Molecular Psychiatry (1997) 2, 247–250

 1997 Stockton Press All rights reserved 1359–4184/97 $12.00

ORIGINAL RESEARCH ARTICLE

Evidence of linkage between the serotonin transporter
and autistic disorder
EH Cook Jr1 , R Courchesne2 , C Lord2 , NJ Cox3 , S Yan1, A Lincoln2 , R Haas2 , E Courchesne2,4 and
BL Leventhal1
1
Laboratory of Developmental Neuroscience, Departments of Psychiatry and Pediatrics, MC 3077, University of Chicago,
5841 S Maryland Avenue, Chicago, IL 60637; 2Autism and Brain Development Research Laboratory, Children’s Hospital
Research Center, 8110 La Jolla Shores Drive, La Jolla, CA 92037; 3Department of Medicine, University of Chicago, 5841
S Maryland Avenue, Chicago, IL 60637; 4Neuroscience Department, School of Medicine, University of California at San
Diego, La Jolla CA 92093, USA

The serotonin transporter gene (HTT) is a primary candidate in autistic disorder based on
efficacy of potent serotonin transporter inhibitors in reducing rituals and routines. We initiated
a candidate gene study of HTT in trios consisting of probands with autistic disorder and both
parents. Preliminary transmission/disequilibrium test (TDT) analysis with 86 families revealed
no evidence for linkage or linkage disequilibrium between autistic disorder and a polymor-
phism in the second intron of HTT. However, preferential transmission of a short variant of
the HTT promoter was found in the same 86 trios (TDT x2 = 4.69, 1 d.f., P = 0.030). In further
analyses, we considered haplotypes of the HTT promoter variant and second intron locus as
alleles in a multiallelic TDT. Results confirmed the significance of the effect of this region
(TDT x2 = 11.85, 4 d.f., P = 0.018). This provides preliminary evidence of linkage and associ-
ation between HTT and autistic disorder.
Keywords: linkage disequilibrium; linkage; autism, infantile; monoamines; transporter; serotonin; pro-
moter; polymorphism

Introduction autistic disorder associated with anxiety and/or

aggression.12,13 This led us to consider the serotonin
Autistic disorder is characterized by qualitative
transporter as a primary candidate gene in autistic dis-
impairments in reciprocal social interaction and com-
order.
munication, in addition to restricted and repetitive
Two polymorphisms have been reported for the sero-
behaviors.1 The population prevalence of autistic dis-
tonin transporter gene, a variable number tandem
order has been estimated between 5–10 per 10 000.2
repeat (VNTR) in the second intron14 and a
Estimates of the risk of sibling recurrence are much
deletion/insertion polymorphism in the promoter
higher at 4.5%3 to 8.9%,4 resulting in a relative sibling
which regulates expression of the 5-HT transporter in
recurrence risk of 45–178. Twin studies have demon-
transfection assays and lymphoblastoid cell lines.15,16
strated increased concordance in monozygotic twins
We examined possible linkage and linkage disequilib-
relative to dizygotic twins.5 The large decrease in risk
rium between autistic disorder and the serotonin trans-
to dizygotic twins relative to monozygotic twins6 and
porter using the transmission/disequilibrium test
a latent class analysis7 are consistent with several loci
(TDT).17,18
contributing to susceptibility to autistic disorder.
Serotonin (5-HT) has been studied in autistic dis-
order since the finding in 1961 of increased whole Materials and methods
blood 5-HT in the blood of children with autistic dis-
Subjects
order.8 A series of studies has replicated and extended
Consecutive subjects seen at the Autism and Brain
this finding.9 Whole blood 5-HT has been demon-
Research Laboratory, Children’s Hospital Research
strated to be positively correlated with serotonin
Center, La Jolla, CA between July 1990 and March 1993
uptake Vmax in both vervet monkeys10 and humans.11 In
and at the University of Chicago Developmental Dis-
addition, potent serotonin transporter inhibitors have
orders Clinic between June 1994 and October 1996
been demonstrated to be partially efficacious in the
were considered for inclusion in the study. Inclusion
treatment of restricted and repetitive behaviors in
criteria included a diagnosis of autistic disorder based
on Autism Diagnostic Interview – Revised criteria,19
Correspondence: EH Cook, Jr, MC 3077, University of Chicago,
clinical judgment of autistic disorder based on admin-
5841 S Maryland Avenue, Chicago, IL 60637, USA istration of the Autism Diagnostic Observation Sched-
Received 27 January 1997; accepted 7 February 1997 ule,20 intelligence quotient greater than or equal to 35,
 Serotonin transporter gene and autistic disorder
EH Cook Jr et al
248
and blood available for genotyping from the proband
and both parents. Subjects were excluded if they had
evidence of a known etiology for a developmental dis-
order after neurological examination including exam-
ination of dysmorphology and neurocutaneous signs
and appropriate laboratory testing. One subject was
excluded because of an interstitial chromosome
15 q11–13 duplication. Subjects participated after pro-
viding written informed consent for themselves and
their children, as appropriate. This study was
approved by the institutional review boards of the Uni-
versity of Chicago and Children’s Hospital, San Diego.
