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Cys311 DRD2 molecular variant with schizophrenia using a classical case control study and the haplotype relative risk
SCHIZOPHRENIA RESEARCH ELSEVIER
Schizophrenia Research 25 ( 1997) 117- 121
No association of the Ser/Cys3 11 DRD2 molecular variant with schizophrenia using a classical case control study and the haplotype relative risk Massimiliano Verga a, Fabio Macciardi a,*, Silvia Pedrini a, Susanna Cohen a, Enrico Smeraldi b a Istituto Scientific0 H. San ~uffae~e, ~epffrt~e~i of ~e~r~ps~c~~~~rie Science,~~~versi~.v of Milan, Aiiilan,i&f b School of Medicine, University of Milan, Milan, Italy
Received1 April 1996;accepted10 January 1997
Abstract Arinami et al. (1994) reported an associationbetween the Ser31l/Cys311 variant of the DRD2 gene and schizophreniain a Japanesepopulation. We did not find statistically significantdifferencesin the distribution of the allelefrequenciesbetweenschizophrenics(103) and controls (97) in a case-controlsample(x2=2.07; p =0.150) or in 64 nuclear familieswith the haplotype relative risk (HRR) design{x2 =O. 13;p=O.718). Our resultsseemto exclude a main involvement of this variant of the dopamineD2 receptor genein the etiopathogenesis of schizophrenia. Keywords:
Associationstudy; DopamineD2 receptor gene;Haplotype relative risk; Schizophrenia
1. Introduction
The human DRD2 gene has been cloned and mapped to llq22-23 (Grandy et al., 1989) and it can be considered as a ‘Candidate Gene’, because it has been suggested that a defect of the D2 receptor gene might be a factor in the etiology of schizophrenia (Seeman, 1987). Itokawa et al. (1993) reported a novel polymorphism causing a structural change from serine to cysteine at codon 3 11 of DRD2. This substitution + Corresponding author. Department of Neuropsychiatric Science. Istituto Scientific0 H San Raffaele, University of Milan, School of Medicine, Via Luigi Prinetti 29. 20127 Milan, Italy. Tel.: +39 2 26433224; fax: +39 2 26433265; e-mail: macciard~dsnp.hsr.it 0920-9964/97/517.00 0 1997 Elsevier Science B.V. All rights reserved PII SO920-9964(97)00013-3
was the first sequence variation described in the coding sequence of human dopamine D2 receptor gene and might be of relevance to schizophrenia. Arinami et al. ( 1994) reported that allele frequency of Cys3 11 in Japanese schizophrenics was significantly higher (5.4%) than in controls (1.8%). Moreover, the frequency of the Cys311 variant was highest among patients with onset before age 25 and with a positive family history and three patients were homozygous for Cys31 I, suggesting that the Cys311 homozygosity may substantially increase susceptibility to the disease. However, the patients with Cys311 also showed significantly less severe thought disorder and negative symptoms of the disease than those without Cys311. Up to now, this finding has not been replicated
118
A4. Verga et al. / ~~h~zophren~a
(Asherson et al., 1994; Gejman et al., 1994; Hattori et al., 1994; Laurent et al., 1994; Nanko et al., 1994; Nothen et al., 1994; Sasaki et al., 1996; Chen et al., 1996) and only Shaikh et al. (1994) showed a significant association (p=O.O17) between the Cys311 mutation and schizophrenia exclusively in a group of unselected Caucasian schizophrenics with unknown family history of the disease. To replicate the Arinami et al. ( 1994) finding in an Italian pop~ation, we evaluated the DRD2 genotypes in a first sample of 103 unrelated schizophrenic patients and 97 controls in a classical casecontrol study, and in a second sample of 64 nuclear families following the haplotype relative risk strategy (HRR).
