No Associations between 5-HTT, 5-HT2A gene Polymorphisms and

ORIGINAL ARTICLES

No Associations between 5-HTT, 5-HT2A gene Polymorphisms and Obsessive-Compulsive Disorder in a Korean Population Hye-Ryung Jung, M.S.,1 Joo-Youn Cho, Ph.D.,1 Jae-Yong Chung, M.D.,1 Jung-Ryul Kim, M.D.,1 Kyung-Sang Yu, M.D.,1 In-Jin Jang, M.D.,1 Sang-Goo Shin, M.D.,1 Kyung Jin Lee, M.D.,2 So Young Yoo, M.D.,2 Euitae Kim, M.D.,2 Jun Soo Kwon, M.D.2 Department of Pharmacology and Clinical Pharmacology Unit, 2Department of Psychiatry, Seoul National University College of Medicine and Hospital, Seoul, Korea 1

Abstract

showed an earlier age of onset compared to the females

This study aimed to investigate the possible associations between candidate single nucleotide polymorphisms (SNPs) in the 5-HTT and 5-HT2A genes and the susceptibility to and clinical features of OCD. We screened the SNPs in the 5-HTT, 5-HT2A and DRD2 genes in one hundred and forty eight healthy volunteers by two dimensional gene scanning (TDGS). We chose candidate SNPs which were newly detected by TDGS or had previously been linked with psychiatric disorders such as schizophrenia or depression. The 5-HTT gene-linked polymorphic region (HTTLPR), 17 bp variable number of tandem repeats in the second intron (VNTR), 878C>T, and 1815A>C SNPs in the 5-HTT gene, and 102T>C SNP in the 5-HT2A gene were analyzed by PCR or PCR-RFLP as appropriate. “One hundred and fifty seven healthy unrelated Korean volunteers were enrolled as a control group.” There were no significant differences in the allele frequencies or genotype distributions between the OCD patients and the control group. However, we found a significant difference in the age of onset according to gender; the males

respectively). This investigation failed to produce evi-

(17.5

6.4 vs. 23.1

11.2 for the males and females,

dence that the 5-HTT and 5-HT2A polymorphisms influence the risk for OCD in Koreans. The inheritance of OCD is not simple and most likely involves a number of susceptibility genes and environmental influences. Key words: 5-HTT, 5-HT 2A, DRD2, OCD, Single nucleotide polymorphism. [ Psychiatry Invest 2006; 3 (1):78-86]

Introduction Obsessive-compulsive disorder (OCD) is a common and severe psychiatric illness whose estimated lifetime prevalence rate is 1 to 3 % of the population.1 Patients afflicted with OCD experience intrusive, disturbing, repetitive thoughts (obsessions) and an uncontrollable urge to repeatedly enact stereotypic rituals (compulsions).2 Twin and family studies have suggested the existence of a genetic component in the etiology of OCD, although the mode of inheritance is unknown.3 It has been theorized that serotonin plays a role in

Correspondence: In-Jin Jang, Department of Pharmacology, College of Medicine, Seoul National University, 28 Yongon-dong, Chongno-gu, Seoul, South Korea (110-799) Tel: +82-2-740-8290, Fax: +82-2-745-7996, E-mail: [email protected]

OCD because of the remarkable efficacy of selective serotonin reuptake inhibitors (SSRIs), including

78

Jang IJ et. al.

clomipramine, fluvoxamine, fluoxetine, sertraline and

It has been suggested that the VNTR region may act as

paroxetine, in treating this disorder.4 This has led to the

a transcriptional regulator of the 5-HTT gene, with the

hypothesis that the pathophysiology of OCD may be

12-repeat allele having stronger enhancer-like proper-

associated with the dysregulation of serotonergic neu-

ties than the 10-repeat allele. Individual repeat ele-

rotransmission. Based on these findings, it was sug-

ments within the VNTR domain were later shown to

gested that serotonin (5-HT) related genes may be

differ in their enhancer activity in the embryonic stem

involved in the pathogenesis of OCD.

cell model, indicating that not only the number of

5

6

By determining the magnitude and duration of the 5-

repeats but also the primary structure of VNTR could

HT synaptic signal, it was found that the 5-HT trans-

affect the transcription of the gene.23 Only two studies

porter (5-HTT) plays a key role in the regulation of

have been conducted to investigate the functional con-

serotonergic neurotransmission, and is therefore con-

sequences of VNTR polymorphisms in native-express-

sidered to be an interesting candidate in neuropsychi-

ing cells, and these found no significant effect of the

atric association studies. 5-HTT is encoded by a single

genotype on either the platelet 5-HT uptake24 or 5-

copy gene located on chromosome 17q12.8 Two well-

hydroxyindoleacetic acid level in the cerebrospinal

known polymorphic regions have been identified in the

fluid.25 However, the latter paper reported a significant-

5-HTT gene: a 44 base pair insertion/deletion in the

ly higher level of norepinephrine metabolite in 12/12

promoter region (5-HTT gene-linked polymorphic

homozygotes.

