No association between the DRD3 Ser9Gly polymorphism and schizophrenia

Abstract

Article Outline

1. Introduction

2. Materials and methods

3. Results

4. Discussion

5. Conclusion

Role of Funding Source

Acknowledgements

References

2.1. Subject recruitment

2.2. Psychiatric assessment

2.3. Genetic analysis

2.4. Statistical analysis

3.1. Case-control study

3.2. DRD3 Ser/Gly polymorphism and therapeutic response to typical neuroleptics

3.3. DRD3 polymorphism and age at onset

3.4. Family-based association analysis

Objective

Methods

Results

Conclusion

2.2.1. Non-responder schizophrenic patients (NR)

2.2.2. Responder schizophrenic patients (R)

Volume 98, Issue 1, Pages 98-104 (January 2008)

13 of 44

ABSTRACT

FULL TEXT

FULL-TEXT PDF (179 KB)

Received 11 January 2007; received in revised form 1 July 2007; accepted 4 July 2007. published online 15 August 2007.

To investigate the association between a Ser9Gly polymorphism of the dopamine D3 receptor gene (DRD3) and schizophrenia.

408 schizophrenic patients and 172 control subjects were compared with regard to their DRD3 Ser9Gly genotypic and allelic frequencies. In addition, we carried out a family-based association study including 183 pedigrees (472 subjects) using the transmission disequilibrium test (TDT).

No significant differences of genotype or homozygosity distribution were identified between patients and controls. When patients were stratified according to gender, response to treatment, age at onset, no significant differences were observed. Neither allele A (Ser), or G (Gly) were preferentially transmitted from parents to affected offspring.

The hypothesis that the DRD3 Ser9Gly polymorphism plays a predisposing role in schizophrenia is not supported by this study.

Keywords: Schizophrenia, DRD3 (dopamine D3 receptor gene), Association study, Neuroleptic response

• Abstract

• 1. Introduction

• 2. Materials and methods

• 2.1. Subject recruitment

• 2.2. Psychiatric assessment

• 2.2.1. Non-responder schizophrenic patients (NR)

• 2.2.2. Responder schizophrenic patients (R)

• 2.3. Genetic analysis

• 2.4. Statistical analysis

• 3. Results

• 3.1. Case-control study

• 3.2. DRD3 Ser/Gly polymorphism and therapeutic response to typical neuroleptics

• 3.3. DRD3 polymorphism and age at onset

• 3.4. Family-based association analysis

• 4. Discussion

• 5. Conclusion

• Role of Funding Source

• Acknowledgment

• References

• Copyright

Schizophrenia is a complex disorder with a prevalence of approximately 1% (Perala et al., 2007). Family, twin and adoption studies confirm that genetic factors play a significant role in the aetiology of this disorder (Kendler and Diehl, 1993, Cannon and Murray, 1998). Yet, no mutations or predisposing DNA sequence variations, have been unequivocally implicated in the disease (Owen and McGuffin, 1993, Tsuang et al., 1990, Kendler, 2005).

The role of the brain dopamine system in the aetiology of schizophrenia has been suggested given the efficacy of the dopamine D2/D3 receptor blockers in treating psychotic symptoms (Carlsson and Lindqvist, 1963). It is also well known that indirect dopamine agonist such as amphetamine induce schizophrenia-like symptoms (Snyder, 1973, Seeman et al., 1976). The dopamine D3 receptor gene (DRD3) maps to chromosome 3q13.3 (Le Coniat et al., 1991) and it is predominantly expressed in limbic areas of the brain. These areas are involved in the regulation of mood, emotions and reward (Sokoloff et al., 1990, Giros, 1991, Le Foll et al., 2005) and are though to be implicated in schizophrenia (Bogerts, 1999).