2. Material
and methods
2. I. Sample
In a classical case-control study, we investigated 103 schizophrenics (61 males and 42 females) recruited from hospitalized patients at the Department of Neuropsychiatric Science of the S. RafIaele Hospital in Milan and 97 healthy controls (48 males and 49 females). The mean age of patients was 37.9 (SD, lo.%), mean age of onset 22.8 (SD, 7.11) while for controls mean age was 53.25 (SD, 16.48). After obtaining informed consent, both parents of 64 additional schizophrenic patients were recruited in order to apply the HRR strategy. All the patients were administered the Diagnostic Interview Schedule, revised version (DIS-R) (Robins et al., 1989; Battaglia et al., I995) and have been diagnosed according to DSMIII-R criteria (American Psychiatric Association, 1987). The controls of the case-control study were both staff members of S. Raffaele Hospital and healthy subjects attending the general hospital laboratories for screening blood testing and randomly selected from the general population. Each control subject had been investigated for the presence of a psychiatric history, for the presence of schizophrenia in his/her first-degree relatives and had been given at least a general medical screening for major illnesses: only subjects that were negative
Research
25 ( 1997j II 7-121
to these investigations have been recruited as controls. All participants in the study were native to Northern Italy. (This sample is part of the EMASS: European Multi-Centre Association Study of the Mol~ular Genetics of ~hizophrenia.) 2.2. DNA analysis
Genomic DNA was prepared from peripheral blood as described in Lahiri and Nurnberg ( 199 1) . PCR-based genotyping of Ser3 1 l/Cys3 11 variant was carried out according to the method described by Arinami et al. ( 1994). PCR products were digested with Saul restriction enzyme. The digested PCR products were resolved in 3.5% agarose gel by electrophoresis, and visualized with ethidium bromide staining under ultraviolet light. The Ser311 allele shows bands of 126,92,53 and 23 bp, whereas the Cys311 allele shows bands of 149, 92 and 53 bp. 2.3. Statistical analysis
We used the experimental design described by Terwilliger and Ott ( 1992) for the HRR strategy to test for deviations from linkage disequilib~um, considering the genotype of the affected offspring as the ‘case’ and the genotype made up of the alleles non-transmitted to the child from its parents as the control sample in an association test (genotype-based haplotype relative risk, GHRR). Under the null hypothesis of no linkage diseq~lib~~ (6=0), the two parental genotypes are independent, and therefore it is possible to consider simply the fate of each parental marker allele instead of the parental genotypes (haplotype-based haplotype relative risk, HHRR). Tr~s~tted and nontransmitted alleles are independently associated under 6 =O, and thus a 2 x 2 table can be constructed with the transmitted and non-transmitted samples: a standard X*-test of independence on this table is a valid test of the hypothesis 6 = 0.
3. Results We confirmed that the Cys311 variant also occurs in our Caucasian pop~ation. Although in
M.
Verga et al. 1 Schizophrenia
the sample of unrelated schizophrenic patients the allele frequency of Cys3 11 was higher (5.3%) than in the control group (2.5%) our results do not show a statistically significant difference in the alleles distribution between the two groups (Table 1). Moreover, we did not hnd significant differences in Cys311 frequency between the control group and patients who developed schizophrenia before the age of 25 or had a family history of the disease (data not shown). Table 1 also presents our results together with the other published reports. With the purpose of making a simple inspection of the results so far obtained, we evaluated whether the risk to develop the disease is differentially increased in Caucasian
Research
25 (1997)
119
I1 7-121
and in non-Caucasian populations due to the Cys3 11 presence. There is a great variability of the observed odds ratios (OR) both in non-Caucasian (range, 0.61-3.09) and in Caucasian (range, 0.61-4.85) populations with the higher OR in Arinami’s (3.09), Laurent’s (2.75) and Shaikh’s (4.85) studies with a not sign&ant test of heterogeneity within the two populations. However, there is also a great variability of the Confidence Interval for the odds ratios, but only in the Arinami’s report is the OR consistently higher than 1 (95% CI = 1.44-6.39). The overall x2 for non-Caucasian (x2= 10.36; df=5; p=O.O6) and for Caucasian (x2 = 7.309; df = 7; p =0.39) populations separately are not significant. Additionally, we also pooled
Table 1 Dopamine D2 receptor genotypes and allele frequencies of Cys311 in case-control studies Authors
References
Genotypes
cys311 frequency
ss
SC
cc
46 105 142 289 93 93 93 93 110 83
4 5 11 11 7 7 7 6 4 5
0 0 3 0 0 0 0 1 0 0
0.023 0.054 0.018 0.035 0.035 0.035 0.040 0.017 0.028
110 63 103 109 108 181 175 133 140 99 261 245 92 92
2 1 3 4 5 3 4 5 7 1 12 9 11 5
0 0 0 0 0 0 0 0 0 0 0 1 0 0
0.008 0.007 0.014 0.017 0.022 0.008 0.011 0.018 0.023 0.005 0.022 0.017 0.053 0.025
p
Naa~-CPucPsinopopulatim
Itokawa et al. (1993)
Biochem. Biophys. Res. Commun. 196(3), 1369-1375
Pts. Ctrl.
