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References
7
1 Rubin DH, Damus C. The relationship between passive smoking and
child health: methodologic criteria applied to prior studies. Yale J Biol
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2 Jarvis MJ, Russel MA, Benowitz NL, Feyerabend C. Elimination of
cotinine from body fluids: implications for noninvasive measurement of
tobacco smoke exposure. Am J Publ Health 1988; 788: 696-98.
3 Wald NJ, Boreham J, Bailey A, Ritchie C, Haddow JE, Knight G.
Urinary cotinine as marker of breathing other people’s tobacco smoke.
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4 Strachan DP, Jarvis MJ, Feyerabend C. The relationship of salivary
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Association between clozapine response
and allelic variation in 5-HT2A receptor
gene
We report allelic association between a polymorphism
(T102C) within the coding region of the 5-HT2A gene
(HTR2A, 13q14-21) and response to clozapine in
schizophrenic patients. Homozygosity for the C102 allele
was more frequent (30/57, 53%) among patients who did
not respond to clozapine than in those who responded
(23/92, 25%). This finding is evidence that allelic variation
of genes which encode neurotransmitter receptors can
influence clinical response to antipsychotic drugs.
Clozapine is a potent neuroleptic which is effective in
40-60% of schizophrenic patients who are refractory to
other drugs. The response to clozapine may involve
genetic variation in the neurotransmitter receptors to
which it binds. Clozapine has high affinity for serotonin
2A, 2C, 6, and 7 receptors and the dopamine D4
receptor. Unlike typical antipsychotics, such as
haloperidol, clozapine has low affinity for the D2 receptor
with subtherapeutic occupancy in vivo.’ The D4 receptor
may be an alternative site of action. However, genetic
studies on clozapine response and genotype at the D4
receptor have failed to show a simple association.2,3
Because clozapine has affinity for serotonin receptors,
especially 5-HT/4 we hypothesised that functional
variation at this receptor’s gene may affect response. We
examined a silent polymorphism in the 5-HT 2A receptor
Langone JJ, Gjika HB, VanVunakis H. Nicotine and its metabolites:
radioimmunoassays for nicotine and cotinine. Biochemistry 1973; 12:
5025-30.
Marbury CM, Hammond SK, Haley NJ. Measuring exposure
to
environmental tobacco smoke in studies of acute health effects.
Am J Epidemiol 1993; 137: 1089-97.
9 Chilmonczyk BA, Salmun LM, Megathlin KN, et al. Association
between exposure to environmental tobacco smoke and exacerbations
of asthma in children. N Engl J Med 1993; 328: 1665-69.
10 Richardson MA. Upper airway complications of cigarette smoking.
J Allergy Clin Immunol 1988; 81: 1032-35.
First Department of Paediatrics (C G Bakoula MD, Y J Kafrista MD,
G D Kavadias PhD, D D Lazopoulou MD, M C Theodoridou MD,
Prof N S Matsaniotis MD) and Department of Forensic Medicine and
Toxicology (K P Maravelias PhD), Athens University, Athens, Greece
Chryssa G Bakoula, First Department of
Paediatrics, Athens University, Children’s Hospital Aghia Sophia,
Correspondence to:
Dr
11527 Athens, Greece
varied between 125 and 600 mg daily and were stable for at least
3 months after clinical stabilisation. Clinical response was
determined by the global assessment score (GAS)5 which
evaluates symptoms and functioning during the 12 weeks after
treatment. The scale ranges from 1 (sickest) to 100 (healthiest)
and is divided into ten equal intervals, anchored at each interval
by defining characteristics. For example, 1 to 10 describes an
individual who needs constant supervision to prevent hurt to self
or others, or makes no attempt to maintain personal hygiene. A
20 point improvement in GAS is equivalent to the patient having
passed through two intervals (eg, from the 1 to 10 to the 30 to 40
interval means the patient no longer requires constant
supervision). This scale is a reasonable operational index of
clinical response, and has been validated by clinical studies.’ 92
patients were classified as responders and 57 as non-responders.
All GAS scores were done blind to the genotype data by a
psychiatrist (RK). The difference in GAS scores measured
(ranging from -10 to 70) was normally distributed, with 59
patients presenting an improvement after medication of between
- 10 and 20 points, 70 patients a 20 to 40 point improvement,
and 20 patients a 40 point improvement.
Patients and controls were genotyped for the T102C
polymorphism by PCR amplification of DNA with
oligonucleotide primers and digestion of the 372 basepair
product by MspI, which cuts the C102 allele only, giving 156 and
216 basepair fragmentDNA was visualised by agarose-gel
electrophoresis followed by staining with ethidium bromide.
