Journal of Neuroscience Research 67:812 822 (2002)

DOI 10.1002/jnr.10173

Differential Expression of the C and T

Alleles of the 5-HT2A Receptor Gene in the
Temporal Cortex of Normal Individuals and
Schizophrenics
Oxana O. Polesskaya1 and Boris P. Sokolov1,2,3*
1

Molecular Neurobiology Branch, National Institute on Drug Abuse, NIH, Baltimore, Maryland
Molecular Neuropsychiatry Section, National Institute on Drug Abuse, NIH, Baltimore, Maryland
3
Department of Psychiatry, Mount Sinai School of Medicine, New York, New York
2

A genetic association between schizophrenia and a silent

C/T(102) polymorphism in the 5-HT2A receptor gene (5HT2AR) has been previously reported; however, the
mechanisms underlying this association remain unknown. Here we developed an improved quantitative
assay for measurements of allele ratios, which revealed
that the expression of allele C in the temporal cortex of
normal heterozygous individuals was significantly lower
than the expression of allele T (allele C to allele T
ratio of 0.8, P 0.0001). Confirming decreased expression of allele C, total levels of 5-HT2AR mRNA and
protein in normal individuals with the C/C genotype were
lower than in individuals with the T/T genotype. Similarly
to normal individuals, allele C to allele T ratio in
heterozygous schizophrenics was reduced (0.8, P
0001). This ratio was independent of neuroleptic treatment history. By contrast, total levels of 5-HT2AR mRNA
correlated inversely with neuroleptic free interval prior to
death (r 0.67, P 0.001) suggesting a reversible
neuroleptic effect. Total levels 5-HT2AR mRNA in neuroleptic free ( 26 weeks) schizophrenics (n 11) were
significantly lower than in controls (P 0.03). The data
suggest that increased prevalence of allele C among
schizophrenics may be due to intrinsically low expression
of this allele, which may contribute to a deficit in
5-HT2AR expression in some schizophrenics.
Published 2002 Wiley-Liss, Inc.

Key words: allele ratio; postmortem brain; 5-HT2A

mRNA; 5-HT2A protein; imprinting

Schizophrenia is a complex disorder of unknown

etiology with significant evidence for multiple susceptibility loci (Williams et al., 1996). The 5-HT2A receptor
gene (5-HT2AR) has been implicated in schizophrenia
based on the functions of the 5-HT2A receptor, its abnormal density in some schizophrenics, and a high affinity
of the receptor for several psychotomimetic or antipsychotic drugs (Meltzer, 1991; Ohuoha et al., 1993; Breier,
1995; Abi-Dargham et al., 1997; Lieberman et al., 1998).
Published 2002 Wiley-Liss, Inc. This article is a US Government work
and, as such, is in the public domain in the United States of America.

A genetic association between schizophrenia and the C

allele of the silent C/T(102) polymorphism of the
5-HT2A receptor gene has been shown in several studies:
the prevalence of allele C was higher than expected
among schizophrenics (Erdmann et al., 1996; Inayama et
al., 1996; Williams et al., 1996, 1997; Spurlock et al.,
1998). Although the odds ratios for allele C and the
combined C/C and C/T genotypes are small (1.3 and
1.7), the attributable fraction is high (0.35), indicating that
the association may be of considerable clinical and therapeutic importance (Williams et al., 1997). The pathogenic
effect of the C allele remains, however, unknown. The
T to C(102) substitution does not alter the amino acid
sequence of the 5-HT2A receptor, which means that the
receptors encoded by the T and C alleles are identical.
It has been hypothesized that alleles T and C may
quantitatively differ in their expression and that this difference may underlie genetic association between the C
allele and schizophrenia. Two previous studies examined
differences between the T and C alleles by comparing
[3H] ketanserin binding in postmortem brains from individuals with different (T/T, T/C, and C/C) genotypes
(Kouzmenko et al., 1997; Turecki et al., 1999). The
results were inconsistent. One study reported no differences between the genotypes (Kouzmenko et al., 1997).
Another study reported lower binding associated with the
C allele (Turecki et al., 1999).
We hypothesized that allelic effect of C/T(102)
polymorphism on expression is small and therefore is
difficult to detect in binding experiments due to limited
specificity of [3H] ketanserin and due to marked variability
in 5-HT2AR expression between individuals. To overcome these limitations, we developed an accurate and
*Correspondence to: Dr. Boris P. Sokolov, Molecular Neuropsychiatry
Section, National Institute on Drug Abuse, NIH, 5500 Nathan Shock Dr.,
Baltimore, MD 21224. E-mail: BSokolov@intra.nida.nih.gov
Received 20 October 2001; Revised 10 December 2001; Accepted 11
December 2001
Published online 7 February 2002

Expression of C and T Alleles of 5-HT2AR and Schizophrenia

highly specific method for measurements of relative levels

of 5-HT2AR mRNA molecules transcribed from the C
or T alleles in heterozygous (C/T) individuals. This
method does not involve comparison of different subjects,
making it possible to avoid effects of variations in demographics and tissue sampling. We applied this method to
examine differential expression of the C and T alleles
in the temporal cortex (Brodmanns area 21 [BA21]) from
normal individuals and schizophrenics. We subsequently
measured total levels of 5-HT2AR mRNA and protein in
different genotype groups using highly specific reverse
transcriptase-polymerase chain reaction (RT-PCR) and
immunoblotting assays.
MATERIALS AND METHODS
Normal Subjects
Postmortem tissue derived from 35 normal individuals
with no history of mental disorders. The genotype groups (T/T,
T/C, and C/C) in normal individuals were well matched for age
(52.6 5.8, 54.7 4.8, and 56.9 4.9 years, respectively) and
brain pH (6.2 0.1, 6.3 0.1, and 6.2 0.1). Postmortem
interval delay (PMI) was not different between the T/C and
C/C groups (13.8 2.7 vs. 13.6 2.4 hours) groups, and was
smaller than in the T/T group (24 4.7 hours). Analysis limited
to cases with PMIs that fell within the same range revealed
directionally the same differences in 5-HT2AR mRNA between
genotypes as did the analysis of the total subject groups (see
Results). The T/T, T/C, and C/C groups were not well
balanced in gender (female/male ratios of 3/2, 6/14, and 4/6,
respectively). However, the differences between genotype
groups were the same in both males and females examined
separately (see Results).
Schizophrenic Patients
Thirty-nine schizophrenic cases (DSM-IIIR/DSM-IV)
were closely matched to normal individuals for gender (f15/m24
vs. f13/m22), age (62 19, range 2595 vs. 55 19, range
16 96) and brain pH (6.3 0.3 vs. 6.2 0.3). One patient has
never been treated with neuroleptics. In other cases, neuroleptic
free intervals before death varied from less than 72 hours to more
than 5 years. PMI among schizophrenics (40 31, range 3110
hours) was greater than that in normal individuals (15 10,
range 3 42 hours). However, exclusion of cases with long PMI
from analyses produced essentially the same results as the analyses
of the total groups (see Results). In addition, no significant effect
of PMI on measurements of 5-HT2AR mRNA using RT-PCR
assay that includes normalization with -actin (see below) was
found (r 0.08, n 70, P 0.51). Similarly, there was no
significant correlation between brain pH and 5-HT2AR mRNA
measurements normalized with -actin (r 0.11, n 62,
P 0.38).
Twenty-four schizophrenics and 10 controls were from
the Mount Sinai Schizophrenia Brain Bank. Other 15 schizophrenic and 15 controls were from the Stanley Foundation
Consortium. Ten additional controls were obtained through the
Institute of Brain, St. Petersburg, Russia. These cases have been
described in detail previously (Hernandez and Sokolov, 2000;
Torrey et al., 2000). There were no significant differences