Arinami et al. (1994)
Lancet 343, 703-704
PtS.
Nanko et al. (1994)
Lancet 343(8904), 1044
Hattori et al. (1994)
Biochem. Biophys. Res, Commun. 202(2), 7577763
Chen et al. ( 1996)
Am. J. Med. C&et. 67.418420
Ctrl. Pts. Ctrl. Pts. Ctrl. PtS.
Ctrl. Asherson et al. (1994)
LanW 343(8904), 1045
PtS.
Ctrl. Gejman et al. (1994)
J. Am. Med. Assoc. 271, 204208
PtS.
Laurent et al. (1994)
Psychiat. Genet. (4), 229-230
Ctrl. Pts. Ctrl.
Nothen et al. ( 1994)
Lancet 343(8908), 1301
PtS.
Shaikh et al. (1994)
Lancet 343(8904), 1045
Ctrl. Pts. Ctrl.
Sasaki et al. (1996)
Am. J. Med. Genet. 67, 415417
PtS.
This report
Ctrl. Pts. Ctrl.
Verga et al.
0.040
S, Ser311; C. Cys311. “p values range from CO.01 to
120
M. Verga et al. / Schizophrenia
results from all the studies together, but again the overall x2 is not significant and the OR is equal to 1.42. In our nuclear families, standard X2-test does not reveal any statistically significant association between schizophrenia and the DRD2 genotypes using the GHRR (genotype-based-haplotype relative risk) design (Table 2(a)). Following the hypothesis that an allele, more than a genotype, could be responsible for a putative association with the disease, we explored this hypothesis again using the HHRR (haplotypebased- haplotype relative risk) design, but again we did not find any association between the DRD2 alleles and schizophrenia (Table 2(b)).
4. Discussion
Our results seem to exclude a main involvement of the Ser3 1l/Cys311 variant of the dopamine D2 receptor in the etiopathogenesis of schizophrenia. This finding in an Italian population is in agreement with other negative reports carried out in different Caucasian and non-Caucasian populations (Table 1). The observed frequency for the Cys311 allele in our schizophrenic patients (5.3%) is similar to Arinami’s (5.4%), while in our respective control groups the difference is more marked (2.5 versus 1.8%). As a first consequence, the risk to develop schizophrenia given the Cys311 allele is increased both in Arinami (OR =3.09) and in our report (OR =2.13), but this effect is statistically significant only for the Japanese group. It might be possible that our sample size is relatively small, mostly if compared to Arinami’s, to detect Table 2 Distribution
of genotypic
and allele frequencies
for DRD2
Research
aXZ=0.53; p=O.466; bXZ=0.13;p=0.718;
df= 1. df=l.
