Positional candidate genes 1. Dysbindin 1 (DNTBP1)

In an attempt to identify the susceptibility genes responsible for the linkage they previously found to chromosome 6p, Straub and co-workers applied a systematic linkage disequilibrium (LD) mapping approach using their original 270 Irish pedigrees ( Straub et al., 1996) (section 1.4.3.4) (Table 1.4). These investigators performed a family-based association analysis of simple sequence length polymorphism (SSLP) markers and analysis of SNP haplotyping in 6p22. They found significant evidence for association with SNPs within the dysbindin (DNTBP1) gene (DNTBP1) with values <0.01 for a number of individual SNP markers and p-values of between 0.001-0.08 for multiple 3-marker haplotypes (Straub et al., 2002).

The human DNTPB1 encodes a 40kDA coiled-coil-containing protein that binds to α-and β-dystrobrevin in muscle and brain tissue to form the dystrobrevin-associated protein complex (Benson et al 2001). This protein complex plays an integral structural role in synapse formation and maintenance and is also thought to be involved in NMDA and GABA receptor signalling (Benson et al., 2001).

Since the original association between DNTBP1 and schizophrenia, several follow-up investigations have been undertaken. In view of the fact that DNTBP1 is located in the centre of their previously reported linkage peak on 6p (Schwab et al., 1995, 2000), Schwab and colleagues analysed the six most positive SNPs from an earlier study by Straub et al. (1996) in a cohort of 78 German and Israeli families, as well as 127 parent-proband trios in an attempt to replicate the finding by Straub and co-workers (Schwab et al., 2003). Evidence for association was observed in the two samples separately, as well as when they were combined (Schwab et al., 2003).

However, a second attempt at replication in 219 Irish cases and 231 control individuals failed to support the involvement of DNTBP1 in schizophrenia (Morris et al., 2003), as did a separate study of 708 DSM-IV diagnosed schizophrenia cases and 711 control subjects from the UK and Ireland (Williams et al., 2004).

However, investigators involved in the later study screened all exons and the promoter region of DNTBP1 and identified novel SNP markers. When these novel SNPs were included in the study, together with markers from the original study, highly significant evidence for association with an identified common risk haplotype was obtained (Williams et al., 2004). These same markers were then examined in the Irish cohort (Morris et al., 2003) and the observed risk haplotype reported in the afore-mentioned study was found to be significantly more common in affected individuals within this group (Williams et al., 2004).

61 The discrepant results in these studies is quite interesting in view of the fact that all these studies were conducted exclusively in European populations, yet the data suggests differences in LD between these populations.

1.4.6.2.2.Catechol-O-methyl transferase (COMT)

The gene encoding catechol-O-methyltansferase (COMT), COMT, has been localised to chr22q11.1-q11.2 by Grossman et al., (1985), at a genomic locus that has been implicated in schizophrenia by linkage studies (Williams et al., 2003) and chromosomal abberations. COMT catalyses the transfer of the methyl group from S-adenosyl-L-methionine to a phenolic hydroxyl group of catechol neurotransmitters, catechol steroids and catechol drugs (Axelrod, 1966; Campbell et al., 1984). In the brain, COMT degrades catechol amines such as norephinepherine, epinephrine and dopamine into O-methyl esters (Guldberg and Marsden, 1975).

Cell fractionation and immunological studies have shown that the COMT enzyme occurs as two distinct forms in mammals: in the cytoplasm as a soluble form (S-COMT) and associated with membranes as a membrane-bound form (MB-COMT) (Assicot and Bohuon, 1971). S-COMT activity is the more prevalent form in all tissues, while MB-COMT generally represents less than 5% of the total COMT activity (Guldberg and Marsden, 1975; Jeffery and Roth, 1984; Grossman et al., 1985). However, in the brain, MB-COMT activity has been reported to be higher than in other tissues (Rivett et al., 1982).