813

between normal control cases from different sources. Protein

samples were available in this study only for 10 cases from the
Stanley Foundation Consortium.
Total RNA, DNA, and protein were isolated from 200 to
500 mg of tissue using TRI Reagent (MRC, Inc., Cincinnati,
OH). RNA was treated with DNAse and reverse-transcribed as
described previously (Hernandez and Sokolov, 2000).
Genotyping of C/T(102) was carried out by amplification
of a 327-bp fragment of the 5-HT2AR gene using primers
5-GGCTTAGACATGGATATTCT-3 and 5-CTGCAGCTTTTTCTCTAGGG-3 followed by digestion with
HpaII. The conditions for PCR were 25 cycles consisting of
94C for 20 seconds, 55C for 30 seconds, and 72C for 50
seconds. The products were separated in 6% polyacrylamide gel
(PAAG) and stained with SYBR Green (FMC, Rockland, ME).
The undigested 327-bp product identified allele T and the
digested products of 216 bp and 111 bp identified allele C.
When genomic DNA was not available, genotypes were determined from mRNA analysis.
Measuring allele ratio (the relative levels of 5-HT2AR
mRNAs expressed by the C or T alleles) in C/T heterozygotes was performed using RT-PCR amplification of 20 ng of
total RNA followed by HpaII digestion, separation in PAAG,
and staining with SYBR Green (Figs. 1, 2). The conditions for
PCR were as described for genomic DNA. The 216-bp and
327-bp DNA fragments (derived from alleles C and T,
respectively) were quantified by fluorescent densitometry
(Storm 800, Molecular Dynamics, Sunnyvale, CA). Allele C
to allele T ratio in mRNA was calculated form these measurements using an equation: allele ratio K 216 bp/327 bp.
K was determined from mean 216 bp/327 bp ratio measured in
genomic DNA from 25 individuals using equation K 216
bp/327 bp 1. This latter equation is based on the fact that real
allele ratio in genomic DNA is invariant, is independent of any
physiological conditions, and always equals 1.0 (reflecting the
fact that there is one copy of allele C per one copy of allele
T in each heterozygous individual). K provides a means for
correction for differences in staining of the 216 -bp and 327-bp
fragments, as well as for possible heteroduplex formation and
subtle differences in amplification rates of the C and T
alleles. Standard error of measurements of allele ratio in genomic
DNA was of 0.04, n 25.
The total levels of 5-HT2AR mRNA were measured as
ratios to -actin using a switch-profile RT-PCR as described
in detail (Hernandez and Sokolov, 2000) or with minor modifications, which replace radioactive labeling by SYBR Green
staining (Fig. 3). This method allows simultaneous amplification
of 5-HT2AR and -actin mRNA in the same reaction with
similar kinetics, yields and overlapping exponential phases, permitting the precise measurements of 5-HT2AR mRNA relative
to -actin mRNA with only a minimal impact of RNA degradation. -actin is a housekeeping gene that is widely used as an
internal standard in gene expression studies (Ma et al., 1994a,b;
Zamorano et al., 1996; Sokolov, 1998; Stanta and Bonin, 1998).
Raw levels of -actin mRNA in controls were not different
between genotypes (612 153 vs. 640 91 vs. 623 127 in
T/T vs. T/C vs. C/C). Primers for 5-HT2AR mRNA were:
5HT2AF (5-TCCAGAATCCCATCCA CCACAGC-3) and

Fig. 1. Assay for measurement of allele ratios (the relative expression of

the C and T alleles). In each heterozygous individual, the two
alleles are expressed in the same cells and thus experience identical
biochemical and physical conditions, which allows us to avoid effect of
any confounding factors. Genomic DNA and cDNA are amplified in
parallel experiments using identical conditions as described in Materials
and Methods. Allele ratio in mRNA is measured as ratio between
216-bp and 327-bp DNA fragments normalized with 216 bp to 327 bp
ratio in genomic DNA. Normalization with genomic DNA is based on

the fact that real allele ratio in genomic DNA in all heterozygous
individuals is the same and equals 1.0 (one copy of allele C per one
copy of allele T). Since the sequences amplified from mRNA and
genomic DNA are identical, equal expression of alleles C and T
implies equal 216:327-bp bands ratios for mRNA and for genomic
DNA. Note, however, that relative intensity of 216-bp band in mRNA
is slightly lower than in genomic DNA, indicating lower expression of
the C allele. The T/T and C/C genotypes are shown on the figure
to facilitate allele identification in the C/T genotype.

Expression of C and T Alleles of 5-HT2AR and Schizophrenia

Fig. 2. Linearity and accuracy of polymerase chain reaction (PCR)

assay for quantitation of allelic ratios. A reconstitution experiment using
varying ratios of cloned DNA fragments of alleles C and T. DNA
fragments containing 1996 bp sequence corresponding to nucleotides
1678 to 318 nt (promoter and part of the first exon, including the C
to T(102) polymorphic site of the C or T alleles of the 5-HT2A
gene were prepared using PCR amplification and cloned. Plasmids
containing sequences of alleles C or T were mixed at different
ratios indicated on the figure. The ratios of alleles in the mixtures were
then measured using PCR followed by HpaII digestion, electrophoresis
(a) and quantification of the ratios (b).