117-121
a significant effect despite the interesting OR that would suggest a risk effect for the Cys311 allele also in our schizophrenic patients. However, in addition to the issue of the dimension of the sample, it is our opinion that we have to also consider other genetic factors when dealing with these types of controversial results obtained in different populations, especially in cases like the DRD2 gene, which shows a great variability also in relation to race and ethnicity (Castiglione et al., 1995). Another possibility is that the positive result obtained in non-Caucasian populations was a chance finding. The great variability of the observed odds ratios both in non-Caucasian and in Caucasian populations could be due to uncontrolled confounding factors specific for each population. Actually, the calculated OR for our HRR sample is quite low despite a good sample size, and this seems to exclude also a minor role of the DRD2 gene in our population. It is relevant that discrepant results also have been reported in alcoholism. Higuchi et al. (1994) found that the Cys3 11 variant is fairly well represented in alcoholic patients. He reported that Japanese alcoholics had the Cys311 allele significantly more often (6.3%) than the Japanese control group (2.9%), but no associations have been reported by Gejman et al. ( 1994) in a Caucasian population. Indeed, the best way of proving true associations between disease and genetic markers is to replicate the original finding, but our failure to replicate the positive association of the Cys3 11 allele and schizophrenia, also with a powerful strategy like the HRR approach, overcomes the potential pitfalls
and schizophrenia
Genotypes”
Transmitted Non-transmitted Total
25 i 1997)
using the haplotype
relative
risk
Allelesb
Ser311
cys3 11
Total
Ser3 11
cys311
Total
59 61 120
5 3 8
64 64 128
123 125 248
5 3 8
128 128 256
M. Verga et al. / Schizophrenia
of admixing populations or the other sources of biases as outlined above. Our lack of a significant result, which might nonetheless benefit of an additional confirmation in an independent and possible larger set of HRR nuclear families to more definitively detect or refute any association with this receptor variant, already casts doubts about a meaningful etiological role of the D2 dopamine gene in schizophrenia.
Acknowledgment
This work has been carried out within the activities of the EMASS (European Multi-Centre Association Study of the Molecular Genetics of Schizophrenia - Head: Prof. P. McGulhn) and of the Italian Research Group for Affective and Schizophrenic Disorders (IRGASD) under the auspices of the Janssen Research Council and Fondazione Italiana per lo Studio della Schizofrenia (FIS).
References American Psychiatric Association ( 1987) Diagnostic and Statistical Manual of Mental Disorders, 3rd Edn. revised. American Psychiatric Association, Washington. Arinami, T., Itokawa, M., Enguchi, H., Tagaya, H., Yano, S., Shimizu, H., Hamaguchi and Toru, M. (1994) Association of dopamine D2 receptor molecular variant with schizophrenia. Lancet 343, 703-704. Asherson, P., Williams, N., Roberts, E., McGulIin, M. and Owen, M. (1994) DRD2 Ser311 Cys311 polymorphism in schizophrenia. Lancet 343(8904), 1045. Battaglia, M., Bemardeschi, L., Franchini, L., Bellodi, L. and Smeraldi, E. (1995) A family study of schizotypal disorder. Schizophr. Bull. 21(l), 33-45. Cast&hone, C.M., Deinard, A.S., Speed, W.C., Sirugo, G., Rosenbaum, H.C., Zhang, Y., Grandy, D.K., Grigorenko, E.L., Bonne-Tamir, B., Pakstis, A.J., Kidd, J.R. and Kidd, K.K. (1995) Evolution of haplotypes at the DRD2 locus. Am. J. Hum. Genet. 57, 144551456. Chen, C.H., Chien, S.H. and Hwu, H.G. ( 1996) No association of dopamine D2 receptor molecular variant Cys311 and schizophrenia in Chinese patients. Am. J. Med. Genet. (Neuropsychiat. Genet.) 67, 418420. Gejman, P.V., Ram, A., Gelemter, J., Friedman, E., Cao, Q., Pickar, D., Bhrm, K., Noble, E.P., Kranzler. H.R., O’Malley,
Research
25 (1997)
117-121
121
