=.06) in the SLC1A1 supports a role for SLC1A1 in early-onset OCD.
overall sample and significant overtransmission of Consistent with this finding, researchers have also found
rs3780412 to male probands (P = .009) but not to fe- evidence of association at rs301434 and rs301435 in a

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 Table 1. TDT Results for SNP Markers in the Overall Sample and in Male and Female Probands Separately

Transmitted From
Heterozygous Parent

Polymorphism Name AB Assay Name Type of SNP Major Allele Minor Allele !2 P Value
Overall Sample
rs10814987 C___3026341_10 G/T 30 27 0.16 .69
rs10739062 C___3026344_10 C/G 35 46 1.49 .22
rs1980943 C___1459297_10 A/G 33 31 0.06 .80
rs10115600 C___1459308_10 C/G 31 23 1.19 .28
rs3780412 C__11261320_10 A/G 43 26 4.19 .04
rs301430 C____797982_1_ T/C 24 42 4.91 .03
rs301979 C___3059669_1_ C/G 29 33 0.26 .61
rs301434 C___3059680_10 A/G 35 30 0.38 .54
rs301443 C___3059691_10 C/G 29 25 0.30 .59
Male Probands
rs10814987 C___3026341_10 G/T 17 14 0.29 .59
rs10739062 C___3026344_10 C/G 18 23 0.61 .43
rs1980943 C___1459297_10 A/G 17 16 0.03 .86
rs10115600 C___1459308_10 C/G 13 13 0.00 #.99
rs3780412 C__11261320_10 A/G 26 8 9.53 .002
rs301430 C____797982_1_ T/C 15 25 2.50 .11
rs301979 C___3059669_1_ C/G 11 20 2.61 .11
rs301434 C___3059680_10 A/G 19 17 0.11 .74
rs301443 C___3059691_10 C/G 14 13 0.04 .85
Female Probands
rs10814987 C___3026341_10 G/T 13 13 0.00 #.99
rs10739062 C___3026344_10 C/G 17 23 0.90 .34
rs1980943 C___1459297_10 A/G 16 15 0.03 .86
rs10115600 C___1459308_10 C/G 18 10 2.29 .13
rs3780412 C__11261320_10 A/G 17 18 0.03 .87
rs301430 C____797982_1_ T/C 9 17 2.46 .12
rs301979 C___3059669_1_ C/G 18 13 0.81 .37
rs301434 C___3059680_10 A/G 16 13 0.31 .58
rs301443 C___3059691_10 C/G 15 12 0.33 .56

Abbreviations: AB, Applied Biosystems; SNP, single nucleotide polymorphism; TDT, transmission disequilibrium test.

Table 2. Intermarker Linkage Disequilibrium*

Marker 1 2 3 4 5 6 7 8 9
1 (rs10814987) 0.00 0.02 0.01 0.00 0.00 0.00 0.01 0.00
2 (rs10739062) 0.11 0.11 0.11 0.01 0.00 0.02 0.02 0.00
3 (rs1980943) 0.24 0.39 0.10 0.01 0.02 0.01 0.00 0.01
4 (rs10115600) 0.16 0.75 0.63 0.01 0.02 0.08 0.01 0.04
5 (rs3780412) 0.02 0.12 0.18 0.15 0.15 0.20 0.19 0.05
6 (rs301430) 0.04 0.03 0.14 0.34 0.64 0.18 0.00 0.00
7 (rs301979) 0.04 0.36 0.18 0.29 0.87 1.00 0.28 0.05
8 (rs301434) 0.11 0.19 0.07 0.14 0.47 0.06 0.93 0.16
9 (rs301443) 0.04 0.06 0.08 0.45 0.27 0.01 0.53 0.55

*D! values are shown below the blank cells and r 2 values are shown above the blank cells.

family-based association study of OCD (Paul Arnold,
MD, e-mail communication, 2005). Furthermore, both
studies find evidence of association primarily for male
probands with OCD.
We also discovered a rare 11-bp deletion in the 3!
flanking region of SLC1A1 in a large, multigenerational
family (Figure 2). This deletion is located in a noncod-
ing region of an uncharacterized gene, C9ORF68, that
seems to be alternatively spliced. Figure 3 shows the
location of the deletion and the genetic organization of
C9ORF68. As shown in Figure 2, this deletion does not
perfectly cosegregate with OCD in this family: 2 unaf-
fected individuals have the deletion and 1 affected indi-
vidual does not. It is possible that the deletion disrupts
splicing for C9ORF68, assuming that this putative gene
makes a functional protein product, or alters a regula-
tory element for one of the genes in this region. How-
ever, given the size of the deletion and the only modest