Eighty-six trios, consisting of a proband with autistic
disorder and both parents were included in the study.
Age of probands ranged from 2.5 to 36 years (mean 8.5,
standard deviation 7.6). Eighty subjects were male and
six were female. Sixty-eight subjects were Caucasian,
five were African-American, three were Hispanic-
American, and ten were Asian-American.
HTT 2nd intron VNTR analysis
DNA was extracted from whole blood using the Pure
Gene DNA Isolation Kit (Gentra Systems, Minnea-
polis, MN, USA). Primers HTT2X (59-
TGGATTTCCTTCTCTCAGTGATTGG-39) and HTT2Y
(59-TCATGTTCCTAGTCTTACGCCAGTG-39) amplified
345-bp (9 copy), 360-bp (10 copy), or 390-bp fragments
containing the HTT 2nd intron variant. The primers
were synthesized on an Applied Biosystems 380B DNA
synthesizer at the Cancer Research Center at the Uni-
versity of Chicago. PCR was performed in a 75-ml vol-
ume containing approximately 400 ng of genomic tem-
plate, 0.5 mM of each primer, 200 mM of each dNTP,
2 units of Taq polymerase (AmpliTaq; Perkin-Elmer,
Norwalk, CT, USA), 2 mM MgCl2, 10 mM Tris-HCl,
50 mM KCl, and 0.001% gelatin. Samples were ampli-
fied in a GeneAmp PCR System 9600 (Perkin Elmer),
beginning with an initial step of 2 min at 95°C, fol-
lowed by 40 cycles of 15 s at 95°C, 30 s at 57°C, and
60 s at 72°C, followed by a 10-min final extension at
72°C. PCR product (75 ml) was concentrated to 10 ml by
vacuum centrifugation (SpeedVac; Savant, Holbrook,
NY, USA). Eight microliters of concentrated PCR pro-
duct were mixed with 3 ml of Sigma loading buffer. Ten
microliters of that mixture were separated at room tem-
perature on 2% agarose (Perkin Elmer) gels containing
0.5 mg ml−1 ethidium bromide in TBE buffer (89 mM
Tris, 89 mM borate, 2 mM EDTA buffer, pH 8.0). Each
gel contained two lanes of a 100-bp ladder (Gibco BRL,
Gaithersburg, MD, USA). In the cases where fragments
were not visible on agarose after 40 cycles of PCR, a
fluorescently-labeled primer (Applied Biosystems) was
used to amplify PCR products which were sized after
injection on an ABI Prism 310 Genetic Analyzer
(Applied Biosystems, Foster City, CA, USA).
HTT promoter variant analysis
Two different primer sets were used to amplify the
region containing the HTT promoter variant. The first
set (stpr5, 59-GGCGTTGCCGCTCTGAATTGC-39, and
stpr3, 59-GAGGGACTGAGCTGGACAACCCAC-39)15
amplified a 484/528-bp fragment. A second set
(HTTp2A, 59-TGAATGCCAGCACCTAACCC-39, and
HTTp2B, 59-TTCTGGTGCCACCTAGACGC-39) success-
fully amplified a 406/450-bp fragment from samples
that failed to amplify with the first set. The primers
were synthesized as above. PCR was performed in a
10-ml vol containing approximately 50 ng of genomic
template, 1 mM of each primer, 200 mM each of dATP,
dCTP, and dTTP, 100 mM each of dGTP and 79-deaza-
dGTP, 0.6 units of Taq polymerase (AmpliTaq; Perkin
Elmer), 1.5 mM MgCl2, 5% DMSO, 10 mM Tris-HCl,
50 mM KCl, and 0.001% gelatin. Samples were ampli-
fied using a Perkin Elmer GeneAmp PCR System 9600,
through 40 cycles consisting of 30 s at 95°C, 30 s at
61°C, and 1 min at 71°C, followed by 10 min at 72°C.