Since the first report of an association between the DRD3 Ser9Gly polymorphism and schizophrenia was published (Crocq et al., 1992), subsequent studies have yielded variable results. Several independent case-control studies supported the findings of an association between increased homozygosity of the DRD3 Ser9Gly polymorphism and schizophrenia (Mant et al., 1994, Asherson et al., 1996, Spurlock et al., 1998). While some studies have shown an association with allele 1 (Ser) (Nimgaonkar et al., 1996, Shaikh et al., 1996), others showed higher frequency of allele 2 (Gly) and genotype 2-2 in patients compared to controls (Kennedy et al., 1995, Ebstein et al., 1997). In contrast, studies from Germany (Nothen et al., 1993, Rietschel et al., 1996), France (Laurent et al., 1994), Italy (Di Bella et al., 1994), Spain (Durany et al., 1996), Sweden (Jonsson et al., 1993), Japan (Tanaka et al., 1996), China (Chen et al., 1997), Ireland (Hawi et al., 1998), Canada (Joober et al., 2000) and Netherlands (Hoogendoorn et al., 2005) failed to detect evidence of either allelic or genotypic association or excess of homozygosity. To resolve the controversy, several meta-analyses were conducted, but they didn't yield clear conclusions (Nimgaonkar et al., 1996, Shaikh et al., 1996, Dubertret et al., 1998, Williams et al., 1998, Jonsson et al., 2003, Jonsson et al., 2004).

Using family-based strategy,Williams et al. (1998) found a significant excess of homozygosity in transmitted alleles compared to untransmitted alleles, whereas several other studies failed to show association/linkage between schizophrenia and this polymorphism (Macciardi et al., 1994, Sabate et al., 1994, Rothschild et al., 1996, Hawi et al., 1998, Prasad et al., 1999, Kremer et al., 2000).

Several studies have also investigated the DRD3 Ser9Gly polymorphism with subgroups of schizophrenic patients having a particular profile of symptoms, age at onset, family history (Gaitonde et al., 1996, Nimgaonkar et al., 1996, Serretti et al., 1999), therapeutic response to neuroleptic drugs (Nimgaonkar et al., 1996, Shaikh et al., 1996, Ebstein et al., 1997, Joober et al., 2000, Dahmen et al., 2001, Szekeres et al., 2004) and those with spontaneous or neuroleptic-induced tardive dyskinesia (Lovlie et al., 2001, Steen et al., 1997, Basile et al., 1999, Zhang et al., 2003, Chong et al., 2003).

In the present study, we conducted a case-control study with a large number of schizophrenic patients (N=408) and unrelated controls (N=172), and a large family-based association study including 183 families (472 individuals) to examine the association of the DRD3 Ser/Gly polymorphism with schizophrenia.

For the case-control study, a total of 408 unrelated Caucasian schizophrenic patients and 172 controls were genotyped. Recruitment of schizophrenic patients was performed in Canada (n=244 cases), Tunisia (n=97) and Hungary (n=67). In Canada, three institutions provided these patients: Douglas Hospital, Clinique Jeunes Adultes of L.H. Lafontaine Hospital and the Schizophrenia Clinic of the Royal Ottawa Hospital. In Hungary, patients were recruited at Budapest psychiatric hospital, and in Tunisia, patients were recruited in Razi-psychiatric hospital. The control subjects were recruited in Canada (n=118) (Douglas Hospital and Royal Ottawa hospitals) and from Razi-hospital in Tunisia (n=54).

For the family-based study, we recruited a large sample of Caucasian families from three distinct populations: Canadian, Tunisian, and Hungarian. A total of 183 pedigrees including 472 individuals were genotyped. All probands from the family-based study were also part of the case-control study.

All participating subjects gave their informed consent. The project was approved by the Research Ethics Board of each participating institution.

All patients were evaluated using the diagnostic Interview for Genetic Studies (DIGS). Diagnoses were made according to DSM-III-R or DSM-IV based on information extracted from the DIGS as well as complementary information from medical files.

