Mais les résultats doivent être attendus longtemps et il n'y a généralement pas de temps metronidazole prix L'autre cas, c'est que l'achat d'un ou d'un autre antibiotique dans une pharmacie classique nécessite des dépenses matérielles considérables et pas toutes les personnes ne peuvent acheter des produits pharmaceutiques aussi coûteux.
Pii: s0304-3940(99)00921-0
A cluster of single nucleotide polymorphisms in the 5H-leader of
the human dopamine D3 receptor gene (DRD3) and its
Sinthuja Sivagnanasundarama,1, Alex G. Morrisa,1, Emma J. Gaitondeb, Peter
J. McKennac, John D. Mollonb, David M. Hunta,*
aDepartment of Molecular Genetics, Institute of Ophthalmology, University College London, Bath Street, London, EC1V 9EL, UK
bDepartment of Experimental Psychology, University of Cambridge, Downing Street, Cambridge, CB2 3EB, UK
Received 26 August 1999; received in revised form 19 November 1999; accepted 19 November 1999
The association between schizophrenia and the Ser9Gly variant of the dopamine D3 receptor gene (DRD3) has been
the subject of numerous studies. Under meta-analysis this site, or one or more in linkage disequilibrium with it, appears
to contribute a small increase to the relative risk of schizophrenia. In this study, 768 bp of the 5H-leader region of DRD3
mRNA was screened for polymorphisms to assess their contribution to the association of DRD3 with schizophrenia. A
cluster of three single nucleotide polymorphisms (SNPs) was identi®ed in tight linkage disequilibrium with each other
and with the Ser9Gly polymorphism. One of the 5H-leader SNPs encodes a Lys9Glu variant within a 36 amino acid residue
stretch of an upstream open reading frame (uORF). Two common haplotypes are found in the population examined; one
is linked to the Ser9 coding variant and the other to the Gly9 variant. A panel of 73 schizophrenic patients and 56 matched
controls recruited from the East Anglia region of the United Kingdom was screened for disease association at these sites.
Since the 5H-leader and coding sites are in tight disequilibrium, the combined genotype of all 4 sites was scored for each
patient. A signi®cant association was seen between disease and the frequency distribution of these genotypes
(x2 13:19, d:f: 3, P 0:0042; Cochran method for sparse cells applied). A 20% excess of one of the heterozygous
genotypes, in which the sequences differ at three of the four SNPs, including Ser9/Gly9 in the receptor and Lys9/Glu9 in
the uORF, was found in the patient group. An absence of association of disease with the Ser9Gly polymorphism had
previously been reported for this panel. This suggests that these SNPs and the corresponding coding changes may exert
a combined or synergistic effect on susceptibility to schizophrenia. q 2000 Elsevier Science Ireland Ltd. All rights
reserved. Keywords: 5H-leader; Dopamine receptor; Schizophrenia; Single nucleotide polymorphism; Association; Common complex disease
The dopamine hypothesis of schizophrenia has been the
residue 9 whereas the G substitution (allele 2) generates an
subject of numerous studies. In particular, the suggestion
MscI (or BalI) restriction enzyme site and encodes glycine
that the dopamine D3 receptor gene (DRD3) is a candidate
at residue 9. An excess frequency of homozygotes for both
for increased susceptibility to the disease comes from the
alleles was originally reported in schizophrenic patients [2].
high af®nity of D3 receptors for neuroleptic drugs [12,13].
This ®nding has been followed up by numerous replication
Higher levels of DRD3 binding have been found in the
studies, some of which have con®rmed this conclusion.
mesolimbic system of schizophrenics [8]. A common
Others however have not supported it, including our study
variant of a single nucleotide polymorphism (SNP) of A/G
[3] which employed the same panel of cases and controls
at position 25 of the DRD3 coding sequence has been iden-
that has been used in this study. A meta-analysis [16] in
ti®ed [7]. The A substitution (allele 1) encodes a serine at
which over 30 of these case-control studies have been exam-
ined, concluded that a relationship between schizophrenia
* Corresponding author. Tel.: 144-171-608-6820; fax: 144-0-
and homozygosity for the Ser9Gly polymorphism is present.
An MspI polymorphism located 40 kb downstream of the
E-mail address: [email protected] (D.M. Hunt)
1 These two authors contributed equally to this study.
0304-3940/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved.
S. Sivagnanasundaram et al. / Neuroscience Letters 279 (2000) 13±16
MscI site has also been identi®ed, but was not associated
with the MscI site or with disease [4].
