The 3′ region of the DRD2 gene is involved in genetic susceptibility to schizophrenia

Schizophrenia Research 67 (2004) 75 – 85 www.elsevier.com/locate/schres

The 3Vregion of the DRD2 gene is involved in genetic susceptibility to schizophrenia Caroline Dubertret a,b, Laurent Gouya c, Naima Hanoun b, Jean-Charles Deybach c, Jean Ade`s a,d, Michel Hamon b, Philip Gorwood a,d,* a

Service de Psychiatrie Adulte, Faculty of Bichat-Claude Bernard, Louis Mourier Hospital (AP-HP), 178 rue des Renouillers, 92701 Colombes Cedex, France b INSERM U288, IFR des neurosciences (IFR 70), Faculty of Pitie´-Salpeˆtrie`re Hospital, 47 Bd de l’hoˆpital, 75013 Paris, France c Service de Biochimie, Fe´de´ration de Ge´ne´tique Mole´culaire, INSERM U409, Faculty of Bichat-Claude Bernard, Louis Mourier Hospital (AP-HP), 178 rue des Renouillers, 92701 Colombes Cedex, France d CNRS UMR 7593, Faculty of Pitie´-Salpeˆtrie`re Hospital, 47 Bd de l’hoˆpital, 75013 Paris, France Received 21 March 2003; accepted 19 July 2003

Abstract The gene coding for the D2 dopamine receptor (DRD2) is considered as one of the most relevant candidate genes in schizophrenia. Previous genetic studies focusing on this gene yielded conflicting results, for example because of differences in methodology (linkage versus association studies) and variability in the loci analyzed (the DRD2 gene having many polymorphic sites). We used a progressive strategy with two different approaches (case-control and transmission disequilibrium test) and investigated six genetic polymorphisms spanning the DRD2 gene in 103 patients with DSM-IV criteria of schizophrenia, their 206 parents and 83 matched healthy control subjects. We found a significant excess of the A2 allele in subject with schizophrenia compared to unaffected controls. An excess of transmission of the A2 allele (and haplotypes containing this marker) from the parents to the affected children was also observed. Interestingly, the TaqI A1/A2 polymorphism, located 9.5 kb downstream from the DRD2 gene, maps in a novel gene, untitled ‘‘X-kinase’’, and leads to a 713 Glu ! Lys substitution in exon 8. As the analysis of the other markers within the DRD2 gene does not improve the strength of the association, our data are in favor of a specific role of the 3Vchromosomic region of the DRD2 gene in the vulnerability to schizophrenia. D 2003 Elsevier B.V. All rights reserved. Keywords: Schizophrenia; DRD2 gene; TDT; Case/control association study; Age at onset; Treatment response

1. Introduction

* Corresponding author. Service de Psychiatrie Adulte, Hoˆpital Louis Mourier, 178 rue des Renouillers, 92701 Colombes Cedex, France. Tel.: +33-1-47-60-64-16; fax: +33-1-47-60-67-40. E-mail address: [email protected] (P. Gorwood). 0920-9964/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0920-9964(03)00220-2

Numerous biochemical and pharmacological features support the idea that dopamine is a key neurotransmitter in schizophrenia (for review, see Seeman, 1987). In humans, the D2 receptor gene is located on chromosome 11q22 –23 (Grandy et al., 1989). Three

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Table 1 Association studies between DRD2 gene polymorphisms and schizophrenia Polymorphism