Controls were assessed using structured clinical interview for DSM Diagnoses (SCID I and II).

In a 193 cases, we were able to document the quality of long-term response to typical neuroleptic through direct interviews and reviews of medical charts. The criteria for response or resistance to conventional neuroleptics were the same as described by (Joober et al., 2000) as follows:

None had experienced remission of psychotic symptoms within the past 2 years. In the preceding 5 years, all NR patients had undergone at least three periods of treatment with conventional neuroleptics from at least two distinct families of drugs at a dose equal to, or greater than, 750 mg chlorpromazine (CPZ) equivalents on monotherapy, or 1000 mg CPZ equivalents when a combination of neuroleptics was used for a continuous period of at least 6 weeks, and which resulted in no significant decrease in symptoms. Finally, all patients were unable to function without supervision in all, or nearly all, domains of social and vocational activities and had a Global Assessment Score <40 within the last 12 months. At the time of enrollment, a minimal score of 4 (moderate to severe) on at least three of the five BPRS items (3, 4, 12, 13, 15), a total BPRS score of at least 45 and/or a CGI (Clinical global Impression) score of at least 5 (markedly ill) were required.

All patients had been admitted at least once to a psychiatric institution because of an acute psychotic episode. During each hospitalization, patients experienced a full or partial remission in response to treatment with conventional neuroleptics within 6–8 weeks of continuous treatment. All patients were able to function autonomously with only occasional supervision in all, or nearly all, domains of social and vocational activities. None of the (R) patients had to be admitted to hospitals because of the exacerbation of their psychosis while under continuous neuroleptic treatment. All R patients had at least one psychotic relapse when neuroleptic medication was reduced or discontinued. Remission was defined as a complete or quasi-complete disappearance of schizophrenic symptoms, with limited residual symptoms, based on the treating psychiatrist clinical evaluation and hospital records. At the time of enrollment, total BPRS scores were less than 30 with no more than one item scoring 4 and/or a CGI score less than 3 (borderline mentally ill).

The age at disease onset was determined as the age at onset of first psychotic symptoms and was based on interviewing patients and information derived from medical files when available. Patients were dichotomized into two groups: those with early onset (<25 years) and those with later onset (>25 years) (Krebs et al., 2000, Tiao-Lai and Chien-Te, 2005).

Blood samples were collected, and genomic DNA was isolated following standard protocol.

A 462-basepair fragment of the first exon of DRD3 gene was amplified by PCR with primer pair described by (Lannfelt et al., 1992) in a PTC-100 (MJ Research, Watertown, MA, USA) thermal cycler. A 25-l amplification mixture contained 150–300 ng DNA, 0.5 M of each primer, 0.2 of each dNTP, 1.75 mM MgCl2, 100 mM Tris–HCl, 500 mM KCl, 0.8% NP40, 0.5 U of Taq DNA polymerase (MBI Fermentas, Vilnius, Lithuania). Cycling conditions were: denaturation of 95 °C for 2 min, 35 cycles with a profile of 94 °C for 30 s, 60 °C for 30 s, 72 °C for 30 s, followed by final elongation at 72 °C for 5 min. A volume of 5 l of PCR products was then digested overnight in a total volume of 7.5 l at 37 °C with 0.5 U of MlsI restriction endonuclease (MBI Fermentas). Digestion products were electrophoresed on 2.5% agarose gel (Prona, Spain) and visualised by ethidium bromide staining. Band sizes were compared with DNA ladder (MBI Fermentas). Digestion resulted in a 304-bp fragment for allele 1 (Ser-9), and in 206-bp and 98-bp fragments for allele 2 (Gly-9). Additionally two constant bands of 111 bp and 47 bp were detected resulting from two non-polymorphic restriction sites.