5HT2AR (5-CCCTGGATCCCTATGGATCGACC-3).
Primers for -actin mRNA were: BA3 (5-GAAACTACCTTCAACTCC-3) and BA7 (5-CCTGTAACAATGCATCTC 3; radioactive assay) or: BA1 (5-ATGAAACTACC
TTCAACTCC-3) and BAR (5-ATCCTGTAACAATGCATCTC-3; nonradioactive assay). The RT-PCR products
were verified by sequencing. The PCR reaction (20 l) contained 0.08 g of cDNA and a mixture of primers for 5-HT2A
and -actin mRNAs. In the radioactive assay primers for PCR
were 5-end-labeled with [32]P. The conditions for PCR were:
eight cycles consisting of 94C for 30 seconds, 68C for
1 minute, and 72C for 50 seconds followed by 21 cycles
consisting of 94C for 30 seconds, 52C for 1 minute, and 72C
for 50 seconds. After separation by electrophoresis in 6% PAAG,
DNA fragments were quantified by incorporated radioactivity as
described previously (Hernandez and Sokolov, 2000) or by
SYBR Green staining (Fig. 3). Linearity and accuracy of RTPCR assay was established by withdrawal aliquots from PCR
reaction after consecutive cycles (Fig. 3). Measured ratio of
5-HT2AR to -actin mRNA was constant between 24 and 30

815

Fig. 3. Linearity and accuracy of reverse transcriptase-polymerase chain

reaction (RT-PCR) assay for nonradioactive quantitation of total
5-HT2AR mRNA level as a ratio to -actin mRNA level. The kinetics
of 5-HT2AR and -actin mRNAs coamplification. Aliquots were
withdrawn from PCR reaction after consecutive cycles and separated in
6% polyacrylamide gel. After staining with SYBR Green, DNA fragments were quantified by fluorescent densitometry. a: Photograph of
the gel. Total number of cycles (eight cycles of 5-HT2AR mRNA
preamplification plus n cycles of coamplification) is indicated. b: Plot of
fluorescence at the respective cycles (measured as arbitrary units using
Storm 800, Molecular Dynamics).

cycles (total) in PCR. Up to four independent measurements

were performed for each sample. Note that this assay does not
distinguish between alleles C and T.
The 5-HT2AR Polypeptide Measurements
Western blotting was carried out using 20 g of total
protein per lane of 10% PAAG and affinity-purified antibody for
the 5-HT2A receptor polypeptide with examination of protein
samples by electrophoresis in 10% PAAG and Coomassie blue
staining to confirm equal protein loading. The antigen/antibody
complex was detected and quantified using Amplified Alkaline
Phosphatase Immun-Blot system (Bio Rad Laboratories, Richmond, CA). Linear relationship was observed in the range of 12
to 42 g of total protein (Fig. 4). Up to three independent
measurements were performed for each of the samples.
Data Analysis
Differences in total levels of 5-HT2AR mRNA were
examined using measurements normalized with endogenously

816

Polesskaya and Sokolov

heterozygous (C/T) individuals. Differences between schizophrenics and controls were examined using t-test (two-tailed).
Gender differences were examined using analysis of covariance
(ANCOVA) with genotype as the covariate. The effects of PMI,
brain pH, and age were examined using Pearsons ProductMoment correlation analysis. The effect of withdrawal from
neuroleptic was examined using Pearsons Product-Moment
correlation analysis. The logarithmic model (5-HRTAR mRNA
vs. log(10) of continuous neuroleptic free interval) was used based on
previous observations in BA9 (Hernandez and Sokolov, 2000).
Only cases with well-documented neuroleptic free intervals prior
to death (n 26) were used for correlation analysis. Statistical
analyses were performed using SPSS Base 10.0 (SPSS Inc., Chicago, IL) and GB-Stat (Dynamic Microsystems, Inc., Silver Spring,
MD) statistical programs. Data are reported as mean S.E.M.

RESULTS

Fig. 4. Linearity and accuracy of immunoblotting for quantitation of

5-HT2AR polypeptide using antibody specific for N-terminal part of
5-HT2AR. Varying amounts of total proteins from human postmortem
tissue (Brodmanns area 21 [BA21]) were run in sodium dodecylsulfate
(SDS) polyacrylamide gel and transferred to a nylon membrane. The
membrane was then incubated with 5-HT2A receptor-specific antibody. The antigen/antibody complex was detected and quantified
using Amplified Alkaline Phosphatase Immun-Blot system. Linear relationship was observed in the range of 12 to 42 g of total protein.
a: 5-HT2A receptor band detected by antibody. b: Plot of relative
5-HT2A receptor band intensity.

expressed internal standard (-actin mRNA). Normalizing with

-actin is a well-established and widely used approach to control
for variability in factors that may influence mRNA measurements, i.e., efficiency of RT-PCR, pipetting errors, staining
efficiency, specific radioactivity of radioisotopes, or overall
RNA degradation (Ma et al., 1994a,b; Zamorano et al., 1996;
Sokolov, 1998; Stanta and Bonin, 1998). Raw measurements of
5-HRTAR and -actin mRNA derived from coamplification
experiments were also examined to confirm that changes reported for 5-HRTAR mRNA normalized with -actin were
indeed due to changes in 5-HRTAR but not due to changes in
-actin.
Statistical Analysis
Significance of difference in allele ratio in mRNA from
heterozygous (C/T) individuals from expected value of 1.0 was
examined using one sample t-test. Additional paired samples
t-tests comparing allele ratios in mRNA with allele ratios in
genomic DNA were performed to confirm the results of the one
sample t-tests. Differences between normal homozygous individuals with C/C and T/T genotypes were examined using
Students t-test. One-tailed t-test was used, since the hypothesis
was directional (C/CT/T) and based on CT allelic ratios in

Normal Individuals
Improved Method for Measurements of Relative Expression of Two Alleles (Allele Ratios) in
Heterozygous Individuals. In each heterozygous individual, two alleles are expressed in the same cells and
thus experience identical biochemical and physical conditions. Therefore, paired comparisons of the two alleles in
the same individuals allow us to avoid effects of variations
in demographics, tissue sampling, overall RNA degradation, etc. Accordingly, a highly specific and accurate
method was developed here to compare relative expression of the C and T alleles in heterozygous (T/C)
individuals by measuring allele ratios. The most commonly used approach for quantifying allele ratios is the use
of PCR amplification followed by restriction endonuclease digestion that distinguishes the polymorphic site (Ohlsson and Ritzen, 1995). This approach is not trivial, because small differences in PCR amplification rate and the
variable presence of heteroduplexes in the PCR products
can skew interpretation of the results. A method that uses
addition of a radiolabelled PCR primer at the final cycle
was developed to circumvent the problem of heteroduplex
formation (Uejima et al., 2000). This approach does not
circumvent problems that may arise from small differences
in PCR amplification. We developed here a simple nonradioactive method, which allows us to circumvent the
problems of both the heteroduplex formation and of unequal PCR. The method employs normalizing measurements of allele ratio in mRNA with measurements of
allele ratio in genomic DNA. (Fig. 1, see Materials and
Methods for details). Real allele ratio in genomic DNA is
constant and equals 1.0 in all heterozygous individuals
providing a means for correcting errors in measurements
of allele ratios arising from heteroduplex formation, unequal amplification, etc. Reconstitution experiments demonstrated the linearity and accuracy of the method even
for small variations in allele ratio (Fig. 2).
The Relative Expression of the C and T
Alleles in Heterozygous (T/C) Normal Individuals.
Examining mRNA from 15 normal heterozygotes produced allele C to allele T ratio of 0.79 0.01, which