Author

Sample size Patients

 141CIns/Del

TaqI B1/B2 (GT)n STRP S311C

TaqI A1/A2

Arinami et al., 1997 Inada et al., 1999 Jo¨nsson et al., 1999 Ohara et al., 1998 Sto¨ber et al., 1998 Li et al., 1998 Li et al., 1998 Tallerico et al., 1999 Breen et al., 1999 Hori et al., 2001 Himei et al., 2002 Dubertret et al., 2001 Dubertret et al., 2001 Virgos et al., 2001 Arinami et al., 1994 Arinami et al., 1996 Arinami et al., 1996 Hattori et al., 1994 Nanko et al., 1994 Gejman et al., 1994 Laurent et al., 1994 Asherson et al., 1994 Shaikh et al., 1994 Sobell et al., 1994 No¨then et al., 1994 Crawford et al., 1996 Ohara et al., 1996 Tanaka et al., 1996 Chen et al., 1996 Sasaki et al., 1996 Fujiwara et al., 1997 Verga et al., 1997 Verga et al., 1997 Kaneshima et al., 1997 Spurlock et al., 1998 Serretti et al., 2000 Hori et al., 2001 Himei et al., 2002 Comings et al., 1991 Lee et al., 1995 No¨then et al., 1993 Campion et al., 1993 Sanders et al., 1993 Jo¨nsson et al., 1996 Dollfus et al., 1996 Dubertret et al., 2001 Dubertret et al., 2001

Method Controls

260 234 129 170 260 151

312 94 176 121 290 145

50 439 241 190 50

54 437 201 103 50

262 156 135 291 100 100 106 113 112 147 338 179 168 153 106 114 273 52 103

278 200 279 579 100 100 34 184 64 100 1914 138 162 121 106 88 255 26 97

78 373 366 241 190 87 77 60 80 55 104 49 50

112 413 267 201 103 108 68 60 80 51 67 161 50

Trios

229

50

64

1: Association, 2: Transmission Disequilibrium Test or Haplotype Relative Risk.

50

1 1 1 1 1 2 1 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 2

Significancy allele wise 0.001 0.04 0.04 0.04 NS NS NS NS 0.02 NS NS 0.03 NS 0.035 0.003 NS 0.001 NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS 0.05 NS NS NS 0.002 NS NS NS NS NS 0.04 0.025

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of its most studied polymorphisms are the deletion of cytosine 141 in the promoter region upstream from the transcription start site (  141CIns/Del) (Arinami et al., 1997), the missense variant 960C ! G in exon 7 which leads to a 311Ser ! Cys (S311C) substitution (Itokawa et al., 1993), and a TaqI A1/A2 single nucleotide polymorphism (SNP) localized 9.5 kb downstream from the DRD2 gene (Grandy et al., 1993). Numerous studies investigated the role of the DRD2 gene in the risk for schizophrenia. To date, linkage analyses have provided no support to this hypothesis (see Grassi et al., 1996), and association studies between DRD2 gene and schizophrenia yielded controversial data (Table 1). We previously examined two polymorphisms, TaqI A1/A2 and TaqI B1/B2, flanking the DRD2 gene in 50 trios, and found a significant excess of the B2A2 haplotype in subjects with schizophrenia, using family-based association studies (Dubertret et al., 2001). Moreover, a recent meta-analysis of the Taq1 A1/A2 polymorphism of the DRD2 gene in schizophrenia, based on seven different case/control studies, clearly showed that the allelic distribution is significantly different in patients and controls (Dubertret et al., 2001).

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Conflicting results in association and linkage studies between schizophrenia and the DRD2 gene polymorphisms might be explained by the various methodological approaches which were used, the sensitivity of association analysis contrasting with the specificity of linkage studies (Gorwood, 1999). Genetic heterogeneity, differences in the recruitment modalities and selected diagnostic criteria, as well as variability of allele frequencies in control populations may also be involved. Family-based association tests are extensively used for identification of genes involved in complex diseases, because they avoid the stratification bias of association study (Gorwood, 1999). Moreover, the former method, frequently regarded as a reference test (Risch and Merikangas, 1996), could be more efficient than linkage analysis, particularly when the impact of vulnerability genes is small, as it would be expected for complex diseases. However, in order to use association analyses in a relevant manner, a dense map of markers is required, because linkage disequilibrium varies over relatively small distances. To further investigate the potential role of the DRD2 gene in schizophrenia susceptibility, we analyzed six polymorphisms spanning the entire coding region of the DRD2 gene in a sample of 103 trios.