7

region, HTTLPR)9 and a 17 bp variable number of tan-

In recent years, evidence has accumulated that, in addition to the serotonergic system, the dopaminergic

dem repeats in the second intron (VNTR).10 In vitro transfection studies have demonstrated that

system might be involved in OCD. 26 The role of

the long (L) and short (S) variants of the promoter

dopamine in the pathophysiology of OCD is supported

polymorphism differentially modulate the transcription

by preclinical and clinical evidence. Preclinical evi-

of the 5-HTT gene, with the S variant being less effi-

dence includes the induction of stereotypies in experi-

cient. These findings were confirmed in peripheral

mental animal models through increased dopaminergic

native-expressing cells, which showed that lym-

transmission.27 In fact, the putative animal models for

phoblasts of L/L homozygotes have a higher rate of 5-

OCD depend primarily on changes in the dopaminergic

HTT mRNA transcription, 5-HTT ligand binding and 5-

system; the DRD2 being of primary interest, because

HT uptake than those containing at least one copy of

rats treated chronically with the selective DRD2 ago-

the S allele. HTTLPR alleles were shown to affect

nist, quinpirole, develop compulsive checking behav-

platelet 5-HT uptake, binding and content in the

ior.28 Clinical evidence includes the observation that

same manner. Further studies demonstrated that HTTL-

insults to basal ganglia structures, which are intimately

PR had analogous functional effects on 5-HTT expres-

linked to rich dopaminergic innervations, are associat-

sion in brain cells and tissues, affecting the transcrip-

ed with the emergence of obsessive-compulsive behav-

tion rate, abundance, and function of neuronal 5-

ior.29 Furthermore, pharmacologic agents influencing

HTT protein, although in some of these studies no evi-

the dopaminergic system, such as methylphenidate,

dence was found for the allele-specific functional dif-

cocaine, and bromocriptine, have been shown to induce

ferences.18,19,20

obsessive-compulsive symptoms. 30 Finally, imaging

9

11

12

15

16

13

14

17

The allele-dependent differential enhancer activity of

studies of the neurobiological processes in OCD have

the polymorphic region in intron 2 was demonstrated

pointed consistently to abnormalities in the cortico-tri-

as different levels of reporter gene (luciferase) expres-

atal-halamo-ortical circuits (especially the caudate

sion in embryonic stem cells21 and in mouse embryos.22

nucleus).31 There is a considerable amount of experi-

79

Serotonin genes and obsessive-compulsive disorder

mental evidence supporting the hypothesis that the A

dopaminergic system plays a pivotal role in the func-

B

C

tion of these cortico-triatal-halamo-ortical circuits, by

D6.1 (DRD2, exon 6, 939T>C)

fine-tuning the patterns of activity in the direct and

T/C

indirect pathways.32 Therefore, this study aimed to identify new single nucleotide polymorphisms in the 5-HTT, 5-HT2A, and DRD2 genes in the general Korean population, and to investigate the possible association between the candi-

FIGURE 1. Two-dimensional gene scanning (TDGS) of 5-HTT (SLC6A4), 5-HT2A (HTR2A) and DRD2. The entire coding regions and exon-intron junctions were amplified by two-step PCR, as described in Materials and Methods. Thirty-two short PCR fragments were distributed in two-dimensional gels according to their molecular weight and melting temperature. (A) A TDGS sample from control subject with the gene symbols (S, H, and D) and appropriate exon numbers. Gene symbols stand for SLC6A4, HTR2A, and DRD2 genes, respectively. (B) A TDGS sample shows heteroduplex bands in exon 12 of SLC6A4 and in exon 6 of DRD2. (C) The genetic variation in exon 6 was identified as 939T/C by nucleotide sequencing.

date SNPs in the 5-HTT and 5-HT2A genes and the susceptibility to and clinical features of OCD.