Expression of C and T Alleles of 5-HT2AR and Schizophrenia

817

Fig. 5. Decreased expression of allele C compared with allele T in

Brodmanns area 21 from heterozygous (C/T) individuals. Note that
real allele ratio in genomic DNA in all heterozygous individuals equals
1:1 (one copy of allele C per one copy of allele T). Accordingly,
measured ratio of 216/bp to 327 bp bands in genomic DNA was taken
as indicating allele ratio of 1:1 and was then used to compute allele ratio
in mRNA. Data for genomic DNA are given for the combined normal
and schizophrenic groups because allele ratio in genomic DNA by its
nature is independent of any physiological conditions. Note that the

ratio of allele C to allele T in mRNA was lower than ratio of 1:1

in each of the diagnostic or drug treatment groups studied. *** and *
denote significant (one sample t-test) difference from 1:1 ratio at P
0.001 and P 0.02 correspondingly (total group: t 13.02, df 30,
P 0.001; normal individuals: t 14.79, df 14, P 0.001;
schizophrenics, neuroleptic free 26 weeks: t 4.85, df 3, p
0.017; schizophrenics drug free 26 weeks: t 6.03, df 11, P
0.001). Data are mean S.E.M. W, weeks.

was significantly lower (t 14.79, df 14, P 0.0001)

than expected ratio of 1.0 (Fig. 5), indicating that the
expression of the C allele was 20% lower than the
expression of the T allele. Additional paired samples
t-test for those cases, for which both the mRNA and
genomic allele ratio measurements were available, confirmed significant difference between allele ratios in
mRNA and in genomic DNA (t 3.37, df 5, P
0.02). Allele C to allele T ratio in mRNA was decreased in all normal heterozygous individuals regardless of
gender, age, PMI, or brain pH (data not shown).
Total Expression of the 5-HT2AR Receptor
Gene in Normal Individuals with the T/T, T/C, and
C/C Genotypes. Decreased expression of the C allele was confirmed by comparison of total levels of
5-HT2A receptor mRNA and protein between normal
individuals with different (T/T, T/C, or C/C) genotypes.
Consistent with lower expression of the C allele compared to the T allele, total levels of 5-HT2AR mRNA
(normalized with -actin ) in individuals with the C/C
genotype were lower than in individuals with the C/T
genotype who had lower levels than individuals with the
T/T genotype (Fig. 6). Difference between the T/T and
C/C genotypes was statistically significant (t 2.41, df
13, P 0.015). Examining the raw (not normalized with
-actin ) measurements of 5-HT2AR mRNA confirmed,
directionally, lower 5-HT2AR expression in the C/C

genotype (653 197) compared with the T/T genotype

(765 60). By contrast, raw measurements of -actin
mRNA were not different between the C/C (623 127)
and T/T (612 153) genotypes. (Although extreme
caution should be applied to interpreting data for raw
measurements of 5-HT2AR and -actin mRNAs, these
analyses demonstrate that differences between the T/T
and C/C genotypes in 5-HT2AR levels normalized with
-actin were indeed due to differences in 5-HT2AR but
not in -actin expression.)
Separate analyses of allele ratios and total 5-HT2AR
mRNA levels in normal subjects from different sources
(Mount Sinai Brain Bank, Stanley Consortium and Institute of Brain) were all consistent with lower expression of
allele C compared to allele T (data not shown).
Differences in 5-HT2AR mRNA Levels Between Genotypes are Unlikely to be Associated with
Variations in Demographics.
Age. The genotype groups (T/T, T/C, and C/C) were
closely matched for age (52.6 5.8, 54.7 4.8, and
56.9 4.9 years). Nevertheless, because of a trend (not
statistically significant, r 0.27, n 35, P 0.12)
toward to a decrease in 5-HT2AR mRNA with age, and
because of the slightly lower age in the T/T group, the
data were reexamined excluding all cases with age greater
than 70 years. In this smaller cohort of cases, mean age
in the T/T group (52.6 5.8, n 5) was not lower than

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Polesskaya and Sokolov

Fig. 6. Total levels of 5-HT2AR mRNA in normal individuals and in

neuroleptic-free (26 weeks) schizophrenics with different genotypes. *Denotes significant difference (t 2.41, df 13, p 0.015)
between normal C/C and T/T homozygotes. #Denotes marginally
significant (t 2.03, df 25, P 0.05) difference between normal
heterozygous (C/T) individuals and heterozygous (C/T) schizophrenics. Note that the level of 5-HT2AR mRNA in the combined genotype
(T/TT/CC/C) group of neuroleptic free schizophrenics was
lower than in normal individuals (t 2.20, df 44, P 0.03).

in the T/C (51.9 4.5, n 15) and C/C (44.5 3.3,

n 8) groups, while the level of 5-HT2AR mRNA in the
T/T group (1.41 0.30) was still higher than in the T/C
(1.12 0.20) and C/C (0.74 0.15) groups. These data
strongly indicate that differences between genotype groups
in 5-HT2AR mRNA were not associated with age.
Brain pH. The above differences between genotypes are
unlikely to be associated with variations in brain pH. The
genotype groups (T/T, T/C, and C/C) were closely
matched for brain pH (6.2 0.1, 6.3 0.1, and 6.2
0.1). In addition, there were no significant interactions
between 5-HT2AR mRNA levels measured as ratios to
-actin mRNA and brain pH (r 0.02, n 35, P
0.92), strongly indicating that differences between genotype groups in total 5-HT2AR mRNA were not associated with brain pH.
PMI. There was no correlation between PMI and
5-HT2AR mRNA level normalized with -actin mRNA
(r 0.22, n 35, P 0.20). PMI was not different
between the T/C (13.6 2.4) and C/C (13.8 2.7)
groups. Thus, the difference between the T/C and C/C
groups may not be related to PMI. PMI in the T/T group
(24.0 4.7 hours) was greater than in the C/C group.
Therefore, we performed additional analysis, which was
restricted to cases who had PMIs that fell within the same
range (C/C, 17.4 2.8, range 10 27 hours, n 7; T/T,
20.0 3.2, range 1326 hours, n 4). In this smaller
cohort of cases, the results were similar to the results in the
total subject groups, i.e., levels of 5-HT2AR mRNA in
the C/C genotype (0.78 0.16) were lower than in the
T/T genotype (1.14 0.16). These data argue against a
possibility that differences in 5-HT2AR mRNA levels
observed between different genotypes were associated
with PMI.

