BRITISH JOURNAL OF PSYCHIATRY (2007), 191, 402^407. doi: 10.1192/bjp.bp.107.036129

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Genotype effects of CHRNA7CHRNA7,, CNR1CNR1 andand COMT SNPs rs737865 and rs165599 within this . The SNP rs4680 alters enzyme activ- in : interactions with tobacco ity of COMT (Chen et aletal, 2004), whereas the GGG haplotype of SNPs rs737865– 4680–165599 has been reported to be asso- and cannabis use ciated with lower expression of COMTCOMT messenger RNA in human brain tissue STANLEY ZAMMIT, GILLIAN SPURLOCK, HYWEL WILLIAMS, (Bray(Bray et aletal, 2003) and with increased risk NADINENORTON,NIGELWILLIAMS,MICHAELC.O’DONOVANNADINE NORTON, NIGEL WILLIAMS, MICHAEL C. O’DONOVAN of schizophrenia (Shifman et aletal, 2002).,2002). andMICHAEL J. OWEN Main genotype and haplotype effects of COMTCOMT in this sample have been previously reported, with no evidence found for any Background Genetic variations might Schizophrenia is associated with increased association with schizophrenia (Williams modify associations between schizo- use of cannabis and tobacco compared with et aletal, 2005).,2005). the general population, although reasons phrenia and cannabis or tobacco use. for these associations have not been clearly METHOD Aims Toexamine whether variants elucidated. There is some evidence that people with schizophrenia may use tobacco Participants within the cannabinoid receptor (CNR1)) to alleviate neurophysiological deficits as- A sample of unrelated individuals with andand aa77 nicotinic receptor (CHRNA7) sociated with this disorder (Adler et aletal,, schizophrenia was recruited from out- are associated with schizophrenia, and 1993; Olincy et aletal, 1998), and that this is patient and in-patient clinical settings and whether these effects vary according to mediated through effects at the aa77 nicotinicnicotinic from volunteer support organisations with- cannabis or tobacco use. We also receptor (CHRNA7) (Gray etet in the UK. These individuals were assessed alal, 1996; Stevens et aletal, 1998). An associa- using the Schedule for Assessment of Neu- examined a putative interaction between tion between schizophrenia and a putative ropsychiatric Disorders semi-structured in- 15 8158 cannabis and Val Met within the functional variant, –86C/T, within the terview (SCAN; Wing et aletal, 1990),1990) catechol-catechol-OO-methyltransferase gene CHRNA7 gene (CHRNA7CHRNA7) has been re- together with case-note review wherever ((COMT). ported (Leonard et aletal, 2002) and warrants possible. The Operational Criteria Check- further exploration. list (OPCRIT; McGuffin et aletal, 1991) and MethodMethod Genotype effects of CHRNA7CHRNA7 The main psychoactive compound with- Global Assessment Scale (GAS; Endicott andand CNR1CNR1were studied in a case ^ control in cannabis is delta-9-tetrahydrocannabinol et aletal, 1976) were also completed. High ((DD99-THC), which acts through the CNR1 levels of reliability (kk440.8) were achieved sample of 750 individuals with cannabinoid receptor. An increased inci- between raters for diagnoses and rating schizophrenia and 688 controls, with dence of psychotic disorders in people using scale items. Controls were unrelated blood interactions for these genes studied in cannabis has been observed (Arseneault donors ascertained from the same regions small subsamples. A case-only design of et aletal, 2002; Zammit et aletal, 2002) and a as the majority of the patients. Given the 493 ofthe schizophreniagroupwasusedtoschizophrenia groupwasusedto putative interaction between cannabis use prevalence of schizophrenia and the fact and variation within the catechol-OO-- that people taking regular medication can- examine interactions between cannabis methyltransferase (COMTCOMT) gene on risk of not be blood donors in the UK, it was not use andanduse COMT.. psychosis has also been reported (Caspi etet deemed necessary to screen the control alal, 2005). Findings from relatively small group for schizophrenia to retain statistical There was no evidence of ResultsResults studies examining association between power (Owen et aletal, 1997). Ethical approval association between schizophrenia and CNR1CNR1 genetic variation – most commonly was granted for this study and informed CNR1CNR1 (OR(OR¼0.97,95% CI 0.82^1.13) or at the single nucleotide polymorphism consent was obtained from all participants. CHRNA7CHRNA7 (OR(OR¼1.07,95% CI 0.77^1.49) (SNP) rs(SNP)rs10493531049353 – and schizophrenia have All study participants were White, with been inconsistent, and it was considered both parents born in the UK or Ireland. All genotypes, or of interactions between worth while to examine this in a substan- cases of schizophrenia satisfied DSM–IV tobacco use and CHRNA7,orcannabisuse tially larger sample than has been studied criteria (American Psychiatric Association, andand CNR1CNR1oror COMT genotypes. thus far.thusfar. 