PCR products were separated on 2% agarose as above.
In the cases where fragments were not visible on aga-
rose after 40 cycles of PCR with the 5-HTTp2A/B
primer set, 50–55 cycles of PCR with AmpliTaq Gold,
a heat-activated DNA polymerase (Perkin Elmer), and
2.5 mM MgCl2 were performed with an initial acti-
vation step of 12 min at 95°C.
Data analysis
The transmission/disequilibrium test was applied to
each of the two HTT loci to test for linkage and linkage
disequilibrium.17,18 In further analyses, we considered
a haplotype of the HTT promoter variant and the HTT
second intron VNTR loci. In constructing the haplo-
types, we considered the probability of recombination
to be negligible due to the close proximity of the mark-
ers (> 15 kb).21
Results
All trios were completely genotyped at the two HTT
polymorphisms. Preliminary TDT analysis with 86
trios of the planned 350 families revealed no evidence
for linkage or linkage disequilibrium between autistic
disorder and the VNTR in the second intron of HTT
(TDT x2 = 4.25, 2 d.f., P = 0.119). However, preferential
transmission of the short variant of the HTT promoter
was found (TDT x2 = 4.69, 1 d.f., P = 0.030).
We then applied a TDT to the resulting haplotypes,
considering the haplotypes as alleles in a multiallelic
TDT. Results confirmed the significance of the effect of
this region (TDT x2 = 11.85, 4 d.f., P = 0.018). Trans-
mission data for alleles and haplotypes are presented
in Table 1 from heterozygous parents only.
Discussion
Preliminary analysis of two serotonin transporter gen-
etic markers revealed evidence of linkage between
haplotypes of the two markers and autistic disorder.
Individually, the short variant of the HTT promoter
variant was found to be preferentially transmitted. The
short variant of the HTT promoter variant has recently
been reported to be a quantitative trait locus for anxi-
ety.16 Anxiety is often a prominent component of autis-
tic disorder and anxiety disorders have been found to

Table 1 Transmission/disequilibrium test of HTT and autis-
tic disorder
Transmitted Not transmitted
Serotonin transporter 2nd intron VNTR
9 copy 0 5
10 copy 32 36
12 copy 40 31
TDT x2 = 4.25, 2 d.f., P = 0.119
Serotonin transporter promoter variant
Short 48 29
Long 29 48
TDT x2 = 4.69, 1 d.f., P = 0.030
Haplotypes of serotonin transporter 2nd intron VNTR and
promoter variant
9 copy, long 0 5
10 copy, short 14 5 serotonin transporter may serve as a susceptibility
10 copy, long 16 29
12 copy, short 27 18
12 copy, long 22 22
TDT x2 = 11.85, 4 d.f., P = 0.018
be increased in relatives of probands with autistic dis-
order.22,23
One would not have predicted the short variant to
be preferentially transmitted based on the limited data
connecting hyperserotonemia and increased platelet
serotonin transporter function, since the HTT promoter
short variant was associated with reduced expression
in transfected cell lines15 and in lymphoblastoid cell
lines.16 Study of the relationship of the HTT promoter
variants and central serotonin function will be required
to fully define the relationships between HTT promoter
genotype and HTT mRNA expression and serotonin
transporter function.
Although most of the information in the haplotype
analysis was provided by the promoter variant, the
finding of a contribution by the HTT VNTR marker may
indicate that the HTT VNTR marker has an effect on
gene expression or that it is in linkage disequilibrium
with another variation in the gene. Although studies
of the relationship of the HTT promoter variants and
function have shown robust differences in different
tissues, it is possible that the HTT promoter variant is
serving as a marker in linkage disequilibrium with a
genomic variant which is contributing to susceptibility
to autistic disorder.
The haplotype analysis was performed to avoid the
problem of multiple testing of the two HTT variants.