In all the above studies, it is the frequency of the two
variant types of receptor gene that have been assessed in
cases and controls, whereas the important factor may be the
level of receptor protein production. This is determined by
upstream regulatory elements, both in the promoter and in
the 5H-leader. To address this problem, we have sequenced
768 bp of the 5H-upstream region of human DRD3. Reverse
transcription PCR (RT PCR) of this upstream region, using
polyA1 mRNA from human adult brain, demonstrated that
the 768 bp stretch is situated in the 5H-leader region of the
mRNA. This region was then screened for polymorphisms
using both single stranded conformation polymorphism
Fig. 1. Sequence of the 5H-leader of DRD3 mRNA together with
(SSCP) and sequencing analysis. Three novel SNPs were
174 bp of the coding sequence. The arrows above and below the
identi®ed and their frequency, and that of the MscI poly-
sequence indicate the forward (F) and reverse (R) primers used.
morphism, were scored in unrelated schizophrenic patients
The position from the translation start site of the 5H-end of each
primer is given with the PCR fragment number. The base substi-
As previously reported [3], a panel of unrelated patients
tution for each SNP is shown, including the 125 change in the
DRD3 coding sequence that gives rise to Ser9Gly. The 36 amino
of European descent with chronic schizophrenia and age,
acids encoded by the uORF are shown with the coding change
sex, and ethnically matched controls was recruited from
East Anglia, United Kingdom. Informed consent was
obtained, subjects were interviewed using the Present
region of DRD3 in DNA samples obtained from patients and
State Examination [17] and diagnoses were made using a
controls. Overlapping PCR products were generated from
structured interview with DSM-IIIR criteria to assess recent
four different PCR reactions. The primer pairs used are
or chronic psychopathology. Data were also gathered on
shown in Fig. 1. The nature of the base changes responsible
personal and family psychiatric and medical history; 22%
for the band shifts was determined by sequencing. For each
had a ®rst- or second-degree relative with schizophrenia or
different fragment detected, the sequence of the correspond-
schizoaffective disorder. Controls were given a semi-struc-
ing PCR product was con®rmed with a minimum of 20
tured interview and subjects with any history of major
individuals comprising at least 10 patients and 10 controls.
illness, either in themselves or in a ®rst degree relative,
Statistical comparisons of the patients and controls in 2 £ m
were excluded. Symptoms were rated on an ordinal scale
contingency tables as well as the estimates of relative risk
for response to clozapine. DNA was extracted from blood
were carried out using standard methods [10]. The SPSSw
using the Nucleon II kit (Scotlab Inc., Shelton, CT).
program was used for the Mann±Whitney±Wilcoxon Rank
Seven high-density gridded membranes of a human PAC
Sum test for association of clozapine response ratings with
library (RPC11) from the UK HGMP resource centre were
screened with a 219 bp fragment generated by PCR from the
The sequence to 2768 was found to correspond to
5H end of the coding region of the DRD3 gene, using primer
mRNA. Unfortunately, all attempts to identify precisely
pair exon1F and exon1R (Fig. 1). A positive signal was
the transcription start site with 5H-RACE were unsuccessful.
obtained for clone 221/M4. The 5H-¯anking region of
The translation start site in exon 1 was previously deduced
DRD3 was then ampli®ed from this clone using the method
from an open reading frame in the sequence of a cDNA
of single-primer walking PCR [11], initially with primer
clone situated 69 bp downstream of an in-frame termination
exon1R to extend upstream. The ampli®cation products
codon [13]. Our extended sequence (Fig. 1) contains an
were cloned into pTAg (R&D Systems) and sequenced
upstream open reading frame (uORF) between 2228 and
using exon1R as a sequencing primer and ABI PRISMe
2120 that would encode a putative 36 residue peptide; the
dye terminator cycle sequencing. Sequencing was then
presence of Kozak sequences [6] at the putative start site
extended to the 768 bp stretch, as well as to 174 bp of the
strengthens the case for the translation of this peptide. No
5H-coding sequence, using primers designed to the new
other ORFs were found in this region of the mRNA. SSCP
sequence. All sequencing was carried out on an ABI
analysis of PCR products 2 and 3 each gave identical band-
Model 373a sequencer. The extent of the 5H-leader upstream
ing patterns across all individuals screened, indicating that
of the translation start site was examined by generating
no polymorphic sites are present. In contrast, fragments 1
DRD3 cDNA from adult human brain polyA1 mRNA
and 4 showed a range of banding patterns. PCR product 1
(Clonteche) with a Promegae reverse transcription-poly-
had a single polymorphic site at 2707 (C/G), whereas PCR
merase chain reaction (RT PCR) kit and primers 1F and
product 4 had two polymorphic sites at 2343 (A/G) and
exon1R (Fig. 1). An isotopic PCR-based SSCP method [5]
2204 (A/G). Assuming random mating, the observed geno-
was used to screen from 2768 to 2150 bp of the 5H-¯anking
typic frequencies for each SNP are not signi®cantly different
S. Sivagnanasundaram et al. / Neuroscience Letters 279 (2000) 13±16
from the expected frequencies, suggesting that each is in
tested by using a 2 £ m x 2 test, for which all cells with
Hardy±Weinberg equilibrium. The Ser9Gly polymorphism
expected values ,5 were collapsed [11]. A signi®cant
in this panel of patients and controls had previously been
difference between cases and controls (x2 13:19,
found to be in Hardy±Weinberg equilibrium [3].
d:f: 3, P 0:0042) suggests that there may be an associa-
The identities of all three upstream polymorphic sites
tion between these four SNPs and schizophrenia. The rela-
were scored for the 73 patient and 56 control DNA samples.
tive risk of the two common genotypes was calculated using
The remaining 11 patient and 21 control samples [3] proved
the odds ratio. The relative risk for individuals that are
refractory to PCR ampli®cation for at least one of the frag-
homozygous for the GGAA haplotype is 1.15 (C.I. 95%
ments. The level of linkage disequilibium between all four
0.54±2.5), which suggests that this genotype does not affect
SNP sites, including the Ser9Gly coding A/G variant at
the susceptibility to schizophrenia. The relative risk of the
125, was analyzed by the EH program [15], which indi-
C/G G/G G/A A/G genotype, which corresponds to hetero-
cated that the four SNPs are in tight linkage disequilibrium
zygotes for the two most common haplotypes, is 3.13 (C.I.