Fig. 1. Organization of the DRD2 and X-kinase genes and molecular locations of the six polymorphisms studied. *: Polymorphisms located in functional regions of the gene (promoter and exons).

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They corresponded to five single nucleotide polymorphisms (SNPs) (  141CIns/Del, Taq1 B1/B2, TaqI D1/D2, S311C, Taq1 A1/A2) and one six-allele (GT)n short tandem repeat polymorphism (STRP) (Fig. 1). SNPs can be analyzed independently and combined in haplotypes. Haplotype analysis is a powerful strategy for resolving the controversial issue of association studies based on individual polymorphisms (Kidd et al., 1998), and is recognized as a validated approach for mapping complex disease genes on the basis of SNPs (Martin et al., 2000). It can also contribute to delimit the gene region where the functional loci in linkage disequilibrium with the studied polymorphisms are located (Daly et al., 2001; Johnson et al., 2001). In this view and according to our haplotype study, we further analyzed the genomic structure of the 3Vchromosomic region of the DRD2 gene. Genetic studies in schizophrenia are most often impaired by phenotypic heterogeneity. To take into account the phenotypic heterogeneity of schizophrenia, we analyzed different clinical markers that may influence the genetic loading of subjects’ vulnerability to schizophrenia, such as age at onset (Gorwood et al., 1995), and treatment response (Rao et al., 1994).

including hallucinations, delusions, thought disorders, and/or catatonic behavior (20.73 F 8 years old, mean F S.D.). Response to the current neuroleptic treatment was evaluated with May et al. (1988) scale on the basis of rapidity and quality of symptom remission, necessity for prolonged hospitalization, ability for autonomy and social rehabilitation. ‘‘Treatmentresponding patients’’ had a clinical remission under current antipsychotic treatment in less than 1 month, with potential residual symptoms, which were compatible with social rehabilitation discharge from hospital (n = 53, score: 1 –3). Conversely, ‘‘treatment refractory’’ patients had no, or only partial, clinical remission despite several trials with different antipsychotic drugs given for at least 1 month, and required permanent daily care, or allowed discharge with several measures to cope with the lack of autonomy and social rehabilitation (n = 50, score: 4 –6). Written informed consent was obtained from all subjects of the three samples (controls, patients and parents). The study protocol was approved by the French Comite´ National d’Ethique and the Commission Nationale Informatique et Liberte´. 2.2. Genotyping

2. Subjects and methods 2.1. Subjects We recruited (i) 83 French healthy control subjects (19% women) among anonymous blood donors, (ii) 103 subjects with schizophrenia (21% women) admitted in a psychiatric department of a French university hospital, and (iii) both parents (n = 206) of the patients. Controls and patients were evaluated by a trained psychiatrist using the Diagnostic Interview for Genetic Study (DIGS) (Nurnberger et al., 1994), a semi-structured interview that assesses DSM-IV criteria of schizophrenia and other psychiatric diseases. Twentyfive patients had prominent positive symptoms (paranoid type) (24.2%), 35 showed mainly negative symptoms (disorganized type) (34.0%), 42 exhibited both positive and negative symptoms (40.8%), and one subject had a catatonic form of schizophrenia (1.0%). Age at onset was defined as the age at which the first symptom of schizophrenia was documented,

We collected blood samples from the 83 controls and the 103 trios, and genotyped six polymorphisms of the DRD2 gene. For all PCR reactions, 100 ng of genomic DNA was amplified in 50 Al of reaction mixture containing 50 mM KCl, 2 mM MgCl2, 15 mM Tris – HCl (pH 8.4), 0.2 mM of each desoxyribonucleoside-5V-triphosphate, 10 pmol of each primer, and one unit of Taq polymerase (Amersham Pharmacia, Uppsala, Sweden). The reaction was performed at 95 jC for 3 min followed by 35 cycles of 95 jC for 30 s, specific annealing temperature for 30 s and 72 jC for 30 s. Twenty microliters of PCR products were then digested by the appropriate restriction enzyme according to the manufacturer’s recommendations and electrophoresed on a 3% standard agarose gel. Primers, individual annealing temperatures, restriction enzymes, as well as details for each polymorphism are listed in Table 2. The genotype distribution of the different polymorphisms (with rare allele above 5%) tested satis-