Methods and Materials 1. SNP scanning A total of 148 healthy unrelated Korean volunteers living in the Seoul metropolitan area (107 males and 41 females, 21.6 1.6 years old) were scanned for the 5-

slot of a 2-dimensional gel. Electrophoresis was per-

HTT (SLC6A4), 5-HT2A (HTR2A) and DRD2 genes. All

formed in an automated 2-dimensional electrophoresis

of the volunteers gave their written informed consent to

system and the gels were stained with ethidium bro-

participate in this study, which was approved by the

mide. The spot patterns were interpreted visually for

Institutional Review Board of Seoul National

the appearance of four spots rather than one, indicating

University Hospital.

the presence of a heterozygous mutation or polymor-

The mutations or polymorphisms of the 5-HTT, 5-

phism (Figure 1, A and B). Each sample was analyzed

HT 2A and DRD2 genes were identified by Two-

only once, under the same conditions, and those frag-

Dimensional Gene Scanning (TDGS) analysis. The

ments that were absent or faint were repeated by one

GenBank accession numbers used as a reference

dimensional gradient gel electrophoresis. Those frag-

sequence in this study for the 5-HTT, 5-HT 2A and

ments that showed a four spot pattern that could be rec-

DRD2 genes were NM_001045, NM_000621, and

ognized as a previously detected polymorphism on the

NM_000795, respectively. The PCR primer sets used

basis of their characteristic configurations were

for the TDGS analysis of the above genes were

assigned as such. New variants were subjected to

designed using similar algorithms to those used in a

sequence analysis (Figure 1, C). The sequence analysis

previous report. In brief, the entire coding regions and

was either carried out by ourselves on a Beckman

exon-intron junctions were amplified from genomic

CEQ2000 sequencer (75% of fragments) or contracted

DNA in a 7-plex long distance PCR. Individual exons

out to Davis Sequencing (Davis, CA, USA) (25% of

or parts of exons were amplified in four multiplex

fragments).

33

groups of eight, fourteen and ten fragments in the 5-

2. SNP genotyping

HT2A, 5-HTT and DRD2 genes, respectively, using the long distance 7-plex PCR products as a template. The

One hundred and fifty seven healthy unrelated

products of the four multiplex groups were combined,

Korean volunteers participated in this study as a con-

mixed with sample buffer, and loaded directly into the

trol group. We also studied one hundred and three

80

Jang IJ et. al.

TABLE 1. 5-HTT, 5-HT2A primer sequences and PCR-RFLP conditions used in genotyping Gene

Amplified Region

SNP

Primer sequence

5-HTT

Promoter

HTTLPR

5’-TGAATGCCAGCACCTAACCC-3’

Annealing temperature

Restriction enzyme

RFLP condition

55

5’-TTCTGGTGCCACCTAGACGC- 3’ 5’-GCTGTGGACCTGGGAATGT-3’

Intron2

VNTR

Exon 12

1815A>C

5-HT2A

61

5’-GACTGAGACTGAAAAGACAT-3’ 5’-CCGCCACAACTACGACTTTT-3’

62

DraI

5’-AAACCTATGCACAGCCCAAG-3 Exon 1

5’-TCTGCTACAAGTTCTGGCTT-3’

102T>C

37.5 for 4 hrs

60

MspI

5’-CTGCAGCTTTTTCTCTAGGG -3’

37.5 for 3 hrs

patients who met the Diagnostic and Statistical Manual

in a GeneAmp PCR System 2400 (Perkin Elmer,

of Mental Disorders (DSM-IV) criteria for OCD on the

Boston, USA). The details of the primer sequences,

Structured Clinical Interview for Axis I Disorders

annealing temperatures, and RFLP conditions used for

(SCID-I).

The controls did not undergo diagnostic

genotyping are summarized in (Table 1). The oligonu-

interviews, and were considered as being representa-

cleotides used for PCR were commercially synthesized

tive of the local general population. The subjects were

at Bioneer Co. Ltd. (Daejeon, Korea).