1994) for consensus lifetime diagnosis of The main aims of our study were to in- the disorder, made by two independent Conclusions NeitherNeither CNR1nornor vestigate whether variations at –86C/T raters. The following phenotypes, deter- CHRNA7CHRNA7 variation appearsto alter therisk withinwithin CHRNA7CHRNA7 and at rs1049353 within minedmined a prioriapriori, were examined in relation of schizophrenia.Furthermore, our CNR1CNR1 were associated with schizophrenia, to –86C/T and rs1049353 genotype: and whether these relationships differed ac- results do not supportthe presence of cording to use of tobacco or cannabis. We (a)(a)ageage at onset, defined as the age at which differenteffects of cannabis use on also investigated whether there was any evi- psychiatric help for psychotic symp- schizophrenia according to variation within dence of an interaction between cannabis toms was first sought; COMTCOMT.. use and the Val158158Met polymorphism (SNP rs4680) within COMTCOMT, as previously (b)(b)worst-everworst-ever GAS score, ranging from 0 Declaration of interest None. reported (Caspi et aletal, 2005), as well as with (most severe) to 100 (least severe);

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(c)(c)poorpoor outcome, defined as either non- (Applied Biosystems, Foster City, California, ValVal158158Met was 3.5 (Caspi et aletal, 2005),,2005), continuous or continuous course of USA) using an ABI3100 sequencer. Details and our case-only approach had more than illness;illness; of primers and reaction conditions are pro- 90% power to detect an interaction odds (d)(d)standardised,standardised, continuous symptom vided in Appendix 1 at http://www.cardiff. ratio of as low as 1.5, at aa¼0.05.0.05. dimensions obtained by factor analysis ac.uk/medicine/psychological_medicine/pub_ data/comt.htm. of OPCRIT psychosis items, corre- Sensitivity analysis sponding to paranoid delusions and Some participants were likely to have hallucinations; disorganised symptoms; negative symptoms; and first-rank AnalysisAnalysis started using tobacco or cannabis after the delusions.delusions. The reference participants for the analyses onset of schizophrenia and it is possible were those with genotypes that were CC that this could obscure and complicate Data on tobacco and cannabis use were homozygous for –86C/T, GG homozygous interpretation of results from this study. obtained from interview and case-note for rs1049353, AA homozygous for Examination of the association between records for 657 of the schizophrenia group. rs737865, AA homozygous for rs165599, schizophrenia and genotypes was therefore Questions regarding the age at which the and homozygous for the Met allele at repeated with analyses restricted to cases person first started using cannabis or ValVal158158Met within COMTCOMT. Only 0.4% of where the onset of substance use was re- tobacco were only introduced during the our participants were homozygous for ported to be at least 1 year prior to age at latter part of the sample recruitment, and the T allele at the –86C/T locus, and schizophrenia onset. these data were therefore available for only they were therefore grouped with the C/T 22% of cases in which the person reported heterozygotes. ever using these substances. Substance use RESULTSRESULTS Logistic regression was used to examine data were not collected initially for the con- associations between dichotomous out- trol group, and unfortunately most mem- There were 838 participants with schizo- comes and genotypes. A dominance genetic bers of the control group were not asked phrenia who were genotyped for any of model, as described above, was examined at the initial interview for permission to CNR1CNR1 ((nn¼797),797), CHRNA7CHRNA7 ((nn¼750) or750)or for –86C/T, whereas additive models were contact them again for further information. COMTCOMT ((nn¼575). Data on cannabis and used for the CNR1CNR1 andand COMTCOMT variantsvariants As a result of this, cannabis use data were tobacco use were missing for 96 (11.5%) (Lewis, 2002). For the study of continuous available for only 116 controls and tobacco and 107 (12.8%) of these respectively. Of phenotypic outcomes, linear regression was use data for 49 controls. those with substance use data, 276 parti- used. However, for age at onset, where cipants (37.2%) had ever used cannabis, assumptions of normality were not met, data and 531 (72.6%) had ever used tobacco. Genotyping were ln-transformed prior to regression TheThe CHRNA7CHRNA7 promoter polymorphism modelling. Statistical interactions on a –86C/T was genotyped as a