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Table 2 Reaction conditions for PCR assays and restriction enzyme analysis Polymorphisms

Localization

Primers

AT

PCR product and restriction fragments sizes

Restriction enzymes

References

 141CIns/Del

5V UTR

67

290 (150 + 140)

BstNI

a

TaqI B1/B2

Intron 1

54

442 (182 + 238 + 22)

aTaqI

a

TaqI D1/D2

Intron 2

55

419 (272 + 147)

aTaqI

Kidd et al. (1998)

(GT)n STRP

Intron 2

58

12 to 17 repeats

s311C

Exon 7

61

TaqI A1/A2

3V: 9.4 kb to DRD

5V-GAAGACTGGCGAGCAGACGGTGAG-3V 5V-CGGTTCGGCACTGAAGCTGGACAG-3V 5V-GATACCCACTTCAGGAAGTC-3V 5V-CAGTAAAGAACTAGGAGTCAG-3V 5V-CCCAGCAGGGAGAGGGAGTA-3V 5V-GACAAGTACTTGGTAAGCATG-3V 5V-GGA GGG CGG TGC GGT CAT-3V 5V-CTGGCAGGAGCACGTTTCTCATAC-3V 5V-ACCAGCTGACTCTCCCCGACCGGT-3V 5V-GGAAGGACATGGCAGGGAATGGGA-3V 5V-CCGTCGACGGCTGGCCAAGTTGTCTA-3V 5V-CCGTCGACCCTTCCTGAGTGTCATCA-3V

149 + 92 + 53 (126 + 23 + 92 + 53) 310 (180 + 130)

62

a

Sau96I aTaqI

Arinami et al. (1994) Grandy et al. (1993)

At: Annealing temperature; 5V UTR: 5V untranslated region. a Reaction conditions for optimal PCR assays were determined in this work.

fied Hardy –Weinberg equilibrium in the samples of controls ( p>0.17), parents ( p>0.17), and subjects with schizophrenia ( p>0.24). 2.3. Analysis of the 3V genomic region of the DRD2 gene 2.3.1. Cloning of the X-kinase transcript In the 3V chromosomic region of the DRD2 gene, several exons were predicted with the GenomeScan program available on the UCSC Genome Browser. Three primer pairs were thus generated using genomic sequence in the predicted exons. PCR fragments were sequenced with an ABI 3100 automated sequencer, and the 5V region of the gene was amplified using a 5V Race strategy (5VRace System, Life Technologies). Human brain mRNA was reverse transcribed with Superscript II Reverse Transcriptase and a gene-specific primer. RT-PCR amplification was then performed using universal amplification primer and a gene-specific primer according to the manufacturer protocol. 2.3.2. Sequence analysis The X transcript was deduced from the sequenced overlapping RT-PCR fragments and compared to the genome template using DNAsis (version 2.1) to determine the exon/intron structure. The SMART program was used to identify known domains in the protein.