S., Hamer, D.H., Whitsitt, F., Rao, P., DeLisi, L.E., Virkkunen, M., Linnoil, M., Goldman, D. and Gershon, E.S. ( 1994) No structural mutation in the dopamine D2 receptor gene in alcoholism or schizophrenia. Analysis using denaturing gradient gel electrophoresis. J. Am. Med. Assoc. 271, 204-208. Grandy, D.K., Litt, M., Allen, M., Bunzow, J.R., Marchionni, M., Makam, H., Reed, L., Magenis, R.E. and Civelli, 0. ( 1989) The human dopamine D2 receptor gene is located on chromosome 11 at q-22 q-23 and identifies a TaqI RFLP. Am. J. Hum. Genet. 45, 778-785. Hattori, M., Nanko, S., Dai, X.Y., Fukuda, R. and Kazamatsuri, H. (1994) Mismatch PCR RFLP detection of DRD2 Ser31lCys polymorphism and schizophrenia. B&hem. Biophys. Res. Commun. 29(202), 757-763. Higuchi, S., Muramatsu, T., Murayama, M. and Hayashida, M. (1994) Association of structural polymorphism of the dopamine D2 receptor gene and alcoholism. Biochem. Biophys. Res. Commun. 204( 3), 119991205. Itokawa, M., Arinami, T., Futamura, N., Hamaguchi, H. and Toru, M. ( 1993) A structural polymorphism of human dopamine D2 receptor, D2 (Ser311-Cys). B&hem. Biophys. Res. Commun. 196(3), 1369-1375. Lahiri, D.K. and Numberg, J.1. Jr. (1991) A rapid non-cnzymatic method for the preparation of HMW DNA from blood for RFLP studies. Nucleic Acids Res. 19, 5444. Laurent C., Bodeau-Pean, S., Campion, D., D’Amato, T., Jay, M., Dollfus, S., Thibault, F., Petit, M., Samolyk, D., Martinez, M. and Mallet, J. (1994) No major role for the dopamine D2 receptor Ser Cys311 mutation in schizophrenia. Psychiat. Genet. (4), 2299230. Nanko, S., Hattori, M., Dai, X.Y., Fukuda, R. and Kazamatsuri, H. (1994) DRD2 Ser311 Cys311 polymorphism in schizophrenia. Lancet 343(8904), 1044. Nothen, M.M., Wildenauer, D., Cichon, S., Albus, M., Maier, W., Minges, J., Lichtermann, D., Bondy, B., Rietschel, M., Komer, J., Fimmers, R. and Propping, P. (1994) Dopamine D2 receptor molecular variant and schizophrenia. Lancet 343(8908), 1301-1302. Robins, L., Helzer, J.E., Cottler, R. and Goldring, E. (1989) NIMH Diagnostic Interview Schedule: version III-R (DIS-R). Washington University School of Medicine, St. Louis, MO. Sasaki, T., Macciardi, F.M., Badri, F., Verga, M., Meltzer, H.Y., Lieberman, J., Howard, A., Bean, G., Joffe, R.T., Hudson, C.J. and Kennedy, J.L. ( 1996) No evidence for association of dopamine D2 receptor variant (Ser311 Cys311) with major psychosis. Am. J. Med. Genet. (Neuropsychiat. Genet.) 67, 415417. Seeman, P. (1987) Dopamine receptors and the dopamine hypothesis of schizophrenia. Synapse 1, 133-152. Shaikh, S., Collier, D., Arranz, M., Ball, D., Gill, M. and Kerwin, R. ( 1994) DRD2 Ser311 Cys311 polymorphism in schizophrenia. Lancet 343(8904), 104-1046. Terwilliger, J.D. and Ott, J. (1992) A haplotype-based ‘Haplotype Relative Risk’ approach to detecting allelic associations. Hum. Hered. 42, 337-346.
Schizophrenia Research 25 ( 1997) 117- 121
No association of the Ser/Cys3 11 DRD2 molecular variant with schizophrenia using a classical case control study and the haplotype relative risk Massimiliano Verga a, Fabio Macciardi a,*, Silvia Pedrini a, Susanna Cohen a, Enrico Smeraldi b a Istituto Scientific0 H. San ~uffae~e, ~epffrt~e~i of ~e~r~ps~c~~~~rie Science,~~~versi~.v of Milan, Aiiilan,i&f b School of Medicine, University of Milan, Milan, Italy
Received1 April 1996;accepted10 January 1997
Abstract Arinami et al. (1994) reported an associationbetween the Ser31l/Cys311 variant of the DRD2 gene and schizophreniain a Japanesepopulation. We did not find statistically significantdifferencesin the distribution of the allelefrequenciesbetweenschizophrenics(103) and controls (97) in a case-controlsample(x2=2.07; p =0.150) or in 64 nuclear familieswith the haplotype relative risk (HRR) design{x2 =O. 13;p=O.718). Our resultsseemto exclude a main involvement of this variant of the dopamineD2 receptor genein the etiopathogenesis of schizophrenia. Keywords:
Associationstudy; DopamineD2 receptor gene;Haplotype relative risk; Schizophrenia
1. Introduction
The human DRD2 gene has been cloned and mapped to llq22-23 (Grandy et al., 1989) and it can be considered as a ‘Candidate Gene’, because it has been suggested that a defect of the D2 receptor gene might be a factor in the etiology of schizophrenia (Seeman, 1987). Itokawa et al. (1993) reported a novel polymorphism causing a structural change from serine to cysteine at codon 3 11 of DRD2. This substitution + Corresponding author. Department of Neuropsychiatric Science. Istituto Scientific0 H San Raffaele, University of Milan, School of Medicine, Via Luigi Prinetti 29. 20127 Milan, Italy. Tel.: +39 2 26433224; fax: +39 2 26433265; e-mail: macciard~dsnp.hsr.it 0920-9964/97/517.00 0 1997 Elsevier Science B.V. All rights reserved PII SO920-9964(97)00013-3