restriction- multiplicative scale between substance use CHRNA7 fragment length polymorphism using the and genotype on risk of schizophrenia were The –86C/T genotypes were in Hardy– restriction enzyme Hph1Hph1 (New England investigated using a likelihood ratio test Weinberg equilibrium in both the schizo- Biolabs, Ipswich, Massachusetts, USA). The within the logistic regression models. For phrenia group (ww22¼0.01,0.01, PP¼0.76) and the primers were 5’’ -agtacctcccgctcacacctcg-3’’ ValVal158158Met, however, as no association was control group (ww22¼0.01,0.01, PP¼0.92). As0.92).As and 5and5’’ -atgttgagtcccggagctg-3’’ as used by observed between this SNP and cannabis shown in Table 1, there was no evidence LeonardLeonard et aletal (2002). The product was am- use in the Dunedin cohort (Caspi et aletal,, for any association between –86C/T geno- plified using the GC-RICH PCR System 2005), we used a case-only approach to type and schizophrenia (CT/TT genotypes (Roche Diagnostics, Basel, Switzerland), investigate possible gene–environment OROR¼ 1.07, 95% CI 0.77–1.49; PP¼0.70).0.70). and the 272 base pairs fragment was interactions because this is statistically There was little evidence of any difference digested with Hph1Hph1 resulting in two frag- more powerful (Khoury & Flanders, in the effect of genotype on schizophrenia ments of 79 bp and 193 bp with the T allele. 1996). The case-only analysis was also used between those who smoked (schizophrenia The products were run out on a 1.5% for rs737865 and rs165599 within COMTCOMT.. groupgroup nn¼473, controls nn¼24; OR24;OR¼3.0,3.0, agarose gel and visualised using ethidium Haplotypes for COMTCOMT were examined 95% CI 0.4–22.9) and those who did not bromide.bromide. using UNPHASED, version 3.0 (Dudbridge, (schizophrenia group nn¼186, controls TheThe CNR1CNR1 polymorphism rs1049353 2003).2003). nn¼25; OR25;OR¼1.7, 95% CI 0.4–7.7; was genotyped by fluorescence polarisation This study had greater than 95% power interaction likelihood ratio test ww22¼0.21,0.21, using an AcycloPrime kit (PerkinElmer, to detect an additive genetic effect with an d.f.d.f.¼1,1, PP¼0.65). As tobacco use data were Waltham, Massachusetts, USA) and the odds ratio of 1.4 or above at aa¼0.05 for0.05for available only for a small proportion of the output was read on an LJL Biosystems thethe CNR1CNR1 andand COMTCOMT variants examined. control group, a more powerful case-only (Sunnyvale, California, USA) plate reader. This study also had greater than 95% analysis was also used, and this also failed A 297 bp amplimere was amplified using power to find an association between to provide any evidence for interaction primers 5primers5’’ -ttccctcttgtgaaggcact-3’’ and 5and5’’ -- –86C/T variation and schizophrenia based ((nn¼659; odds ratio for tobacco use by tcattgagcatggtaaagtt-3’’ . The SNP was at on frequencies of CC genotype of 0.91 in CHRNA7CHRNA7 genotype 0.89, 95% CI 0.53– position 125. The extension primer used the control group and 0.84 in the schizo- 1.48).1.48). in the fluorescence polarisation assay was phrenia group, as observed by Leonard etet There were 123 in the schizophrenia 55’’ -catggttaccttggcaatcttgac-3’’ . The.The COMTCOMT alal (2002). The interaction odds ratio group with data relating to age of first markers were genotyped using SNaPshot previously reported for cannabis and using tobacco, and 104 (85%) of these

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claimed to have started using tobacco prior Ta b l e 11Tab Association between CHRNA7 (^(^86C/T) 86C/T) and CNR1 (rs1049353) genotypes and schizophrenia to the onset of schizophrenia. In the sensi- tivity analysis there was similarly little evi- Number (%) by genotypeTotal OR (95% CI) dence of any difference in the effect of genotype on schizophrenia between non- ^86C/T CCCT TT smokers and those smoking prior to illness Control group 548 (88.7)548(88.7) 68 (11.0)68(11.0) 2 (0.3)2(0.3)618 1.0 onset (onset(nn¼110; OR110;OR¼2.7, 95% CI 0.3–22.3; Schizophrenia group 660 (88.0)(88.0)660 87 (11.6)87(11.6) 3 (0.4)3(0.4) 7507501.07 (0.77^1.49)11 22 interactioninteraction ww ¼0.1, d.f.0.1,d.f.¼1,1, PP¼0.73).0.73). rsrs10493531049353 GGGA AA Another way of presenting these data is Control group 335 (48.7) 298 (43.3)298(43.3) 55 (8.0)(8.0)55 688688 1.0 to examine the relationship between Schizophrenia group407 (51.1)319(40.0) 319 (40.0) 71 71(8.9) (8.9)797 0.97 (0.82^1.13)22 tobacco use and schizophrenia stratified by –86C/T genotype. Tobacco use was 1. In CT/C T/ T T v. CC.CC. strongly associated with schizophrenia in 2. Per A allele. the whole sample (OR¼4.4. 