A functional polymorphism in COMT, in which the high (H) and low (L) activity alleles encode a valine or methionine amino acid residue, respectively, at codon 158 of the MB-COMT and codon 108 of S-COMT, has been described (Val/Met polymorphism). There is a three- to four-fold reduction in enzyme activity between the variants encoded by the H/H and L/L genotypes, with heterozygotes (H/L) showing intermediate enzyme activity. This polymorphism is represented by a G to A (Lachman et al., 1996) and has been the focus of numerous association studies in schizophrenia (Table 1.5), but have yielded ambiguous results (Li et al., 1996 Chen et al., 1997; Kunigi et al., 1997; Strous et al., 1997; Ohmori et al., 1998; de Chaldee et al., 2001; Egan et al., 2001; Liou et al., 2001; Norton et al., 2002, Williams et al., 2005; Tsai et al., 2006). Whereas studies by Ohmori and co-workers (150 schizophrenia patients and 150 control individuals) and Kremer and co-workers (276 cases and 77control individuals) found significant evidence for association between the L allele and schizophrenia, several other studies failed to find any association (Strous et al., 1997; Chen et al., 1997;

Norton et al., 2002; Williams et al., 2005; Tsai et al., 2006). Moreover, a number of family-based association studies have provided weak and inconsistent evidence that the H allele may be involved (Table 1.6).

The largest reported case-control association study investigating the role of COMT in schizophrenia was performed using over 700 patients and more than 7000 control individuals (Shifman et al., 2002). These investigators not only tested the Val/Met polymorphism for association, but also several other SNPs across the COMT gene in an Ashkenazi Jewish cohort (Shifman et al., 2002). Interestingly, the Val/Met polymorphism by itself only showed modest evidence for association, however, when it was analysed as part of a haplotype study that included two non-coding SNPs, a high level of significance was achieved (p=9.5x10^-8). These two

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non-coding SNPs (one in intron 1 of MB-COMT [rs737865] and the other near the 3’ UTR [rs165599]) were themselves significantly associated with schizophrenia, with rs165599 showing the highest levels of significance (Shifman et al., 2002) (Fig 1.9). Even though this study reached statistical significance, it remains unreplicated. The data generated in this study suggests that if an association exists between the COMT locus and schizophrenia, it cannot wholly be explained by the Val/Met polymorphism (Owen et al., 2004).

Given that COMT degrades dopamine, that most treatments for schizophrenia are dopamine blockers and that a deletion of chr22q11, which includes COMT, are all associated with increased schizophrenia risk, Bray and colleagues hypothesized that the COMT haplotypes associated with schizophrenia in the study by Shifman et al., (2002) would also be associated with lowered COMT mRNA expression levels (Bray et al., 2003). These investigators made use of SNPs within an expressed sequence as a tag for mRNA transcribed from each chromosomal allele and applied quantitative methods of allele discrimination to mRNA from individuals who are heterozygous for the marker polymorphism to measure relative allelic expression. They applied this principle to investigate the possible cis-acting mechanisms that affect expression of COMT in the human brain using 23 heterozygous individuals. Their data showed that the COMT haplotype implicated in schizophrenia is indeed associated with lowered COMT expression. Furthermore, they showed that the SNP rs165599 (3’ UTR SNP), which gave the best evidence for association in the study by Shifman and co-workers, is transcribed in the human brain and exhibits allelic expression differences, with lower expression of the schizophrenia-associated allele (Bray et al., 2003). These results support the hypothesis that the COMT haplotype implicated in schizophrenia susceptibility may exert its effect by the down-regulation of COMT and is also compatible with the hyperdopaminergic hypothesis of schizophrenia.

Fig 1.9: Location of SNPs in COMT investigated in the study by Shifman et al., 2002. The capital letters represent each of the SNPs as follows: A(rs737685); B(rs174686); C(rs740603); D(rs6269); E(rs6270);

F(rs4633); G(rs6267); H(rs165688); I(rs174689); J(rs362204); K(rs165599). Solid blue rectangles represent transcribed regions, whereas open rectangles represent untranslated regions.

Exon 1 95-112bp

Exon 2 90 bp

Exon 3 289 bp

Exon 4 194 bp

Exon 5 132 bp

Exon 6 472 bp

A B C D E FG H I J K

63 Table 1.6: Association studies of COMT Val/Met polymorphism in schizophrenia

Abbreviations: COMT, Catechol-O-methyltransferase; TDT, Transmission disequilibrium test; Val, Valine

Study reference Study Design Findings Statistical values

Li et al., 1996 TDT of 178 Han Chinese parent/schizophrenic offspring trios consisting of schizophrenic patients and their parents

Val-108 allele predominantly transmitted to affected

offspring p=0.005

Daniels et al., 1996 Case-control association study of 78 unrelated

schizophrenia patients and unrelated controls No association χ² =0.12, 1df, p=0.81 (allele)