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 Table 3. Transmission Disequilibrium Test Results for rs301430-rs301979 Haplotypes in Different Proband Groups

Proband Group Haplotype Transmitted Untransmitted !2 P Value

Overall sample T/C 13 27 4.90 .03
C/C 22 14 1.78 .18
T/G 22 16 1.89 .17
Males T/C 4 18 8.91 .003
C/C 13 8 1.19 .28
T/G 16 7 3.52 .06
Females T/C 9 9 0.00 #.99
C/C 9 6 0.60 .44
T/G 6 9 0.60 .44

conservation found at its location, it is unlikely that it is

contributing to OCD susceptibility in this family. Fur-
ther study is needed to determine whether this deletion
is altering the function or expression of C9ORF68 or
other genes in the region.
Caution should be exercised in interpreting the nomi-
nally significant association findings at rs3780412,
rs301430, and rs301979. First, use of the TDT, which
tests for association in the presence of linkage, in mul-
tiplex families in a genomic region with evidence of link-
age raises the possibility that linkage may confer a sig-
nificant finding in the absence of true association.42 To
assess whether this may be the case, we applied the FBAT
with the exclusion of linkage effects.42 The resulting de-
creases in significance were proportional to the loss of
power, suggesting that linkage does not solely account
for the finding. Second, because of the strong evidence
for SLC1A1 as a positional and functional candidate gene
for OCD,17,18,23,24 a correction for multiple testing was not
performed. The most conservative correction at this lo-
cus, the Bonferroni method, would eliminate the signifi-
cant findings in the overall sample but leave significant
findings in the group of male probands. Correcting for
association testing across the genome,43 which has not
been pursued in this sample, would eliminate any sig-
nificant findings whatsoever. However, these nominally
significant findings must be considered in the context of
low power to detect the relatively weak association ex-
pected at a marker in a complex genetic disorder. The
parallel findings in the accompanying study by Arnold
and colleagues also support the case for SLC1A1 as a pri-
mary candidate gene in OCD.
These association findings in 2 studies present a chal-
lenge for additional research on the possible involve-
ment of SLC1A1 in OCD. None of the 5 SNPs associated
in one or the other study seems likely to itself confer sus-
ceptibility. Four of the SNPs (rs3780412, rs301979,
rs301434, and rs301435) are located in introns, and 1
SNP (rs301430) is a synonymous coding SNP. In addi-
tion, no single SNP was significantly associated in both
studies, despite both studies genotyping at least 1 SNP
that was associated in the other study. Therefore, to ac-
count for these findings, functional variation in this re-
gion must be in varying degrees of LD with these SNPs
in different populations or multiple functional variants
must be present and account for the association with dif-
ferent SNPs. Similar patterns of association findings at
different markers across populations have been noted at
SLC1A1
C9ORF68
C9ORF68
C9ORF68
Figure 3. Location of the 11–base pair (bp) deletion. The genomic
organization of the solute carrier family 1, member 1 gene (SLC1A1) and the
isoforms of chromosome 9 open reading frame 68 (C9ORF68) are shown.
The horizontal lines represent the genomic sequence of each gene; vertical
bars, exons, with tall bars representing translated regions and short bars
representing untranslated regions. The location of the observed 11-bp
deletion is indicated by the red vertical line.
other genes implicated in psychiatric disorders, such as
the serotonin transporter gene in autism,44,45 where mul-
tiple common and uncommon functional alleles have been
reported.46 Compared with previous studies, the pre-
sent study and that of Arnold and colleagues achieved
improved coverage of the SLC1A1 gene region; how-
ever, the low intermarker LD in portions of this ge-
nomic region (Table 2) makes it difficult to clarify the
extent of association. It may eventually be necessary to
genotype all the common variation in this region to
achieve adequate coverage. Further screening of the cod-
ing region and surrounding gene regulatory regions for
uncommon variation is also warranted, although an ini-
tial screen in the probands from the original genome scan
revealed no amino acid variation.20 Finally, expression
in the renal tubules may allow assessment of potentially
functional variation in SLC1A1 by correlation with uri-
nary levels of aspartate and glutamate.22
Association findings at SLC1A1 add to existing ge-
netic, neuroimaging, and neurochemical evidence of glu-
tamatergic dysfunction in OCD. Identification of func-
tional variation is necessary to clarify the relationship
between SLC1A1 and the glutamate abnormalities in OCD;
however, the regional distribution and function of EAAC1
in the brain match neuroimaging and neurochemical
findings. The glutamate transporter EAAC1 shows
particularly high expression in the striatum but is also
expressed postsynaptically on glutamatergic and
&-aminobutyric acidergic neurons throughout the cor-
tex, including the cingulate cortex.47 Antisense RNA to
SLC1A1 substantially decreases &-aminobutyric acid syn-
thesis in the rat hippocampus, suggesting that EAAC1
uptake may provide a pool of glutamate for conversion
to &-aminobutyric acid.48 Some evidence suggests that
tonic glutamatergic signaling from the ventral prefron-