34

enrolled in the study only after providing written con-

3. Chart reviews

sent, and the study protocol was approved by the Institutional Review Board of Seoul National

Specific demographic data, including current age,

University Hospital.

age at onset of OCD and gender, was obtained from the

Whole blood (8 mL) was obtained from each subject,

OCD patients. In addition, the Yale-rown Obsessive-

and genomic DNA was extracted from peripheral lym-

ompulsive Checklist and Severity Scale (Y-BOCS) was

phocytes using a QIAamp

DNA Blood Mini Kit

implemented in order to assess the typology and severi-

(QIAGEN, Hilden, Germany). Genotypes for the pro-

ty of the OCD symptoms.35 However, this information

moter HTTLPR, intron 2 VNTR, exon 5 878C>T, exon

was not able to be collected for the majority of the

12 1815A>C in the 5-HTT gene and exon 1 102T>C

patients.

polymorphism in the 5-HT2A gene were analyzed by

4. Statistical analysis

PCR, PCR-RFLP, or the SNaPshot method in both the control and OCD patients groups. Briefly, approxi-

The deviation of the allele and genotype frequencies

mately 100 ng of genomic DNA in a total volume of 20

for the various SNPs from Hardy-Weinberg equilibri-

L, were added to PCR mixtures consisting of 0.25 to

um was assessed using Fisher’s exact test. The differ-

0.5 M of each specific primer pair, 10

PCR buffer

ences in the genotype frequencies between the healthy

with 1.5 mM MgCl2, 0.2 mM of deoxyribonucleotide

volunteers and patients were determined using the chi-

triphosphates (dNTPs), and 0.5 U of recombinant Taq

square test. The variations in the genotypes with gen-

DNA polymerase (Takara, Shiga, Japan). After initial

der were analyzed by Kruskal-Wallis ANOVA. The dif-

denaturation at 95

for 5 min, the DNA was amplified

ferences between the ages of onset according to gender

for 1 min, anneal-

were determined with the student t test. A P value of
G, 939T>C and C957T in exon 6 (Table 2). C957T was a polymorphism which was newly detected, and the allele fre-

Results

quency of this SNP was 5.07 %.

1. SNP scanning

2. Candidate polymorphism selection and association analysis

We identified a total of eight SNPs in the coding regions; there were two polymorphisms in the 5-HT2A

Among the 103 patients, 69 were male and 34 were

gene, and three polymorphisms in both the 5-HTT and

female. Their age was 27.0 10.2 (years, mean SD)

DRD2 genes. There were three polymorphisms in the

for the males, and 31.9

5-HTT gene which were 684T>C in exon 3, 878C>T in

the 157 control subjects, 83 were male and 74 were

exon 5 and 1815A>C in exon 12. Among these poly-

female. The age of the males and females was almost

morphisms, 684T>C and 878T>C were not previously

the same (24.1 2.0, overall).

reported. The allele frequencies of these SNPs were

Among the screened SNPs, we chose three candidate

0.34 % (684T>C), 1.01 % (878C>T) and 3.38 %

SNPs which were expected to be related to obsessive-

(1815A>C) (Table 2). The 5-HT2A gene polymorphisms

compulsive disorder. In addition, we searched previous

identified were 102T>C in exon 1 and 744C>T in exon

reports to study the relationship between the genetic

3. For the new polymorphism, 744T>C, only one of the

variations and psychiatric disorders such as depression, schizophrenia, etc. We selected two additional poly-

TABLE 2. Genotype and allele frequencies in the 5-HTT, 5-HT2A , DRD2 by TDGS

Gene

5-HTT

5-HT2A

DRD2

SNP

Amino acid change

8.2 for the females. Among

morphisms in the 5-HTT gene for genotyping; one was a 44bp insertion or deletion in the promoter region

Frequency (%)

(HTTLPR) and the other was various tandem Genotype

T684C synonymous TT TC CC C878T Ser293Phe CC CT TT A1815C Lys605Asn AA AC CC T102C synonymous TT TC CC C744T synonymous CC CT TT C932G Ser311Cys CC CG GG T939C synonymous TT TC CC C957T synonymous CC CT TT

147 (99.32) 1 (0.68) 0 (0) 145 (97.97) 3 (2.03) 0 (0) 138 (97.24) 10 (6.76) 0 (0) 11 (7.43) 35 (23.65) 102 (68.92) 147 (99.32) 1 (0.68) 0 (0) 142 (95.95) 6 (4.05) 0 (0) 67 (45.27) 72 (48.65) 9 (6.08) 133 (89.86) 15 (10.14) 0 (0)