95% CI 3.3– using cannabis, and 64 (90%) of these use in our sample of 493 persons with 6.0;6.0; PP550.001), with no evidence of any reported first use prior to onset of schizo- schizophrenia (OR¼0.98, 95% CI 0.76– interaction when stratified by genotype phrenia. As in the main analysis, there 1.27,1.27, PP¼0.89). Results were almost identi- (CC genotype OR¼2.6, 95% CI 1.4–4.7; was little evidence of any difference in the cal when restricting the analysis to partici- CT/TT genotypes OR¼4.6, 95% CI 0.4– effect of rs1049353 genotype on schizo- pants who first used cannabis at least 1 53.0; interaction likelihood ratio test as phrenia between those who did not use year prior to their illness onset and who above,above, PP¼0.65). Tobacco use was not asso- cannabis and those who claimed to have had first used it by age 18 years or earlier ciated with –86C/T genotype (OR¼0.9,0.9, used cannabis at least 1 year prior to illness ((nn¼338; OR338;OR¼0.76, 95% CI 0.41–1.40; 95% CI 0.5–1.5). onset (onset(nn¼614; OR614;OR¼0.84, 95% CI 0.40– PP¼0.38). Similarly, there was no evidence Results for associations between –86C/T 1.78; interaction ww22¼0.26, d.f.0.26,d.f.¼1,1, PP¼0.61).0.61). that variation at rs737865 or rs165599 genotype and various phenotypes within Presenting these data in another way, was associated with cannabis use in the schizophrenia are presented in Table 2. there was a strong association between case-only analysis, even when restricting There was weak evidence (PP¼0.07) that0.07)that cannabis use and schizophrenia in this the analysis to first use of cannabis at least participants with the CT/TT genotypes sample (OR¼2.6, 95% CI 1.8–3.7; 1 year prior to illness onset and first use by had a younger age of onset, by approxi- PP550.001), with no evidence of any differ- age 18 years or earlier (rs737865, mately 2 years on average, than those ence when stratified by rs1049353 geno- OROR¼1.09, 95% CI 0.56–2.00; rs165599, homozygous for the C allele. type (GG genotype OR¼2.3, 95% CI 1.2– OROR¼1.09, 95% CI 0.57–2.08). There was 4.4; GA genotype OR¼3.1, 95% CI 1.3– no evidence of overall haplotype associa- CNR1CNR1 7.2; AA genotype OR¼1.1, 95% CI 0.2– tion with cannabis use (ww22¼4.7, d.f.4.7,d.f.¼7,7, Genotypes at rs1049353 were in Hardy– 4.6; interaction likelihood ratio test as PP¼0.69) or of specific association with the Weinberg equilibrium in both the schizo- above,above, PP¼0.33). There was no evidence rs737865–4680–165599 GGG haplotype phrenia group (ww22¼ 0.56,0.56, PP¼0.44) and0.44)and for any association between rs1049353 ((ww22¼0.001, d.f.0.001,d.f.¼1,1, PP¼0.98).0.98). controls (controls(ww22¼1.0,1.0, PP¼0.36). As shown in genotype and various phenotypes within Table 1, there was no evidence for any schizophrenia (Table 2). DISCUSSION association between rs1049353 genotype and schizophrenia (odds ratio for linear COMT There was no evidence for any association trend of genotypes 0.97, 95% CI 0.82– There was no evidence for any association betweenbetween CHRNA7CHRNA7 oror CNR1CNR1 genotypegenotype 1.13;1.13; PP¼0.66).0.66). between Val158158MetMetgenotypegenotype and cannabis and schizophrenia in our sample, and also There was little evidence of any differ- ence in the effect of rs1049353 genotype Ta b l e 22Tab Effect estimates for phenotype characteristics according to CHRNA7 (^86C/T) and CNR1 on schizophrenia between those who did (rs1049353) genotypes in participants with schizophrenia not use cannabis (schizophrenia group nn¼445, controls nn¼93; OR93;OR¼1.04, 95%1.04,95% ^86C/T11 rs104935322 CI 0.73–1.47) and those who did (schizo- phrenia group nn¼261, controls nn¼23;23; bb (95% CI)CI)(95% PP bb (95% CI)CI)(95% PP OROR¼0.92, 95% CI 0.48–1.75; interaction ww22¼0.11, d.f.0.11,d.f.¼1,1, PP¼0.74). As cannabis Age at onset33 770.06 (770.13 to 0.01) 0.070.070.01 (770.02 to 0.05)0.43 use data were again available for only a GAS scorescoreGAS 770.9 ((0.9 772.7 to 0.9)0.34 0.02 (770.86 to 0.90)0.97 small proportion of the control group, a Paranoid delusions 770.05 ((0.05 770.3 to 0.2) 0.700.70 770.09 ((0.09 770.21 to 0.03)0.03)0.21 0.140.14 case-only analysis was used, and this also Disorganised symptoms0.11 (770.1 to 0.4)0.40 0.07 (770.05 to 0.19)0.26 failed to provide any evidence for inter- Negative symptoms 0.01 (770.2 to 0.3)0.3)0.2 0.96 0.03 (770.09 to 0.15)0.61 action (action(nn¼706; odds ratio for cannabis First-rank delusions 770.01 (770.3 to 0.2) 0.910.91 770.03 (770.15 to 0.09)0.62 use byuseby CNR1CNR1 genotype 0.83, 95% CI Poor outcome OR¼1.30 (0.8 to 2.1)0.29 OR ¼1.03 (0.81 to 1.29)0.83 0.65–1.05). As part of the sensitivity analysis, there GAS,Global Assessment Scale. 1. Effect estimate is comparing CC with CT/TT genotypes for ^86C/T. were 71 individuals in the schizophrenia 2. Effect estimate is per A allele for CNR1.. group with data relating to age of first 3. ln transformed.