χ² =0.32, 2df, p=0.83 (genotype) Chen et al., 1997 Case-control association study of 177 unrelated Chinese

schizophrenic patients and 99 unrelated Chinese controls No association χ² =0.01, 2df, p=0.99 (genotype) χ² =0.000, 1df, p=1.00 (allele) Strous et al., 1997b Case-control association study of 42 unrelated

Caucasian schizophrenia patients and 87 unrelated Caucasian controls from the U.S

No association p=0.07 (genotype)

p=0,15 (allele) Ohmori et al., 1998 Case-control association study of 150 unrelated

Japanese schizophrenia patients and 150 unrelated Japanese controls

Val-108 allele associated with schizophrenia χ² =4.83, 1df, p=0.028 (allele) χ² =7.26, 2df, p=0.026 (genotype) Egan et al., 2001 TDT of 104 Caucasian parent/schizophrenic offspring

trios Val-108 allele transmitted more frequently to affected

offspring χ² =4.57, p=0.03

Liou et al., 2001 Case-control association study of 198 unrelated Chinese schizophrenia patients and 188 unrelated Chinese controls

No association between COMT and schizophrenia, but significant differences in age of onset among the different genotypes

χ²=5.501; p=0.005

Kremer et al., 2002 TDT of 194 Palestinian Arab parent/ schizophrenic offspring trios

Case-control association study of 276 unrelated Palestinian Arab schizophrenia patients and 77 unrelated Palestinian Arab controls

Val-108 allele associated with schizophrenia in case-control study. Association stronger in females.

No preferential transmission of either allele in TDT

χ²=3.935; 1df, p=0.047 χ²=5.89; 1df, p=0.015 (females) χ²= 0.14, p>0.05

Norton et al., 2002 Case-control association study of 346 unrelated Caucasian schizophrenia patients and 334 unrelated schizophrenia controls

No association χ²=0.73; 1df; p=0.55

Glatt et al., 2003 Meta-analysis of 14 case-control association studies (2205 cases, 2236 controls and 5 family-based studies Sazci et al., 2004 Case-control association study of 297 unrelated Turkish

schizophrenia patients and 341 unrelated Turkish controls

Val-108 allele and Val/Val genotype associated with

schizophrenia. χ² =13.03, p=0.001 (allele)

χ² =4.048, p=0.020 (genotype) Williams et al., 2005 Case-control association study of 709

unrealated Caucasian schizophrenia patients and 710 unrelated Caucasian controls and TDT analysis of 488 parent/offspring trios

No association found in case-control studies No preferential transmission of either allele found in family-based studies

p=0.9 p=0.75

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1.4.6.2.3.Proline dehydrogenase 2 (PRODH2)

The high frequency of schizophrenia in patients with 22q11DS has led to the hypothesis that sequence variation within one or several genes in the deleted region of chr22q11 (Fig 1.5) might contribute to schizophrenia in the general population (Egan et al., 2001). The overwhelming majority of chr22q11 deletions are 3Mb in size whereas approximately 8% involve a smaller 1.5Mb deletion. Karayiourgou et al. reported a schizophrenic patient carrying this smaller 1.5Mb deletion and thus defined the “schizophrenia critical region”

on chr22q11 (Karayiourgou et al., 1995). Linkage disequilibrium mapping of this 1.5Mb region in schizophrenic patients identified a segment containing the gene encoding proline dehydrogenase (PRODH) (Lui et al., 2002). PRODH is a mitochondrial enzyme that converts proline to ∆-1-pyrroline-5-carboxylate and is involved in the transfer of redox potential across the mitochondrial membrane (Gogos et al., 1999). ∆-1-Pyrroline-5-carboxylate can be converted to glutamate and GABA, both of which are candidate neurotransmitter systems implicated in schizophrenia (Pearlson 2000). Additionally, mice with an inactivated PRODH gene have abnormal sensorimotor abnormalities (discussed in section 1.1.4.1.) similar to schizophrenic patients (Gogos et al., 1999).

Liu and colleagues analysed polymorphisms in PRODH and found an association between the PRODH^*1945T/C SNP and schizophrenia using TDT in 107 independent North American triads. These investigators subsequently revealed an association with a two marker haplotype, PRODH^*1945/1766, which was significantly more associated with schizophrenia than either SNP individually, when analysed using the North American triads and a case-control sample of 109 unrelated Afrikaner schizophrenic patients and 75 unrelated Afrikaner control individuals) (Liu et al., 2002). However, Fan and colleagues found no evidence of preferential transfer of PRODH^*1945T/C alleles to affected offspring using both TDT and HRR methods in 166 family trios from east China. This study (Fan et al., 2003).