In this study, the more functionally relevant marker
was in linkage with autistic disorder. Several lines of
evidence suggest that the serotonin transporter is the
most logical candidate gene based on existing evi-
dence. However, many other candidates may be con-
sidered with only slightly weaker evidence in autistic
disorder. Therefore, evidence for linkage in this study
must be considered very preliminary. We are continu-
ing to collect subjects in this study and beginning
Serotonin transporter gene and autistic disorder
EH Cook Jr et al
249
to collect a separate replication sample. More
importantly, independent replication by other investi-
gators would be necessary before linkage would be con-
sidered to have been demonstrated in autistic disorder.
A case-control association study has provided pre-
liminary evidence of association between the HTT
second intron24 and HTT promoter25 variants and
bipolar mood disorder. Both our finding in autism and
the finding in bipolar mood disorder are preliminary
and require replication. If both are replicated, it is
possible that autistic disorder may share common risk
at this locus, but that additional genetic and/or
environmental factors may be required for the develop-
ment of either syndrome.
In summary, the short variant at the serotonin trans-
porter promoter locus was found to be preferentially
transmitted from parents to probands with autistic dis-
order. This provides preliminary evidence that the
locus in autistic disorder. If replicated, the finding may
contribute to identification of other susceptibility loci
which act additively or in a multiplicative manner. In
addition, study of the relationship of genotypes at the
serotonin transporter locus to response to potent sero-
tonin transporter inhibitors may be feasible. Repli-
cation of this preliminary finding of linkage between
the serotonin transporter locus and autistic disorder
may be performed in other samples with a single affec-
ted child and both parents or in the several relatively
large samples of affected sibling pairs.
Acknowledgements
Amy Jersild, Cory Shulman, Elizabeth Moreno, Mat-
thew Leventhal, Jeremy Veenstra-Vander Weele, Pam
DiLavore, Justine Levin, and Deborah Olkon provided
expert technical assistance. This study was supported
in part by NIMH R01 MH52223 (EHC), NINDS 5-R01-
NS-19855 (EC), the Jean Young and Walden W Shaw
Foundation (BLL), Harris Foundation (BLL), the Brain
Research Foundation (EHC), and NIMH K05MH01196
(CL).
References
1 American Psychiatric Association, Diagnostic and Statisti-
cal Manual of Mental Disorders (4th edn). American Psy-
chiatric Association Press: Washington, DC, 1994.
2 Bryson S, Clark B, Smith T. First report of a Canadian
epidemiological study of autistic syndromes. J Child Psy-
chol Psychiat 1988; 29: 433–445.
3 Ritvo E, Jorde L, Mason-Brothers A, Freeman B, Pingree
C, Jones M, McMahon W, Petersen P, Jenson W, Mo A. The
UCLA-University of Utah epidemiologic survey of autism:
recurrence risk estimates and genetic counseling. Am J
Psychiatry 1989; 146: 1032–1036.
4 Jorde L, Hasstedt S, Ritvo E, Mason-Brothers A, Freeman
B, Pingree C, McMahon W, Peterson B, Jenson W, Moll A.
Complex segregation analysis of autism. Am J Hum Genet
1991; 49: 932–938.
5 Bailey A, Le Couteur A, Gottesman I, Bolton P, Simonoff
E, Yuzda E, Rutter M. Autism as a strongly genetic dis-
 Serotonin transporter gene and autistic disorder
EH Cook Jr et al
250
order: evidence from a British twin study. Psychol Med
1995; 25: 63–78.
6 Hallmayer J, Hebert J, Spiker D, Lotspeich L, McMahon
W, Petersen P, Nicholas P, Pingree C, Lin A, Cavalli-Sforza
L, Risch N, Ciaranello R. Autism and the X chromosome:
mulipoint sib-pair analysis. Arch Gen Psychiatry 1996; 53:
985–989.
7 Pickles A, Bolton P, Macdonald H, Bailey A, Le Couteur
A, Sim CH, Rutter M. Latent-class analysis of recurrence
risks for complex phenotypes with selection and measure-
ment error: a twin and family history study of autism. Am
J Hum Genet 1995; 57: 717–726.
8 Schain RJ, Freedman DX. Studies on 5-hydroxyindole
metabolism in autistic and other mentally retarded chil-
dren. J Pediatrics 1961; 58: 315–320.