2.3.3. RNA analysis Commercial human mRNAs were obtained from Clontech. RT-PCR analysis of X-kinase expression in human tissues was carried out using primers in exons 3 and 5 leading to the amplification of a 217-bp fragment. 2.3.4. Statistical analyses Pairwise linkage disequilibrium (LD) was assessed for all combinations of the 6 polymorphisms spanning the DRD2 gene according to Thompson et al.’s (1988) method. With this method, the ADVA (D/Dmax) value ranges between 0 and 1, and greater values indicate stronger linkage disequilibrium. Linkage disequilibrium (i.e., when two markers are transmitted together more frequently than by chance only) was assessed as previously described (Hauge et al., 1991; Castiglione et al., 1995; Gelernter and Kranzler, 1999). Allele frequencies were compared between patients and controls by standard v2 tests. We then attempted to confirm significant association results in the family sample using the ‘‘transmission disequilibrium test’’ (TDT) (Spielman et al., 1993) using a McNemar v2 test. The informativity of the 103 trios have been calculated on the bases of parental genotypic distribution for all studied polymorphisms (TaqI A1/A2 polymorphism: informativity = 0.60, with 67 heterozygous parents; TaqI B1/B2 polymorphism: informativity = 0.50, with 51 heterozygous parents; TaqI D1/D2 polymorphism: informativity = 0.74, 98 heterozygous parents;  141CIns/Del polymorphism: informativity = 0.40,

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39 heterozygous parents; S311C polymorphism: informativity = 0.15, 13 heterozygous parents). Analyses were performed for each polymorphism independently, then for multiple-loci haplotypes constructed with all markers. P value assesses the significance of transmission distortion for specific haplotypes. When the number of markers is increased, a higher proportion of the families becomes ambiguous with respect to the resolution of haplotypes, and fewer families can be used to estimate haplotype frequency. For global p value, which assesses the significance of transmission distortion for all haplotypes, the TDT using multiple linked markers were analyzed according to Zhao et al. (2000). Treatment response was analyzed with TDT in each subgroups. The quantitative trait of age at onset was finally analyzed using a quantitative trait loci-transmission disequilibrium (QTDT) test (Abecasis et al., 2000). QTDT incorporates variance components’ methodology in the analysis of family data and includes exact estimation of p-values for analysis of small samples and non-normal data.

Table 3 Distribution of the most common allele of six polymorphisms of the DRD2 gene in a French sample of controls (n = 83) and schizophrenic subjects (n = 103) using case/control association study Markers

 141CIns/Del TaqI B1/B2 TaqI D1/D2 S311C TaqI A1/A2 (GT)n STRP

Major allele

Major allele frequency in Cases

Controls

Ins B2 D1 Ser A2 15

0.90 0.88 0.61 0.97 0.83 0.49

0.72 0.65 0.62 0.98 0.60 0.35

P

0.84 0.04 0.70 0.58 0.04 0.60

allele of TaqI B1/B2 SNP was observed in the group of patients with schizophrenia compared to unaffected controls (OR = 1.71 [1.03 – 2.85] and OR = 1.81 [1.02 –3.22], respectively). Allelic distribution of other polymorphisms did not differ between patients and controls. An excess of transmission of the A2 allele only from heterozygous parents to offspring with schizophrenia was observed with TDT analyses (v2 = 6.58, df = 1, p = 0.01).

3. Results

3.3. Haplotype analyses

3.1. Linkage disequilibrium relationship at the DRD2 locus Linkage disequilibrium was carried out on a pairwise basis for all the SNPs in the 206 unrelated parents. There was strong evidence for linkage disequilibrium for the majority of SNPs, with the exception of the  141CIns/Del and S311C polymorphisms. The low frequency of the rare allele of these two SNPs reduced the ability to detect linkage disequilibrium between that rare allele and the common allele of other system. A total of 37 haplotypes were detected, with one common haplotype (39%) containing the most common allele of each polymorphism. Seven haplotypes were observed in more than 3% of the sample, and accounted for 76% of all the haplotypes detected (data available upon request).