Gender. Levels of 5-HT2AR mRNA in normal males

were lower than in normal females in all three genotype
groups, reaching near significance (F1,32 3.54, P 0.07,
ANCOVA, controlling for genotype). The relationship
between different genotypes in respect to 5-HT2AR
mRNA levels was directionally the same (T/T T/C
C/C) in both males and females examined separately
(females, 1.69 0.44 1.44 0.5 0.92 0.2; males,
0.99 0.01 0.97 0.12 0.68 0.15).
Expression of the 5-HT2A Receptor Gene at
the Protein Level. Protein expression of the 5-HT2A
receptor gene was measured using Western blotting and
antibodies specific to the 5-HT2AR polypeptide (Fig. 4).
Measurements of 5-HT2AR polypeptide and measurements of 5-HT2AR mRNA were highly correlated (r
0.69, n 10, P 0.029). The relationship between
different genotypes at the protein level was the same as at
the mRNA level (T/T C/T C/C) and was consistent with lower expression of the C allele compared
with the T allele. Specifically, mean levels of 5-HT2AR
polypeptide in the T/T, T/C, and C/C groups were of
35.0 3.8 (n 4), 22.2 1.3 (n 3), and 19.5 3.9
(n 3), respectively. Because of the small group sizes, the
two genotype groups carrying allele C (genotypes C/C
and T/C) were combined for the purpose of statistical
analysis and compared with the T/T group. The difference between the combined C/C T/C group (n 6)
and the T/T group (n 4) was statistically significant (t
3.71, P 0.003, one-tailed t-rest). Note that the difference between the T/T and C/C homozygotes was also
significant (t 2.27, P 0.018).
Thus, measuring allele ratio in heterozygous individuals, as well as measuring total levels of 5-HT2AR mRNA
and 5-HT2AR polypeptide in individuals with different
genotypes, strongly indicates that the expression of allele
C allele is lower than the expression of allele T
resulting in gene-dose-dependent decrease in total levels
of 5-HT2AR mRNA and protein in normal allele C
carriers.
Schizophrenic Individuals
The Relative Expression of the C and T
Alleles (Allele Ratio) in Heterozygous (T/C)
Schizophrenics. Ratio of allele C to allele T in
mRNA from heterozygous (T/C) schizophrenics was
0.76 0.03 (n 16). This ratio was significantly lower
than the ratio of 1.0 expected if the expression of the
C and T alleles was equal (t 7.69, df 15,
P 0.0001), indicating that similarly to normal individuals, the expression of allele C in heterozygous schizophrenics was lower than the expression of allele T.
Additional paired samples t-test for those schizophrenic
cases for which both the mRNA and genomic allele ratio
measurements were available confirmed significant difference between allele ratios in mRNA and genomic DNA
(t 4.03, df 8, P 0.004). Thus, the difference in
expression of the C and T alleles (CT) in
heterozygous individuals is independent of the diagnosis

Expression of C and T Alleles of 5-HT2AR and Schizophrenia

Fig. 7. Correlation between levels of 5-HT2AR mRNA in schizophrenics and neuroleptic free intervals prior to death (r 0.67, n
23, P 0.001).

and apparently reflects intrinsic differences in functional

activity of the C and T alleles.
Total Expression of the 5-HT2A Receptor
Gene in Schizophrenics with the T/T, T/C or C/C
Genotypes. Consistent with a previous finding in the
frontal cortex (Hernandez and Sokolov, 2000), total levels
of 5-HT2AR mRNA (normalized with -actin ) in BA21
from schizophrenics correlated inversely with neuroleptic
free intervals prior to death (r 0.67, P 0.001),
indicating, indirectly, a neuroleptic effect which may confound comparison of different genotypes in schizophrenics. Additional correlational analysis controlling for factors
that indicate significant interaction with 5-HT2AR (i.e.,
genotype), or a trend toward an interaction with
5-HT2AR level (i.e., age and gender) confirmed significant inverse correlation between 5-HT2AR mRNA and
neuroleptic free interval (r 0.63, P 0.003).
Neuroleptic effect appears to be washed out 26
weeks after withdrawal from neuroleptic (Fig. 7). Accordingly, to minimize possible neuroleptic effect, analysis of
total 5-HT2AR mRNA levels in schizophrenics with different genotypes was limited to those patients (n 11),
who had never been treated or who were free of neuroleptic for at least 26 weeks prior to death. This cut-off
produced groups which were not sufficient for adequate
analysis of differences between T/T, T/C, and C/C genotypes among schizophrenics.
Comparison of combined (T/T T/C C/C)
group of neuroleptic free schizophrenics with combined
(T/T T/C C/C) group of normal individuals indicated that mean level of 5-HT2AR mRNA was significantly decreased in schizophrenics (0.58 0.12 vs. 1.05
0.11, t 2.20, df 44, P 0.03). Further analysis
limited to the T/C genotype indicated that in neuroleptic free schizophrenics with the T/C genotype, mean
level of 5-HT2AR mRNA normalized with -actin was
lower than in controls with the T/C genotype (0.50
0.14, n 7 vs. 1.11 0.17, n 20), reaching marginal

819

significance (t 2.03, df 25, P 0.05; Fig. 6).