was the first sequence variation described in the coding sequence of human dopamine D2 receptor gene and might be of relevance to schizophrenia. Arinami et al. ( 1994) reported that allele frequency of Cys3 11 in Japanese schizophrenics was significantly higher (5.4%) than in controls (1.8%). Moreover, the frequency of the Cys311 variant was highest among patients with onset before age 25 and with a positive family history and three patients were homozygous for Cys31 I, suggesting that the Cys311 homozygosity may substantially increase susceptibility to the disease. However, the patients with Cys311 also showed significantly less severe thought disorder and negative symptoms of the disease than those without Cys311. Up to now, this finding has not been replicated
118
A4. Verga et al. / ~~h~zophren~a
(Asherson et al., 1994; Gejman et al., 1994; Hattori et al., 1994; Laurent et al., 1994; Nanko et al., 1994; Nothen et al., 1994; Sasaki et al., 1996; Chen et al., 1996) and only Shaikh et al. (1994) showed a significant association (p=O.O17) between the Cys311 mutation and schizophrenia exclusively in a group of unselected Caucasian schizophrenics with unknown family history of the disease. To replicate the Arinami et al. ( 1994) finding in an Italian pop~ation, we evaluated the DRD2 genotypes in a first sample of 103 unrelated schizophrenic patients and 97 controls in a classical casecontrol study, and in a second sample of 64 nuclear families following the haplotype relative risk strategy (HRR).
2. Material
and methods
2. I. Sample
In a classical case-control study, we investigated 103 schizophrenics (61 males and 42 females) recruited from hospitalized patients at the Department of Neuropsychiatric Science of the S. RafIaele Hospital in Milan and 97 healthy controls (48 males and 49 females). The mean age of patients was 37.9 (SD, lo.%), mean age of onset 22.8 (SD, 7.11) while for controls mean age was 53.25 (SD, 16.48). After obtaining informed consent, both parents of 64 additional schizophrenic patients were recruited in order to apply the HRR strategy. All the patients were administered the Diagnostic Interview Schedule, revised version (DIS-R) (Robins et al., 1989; Battaglia et al., I995) and have been diagnosed according to DSMIII-R criteria (American Psychiatric Association, 1987). The controls of the case-control study were both staff members of S. Raffaele Hospital and healthy subjects attending the general hospital laboratories for screening blood testing and randomly selected from the general population. Each control subject had been investigated for the presence of a psychiatric history, for the presence of schizophrenia in his/her first-degree relatives and had been given at least a general medical screening for major illnesses: only subjects that were negative
Research
25 ( 1997j II 7-121
to these investigations have been recruited as controls. All participants in the study were native to Northern Italy. (This sample is part of the EMASS: European Multi-Centre Association Study of the Mol~ular Genetics of ~hizophrenia.) 2.2. DNA analysis
Genomic DNA was prepared from peripheral blood as described in Lahiri and Nurnberg ( 199 1) . PCR-based genotyping of Ser3 1 l/Cys3 11 variant was carried out according to the method described by Arinami et al. ( 1994). PCR products were digested with Saul restriction enzyme. The digested PCR products were resolved in 3.5% agarose gel by electrophoresis, and visualized with ethidium bromide staining under ultraviolet light. The Ser311 allele shows bands of 126,92,53 and 23 bp, whereas the Cys311 allele shows bands of 149, 92 and 53 bp. 2.3. Statistical analysis
We used the experimental design described by Terwilliger and Ott ( 1992) for the HRR strategy to test for deviations from linkage disequilib~um, considering the genotype of the affected offspring as the ‘case’ and the genotype made up of the alleles non-transmitted to the child from its parents as the control sample in an association test (genotype-based haplotype relative risk, GHRR). Under the null hypothesis of no linkage diseq~lib~~ (6=0), the two parental genotypes are independent, and therefore it is possible to consider simply the fate of each parental marker allele instead of the parental genotypes (haplotype-based haplotype relative risk, HHRR). Tr~s~tted and nontransmitted alleles are independently associated under 6 =O, and thus a 2 x 2 table can be constructed with the transmitted and non-transmitted samples: a standard X*-test of independence on this table is a valid test of the hypothesis 6 = 0.