We constructed six-marker haplotypes with all the polymorphisms studied previously, and found a tendency for global distortion of transmission (global p value = 0.05). TDT analyses only revealed a significant excess of transmitted Ins-B2-D2-CA2S311-A2 haplotype (v2 = 4.26, df = 1, p = 0.04) and a deficit of transmitted Ins-B2-D1-CA3-S311-A1 haplotype (v2 = 6.25, df = 1, p = 0.01) from heterozygous parents to affected offspring. The use of haplotype analyses did not increase the significancy compared to the single-locus analysis (and particularly the TaqI A1/A2 polymorphism), indicating that the finding of haplotype transmitted in excess is largely driven by excess of transmission of the A2 allele. Indeed, when excluding the A1/A2 polymorphism, no haplotype was found transmitted in excess to affected proband (global p value = 0.62).

3.2. Analysis of individual markers

3.4. Phenotypical heterogeneity

With the case/control study (Table 3), a significant excess of A2 allele of the TaqI A1/A2 SNP and B2

TDT analyses showed a higher transmission of A2 allele in the subsample of patients with good treatment

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responses (v2 = 6.43, df = 1, p = 0.01). This excess of transmission from parents to patients with poor treatment responses was not observed (v2 = 1.12, df = 1, p = 0.29). QTDT analyses confirmed the impact of the A2 allele on age at onset. Patients with the A2 allele had a significantly later age at onset ( p < 0.0001), and, independently, the A2 allele was transmitted with an older age at onset ( p = 0.01) according to the QTDT approach. 3.5. Analysis of the 3Vgenomic region of the DRD2 gene 3.5.1. Cloning of the X-kinase transcript We obtained the X-kinase cDNA sequence by RT-PCR and 5V Race analysis of total human brain mRNA. Overlapping cDNA products were aligned to identify the longest 2295 bp Open Reading Frame (ORF) of human X-kinase. The genomic structure determined by aligning the cDNA sequence with the human genomic DNA sequence indicated that the ORF is made by eight exons and spans over 13 kb (Fig. 1). RT-PCR analysis showed that the X-kinase gene is expressed in a wide range of tissues including whole brain from human adult and fetus, cerebellum, spinal cord, placenta, and liver (data not shown). The TaqI A1/A2 SNP maps in the exon 8 at nucleotide 2140 of the cDNA and leads to an E713K substitution. The DRD2 and Xkinase genes have an inverse orientation and 9.4 kb separate the two stop codons (Fig. 1). 3.5.2. Structure of the X-kinase protein The protein encoded is predicted to contain 765 amino acids with a molecular weight of 84.5 kb. A putative serine, threonine, tyrosine kinase domain characterize the structure of the protein, analyzed by SMART program, and 11 ankyrin repeats. These data led us to call this protein ‘‘X-kinase’’ until further characterizations is achieved.

4. Discussion Two SNPs (TaqI B1/B2 and TaqI A1/A2) of the DRD2 gene appeared to be significantly associated with schizophrenia in our sample (using case/control

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association study), but an excess of transmission from the parents to the affected children was observed only for the A2 allele of TaqI A1/A2 polymorphism or for haplotypes containing this allele (according to the TDT method). Considering phenotypic heterogeneity, the A2 allele was transmitted with an older age at onset (QTDT) and better treatment response (TDT). The other five markers studied within the DRD2 gene did not increase the strength of the association. Finally, we showed that the TaqI A1/A2 polymorphism maps in the last ankyrin repeat of the X-kinase gene, which is expressed in the human brain, and is potentially functional because it leads to an acid to basic amino acid substitution (E713K). This study thus provides convergent evidence for a significant role in the vulnerability to schizophrenia of the 3V genomic region of the DRD2 gene, where the TaqI A1/A2 polymorphism is located, in a novel X-kinase gene that codes for a phosphotransferase containing ankyrin repeat domains. The gene coding for the DRD2 has been largely investigated in schizophrenia, the majority of initial positive studies being not further replicated. Firstly, stratification bias is an important potentially confounding factor for case/control studies, specifically when the studies are based on heterogeneous populations and when the allele frequency varies between those populations (Gorwood, 1999). We thus used the TDT test, which avoids this bias and gives additional information in case of clear-cut association (Spielman and Ewens, 1996). Surprisingly, only one negative TDT analysis (Li et al., 1998) was conducted for the DRD2 gene in schizophrenia prior to our own study. Secondly, phenotypical and genetic heterogeneity may also be involved. The impact of the A2 allele or Ins-B2-D2-CA2-S311-A2 haplotype detected in our sample was especially observed in a subgroup of patients, namely those with a later age at onset and better treatment response. Such clinical characteristics are rarely analyzed and could have been diversely represented in previous association studies. Thirdly, the studied polymorphisms may not be directly involved in the vulnerability to schizophrenia but in linkage disequilibrium with a functional polymorphism. Previous significant association between the S311C and  141CDel/Ins polymorphisms of the DRD2 gene and schizophrenia (Table 1) may reflect the implication of the TaqI A1/A2 polymorphism.