Allele and genotype frequencies were compared using a standard Chi-square test (χ2). We used the fbat program to analyze transmission/non-transmission of each allele from parents to affected children (Horvath et al., 2001). Fbat is a unified approach to family association study based on the original Transmission disequilibrium Test (TDT) (Spielman et al., 1993, Rabinowitz and Laird, 2000).

Genotypes in the patient and control population were in Hardy–Weinberg equilibrium (for patients: χ2=1; df=2 ; p=0.31 ;and for controls: χ2=2; df=2 ; p=0.15).

Table 1 summarizes the allelic and genotype distribution in patients and controls separated according to gender and center of recruitment. There were no differences between patients and control subjects regarding Genotype (χ2=2.15; df=2; p=0.34) and allelic frequencies (χ2=1.39; df=1; p=0.23). No excess of homozygosity was observed in patients compared to controls (χ2=0; df=1; p=1). When patients and controls were stratified according to gender, there were no significant differences in either allelic or genotypic distributions between the two groups.

Table 1.

Genotype and allelic distribution of DRD3 Ser/Gly polymorphism in patients and controls separated according to their sex and center of recruitment


			Males						Females						All (males+females)
			Genotypes				Alleles		Genotypes				Alleles		Genotypes				Alleles
			AA	AG	GG	All genotypes	A	G	AA	AG	GG	All genotypes	A	G	AA	AG	GG	All genotypes	A	G
Controls	Canada	Montreal	15	19	2	36	49	23	25	25	3	53	75	31	40	44	5	89	124	54
		Ottawa	8	6	2	16	22	10	10	3	0	13	23	3	18	9	2	29	45	13
		Montreal+Ottawa	23	25	4	52	71	33	35	28	3	66	98	34	58	53	7	118	169	67
	Hungary		0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	Tunisia		10	25	6	41	45	37	5	6	2	13	16	10	15	31	8	54	61	47
	Total controls		33	50	10	93	116	70	40	34	5	79	114	44	73	84	15	172	230	114
Patients	Canada	Montreal	56	63	20	139	175	103	22	26	7	55	70	40	78	89	27	194	245	143
		Ottawa	16	20	7	43	52	34	1	6	0	7	8	6	17	26	7	50	60	40
		Montreal+Ottawa	72	83	27	182	227	137	23	32	7	62	78	46	95	115	34	244	305	183
	Hungary		16	22	4	42	54	30	14	10	1	25	38	12	30	32	5	67	92	42
	Tunisia		24	41	9	74	89	59	9	11	3	23	29	17	33	52	12	97	118	76
	Total patients		112	146	40	298	370	226	46	53	11	110	145	75	158	199	51	408	515	301

Genotype and allelic frequencies were compared between responder and non-responder patients. We did not find any difference in allelic (χ2=0.63; df=1; p=0.42), genotype (χ2=0.9; df=2; p=0.63), or homozygosity (χ2=0.46; df=1; p=0.5) distributions (Table 2).

Table 2.

Allelic and genotype distribution in patients diagnosed with schizophrenia and separated according to the quality of response to conventional neuroleptics


	Alleles		Total	Genotypes			Total
	A	G		AA	AG	GG
Non-responders	81	43	124	26	29	7	62
Responders	160	102	262	46	68	17	131
Total	241	145	386	72	97	24	193

When responders or non responders were compared with control subjects, no significant differences were found regarding genotypic, allelic, or homozygosity distributions.

When patients were stratified based on age at onset (below or over 25 years), there was no difference between the two groups regarding allele or genotype distribution (χ2=0.67; df=2; p=0.71) (Table 3).

Table 3.

Genotype distribution in patients diagnosed with schizophrenia and separated according to their age at onset

(χ2=0.67; df=2; p=0.71).

We performed the TDT test using the Fbat program under dominant and recessive models.

No preferential transmission of either one of the two alleles from parents to affected offspring was observed (Table 4).

Table 4.