Williams and colleagues undertook a detailed analysis of PRODH using a large case-control cohort (368 unrelated Caucasian schizophrenic patients from the U.K and Ireland and 368 unrelated matched Caucasian control individuals, a sample of VCFS probands with and without schizophrenia and a sample of 55 proband trios with juvenile-onset schizophrenia) (Williams et al., 2003). They found none of the SNPS employed in the Liu et al. study, nor nine newly identified cDNA varients, to be associated with schizophrenia susceptibility (Williams et al., 2003).

Thus, despite several lines of evidence suggesting that PRODH is likely to be a promising candidate gene for schizophrenia, given the lack of replication of the original findings in studies employing 95% power to replicate, PRODH, as other schizophrenia implicated genes, may not be ubiquitously associated with increased schizophrenia risk (Owen et al., 2004).

1.4.6.2.4. Neuregulin 1 (NRG1)

Neuregulin 1 (NRG1), a member of a family of four growth factors, the neuregulins, plays multiple roles in a number of organs including the nervous system, heart and breast (Lemke et al., 1996; Ozaki et al., 2000; Falls et al., 2003). In the brain, NRG1 is involved in synapse formation, activity-dependent synaptic plasticity and

65 regulation of NMDA, GABAA and acetylcholine receptor subunit expression (Ozaki et al., 1997; Yang et al., 1998; Rieff et al., 1999; Liu et al., 2001, Liu Stefansson et al., 2002). Neuregulin 1 also regulates the proliferation and migration of Schwann cells (Dong et al., 1995; Lemke, 1996; Gassmann and Lemke,1997) and neurons within the brain (Rieff et al., 1999; Schmid et al., 2003). It has been suggested that NRG1 may mediate schizophrenia by changing expression profiles of a wide variety of neuroreceptors, particularly NMDA and glutametergic receptors (Stefansson et al., 2002).

The neuregulin1 gene (NRG1) has been localised to chr8p12-22, which has been implicated in schizophrenia susceptibility in a number of independent studies in varying population groups (Pulver et al., 1995; Kender et al., 1996; Levinson et al., 1998, Brustowicz et al., 1999; Gurling et al., 2001). Stefansson and colleagues conducted a genome-wide scan for schizophrenia linkage in a large Icelandic population and detected suggestive evidence for linkage to chr8p12-21 (Stefansson et al., 2002). This was followed up with systematic LD analysis around the linked region on 8p and three “at-risk” haplotypes (HapA, HapB and HapC) were identified. Each “at-risk” haplotype shared a 290kb core haplotype of seven markers (5 SNPs and 2 microsatellites), upstream from the first 5’ exon of NRG1, that showed an estimated frequency of 7.5% in the general population and 15.4% among schizophrenic patients (Stefansson et al., 2002) (Fig 1.10). This core “at-risk” haplotype is defined by a minimum haplotype of one SNP (SNP8NRG221533) and two microsatellites (478B14-848 and 420M9-1395). Following the identification of the “core at-risk haplotype” in an Icelandic population, Stefansson and co-workers replicated this finding in a Scottish cohort of 609 schizophrenic patients and 618 control individuals, suggesting that the “at-risk” haplotype is not specific to the Icelandic population (Stefansson et al., 2003)

Since these initial studies, several investigations of NRG1 involvement in schizophrenia have been completed.

Several of these studies provided further evidence for the association of NRG1 and schizophrenia risk.

Williams and colleagues genotyped SNP8NRG221533, 478B14-848 and 420M9-1395 (defined by Stefansson et al., [2002] as the minimum “core at-risk” haplotype) in their sample of 709 unrelated Caucasian schizophrenic patients and matched control individuals born in the U.K. and Ireland (Williams et al., 2003).

None of the three analysed markers achieved significant allelic association with schizophrenia by themselves, however, when testing for haplotype association, the previously described “at-risk” haplotype was significantly more common in the patients than in the control group (Williams et al., 2003).