9 Cook E, Leventhal B. The serotonin system in autism. Curr
Opin Pediatr 1996; 8: 348–354.
10 Brammer GL, McGuire MT, Raleigh MJ. Vervet monkey
(Cercopithecus aethiops sabaeus) whole blood serotonin
level is determined by platelet uptake sites. Life Sci 1987;
41: 1539–1546.
11 Cook E, Arora R, Anderson G, Berry-Kravis E, Yan S-Y,
Yeoh H, Sklena P, Charak D, Leventhal B. Platelet sero-
tonin studies in hyperserotonemic relatives of children
with autistic disorder. Life Sci 1993; 52: 2005–2015.
12 Gordon C, State R, Nelson J, Hamburger S, Rapoport J. A
double-blind comparison of clomipramine, desipramine,
and placebo in the treatment of autistic disorder. Arch
Gen Psychiatry 1993; 50: 441–447.
13 McDougle C, Naylor S, Cohen D, Volkmar F, Heninger G,
Price L. A double-blind, placebo-controlled study of flu-
voxamine in adults with autistic disorder. Arch Gen Psy-
chiatry 1996; 53: 1001–1008.
14 Lesch K-P, Balling U, Gross J, Strauss K, Wolozin B, Mur-
phy D, Riederer P. Organization of the human serotonin
transporter gene. J Neural Transm 1994; 95: 157–162.
15 Heils A, Teufel A, Petri S, Stoeber G, Riederer P, Bengel
D, Lesch KP. Allelic variation of human serotonin trans-
porter gene expression. J Neurochem 1996; 66: 2621–2624.
16 Lesch K-P, Bengel D, Heils A, Sabol SZ, Greenberg BD,
Petri S, Benjamin J, Müller CR, Hamer DH, Murphy DL.
Association of anxiety-related traits with a polymorphism
in the serotonin transporter gene regulatory region.
Science 1996; 274: 1527–1531.
17 Spielman RS, McGinnis RE, Ewens WJ. Transmission test
for linkage disequilibrium: the insulin gene region and
insulin-dependent diabetes mellitus (IDDM). Am J Hum
Genet 1993; 52: 506–516.
18 Spielman R, Ewens W. The TDT and other family-based
tests for linkage disequilibrium and association. Am J
Hum Genet 1996; 59: 983–989.
19 Lord C, Rutter M, Le Couteur A. Autism Diagnostic Inter-
view – Revised: a revised version of a diagnostic interview
for caregivers of individuals with possible pervasive
developmental disorders. J Autism Dev Disord 1994; 24:
659–685.
20 Lord C, Rutter M, Goode S, Heemsbergen J, Jordan H,
Mawhood L, Schopler E. Autism Diagnostic Observation
Schedule: a standardized observation of communicative
and social behavior. J Autism Develop Disord 1989; 19:
185–212.
21 Heils A, Teufel A, Petri S, Seemann M, Bengel D, Balling
U, Riederer P, Lesch K-P. Functional promoter and poly-
adenylation site mapping of the human serotonin (5-HT)
transporter gene. J Neural Transm 1995; 102: 247–254.
22 Piven J, Chase GA, Landa R, Wzorek M, Gayle J, Cloud D,
Folstein S. Psychiatric disorders in the parents of autistic
individuals. J Am Acad Child Adolesc Psychiatry 1991;
30: 471–478.
23 Smalley SL, McCracken J, Tanguay P. Autism, affective
disorders, and social phobia. Am J Med Genet 1995; 60:
19–26.
24 Collier DA, Arranz MJ, Sham P, Battersby S, Vallada H,
Gill P, Aitchison KJ, Sodhi M, Li T, Roberts GW, Smith
B, Morton J, Murray RM, Smith D, Kirov G. The serotonin
transporter is a potential susceptibility factor for bipolar
affective disorder. Neuroreport 1996; 7: 1675–1679.
25 Collier DA, Stober G, Li T, Heils A, Catalano M, Di Bella
D, Arranz MU, Murray RM, Vallada HP, Bengel D et al. A
novel functional polymorphism within the promoter of
the serotonin transporter gene: possible role in suscepti-
bility to affective disorders. Mol Psychiatry 1996; 1:
453–460.