Confirming these results, examining of the raw (not normalized with -actin ) measurements of 5-HT2AR
mRNA indicated nominally lower levels in neuroleptic
free schizophrenics compared with controls, both in the
combined genotype groups (527 168 vs. 770 88), and
in the T/C genotype subgroup examined separately
(534 270 vs. 824 122).
Caution should be applied, however, to these latter
analyses because of the small sample sizes, and because
neuroleptic free schizophrenics had greater PMI
(54.9 15.2 hours) than did normal individuals (13.6
2.4 hours). It should be noted, however, that the results
were directionally the same when the analyses were limited to schizophrenics with PMI that fell within the control range (schizophrenics, 16.6 6.3, n 6; controls,
15.1 1.8, n 35). In this smaller cohort of cases, level
of 5-HT2AR mRNA in the combined genotype group
(T/T T/C C/C) of neuroleptic free schizophrenics was lower than that in controls (0.62 0.18 vs. 1.06
0.11). Similarly, level of 5-HT2AR mRNA in T/C
schizophrenics was lower than that in T/C controls
(0.64 0.3 vs. 1.11 0.17). Together with absence of
correlation between PMI and 5-HT2AR mRNA normalized with -actin (schizophrenics, r 0.02, p 0.92;
normals, r 0.22, P 0.20), these data indicate that
differences in 5-HT2AR mRNA between normal individuals and schizophrenics are unlikely to have been associated with PMI. Another possible confound is difference in age between neuroleptic free schizophrenics
(74.3 43) and normal individuals (55 3.1). Although
no significant correlation between 5-HT2AR mRNA and
age was found in controls (r 0.27, n 35, P 0.12)
and in neuroleptic free schizophrenics (r 0.42, n
11, P 0.19) examined separately, examining combined
subject groups produced nearly significant correlation (r
0.22, n 70, P 0.07). Therefore, we performed
additional analysis of differences between schizophrenics
and controls using cases with similar age (schizophrenics,
76.9 3.2, range 59 96, n 9; controls, 79.2 3.6,
range 60 95 years, n 11). Level of 5-HT2AR mRNA
(normalized with -actin ) was lower in neuroleptic free
schizophrenics than in age-matched controls (0.49 0.12
vs. 0.96 0.18), reaching marginal significance (t 2.06,
df 18, P 0.05). Raw (not normalized with -actin )
level of 5-HT2AR mRNA was also nominally decreased
in neuroleptic free schizophrenics compared with agematched controls (473 208 vs. 889 266), although the
difference did not reach statistical significance (t 1.15,
df 18, P 0.27). Separate analyses of cases from the
Mount Sinai Brain Bank and the Stanley Consortium (data
not shown) indicated similar decreases in 5-HT2AR
mRNA levels neuroleptic free schizophrenics compared
with normal individuals.
It should be noted, that despite reduced total levels of
5-HT2AR mRNA in neuroleptic free heterozygous
(T/C) schizophrenics compared with heterozygous (T/C)
normal individuals (Fig. 5), the difference between the

820

Polesskaya and Sokolov

C and T alleles was preserved among schizophrenics

(allele ratio: 0.74 0.05), indicating that both alleles may
be downregulated in neuroleptic free schizophrenics
compared with normal individuals. Also preserved was the
difference between the C and T alleles in heterozygous schizophrenics who were receiving neuroleptics at
time near death (allele ratio: 0.77 0.04, Fig. 5). These
observations indicate that the difference in expression between alleles C and T (C T) is independent of
both the diagnosis and drug treatment, while total levels of
5-HT2AR may reflect interactions between the genotype,
diagnosis, and drug treatment.
The relationship between total levels of 5-HT2AR
mRNA among genotypes (T/T T/C C/C) expected
from allele T allele C expression was not evident in
neuroleptic free schizophrenics (Fig. 6). This is likely to
be due to very small group sizes in the C/C (n 2) and
T/T (n 2) groups. It is interesting to note, however,
that the absence of T/T T/C C/C relationship in
schizophrenics is consistent with the possible etiological
role of the 5-HT2AR expression deficit in schizophrenia.
That is, because if decreased expression of 5-HT2AR is an
important feature of schizophrenia, than the T/T and T/C
genotypes among schizophrenics should have been limited
to those individuals, who had decreased (compared with
T/T and T/C controls) 5-HT2AR expression. By contrast, the C/C genotype by itself predisposes to low expression of 5-HT2AR. Therefore, the C/C group in
schizophrenics does not have to be limited to those individuals who have lower 5-HT2AR expression than normal C/C heterozygotes.
DISCUSSION
Previous studies have shown genetic association between the C allele of the 5-HT2A receptor gene and
schizophrenia (Inayama et al., 1996; Williams et al., 1996,
1997). In addition, associations with earlier onset and poor
long-term outcome of schizophrenia (Joober et al., 1999),
with lower responsiveness to treatment (Arranz et al.,
1996; Joober et al., 1999), and with susceptibility to tardive dyskinesia (Segman et al., 2001), have been reported.
The mechanisms of these associations remained, however,
elusive.
The current study provides converging lines of evidence that the expression of the C allele in the temporal
cortex is lower than the expression of the T allele.
Decreased expression of the C allele compared with the
T allele was revealed using three independent and
highly specific methods, i.e.: (1) measuring allelic ratios in
heterozygous normal individuals and heterozygous schizophrenics, (2) measuring total levels of 5-HT2AR mRNA
in normal individuals with different genotypes, and (3)
measuring total levels of 5-HT2AR polypeptide in normal
individuals with different genotypes. Decreased expression
of the C allele of 5-HT2A receptor gene is consistent
with decreased 3H-ketanserin binding associated with this
allele reported in one study (Turecki et al., 1999). Another
study reported no effect of T/C(102) polymorphism on
3
H-ketanserin binding (Kouzmenko et al., 1997), which

may possibly reflect complexity of 3H-ketanserin binding

or, perhaps, significant individual variability in 5-HT2AR
expression due to factors not related to the polymorphism.
The current study also provides evidence indicating
overall reduction in 5-HT2AR expression in the temporal
cortex of neuroleptic-free schizophrenics. This finding
extends our previous finding in the frontal cortex from an
overlapping cohort of subjects (Hernandez and Sokolov,
2000), and is consistent with multiple reports of reduced
5-HT2A receptor binding in schizophrenia (Bennett et al.,
1979; Mita et al., 1986; Arora and Meltzer, 1991; Hashimoto et al., 1993; Laruelle et al., 1993; Burnet et al., 1996;
Dean and Hayes, 1996; Kouzmenko et al., 1997). Taken
together, these observations strongly indicate that low
5-HT2AR expression may be an important feature of at
least some cases of schizophrenia. It is conceivable therefore, that genetic association between schizophrenia and
allele C (Erdmann et al., 1996; Inayama et al., 1996;
Williams et al., 1996, 1997; Spurlock et al., 1998) may be
due to intrinsically low expression of this allele. We hypothesize that the presence of allele C may predispose to
low overall expression of 5-HT2AR, which in turn (in
combination with other factors) may predispose to schizophrenia. It should be noted that allelic effect on 5-HT2AR
expression is small. Besides, low overall expression of
5-HT2AR may apparently be caused by multiple mechanisms. Indeed, additional, allele C independent mechanisms may be suggested to explain lower expression of
both alleles in T/C schizophrenics compared with T/C
normal individuals. It should also be noted that low expression of 5-HT2AR may only be a risk factor but not a
cause of schizophrenia (normal C/C homozygotes display
low 5-HT2AR expression but show no clinical features of
schizophrenia). Small effect of allele C on 5-HT2AR
expression, small effect of low 5-HT2AR expression on
the disease, and multiplicity of mechanisms controlling
5-HT2AR expression may explain failure to reveal association between allele C and schizophrenia (Nothen et
al., 1995; Arranz et al., 1996; Ishigaki et al., 1996; Jonsson
et al., 1996; Malhotra et al., 1996a,b; Nimgaonkar et al.,
1996; Sasaki et al., 1996), and failure to reveal altered
5-HT2A receptor binding reported in several studies
(Reynolds et al., 1983; Joyce et al., 1993; Gurevich and
Joyce, 1997; Lewis et al., 1999).
Of note, no evidence on polymorphic imprinting
(complete inactivation of one allele in some but not other
individuals) was found in our sample of 15 heterozygous
controls and 16 heterozygous schizophrenics. This is in
contrast with finding complete imprinting of one allele in
four out of 18 samples recently reported (Bunzel et al.,
1998). It is plausible to suggest, therefore, that polymorphic imprinting of the 5-HT2AR gene may be a relatively
rare event.
The mechanism(s) underlying lower expression of
the C allele compared with the T allele remains to be
established and may possibly involve a difference in the
stability of mRNAs with C or U, or a linkage dis-