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no evidence of any gene–environment in- schizophrenia is mediated by impairments by age 18 years, but not for those using it teractions between tobacco use and in sensory gating or other related physiolo- after this age (Caspi et aletal, 2005). One ex- CHRNA7CHRNA7, or cannabis use and CNR1CNR1 oror gical responses. In the study by Leonard etet planation proposed for this was that there COMTCOMT genotype.genotype. alal (2002), presence of the T allele at – may exist a sensitive or even critical period 86C/T was also associated with reduced in- of risk when the influence of cannabis ex- hibition of the P50 response in the control posure is moderated by COMTCOMT genotype.genotype. CHRNA7 and tobacco use group, and although two other studies did In our study we failed to find evidence for There have been few association studies of not replicate this finding (Gault et aletal,, an interaction between cannabis use and polymorphisms within CHRNA7CHRNA7 andand 2003; Houy et aletal, 2004), one reported an COMTCOMT genotype even when restricting the schizophrenia to date. Leonard et aletal association between P50 sensory gating re- analysis to participants who claimed to (2002) screened the core promoter region sponse and another promoter variant, – have first used cannabis by the same cut- of the full-length gene and reported an 194G/C (Houy et aletal, 2004). There is a clear off period of age 18 years, despite more association between schizophrenia and need for research into CHRNA7CHRNA7 variationvariation than adequate statistical power to replicate variant –86C/T. Although we found no in relation to neurophysiological deficits the original findings. Furthermore, in con- evidence for an association between the in well-designed and adequately powered trast to the findings by Caspi et aletal (2005),(2005), promoter SNP –86C/T and schizophrenia, studies to address this further. cannabis use by age 18 years was actually CT/TT genotypes occurred slightly more less common in participants with schizo- frequently in participants with schizo- phrenia homozygous for the Val allele phrenia than in controls, in a direction con- CNR1CNR1,, COMT and cannabis compared with those heterozygous for this sistent with the findings by Leonard et aletal We found no evidence of association be- allele or homozygous for Met (5.3%, (2002). However, we observed a much tween thetweenthe CNR1CNR1 locus rs1049353 and 6.4% and 8.7% respectively),although this smaller difference in CC frequency of less schizophrenia, consistent with the overall was not significantly different. than 1%, as opposed to the 7% reported findings previously reported for this variant in the original study (Leonard et aletal, 2002).,2002). from two much smaller studies (Leroy et aletal,, People with schizophrenia commonly 2001; Ujike et aletal, 2002), although one of Limitations to the interpretation of display evidence of sensory attention im- these reported an association in a subgroup our results pairments (Adler et aletal, 1982; Leonard etet analysis (Leroy et aletal, 2001). Two studies This study was adequately powered to ex- alal, 1996), including deficits in pre-pulse in- have reported associations between schizo- amine main effects on risk of schizo-

hibition and P50 gating response (Braff & phrenia and variation within an (AAT)nn phrenia, suggesting it is unlikely that Saccuzzo, 1985; Braff et aletal, 1992; Waldo microsatellite approximately 20 kb up- variations in CNR1CNR1 oror CHRNA7CHRNA7 areare et aletal, 1994). Improvements in such neuro- stream of the translational start site of important risk factors for schizophrenia. physiological deficits in people with schizo- CNR1CNR1 (Ujike(Ujike et aletal, 2002; Martinez-Gras Furthermore, this study was adequately phrenia following cigarette smoking have et aletal, 2006). However, different alleles were powered for studies of interactions using a been reported (Adler et aletal, 1993; Olincy etet associated with increased risk in these two case-only design, but this approach is alal, 1998), with similar improvements ob- studies, and the association in one of the dependent on the assumption of no served following nicotine administration studies was again observed only for a genotype–exposure association in the popu- in animal models (Bickford & Wear, subgroup of participants, this time with lation. Forlation.For COMTCOMT this assumption is likely 1995; Stevens et aletal, 1996, 1998). Specific hebephrenic schizophrenia. to be a reasonable one, given that no asso-

agonists of the aa77 receptor (CHRNA7) nor- TheThe CNR1CNR1 gene is located on 6q14–15, ciation with cannabis use was observed in malise sensory gating deficits in animal a region of replicated linkage for schizo- the Dunedin cohort (Caspi et aletal, 2005).,2005). models (Stevens et aletal, 1998), whereas evi- phrenia (Lewis et aletal, 2003). There are four However, this assumption may be less dence for genetic linkage to the P50 deficit SNPs within CNR1CNR1 on HapMap that have likely to hold true for CNR1CNR1 oror CHRNA7CHRNA7,, and, to a lesser extent, to schizophrenia, a heterozygosity in European populations given that cannabis and nicotine act has been reported for band greater than 0.1; three of these are in the through receptors coded for by these genes, 15q14, an area that contains CHRNA7 33’’ untranslated region whereas rs1049353 and also given the sporadic reports of asso- (Coon(Coon et aletal, 1993; Freedman et aletal, 1997;,1997; is a synonymous SNP found within exon ciations between cannabis and tobacco de- LeonardLeonard et aletal, 1998).,1998). 