(P , 10227), as expected for sites that lie within an 732 bp
95% 1.28±7.6). From inspection, this class is 20% over-
stretch of DNA. The combined genotype of all four sites
represented in patients. Since these two genotypes were
was therefore scored for each patient; the corresponding
selected post-hoc for the estimation of relative risk, the
frequencies of each of the 19 combined genotypes found
hypothesis that the heterozygosity for these two haplotypes
in the panel are shown in Table 1. Inspection of the mean
may increase the risk to schizophrenia remains to be tested
genotype frequencies of the pooled cases and controls,
in a separate sample. This avoids applying an overly conser-
reveals that two of the genotypes are much more common
vative multiple test correction for the 19 genotypes found.
than the 17 remaining genotypes. Since one of the common
The presence of multiple haplotypes in the population
genotypes is homozygous at all three upstream sites and for
examined indicates that these SNPs are suf®ciently ancient
Ser9, this identi®es one common haplotype as GGAA.
for recombination to have taken place between sites that are
Given the presence of linkage disequilibrium, the other
common Gly9 coding haplotype can be identi®ed by
The association of clozapine response with the common
subtraction as CGGG. These haplotypes differ therefore at
C/G G/G A/G A/G genotype was assessed by comparing the
two sites (2707 and 2204): one is linked to the Ser9 coding
response ratings of patients on clozapine (n 49) with this
variant and the other to the Gly9 variant of the D3 receptor.
genotype versus all other genotypes. No signi®cant associa-
The A-204G SNP encodes a non-conservative Lys9Glu
tion was found (P 0:24); this genotype is not associated
substitution in the putative 36 residue peptide encoded by
therefore with clozapine response in this sample. We have
formerly found that patients in this panel either taking or not
The frequency distribution of the 19 combined genotypes
taking clozapine did not differ signi®cantly in the allele or
in the patient group compared to the control group was
genotype frequencies of the Ser9Gly variant [3].
a Frequency of 5H-leader and coding SNPs in the DRD3 gene in patients and controls. The number and % frequency in parentheses of
each genotype is shown. The Ser9Gly genotypes were determined by Gaitonde et al. (1996) [3]. x 2 test between 73 patients and 56
controls for all genotypes in the table: P 0:004. Relative risk was estimated by the odds ratio (OR) and 95% con®dence interval (CI) for
the two most common genotypes, which are indicated by asterisks: *OR 1.15 (CI 95%, 0.54±2.5) **OR 3.13 (CI 95% 1.28±7.6).
S. Sivagnanasundaram et al. / Neuroscience Letters 279 (2000) 13±16
The absence of an association between schizophrenia and
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the Ser9Gly polymorphism in this panel of patients and
controls has been reported previously [3], and this subset
[3] Gaitonde, E.J., Morris, A., Sivagnanasundaram, S.,
McKenna, P.J., Hunt, D.M. and Mollon, J.D., Assessment
similarly showed no association with this polymorphism
of association of D3 dopamine receptor MscI polymorph-
(P 0:13). However, when the SNPs in the coding and
ism with schizophrenia: analysis of symptom ratings,
5H-leader are considered together, a signi®cant association
family history, age at onset, and movement disorders.
with disease is seen. A similar combined `super-allele' was
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[4] Griffon, N., Crocq, M.A., Pilon, C., Martres, M.P., Mayerova,
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This work was supported by a project grant (037331)
[16] Williams, J., Spurlock, G., Holmans, P., Mant, R., Murphy,
from the Wellcome Trust. Sinthuja Sivagnanasundaram
K., Jones, L., Cardno, A., Asherson, P., Blackwood, D., muir,
was in receipt of a Wellcome Trust Prize Studentship.
W., Meszaros, K., Aschauer, H., Mallet, J., Laurent, C.,
Pekkarinen, P., Seppala, J., Stefanis, C.N., Papadimitriou,
G.N., Macciardi, F., Verga, M., Pato, C., Azavedo, H.,
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Cambridge University Press, Cambridge, 1974, pp. 189±228.
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Centre Hospitalier Universitaire de Reims PROPHYLAXIE CONTRE LE PALUDISME CHEZ L’ADULTE N (chloroquine) Présentation : comprimé à 100 mg Posologie : 1 cp par jour, le jour du départ, pendant le séjour et 4 semaines après le retour Contre-indications : Rétinopathies . Sujet atteint de maladie coeliaque Effets indésirables : Troubles digestifs . Atteintes visuelles révers