3. Results We confirmed that the Cys311 variant also occurs in our Caucasian pop~ation. Although in
M.
Verga et al. 1 Schizophrenia
the sample of unrelated schizophrenic patients the allele frequency of Cys3 11 was higher (5.3%) than in the control group (2.5%) our results do not show a statistically significant difference in the alleles distribution between the two groups (Table 1). Moreover, we did not hnd significant differences in Cys311 frequency between the control group and patients who developed schizophrenia before the age of 25 or had a family history of the disease (data not shown). Table 1 also presents our results together with the other published reports. With the purpose of making a simple inspection of the results so far obtained, we evaluated whether the risk to develop the disease is differentially increased in Caucasian
Research
25 (1997)
119
I1 7-121
and in non-Caucasian populations due to the Cys3 11 presence. There is a great variability of the observed odds ratios (OR) both in non-Caucasian (range, 0.61-3.09) and in Caucasian (range, 0.61-4.85) populations with the higher OR in Arinami’s (3.09), Laurent’s (2.75) and Shaikh’s (4.85) studies with a not sign&ant test of heterogeneity within the two populations. However, there is also a great variability of the Confidence Interval for the odds ratios, but only in the Arinami’s report is the OR consistently higher than 1 (95% CI = 1.44-6.39). The overall x2 for non-Caucasian (x2= 10.36; df=5; p=O.O6) and for Caucasian (x2 = 7.309; df = 7; p =0.39) populations separately are not significant. Additionally, we also pooled
Table 1 Dopamine D2 receptor genotypes and allele frequencies of Cys311 in case-control studies Authors
References
Genotypes
cys311 frequency
ss
SC
cc
46 105 142 289 93 93 93 93 110 83
4 5 11 11 7 7 7 6 4 5
0 0 3 0 0 0 0 1 0 0
0.023 0.054 0.018 0.035 0.035 0.035 0.040 0.017 0.028
110 63 103 109 108 181 175 133 140 99 261 245 92 92
2 1 3 4 5 3 4 5 7 1 12 9 11 5
0 0 0 0 0 0 0 0 0 0 0 1 0 0
0.008 0.007 0.014 0.017 0.022 0.008 0.011 0.018 0.023 0.005 0.022 0.017 0.053 0.025
p
Naa~-CPucPsinopopulatim
Itokawa et al. (1993)
Biochem. Biophys. Res. Commun. 196(3), 1369-1375
Pts. Ctrl.
Arinami et al. (1994)
Lancet 343, 703-704
PtS.
Nanko et al. (1994)
Lancet 343(8904), 1044
Hattori et al. (1994)
Biochem. Biophys. Res, Commun. 202(2), 7577763
Chen et al. ( 1996)
Am. J. Med. C&et. 67.418420
Ctrl. Pts. Ctrl. Pts. Ctrl. PtS.
Ctrl. Asherson et al. (1994)
LanW 343(8904), 1045
PtS.
Ctrl. Gejman et al. (1994)
J. Am. Med. Assoc. 271, 204208
PtS.
Laurent et al. (1994)
Psychiat. Genet. (4), 229-230
Ctrl. Pts. Ctrl.
Nothen et al. ( 1994)
Lancet 343(8908), 1301
PtS.
Shaikh et al. (1994)
Lancet 343(8904), 1045
Ctrl. Pts. Ctrl.