1. The relatively small size of CNR1CNR1, the,the pendence and CNR1CNR1//CHRNA7CHRNA7 genotypesgenotypes Despite this support, from a variety of limited variation within the gene and its (Greenbaum et aletal, 2006; Hopfer et aletal,, sources, that CHRNA7CHRNA7 is a good candidate linkage disequilibrium structure mean it is 2006). For that reason we also conducted gene for schizophrenia, there is weak evi- unlikely that a substantial effect on schizo- studies of interactions between CNR1CNR1 andand dence at present that variation within this phrenia risk is conferred by variation with- cannabis as well as between CHRNA7CHRNA7 gene is associated with the disorder (Riley in this gene, given our findings and the lack and tobacco using a more traditional et aletal, 2000; XuXu,2000; et aletal, 2001; Leonard et aletal,, of other consistent associations reported to case–control approach, although statistical 2002; Gault et aletal, 2003; LiLi,2003; et aletal, 2004;,2004; date.date. power to exclude anything other than large FanFan et aletal, 2006). However, given the We also failed to find any supporting interaction effects for these two genes using findings from experimental studies of the evidence for a differential effect of cannabis this latter approach was limited. effect of nicotine on neurophysiological use on psychosis risk according to variation Although we genotyped three SNPs in deficits in both animal models and humans, at Val158158Met within COMTCOMT. In the Dunedin COMTCOMT that together form a haplotype re- as described earlier, it may be that any study evidence for an interaction was ob- ported to be significantly associated with association between CHRNA7CHRNA7 andand served only for people first using cannabis schizophrenia (Shifman et aletal, 2002), we

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only genotyped one SNP in each of CNR1CNR1 longitudinal studies may be able to investi- Coon, H., Plaetke, R., Holik, J., et aletal (19 93) Use of aaUse andand CHRNA7CHRNA7. It is not possible therefore gate this with greater confidence in the neurophysiological trait in linkage analysis of schizophrenia. Biological Psychiatry,, 3434, 277^289.,277^289. to rule out causal effects of variants within future.future. these genes that are not in strong linkage In summary, we failed to find any evi- Dudbridge, F. (2003) Pedigree disequilibrium tests for multilocus haplotypes. Genetic Epidemiology,, 2525,115^121. disequilibrium with the SNPs we tested. dence that variation at the CHRNA7CHRNA7 oror However, a strong effect of CNR1CNR1 on riskonrisk CRN1CRN1 locus was associated with schizo- Endicott, J., Spitzer, R. L., Fleiss, J. L., et aletal (19 76) The Global Assessment Scale. A procedure for of schizophrenia seems unlikely, given the phrenia, or that the effect of variation at measuring overall severity of psychiatric disturbance. linkage disequilibrium structure within this these loci was modified by use of tobacco Archives of General Psychiatry,, 33, 766^771.,766^771. gene. Our confidence in ruling out such an or cannabis respectively. Cannabis use Fan,J.B.,Ma,J.,Li,X.W.,Fan, J. B., Ma, J., Li, X.W., et aletal (2006) Population- effect foreffectfor CHRNA7CHRNA7 is lower, although we was not associated with presence of the va- based and family-based association studies of an (AC)n did not feel that the evidence we obtained line allele at Val158158Met within COMTCOMT inin dinucleotide repeat in alpha-7 nicotinic receptor subunit was strong enough to warrant further geno- our sample, therefore our findings do not gene and schizophrenia. Schizophrenia Research,, 84,, 222^227. typing oftypingof CHRNA7CHRNA7 SNPs, especially given not support a previous report of a putative the problems resulting from the partial gene–environment interaction between Freedman, R., Coon, H., Myles-Worsley, M., et al (19 9 7) Linkage of a neurophysiological deficit in duplication of this gene, which makes such COMTCOMT genotype and cannabis use on risk schizophrenia to a locus. Proceedings of studies inherently more difficult. of schizophrenia. the National Academy of Sciences of the USA,, 9494,, A final limitation of our study is that, 587^592.587^592. unlike the longitudinal data collection in ACKNOWLEDGEMENTS Gault, J., Hopkins, J., Berger, R., et aletal (2003) the Dunedin cohort, our case–control de- Comparison of polymorphisms in the alpha7 nicotinic receptor gene and its partial duplication in schizophrenic sign relied on people recalling age of first S.Z. is funded through a Clinician Scientist Award and control subjects. American Journal of Medical use of cannabis and relating this in time from the National Assembly for Wales. Genetics. Part B, Neuropsychiatric Genetics,, 123, 39^49.,39^49. to the date of their first contact with psychi- Gray, R., Rajan, A. S., Radcliffe, K. A., et aletal (19 9 6) atric services. Such data seem inherently REFERENCES Hippocampal synaptic transmission enhanced by low more likely to be misclassified than pro- concentrations of nicotine. Nature,, 383383, 713^716.,713^716. Adler, L. E., Pachtman, E., Franks, R. D., et aletal (19 82) spectively collected data. It is unclear to Greenbaum, L., Kanyas, K., Karni, O., et aletal (2006) Neurophysiological evidence for a defect in neuronal what extent any such misclassification Why do young women smoke? I. Direct and interactive mechanisms involved in sensory gating in schizophrenia. effects of environment, psychological characteristics and might have resulted in an underestimate of Biological Psychiatry,, 1717, 639^654.,639^654. nicotinic cholinergic receptor genes. Molecular Psychiatry,, the association between cannabis use and Adler,L.Adler, L . E.,E. , Hoffer, L.L . D.,Wiser,D. , Wis er, A.,A. , et aletal (19 93) 11, 312^32312^322. 