The 5-HT2A gene was also analysed by single-strand
conformation polymorphism to see whether additional
polymorphisms were present. As part of a systemic screen, DNA
between nucleotides 410 and 606 was amplified with
and
5’-GTATGGGTACCGGTGGCCTCT-3’
primers
5’-GTCCAAACAGCAATGATTTTCA-3’. 25 ng genomic DNA
was amplified in 25 IJoL containing 1.5 mmol/L MgCI2, 200
mmol/L dNTPs, 1 jmoI/L of each primer, and 0-5 U DNA
(HTR2A, 13q14-21) in clozapine-treated patients
classified as responders and non-responders. We screened
the gene for additional variations and report a novel silent
polymorphism, C516T.
gene
The patients were a group of 149 on clozapine (79 female and
70 male). We included 99 normal controls. Patients were all west
European with a DSM-IIIR diagnosis of schizophrenia. All had
shown resistance to two classes of neuroleptic equivalent to at
least 1000 mg chlorpromazine for over 6 weeks. Clozapine doses
*&khgr;2:p=0.001 for clinical response
frequency.
relative to genotype;
tp=0.016 for clinical
response
relative to allele
Table: Genotype and allele frequencies of T102C
in 5-HT 2A gene in clozapine-treated patients
polymorphism
281
polymerase. The PCR conditions were thirty cycles of 45 s at
90°C, 45 s 59°C, and 1 min at 72°C. Unusual mobility was
detected by electrophoresis at 1 W in a 10% non-denaturing
polyacrylamide gel (49:1 acrylamide:bisacrylamide) at room
temperature, followed by silver staining of DNA fragments.
Alleles were detected by digestion with Sau961 and
electrophoresis in agarose gels. Fragments containing the C516
allele showed two bands of 109 and 87 basepairs, whereas the
T516 allele remained uncut (195 basepairs). The C516T
mutation has been reported independently by E H Cook
(Department of Psychiatry, University of Chicago Medical
Center).
Allele frequencies for Tl02 and Cl02 (table) did not
differ significantly between the whole patient group (0-40
for T102 and 0-60 for C102) and the controls (0-41 and
0-59, respectively), and were similar to the population
allele frequencies found by Warren et al,6 which suggest
that the 5-HT2Agene did not have a significant effect on
liability to develop schizophrenia in our sample. However,
the homozygous genotype C102/C102 was significantly
more frequent in the non-responders (53%) than in the
responders (26%), whereas heterozygotes and individuals
homozygous for T102/T102 were significantly more
frequent in responders than in non-responders (table).
The C516T mutation at position 516 occurred at a
frequency of under 2%. Only four heterozygous
individuals were detected among 125 clozapine-treated
patients typed for this polymorphism (4/250 [2%]), three
in the responder (3/148 [2%]) and one in the non-
chromosome 13 that includes the HTR2A locus is linked
schizophrenia (M-W Lin, Institute of Psychiatry,
London). Since the predictive polymorphism, T102C,
does not involve variation in aminoacid sequence of the
protein, we hypothesise that it may affect translation
through the secondary structure and stability of the
mRNA or is in linkage disequilibrium with a functional
variant that affects clozapine binding. An additional
polymorphism (C516T) has been detected, but this is not
associated with response.
to
We thank Dr Tonmoy Sharma, Dr Peter Bush, Dr Michael Launer,
Dr Peter McKenna, and Dr Anne Thornton for providing patients’
samples, and Dr David Curtis and Dr Lyn Pilowsky for critical reading of
the manuscript.
References
1
2
3
4
5
responder group (1/102 [1%]) (not significant).
There is considerable interest in the role of the 5-HT2A
receptor as a site for antipsychotic action.4 Although this
hypothesis is difficult to test directly for clozapine because
of its broad binding specificity, the association we
observed with response supports the view that 5-HT2A
antagonism may be an important mediator of the drug’s
effects.
Our results indicate that allelic variation of the 5-HT2A
gene may be involved in the response to clozapine. This is
interesting because there is evidence that the region of
282
6
Costa DC, Ell PJ, Murray RM, Verhoeff NPLG,
Kerwin RW. Clozapine, single photon emission tomography, and the
D2 dopamine receptor blockade hypothesis of schizophrenia. Lancet
1992; 340: 199-202.
Van Tol HHM, Wu CM, Guan HC. Multiple dopamine D4 receptor
subtypes in the human population. Nature 1992; 358: 149-52.
Shaikh S, Collier D, Kerwin RW, et al. Dopamine D4 receptor
subtypes and response to clozapine. Lancet 1993; 341: 116.
Leysen JE, Janssen PM, Megens AHP, Schotte A. Risperidone: a novel
antipsychotic with balanced serotonin-dopamine antagonism, receptor
occupancy profile, and pharmacologic activity. J Clin Psychiatry 1994;
55 (suppl): 5-12.
Endicott J, Spritzer RL, Fleiss JL, Cohen J. The global assessment
scale. Arch Gen Psychiatry 1976; 33: 766-71.
Warren JT, Peacock ML, Rodriguez LC, Fink JK. An MspI
polymorphism in the human serotonin receptor gene (HTR2):
detection by DGGE and RFLP analysis. Hum Mol Genet 1993; 2: 338.
Pilowsky LS,
Sections of Molecular Genetics and Neuropsychopharmacology,
Institute of Psychiatry, London SE5 8AF, UK (M J Arranz PhD,
D A Collier PhD, M Sodhi MRPharmS, D Ball MRCPsych, G W Roberts PhD,
P Sham MRCPsych, R Kerwin MRCPsych); and Department of Molecular
Neuropathology, SmithKline Beecham Pharmaceuticals, Harlow,
Essex (J Price PhD, G W Roberts)
Correspondence to:
Dr Maria J Arranz