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 tal cortex, which includes the cingulate cortex, de-
creases phasic activity in the striatum, which may ex-
plain the inverse relationship in pediatric OCD between
the glutamatergic signal in the cingulate cortex, where
it is decreased, and in the caudate, where it is in-
creased.23,24,49 The role of EAAC1 in glutamate reuptake
and &-aminobutyric acid synthesis makes it a natural can-
didate protein as part of the putative tonic-phasic dys-
regulation in OCD. Altered SLC1A1/EAAC1 function
could also be involved in perturbation of the glutamate
system in other psychiatric disorders frequently comor-
bid with OCD, especially mood disorders,50 where changes
in the glutamate system have been observed.23,51,52
Our finding of association confined to male probands
supports a body of literature on sex differences in OCD.
Males with OCD differ from females with OCD in terms
of clinical presentation and course. Males are more likely
to have a childhood onset, a chronic course of disease, and
comorbid tic disorder or attention-deficit/hyperactivity dis-
order.9,53-57 Consistent with clinical differences, segrega-
tion analyses suggest that the inheritance of OCD is af-
fected by sex effects.58,59 A variety of previous genetic
association studies60-64 have identified association in one
sex or another, although findings have not previously been
consistent across studies. This pattern of sexually dimor-
phic inheritance may suggest involvement of the X chro-
mosome in disease, but it could also reflect general hor-
monal or developmental sex differences.
In conclusion, we genotyped 9 SNPs spanning the glu-
tamate transporter gene SLC1A1 in a family-based asso-
ciation study of early-onset OCD and identified nomi-
nal evidence of association at 2 of the markers examined,
rs3780412 and rs301430, and a haplotype that includes
a third marker, rs301979. The association was stronger
in males, supporting a sex-specific contribution to OCD
susceptibility. This evidence supports a role for SLC1A1
in the etiology of early-onset OCD and adds further sup-
port to a role for glutamatergic dysfunction in the patho-
genesis of this disorder.23-25 Analyses of possible associa-
tions with the examined markers in subtypes of OCD are
warranted. We also discovered an uncommon 11-bp de-
letion in the 3! flanking region of the gene in 10 mem-
bers of a multigenerational family. Further work is needed
to determine whether this deletion is contributing to OCD
susceptibility in this family.
Submitted for Publication: August 4, 2005; final revi-
sion received December 12, 2005; accepted December 29,
2005.
Correspondence: Jeremy Veenstra-VanderWeele, MD,
Center for Molecular Neuroscience, Medical Research
Building III, Vanderbilt University Medical Center, 465
21st Ave S, Nashville, TN 37232 (jeremy.veenstra-
vanderweele@vanderbilt.edu).
Funding/Support: This work was supported in part by
grants K02 MH01389 (Dr Cook), K20 MH01065 (Dr
Hanna), and R01 MH58376 (Dr Hanna) from the Na-
tional Institutes of Health; the Jean Young and Walden
W. Shaw Foundation (Dr Leventhal); the Harris Foun-
dation (Dr Leventhal); the Brain Research Foundation
(Dr Cook); and the Obsessive Compulsive Foundation
(Drs Cook and Hanna).
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784
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Acknowledgment: We thank Kathy Hennessy and Cheryl
Roe, MS, for their expert technical assistance and the fami-
lies who participated in this study.
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