2. genotype in our case-only analysis, and Normalization of auditory physiology by cigarette Henquet, C., Rosa, A., Krabbendam, L., et aletal (2006) smoking in schizophrenic patients. American Journal of therefore obscured any true interaction ef- An experimental study of catechol-o-methyltransferase PsychiatryPsychiatry,, 150,1856^1861.,1856^1861. fect. It would, however, presumably require Val158Met moderation of delta-9-tetrahydrocannabinol- a substantial amount of misclassification to American Psychiatric Association (1994) Diagnostic induced effects on psychosis and cognition. obscure an interaction effect as strong as and Statistical Manual of Mental Disorders (4th edn)edn)(4th Neuropsychopharmacology,, 3131, 2748^2757. that reported by Caspi and colleagues, (DSM^IV). APA. Hopfer,C. J.,Young, S. E., Purcell, S., et aletal (2006)(2006) Cannabis receptor haplotype associated with fewer whereby cannabis use was associated with Arseneault, L., Cannon, M., Poulton, R., et aletal (2002) cannabis dependence symptoms in adolescents. Cannabis use in adolescence and risk for adult psychosis: a 10-fold increase in risk of psychotic dis- American Journal of Medical Genetics. Part B, longitudinal prospective study. BMJBMJ,, 325325,1212^1213. order in those homozygous for valine but Neuropsychiatric Genetics,, 141141,,895^901. 895^901. Bickford,Bickford,P.C.&Wear,K.D.(1995) P.C. & Wear, K. D. (1995) Restoration of had no effect in those homozygous for Houy, E., Raux, G.,G.,Thibaut, Thibaut, F., et aletal (2004)(2004) TheThe sensory gating of auditory evoked response by nicotine methionine (Caspi et aletal, 2005). This finding promoter ^194 C polymorphism of the nicotinic alpha 7 in fimbria-fornix lesioned rats. Brain Research,, 705705,, receptor gene has a protective effect against the P50 of an interaction effect in the Dunedin co- 235^240. sensory gating deficit. Molecular Psychiatry,, 99, 320^322. hort was observed only in a subgroup of Braff, D. L. & Saccuzzo, D. P. (1985) The time course Khoury, M. J. & Flanders,W. D. (1996) Nontraditional participants – those using cannabis by age of information-processing deficits in schizophrenia. 18 years. Similarly, supportive evidence of epidemiologic approaches in the analysis of gene ^ American Journal of Psychiatry,, 142142,170^174.,170^174. environment interaction: casecase^control ^ control studies with no a putative interaction between cannabis controls! American Journal of Epidemiology,, 144144, 207^213.,207^213. Braff, D. L., Grillon, C. & Geyer, M. A. (1992) Gating use anduseand COMTCOMT on psychotic symptoms, and habituation of the startle reflex in schizophrenic Leonard, S., Adams, C., Breese, C. R., et aletal (19 9 6) following administration of cannabis in an patients.patients. Archives of General Psychiatry,, 49, 206^215.,206^215. Nicotinic receptor function in schizophrenia. experimental setting was again observed Schizophrenia Bulletin,, 2222, 431^445.,431^445. Bray, N. J., Buckland, P. R.,Williams, N. M., et aletal only in a subgroup of participants with (2003) A haplotype implicated in schizophrenia Leonard, S., Gault, J., Moore,Moore,T., T., et aletal (19 9 8) Further schizophrenia, this time those with evi- susceptibility is associated with reduced COMT investigation of a chromosome 15 locus in schizophrenia: dence of pre-existing psychotic traits expression in human brain. American Journal of Human analysis of affected sibpairs from the NIMH Genetics GeneticsGenetics,, 7373,152^161. Initiative. American Journal of Medical Genetics,, 81,, (Henquet(Henquet et aletal, 2006). Although such find- 308^312. ings are biologically plausible and seem Caspi, A., Moffitt, T. E., Cannon, M., et aletal (2005)(2005) Moderation of the effect of adolescent-onset cannabis intuitively appealing, substantially more Leonard, S., Gault, J., Hopkins, J., et aletal (2002)(2002) use on adult psychosis by a functional polymorphism in Association of promoter variants in the alpha7 nicotinic evidence from replication of these findings the catechol-O-methyltransferase gene: longitudinal subunit gene with an inhibitory is required. Our study, although providing evidence of a gene66environment interaction. Biological deficit found in schizophrenia. Archives of General adequate power to observe even a relatively PsychiatryPsychiatry,, 5757,,1117^1127. 1117 ^ 112 7. PsychiatryPsychiatry,, 5959,1085^1096. small association between cannabis use and Chen, J., Lipska, B. K., Halim, N., et aletal (2004)(2004) Leroy, S., Griffon, N., Bourdel, M. C., et aletal (2001)(2001) COMTCOMT genotype in participants with Functional analysis of genetic variation in catechol-O- Schizophrenia and the cannabinoid receptor type 1 methyltransferase (COMT): effects on mRNA, , (CB1): association study using a single-base schizophrenia, may not be the ideal design and enzyme activity in postmortem human brain. polymorphism in coding exon 1. American Journal of to examine such a relationship, and other American Journal of Human Genetics,, 7575, 807^821.,807^821. Medical Genetics,, 105105, 749^752.,749^752.