Sasaki et al. (1996)
Am. J. Med. Genet. 67, 415417
PtS.
This report
Ctrl. Pts. Ctrl.
Verga et al.
0.040
S, Ser311; C. Cys311. “p values range from CO.01 to
120
M. Verga et al. / Schizophrenia
results from all the studies together, but again the overall x2 is not significant and the OR is equal to 1.42. In our nuclear families, standard X2-test does not reveal any statistically significant association between schizophrenia and the DRD2 genotypes using the GHRR (genotype-based-haplotype relative risk) design (Table 2(a)). Following the hypothesis that an allele, more than a genotype, could be responsible for a putative association with the disease, we explored this hypothesis again using the HHRR (haplotypebased- haplotype relative risk) design, but again we did not find any association between the DRD2 alleles and schizophrenia (Table 2(b)).
4. Discussion
Our results seem to exclude a main involvement of the Ser3 1l/Cys311 variant of the dopamine D2 receptor in the etiopathogenesis of schizophrenia. This finding in an Italian population is in agreement with other negative reports carried out in different Caucasian and non-Caucasian populations (Table 1). The observed frequency for the Cys311 allele in our schizophrenic patients (5.3%) is similar to Arinami’s (5.4%), while in our respective control groups the difference is more marked (2.5 versus 1.8%). As a first consequence, the risk to develop schizophrenia given the Cys311 allele is increased both in Arinami (OR =3.09) and in our report (OR =2.13), but this effect is statistically significant only for the Japanese group. It might be possible that our sample size is relatively small, mostly if compared to Arinami’s, to detect Table 2 Distribution
of genotypic
and allele frequencies
for DRD2
Research
aXZ=0.53; p=O.466; bXZ=0.13;p=0.718;
df= 1. df=l.
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a significant effect despite the interesting OR that would suggest a risk effect for the Cys311 allele also in our schizophrenic patients. However, in addition to the issue of the dimension of the sample, it is our opinion that we have to also consider other genetic factors when dealing with these types of controversial results obtained in different populations, especially in cases like the DRD2 gene, which shows a great variability also in relation to race and ethnicity (Castiglione et al., 1995). Another possibility is that the positive result obtained in non-Caucasian populations was a chance finding. The great variability of the observed odds ratios both in non-Caucasian and in Caucasian populations could be due to uncontrolled confounding factors specific for each population. Actually, the calculated OR for our HRR sample is quite low despite a good sample size, and this seems to exclude also a minor role of the DRD2 gene in our population. It is relevant that discrepant results also have been reported in alcoholism. Higuchi et al. (1994) found that the Cys3 11 variant is fairly well represented in alcoholic patients. He reported that Japanese alcoholics had the Cys311 allele significantly more often (6.3%) than the Japanese control group (2.9%), but no associations have been reported by Gejman et al. ( 1994) in a Caucasian population. Indeed, the best way of proving true associations between disease and genetic markers is to replicate the original finding, but our failure to replicate the positive association of the Cys3 11 allele and schizophrenia, also with a powerful strategy like the HRR approach, overcomes the potential pitfalls
and schizophrenia
Genotypes”
Transmitted Non-transmitted Total
25 i 1997)
using the haplotype
relative
risk
Allelesb
Ser311
cys3 11
Total
Ser3 11
cys311
Total
59 61 120
5 3 8
64 64 128
123 125 248
5 3 8
128 128 256
M. Verga et al. / Schizophrenia
of admixing populations or the other sources of biases as outlined above. Our lack of a significant result, which might nonetheless benefit of an additional confirmation in an independent and possible larger set of HRR nuclear families to more definitively detect or refute any association with this receptor variant, already casts doubts about a meaningful etiological role of the D2 dopamine gene in schizophrenia.
Acknowledgment
This work has been carried out within the activities of the EMASS (European Multi-Centre Association Study of the Molecular Genetics of Schizophrenia - Head: Prof. P. McGulhn) and of the Italian Research Group for Affective and Schizophrenic Disorders (IRGASD) under the auspices of the Janssen Research Council and Fondazione Italiana per lo Studio della Schizofrenia (FIS).
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