Expression of C and T Alleles of 5-HT2AR and Schizophrenia

equilibrium with some distant polymorphic regulatory

elements of the 5-HT2AR gene.
In conclusion, the current study reveals functional
difference between the C and T alleles of the
5-HT2A receptor gene which may possibly underlie increased prevalence of the C allele among schizophrenics
and contribute to this disorder. If confirmed, these findings may help to unravel one of the molecular mechanisms
contributing to schizophrenia.
ACKNOWLEDGMENTS
This work was supported in part by grant from the
Stanley Foundation to B.P. Sokolov, and by NIDA-IRP.
Postmortem brains were donated by Drs. V. Haroutunian
and K.L. Davis, and the Stanley Foundation Brain Consortium courtesy of Drs. L.B. Bigelow, J. Cervenak, M.M.
Herman, T.M. Hyde, J.E. Kleinman, J.D. Paltan, R.M.
Post, E.F. Torrey, M.J. Webster, and R.H. Yolken. We
thank I. Hernandez for his expert help in some experiments and D. Walther for synthesizing primers for genotyping. We gratefully acknowledge Drs. J. Joyce, B. Hope,
and E.S. Onaivi for critical reading the manuscript and
helpful suggestions.
REFERENCES
Abi-Dargham A, Laruelle M, Aghajanian GK, Charney D, Krystal J. 1997.
The role of serotonin in the pathophysiology and treatment of schizophrenia. J Neuropsychiatry Clin Neurosci 9:117.
Arora RC, Meltzer HY. 1991. Serotonin2 (5-HT2) receptor binding in the
frontal cortex of schizophrenic patients. J Neural Transm Gen Sect
85:19 29.
Arranz MJ, Lin MW, Powell J, Kerwin R, Collier D. 1996. 5HT 2a
receptor T102C polymorphism and schizophrenia. Lancet 347:1831
1832.
Bennett JP, Jr, Enna SJ, Bylund DB, Gillin JC, Wyatt RJ, Snyder SH. 1979.
Neurotransmitter receptors in frontal cortex of schizophrenics. Arch Gen
Psychiatry 36:927934.
Breier A. 1995. Serotonin, schizophrenia and antipsychotic drug action.
Schizophr Res 14:187202.
Bunzel R, Blumcke I, Cichon S, Normann S, Schramm J, Propping P,
Nothen MM. 1998. Polymorphic imprinting of the serotonin-2A (5HT2A) receptor gene in human adult brain. Brain Res Mol Brain Res
59:90 92.
Burnet PW, Eastwood SL, Harrison PJ. 1996. 5-HT1A and 5-HT2A
receptor mRNAs and binding site densities are differentially altered in
schizophrenia. Neuropsychopharmacology 15:442 455.
Dean B, Hayes W. 1996. Decreased frontal cortical serotonin2A receptors
in schizophrenia. Schizophr Res 21:133139.
Erdmann J, Shimron-Abarbanell D, Rietschel M, Albus M, Maier W,
Korner J, Bondy B, Chen K, Shih JC, Knapp M, Propping P, Nothen
MM. 1996. Systematic screening for mutations in the human
serotonin-2A (5-HT2A) receptor gene: identification of two naturally
occurring receptor variants and association analysis in schizophrenia. Hum
Genet 97:614 619.
Gurevich EV, Joyce JN. 1997. Alterations in the cortical serotonergic
system in schizophrenia: a postmortem study. Biol Psychiatry 42:529
545.
Hashimoto T, Kitamura N, Kajimoto Y, Shirai Y, Shirakawa O, Mita T,
Nishino N, Tanaka C. 1993. Differential changes in serotonin 5-HT1A
and 5-HT2 receptor binding in patients with chronic schizophrenia.
Psychopharmacology (Berl) 112:S3539.
Hernandez I, Sokolov BP. 2000. Abnormalities in 5-HT2A receptor