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AUTHOR’ S P ROOF

Lewis, C. M. (2002) Genetic association studies: design, analysis and interpretation. Briefings in Bioinformatics,, 33,, STANLEY ZAMMIT,ZAMMIT,PhD,GILLIANSPURLOCK PhD, GILLIAN SPURLOCK,,PhD,HYWELWILLIAMS PhD, HYWEL WILLIAMS,,PhD,NADINENORTON PhD, NADINE NORTON,,PhD, PhD, 146^153.14 6 ^ 153. NIGEL WILLIAMS,WILLIAMS,PhD,MICHAELC.O’DONOVAN PhD, MICHAEL C. O’DONOVAN,,PhD,FRCPsych,MICHAELJ.OWEN PhD, FRCPsych, MICHAEL J. OWEN,,MB,PhD, MB, PhD, Lewis, C. M., Levinson, D. F.,Wise, L. H., et aletal (2003) Department of Psychological Medicine,Cardiff University,Cardiff,UK Genome scan meta-analysis of schizophrenia and bipolar disorder, part II: Schizophrenia. American Journal of Correspondence:Correspondence: Dr Stanley Zammit,Department of Psychological Medicine,Cardiff University,Heath Human Genetics,, 7373,34^48.,34^48. Park,Cardiff CF14 4XN,UK.Tel: +44 (0)2920 743058; fax: +44 (0)2920 747839; email: @@ Li, C. H., Liao, H. M. & Chen, C. H. (2004) zammitszammits Cardiff.ac.uk Identification of molecular variants at the promoter region of the human alpha 7 neuronal nicotinic (First received 25 January 2007, final revision 30 May 2007, accepted 6 July 2007) acetylcholine receptor subunit gene but lack of association with schizophrenia. Neuroscience Letters,, 372,, 1^5.1^5. schizophrenia in Southern African Bantu families. Waldo, M. C., Cawthra, E., Adler, L. E., et aletal (19(1994) 94) Martinez-Gras, I., Hoenicka, J., Ponce, G., et aletal American Journal of Medical Genetics,, 96,196^201.,196^201. Auditory sensory gating, hippocampal volume, and (2006)(2006) (AAT)n repeat in the cannabinoid receptor gene, catecholamine metabolism in schizophrenics and their CNR1: association with schizophrenia in a Spanish Shifman, S., Bronstein, M., Sternfeld, M., et aletal siblings. Schizophrenia Research,, 12,93^106., 93^106. population.population. European Archives of Psychiatry and Clinical (2002) A highly significant association between a Neuroscience,, 256, 437^441.,437^441. COMT haplotype and schizophrenia. American Journal of Williams, H. J., Glaser, B.,Williams,B., Williams, N. M., et aletal Human Genetics,, 7171,1296^1302. (2005)(2005) No association between schizophrenia and McGuffin, P., Farmer, A. & Harvey, I. (1991) AA polymorphisms in COMT in two large samples. polydiagnostic application of operational criteria in Stevens, K. E., Freedman, R., Collins, A. C., et aletal American Journal of Psychiatry,, 162162,1736^1738., 1736^1738. studies of psychotic illness. Development and reliability (19 9 6) Genetic correlation of inhibitory gating of of the OPCRIT system. Archives of General Psychiatry,, 4848,, hippocampal auditory evoked response and alpha- Wing, J. K., Babor,T., Brugha, T., et aletal (19 9 0)0)(19 SCAN. 764^770. bungarotoxin-binding nicotinic cholinergic receptors in Schedules for Clinical Assessment in Neuropsychiatry. inbred mouse strains. Neuropsychopharmacology,, 15,, Archives of General Psychiatry,, 4747, 589^593.,589^593. Olincy, A., Ross, R. G.,Young, D. A., et aletal (19 9 8) 152152^162. ^162. Improvement in smooth pursuit eye movements after Xu, J., Pato, M.T.,Torre,C. D., et aletal (2001)(2001) Evidence cigarette smoking in schizophrenic patients. Stevens, K. E., Kem,W. R., Mahnir,V.M., et aletal (19 9 8)8)(19 for linkage disequilibrium between the alpha 7-nicotinic Neuropsychopharmacology,, 1818,175^185., 175^185. Selective alpha7-nicotinic agonists normalize inhibition of receptor gene (CHRNA7) locus and schizophrenia in Owen, M. J., Holmans, P. & McGuffin, P. (1997) auditory response in DBA mice. Psychopharmacology,, Azorean families. American Journal of Medical Genetics,, Association studies in psychiatric genetics. MolecularMolecular 136136,,320^327. 320^327. 105105, 669^674.,669^674. PsychiatryPsychiatry,, 22, 270^273. Ujike, H., Takaki, M., Nakata, K., et aletal (2002)(2002) CNR1,CNR1, Zammit, S., Allebeck, P., Andreasson, S., et al Riley, B. P., Makoff, A., Mogudi-Carter, M., et aletal central cannabinoid receptor gene, associated with (2002)(2002) Self reported cannabis use as a risk factor for (2000)(2000) Haplotype transmission disequilibrium and susceptibility to hebephrenic schizophrenia. Molecular schizophrenia in Swedish conscripts of 1969: historical evidence for linkage of the CHRNA7 gene region to Psychiatry,, 77,,515^518. 515^518. cohort study. BMJBMJ,, 325325,1199.

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