Allele

nucleotide repeats (VNTR) in intron 2. Therefore, we

T 295 (99.66) 1 (0.34) C

studied HTTLPR, VNTR, C878T and A1815C in 5-

C 293 (98.99) 3 (1.01) T

identified in this study (T684C for 5-HTT, C744T for

A 286 (96.62) C 10 (3.38)

were not analyzed in the patients, due to either their

HTT, and T102C in 5-HT2A. The other polymorphisms 5-HT 2A, and C932G, T939C and C957T for DRD2) low frequency or a limitation of resources. To identify

T 57 (19.26) C 239 (80.74)

the SNPs, we used PCR or PCR-RFLP as appropriate. The observed genotype frequency distribution did not

C 295 (99.66) 1 (0.34) T

show a significant deviation from Hardy-Weinberg equilibrium (P > .05)

C 290 (97.97) G 6 (2.03)

We compared the genotype frequencies between the OCD patients and controls by additive, recessive and

T 206 (69.59) C 90 (30.40)

dominant methods. Genotype frequencies did not reach statistical significance in any of the statistical methods

C 281 (94.93) 15 (5.07) T

that we employed (Table 3). The differences in the allele frequencies between the groups were not signifi-

82

Jang IJ et. al.

gene and 957C>T in the DRD2 gene. Among these

TABLE 3. Genotype frequency comparisons between OCD patients and controls OCD Patients

Control Gene

5-HT2A

SNP

Genotype

T102C

5-HTT HTTLPR

VNTR

A1815C

C878T

TT TC CC L/L L/S S/S 12/12 10/12 10/10 AA AC CC CC CT TT

N

%

N

%

39 82 38 8 49 100 121 16 2 141 17 0 156 1 0

24.5 51.6 23.9 5.1 31.2 63.7 87.1 11.5 1.4 89.2 10.8 0 99.4 0.6 0

27 58 18 9 36 58 89 13 0 84 14 0 103 0 0

26.2 56.3 17.5 8.7 35.0 56.3 87.3 12.7 0 85.7 14.3 0 100 0 0

SNPs, 878C>T and 1815A>C in the 5-HTT gene had the amino acid substitutions 293Ser>Phe and

P value

605Lys>Asn, respectively. This investigation failed to obtain evidence that the 5-HTT and 5-HT2A polymorphisms influence the OCD

0.463

risk factors in Koreans. The association between HTTLPR polymorphisms and OCD has been studied

0.352

by several research groups. Billett et al. did not find any association in 72 OCD patients compared to 72

0.463

controls.36 However, a relative increase (not statistically significant, P=0.07) in the prevalence of homozy-

0.401

gous L allele variants was observed in the OCD group. 0.418

We also found that the L allele was more frequent in the OCD patients than in the control group, but this difference did not reach statistical significance. The fre-

TABLE 4. Age of onset comparisons between genotypes Gene

SNP

Genotype

N

5-HT2A

T102C

T/T T/C C/C L/L L/S S/S 12/12 10/10 A/A A/C

25 55 18 9 33 57 84 13 13 79

5-HTT

HTTLPR

VNTR A1815C

Age of onset (years, Mean SD) 19.4 18.2 21.2 20.3 16.1 20.0 19.3 17.5 19.2 19.6

quencies of the L allele were 20.7% and 26.2% in the control group and OCD patients, respectively (Table 3).

P value

7.2 7.9 9.5 9.2 5.7 8.6 8.3 6.7 8.0 10.2

The possible correlation of the 102T>C polymorphism with various psychiatric diseases has been

0.212

extensively studied, but many of these studies failed to obtain evidence of any such association. Frisch et al.

0.061

reported no differences with respect to the genotypic 0.494

and allelic distribution of the 5-HT2A receptor gene in

0.798

75 unrelated OCD patients compared to controls. 37 Similarly, Nicolini et al. found no association between the 5-HT2A receptor gene T102C polymorphism and

cant either (data not shown).

OCD.38 These findings have only partial significance as

We evaluated the genotype distributions and age of

related to OCD. A recent study by Tot. et al. found that

onset according to gender in the patients group.

the TT genotype of the T102C polymorphism and the

Although we could not find any differences in the

AA genotype of the -1438 G/A polymorphism were

genotype distribution, there was a significant difference

observed at a higher rate in patients with severe OCD,

6.4 vs.

as compared to those with moderate or moderate-

11.2 for males and females, respectively;

severe OCD (P = 0.027 and P = 0.03, respectively).39

PC

group may have included patients who were not really OCD patients.

and C878T, in the 5-HTT gene, 744C>T in the 5-HT2A

83

Serotonin genes and obsessive-compulsive disorder

We found that compared to the females, the males

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