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Interestingly, the TaqI A1/A2 polymorphism maps 9.5 kb downstream from the DRD2 gene stop codon, in a new gene predicted by Genome Scan analysis. This prediction was particularly useful as no Express Sequence Tag (EST) was available in this region in public database. The inter-exonic RT-PCR and the subsequent sequencing performed herein revealed two unpredicted exons, the splice site of one incorrect exon and two sequencing mistakes. The predicted protein belongs to the phosphotransferase family with a putative dual specificity as a serine/threonine and tyrosine kinase. Moreover, this protein has 11 ankyrin repeat domains that are usually involved in protein – protein interactions, especially with transmembrane proteins, and occur in a large number of functionally diverse proteins mainly from eukaryotes (Sedgwick and Smerdon, 1999). The TaqI A1/A2 polymorphism maps in the last ankyrin repeat and leads to a E713K substitution that might affect X-kinase interaction with other proteins. The mechanism underlying the association between the A2 allele, or the haplotypes containing this allele, and vulnerability to schizophrenia is still unclear. A direct effect of the TaqI A1/A2 polymorphism on the structure of the DRD2 has been excluded (Gejman et al., 1994). Because no data are yet available regarding the function of the X-kinase and more precisely its interaction with the DRD2, it remains difficult to speculate whether or not its encoding gene could be involved in susceptibility to schizophrenia. Interestingly, in inbred mouse strains, a genome-wide search for catalepsy related genes using quantitative trait loci approach detected a locus close to the distal 3V region of the DRD2 gene (Hitzemann, 1998). These convergent data suggest that the association found between the TaqI A1/A2 polymorphism and schizophrenia cannot be explained through direct modifications of the DRD2 gene. In order to conclude whether or not the DRD2 gene and/or the X-kinase gene are involved in genetic vulnerability to schizophrenia, it will be important to (i) determine the exact function of the X-kinase, and characterize the partners interacting with its different domains, (ii) study the impact of the TaqI A1/A2 polymorphism on the Xkinase function, and (iii) look for new polymorphisms. We are currently sequencing the X-kinase gene and the 3VUTR (untranslated region) of the

DRD2 gene in trios with the aim of assessing further the extent of linkage disequilibrium around the TaqI A1/A2 polymorphism. All polymorphisms inside this region, alone or combined in haplotypes, should be tested by the same statistical approach and by functional investigations.

5. Electronic-database information Accession number and URL for data in this article are as follows: University of California, Santa Cruz (UCSC) Genome Browser, http://www.genome.ucsc.edu. Single Modular Architecture Research Tool (SMART), http://smart.embl-heidelberg.de. GenBank, http://www.ncbi.nlm.nih.gov/. Accession Number (GenBank/EMBL): X-kinase gene Ban It477899 AF525298.

Acknowledgements INSERM (97009) has supported this work. This research received grants from INSERM, the ‘‘Fondation pour la Recherche Me´dicale’’ and Bristol-Meyer Squibb Foundation (unrestricted grant research program). We thank David Tre´gouet for statistical assistance (INSERM U525, Epidemiologic and Molecular Genetics of Cardiovascular Diseases).

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