Results of the FBAT analysis


Model	Allelea	Familiesb	Z-score	P-value
Additive	1(A)	63	0.41	0.67
Additive	2(G)	63	−0.41	0.67
Dominant	1(A)	34	1.45	0.14
Dominant	2(G)	50	0.56	0.57
Recessive	1(A)	50	−0.56	0.57
Recessive	2(G)	34	−1.45	0.14

Allele frequencies 0.650 for allele A and 0.35 for allele G.

Number of informative families. (Analyses performed if ≥9 informative families.

Our data indicate that the DRD3Ser9Gly polymorphism is not associated with schizophrenia, even after stratifying for therapeutic response to typical neuroleptics or age at onset.

In the case-control study, we did not find this polymorphism to be associated with schizophrenia; neither did we observe the excess of homozygosity of either one of the alleles described in European samples (Crocq et al., 1992, Asherson et al., 1996, Spurlock et al., 1998). This is in accordance with several published case-control studies (Jonsson et al., 1993, Nothen et al., 1993, Di Bella et al., 1994, Laurent et al., 1994, Rietschel et al., 1996, Durany et al., 1996, Tanaka et al., 1996, Chen et al., 1997, Hawi et al., 1998, Wong et al., 2000, Elvidge et al., 2001, Hoogendoorn et al., 2005, Staddon et al., 2005), as well as the largest published meta-analysis including 11,066 subjects (Jonsson et al., 2004).

Our results are consistent with most family-based studies which conclude that there is no association between DRD3Ser9Gly variant and schizophrenia (Wiese et al., 1993, Macciardi et al., 1994, Sabate et al., 1994, Rothschild et al., 1996, Hawi et al., 1998, Prasad et al., 1999, Kremer et al., 2000).

However, the possibility that this gene is implicated in schizophrenia cannot be formally excluded without studying multiple other polymorphisms in this locus. In a recent case-control and family-based association study, Talkowski et al reported an association between schizophrenia and a common haplotype spanning intron 1 to the 3′ region of the gene. The Ser9Gly polymorphism was associated with schizophrenia only when it was present in this associated haplotype but not when it was present on other haplotypes (Talkowski et al., 2006). This observation, although needs confirmation in a larger sample, suggest that other polymorphisms in LD with the Ser9Gly polymorphism may contribute to schizophrenia susceptibility.

Regarding the treatment response, and in accordance with several studies (Yang et al., 1993, Kennedy et al., 1995, Durany et al., 1996, Nimgaonkar et al., 1996, Joober et al., 2000, Staddon et al., 2005), we did not find any association between DRD3 Ser9Gly polymorphism and response to conventional anti-psychotic treatment.

The present study using a large sample (408 schizophrenic patients, 172 controls for a case-control analysis and 183 pedigrees including 472 subjects for family-based analyses) adds to the evidence that the DRD3 Ser9Gly polymorphism is not implicated in schizophrenia.

This work was supported in part by a grant from the Fond de Recherche de Santé du Québec and the Canadian Institutes of Health Research to RJ.

We thank the participants and their families and Dr. Heidi Howard for her comments. RJ has received consultant honorarium from Janssen Ortho and Pfeizer Canada.

Age of onset <25 year

Age of onset ≥25 year

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a Douglas Hospital Research Centre, Montréal, Québec, Canada

b Montréal University, Montréal, Québec, Canada

c Notre Dame Hospital Research Centre (CHUM), Montréal, Québec, Canada

d Department of Psychiatry, University of Tunis, Tunisia

e Department of Psychiatry, McGill University, Montréal, Québec, Canada

f National Institute of Psychiatry, Budapest, Hungary

Corresponding author. Douglas Hospital Research Center 6875, Boul LaSalle Verdun, Quebec, Canada H4H 1R3; Department of Psychiatry, McGill University, Montréal, Québec, Canada. Tel.: +1 514 762 3048; fax: +1 514 888 4064.

PII: S0920-9964(07)00300-3

doi:10.1016/j.schres.2007.07.002

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