821

mRNA expression in frontal cortex of chronic elderly schizophrenics

with varying histories of neuroleptic treatment. J Neurosci Res 59:218
225.
Inayama Y, Yoneda H, Sakai T, Ishida T, Nonomura Y, Kono Y, Takahata
R, Koh J, Sakai J, Takai A, Inada Y, Asaba H. 1996. Positive association
between a DNA sequence variant in the serotonin 2A receptor gene and
schizophrenia. Am J Med Genet 67:103105.
Ishigaki T, Xie DW, Liu JC, Nakamura Y, Zhang HY, Tani K, Shimazu
Y, Chen K, Shih JC, Miyasato K, Ohara K. 1996. Intact 5-HT2A
receptor exons and the adjoining intron regions in schizophrenia. Neuropsychopharmacology 14:339 347.
Jonsson E, Nothen MM, Bunzel R, Propping P, Sedvall G. 1996. 5HT 2a
receptor T102C polymorphism and schizophrenia. Lancet 347:1831.
Joober R, Benkelfat C, Brisebois K, Toulouse A, Turecki G, Lal S, Bloom
D, Labelle A, Lalonde P, Fortin D, Alda M, Palmour R, Rouleau GA.
1999. T102C polymorphism in the 5HT2A gene and schizophrenia:
relation to phenotype and drug response variability. J Psychiatry Neurosci
24:141146.
Joyce JN, Shane A, Lexow N, Winokur A, Casanova MF, Kleinman JE.
1993. Serotonin uptake sites and serotonin receptors are altered in the
limbic system of schizophrenics. Neuropsychopharmacology 8:315336.
Kouzmenko AP, Hayes WL, Pereira AM, Dean B, Burnet PW, Harrison
PJ. 1997. 5-HT2A receptor polymorphism and steady state receptor
expression in schizophrenia. Lancet 349:1815.
Laruelle M, Abi-Dargham A, Casanova MF, Toti R, Weinberger DR,
Kleinman JE. 1993. Selective abnormalities of prefrontal serotonergic
receptors in schizophrenia. A postmortem study. Arch Gen Psychiatry
50:810 818.
Lewis R, Kapur S, Jones C, DaSilva J, Brown GM, Wilson AA, Houle S,
Zipursky RB. 1999. Serotonin 5-HT2 receptors in schizophrenia: a PET
study using [18F]setoperone in neuroleptic-naive patients and normal
subjects. Am J Psychiatry 156:7278.
Lieberman JA, Mailman RB, Duncan G, Sikich L, Chakos M, Nichols DE,
Kraus JE. 1998. Serotonergic basis of antipsychotic drug effects in schizophrenia. Biol Psychiatry 44:1099 1117.
Ma YJ, Costa ME, Ojeda SR. 1994a. Developmental expression of the
genes encoding transforming growth factor alpha and its receptor in the
hypothalamus of female rhesus macaques. Neuroendocrinology 60:346
359.
Ma YJ, Hill DF, Junier MP, Costa ME, Felder SE, Ojeda SR. 1994b.
Expression of epidermal growth factor receptor changes in the hypothalamus during the onset of female puberty. Mol Cell Neurosci 5:246 262.
Malhotra AK, Goldman D, Buchanan R, Breier A, Pickar D. 1996a. 5HT
2a receptor T102C polymorphism and schizophrenia. Lancet 347:1830
1831.
Malhotra AK, Goldman D, Ozaki N, Breier A, Buchanan R, Pickar D.
1996b. Lack of association between polymorphisms in the 5-HT2A
receptor gene and the antipsychotic response to clozapine. Am J Psychiatry 153:10921094.
Meltzer HY. 1991. The mechanism of action of novel antipsychotic drugs.
Schizophr Bull 17:263287.
Mita T, Hanada S, Nishino N, Kuno T, Nakai H, Yamadori T, Mizoi Y,
Tanaka C. 1986. Decreased serotonin S2 and increased dopamine D2
receptors in chronic schizophrenics. Biol Psychiatry 21:14071414.
Nimgaonkar VL, Zhang XR, Brar JS, DeLeo M, Ganguli R. 1996. 5-HT2
receptor gene locus: association with schizophrenia or treatment response
not detected. Psychiatr Genet 6:2327.
Nothen MM, Rietschel M, Erdmann J, Oberlander H, Moller HJ, Nober
D, Propping P. 1995. Genetic variation of the 5-HT2A receptor and
response to clozapine. Lancet 346:908 909.
Ohlsson R. HK, Ritzen M. 1995. Genomic imprinting: causes and consequences. Cambridge: Cambridge University Press.

822

Polesskaya and Sokolov

Ohuoha DC, Hyde TM, Kleinman JE. 1993. The role of serotonin in
schizophrenia: an overview of the nomenclature, distribution and alterations of serotonin receptors in the central nervous system. Psychopharmacology (Berl) 112:S515.
Reynolds GP, Rossor MN, Iversen LL. 1983. Preliminary studies of human
cortical 5-HT2 receptors and their involvement in schizophrenia and
neuroleptic drug action. J Neural Transm Suppl 18:273277.
Sasaki T, Hattori M, Fukuda R, Kunugi H, Nanko S. 1996. 5HT 2a
receptor T102C polymorphism and schizophrenia. Lancet 347:1832.
Segman RH, Heresco-Levy U, Finkel B, Goltser T, Shalem R, Schlafman
M, Dorevitch A, Yakir A, Greenberg D, Lerner A, Lerer B. 2001.
Association between the serotonin 2A receptor gene and tardive dyskinesia in chronic schizophrenia. Mol Psychiatry 6:225229.
Sokolov BP. 1998. Expression of NMDAR1, GluR1, GluR7, and KA1
glutamate receptor mRNAs is decreased in frontal cortex of neurolepticfree schizophrenics: evidence on reversible up-regulation by typical
neuroleptics. J Neurochem 71:2454 2464.
Spurlock G, Heils A, Holmans P, Williams J, DSouza UM, Cardno A,
Murphy KC, Jones L, Buckland PR, McGuffin P, Lesch KP, Owen MJ.
1998. A family based association study of T102C polymorphism in
5HT2A and schizophrenia plus identification of new polymorphisms in
the promoter. Mol Psychiatry 3:42 49.
Stanta G, Bonin S. 1998. RNA quantitative analysis from fixed and
paraffin-embedded tissues: membrane hybridization and capillary electrophoresis. Biotechniques 24:271276.

Torrey EF, Webster M, Knable M, Johnston N, Yolken RH. 2000. The

stanley foundation brain collection and neuropathology consortium.
Schizophr Res 44:151155.
Turecki G, Briere R, Dewar K, Antonetti T, Lesage AD, Seguin M,
Chawky N, Vanier C, Alda M, Joober R, Benkelfat C, Rouleau GA.
1999. Prediction of level of serotonin 2A receptor binding by serotonin
receptor 2A genetic variation in postmortem brain samples from subjects
who did or did not commit suicide. Am J Psychiatry 156:1456 1458.
Uejima H, Lee MP, Cui H, Feinberg AP. 2000. Hot-stop PCR: a simple
and general assay for linear quantitation of allele ratios. Nat Genet 25:
375376.
Williams J, Spurlock G, McGuffin P, Mallet J, Nothen MM, Gill
M, Aschauer H, Nylander PO, Macciardi F, Owen MJ. 1996. Association between schizophrenia and T102C polymorphism of the
5-hydroxytryptamine type 2a-receptor gene. European Multicentre
Association Study of Schizophrenia EMASS) Group. Lancet 347:
1294 1296.
Williams J, McGuffin P, Nothen M, Owen MJ. 1997. Meta-analysis of
association between the 5-HT2a receptor T102C polymorphism and
schizophrenia. EMASS Collaborative Group. European Multicentre Association Study of Schizophrenia. Lancet 349:1221.
Zamorano PL, Mahesh VB, Brann DW. 1996. Quantitative RT-PCR for
neuroendocrine studies. A minireview. Neuroendocrinology 63:397
407.