Corvin and colleagues provided additional support for a susceptibility locus for schizophrenia on chr8p12-22 by replicating (Corvin et al., 2004) the two above-mentioned studies (Stefansson et al., 2002; Williams et al., 2003) in an Irish case-control sample. They identified an ‘at-risk’ haplotype that overlaps one of the “at-risk”

haplotypes (HapB) reported in the Icelandic population (Corvin et al., 2004; Stefansson et al., 2002). This refined haplotype (HapBIRE) was found in significant excess in the Irish schizophrenia cases versus control individuals (Corvin et al., 2004). These results were also confirmed in the Scottish schizophrenia cohort described by Stefansson et al . Moreover, this study suggests that the expressed sequence tag (EST) cluster Hs97362 may potentially be a susceptibility gene at the NRG1 locus (Corvin et al., 2004). However,

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A

B

Fig 1.10. continues on next page

67 C

Fig 1.10: Schematic representation of markers used in the study by Stefansson and colleagues (2002). A:.

Microsatellite markers on chromosome 8p. The position of NRG1 is schematically shown (taken from Stefansson et al., 2002). B: Microsatellite markers and SNPs at the 5’ end of NRG1. The 4 haplotypes, HapA-HapC2, were individually found in excess in schizophrenia patients. The blue arrows indicate the three markers that minimally define the core haplotype. The panel on the right indicates the frequencies of each of the four “at-risk” haplotypes in all affected individuals (taken from Stefansson et al., 2002). C: Schematic representation of the truncated forms of the ‘at-risk’

haplotypes. Haplotype frequency of HapBIRE and HapD (the haplotypes that were found to be associated with schizophrenia in the Irish cohort in the study by Corvin et al., 2004) is shown in the panel on the right. The relative physical position of each of the markers on NRG1 is indicated by the black arrows. The positions of the Chinese haplotypes are indicated by solid black lines, while the position of the Scottish/Icelandic haplotype is indicated by the solid blue line (taken from Corvin et al., 2004).

independent studies conducted in the Irish study of high-density schizophrenia families (ISHDSF) (Thiselton et al., 2004) and in a Japanese cohort (Iwata et al., 2004) found no evidence for linkage or association with the previously described “at-risk” haplotypes (Stefansson et al., 2002; Williams et al., 2003; Corvin et al., 2004) at the NRG1 locus.

But, a novel “at-risk” haplotype was identified at this locus in a Han Chinese sample (Li et al., 2004). In this study, five of the seven markers used in the Icelandic sample were genotyped and used for haplotype analysis in family-based (184 parent-offspring trios and 138 affected siblings with at least one parent) and case-control association studies (298 unrelated Han Chinese schizophrenic patients and 336 unrelated Han Chinese control individuals) (Li et al., 2004). Neither the haplotype nor alleles associating with the Icelandic “at-risk”

haplotype were found in excess in the Chinese schizophrenia cases. Three interesting haplotypes where, however, identified, ie. HapCHINA1, HapCHINA2 and HapCHINA3.. HapCHINA1, situated immediately upstream of the Icelandic “at-risk” haplotype was found in excess in schizophrenic patients versus control individuals.

However, this association was lost when family-based methods were used. HapCHINA2 was associated with schizophrenia in both case-control and family-based studies, while HapCHINA3 was only associated in family-based studies (Li et al., 2004).

Hap_CHINA3

HapCHINA1 Hap_CHINA2

Scottish/Icelandic haplotype

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Recently, Kim an co-workers genotyped the seven markers used in the Icelandic sample (Stefansson et al., 2002) in a cohort of 242 unrelated Korean schizophrenia patients and 242 matched controls (Kim et al., 2006). In the above-mentioned study, only SNP8NRG241930 showed a tendacy toward significance in the schizophrenia sample and a significant association was only observed when the schizophrenia group was sub-stratified into patients with auditory hallucinations (section 1.1.1). However, as with the study by Li et al., 2004, this study also did not replicate the haplotype association findings of Stefannson and colleagues. Instead, their results showed that another haploytype containing the opposite alleles reported by Stefansson and co-workers was significantly increased in the control group (Kim et al., 2006).

The failure of the studies by Li and co-workers and Kim and co-workers to replicate the findings in the Icelandic, Scottish and Irish studies (Stefansson et al., 2002, Williams et al., 2003; Corvin et al., 2004) is not surprising as the origins of Northern European and Chinese populations are separated by tens of thousands of years and different relationships between risk haplotypes and disease susceptibility variants may have evolved during this time (Owen et al., 2004). The same unidentified disease-predisposing genetic variant or variants may be present in both European and Chinese populations but are associated with different overlying haplotypes (Li et al., 2004).

There have also been reports of association studies between a non-synonymous NRG1 polymorphism (Arg38Gln), located in exon 2 of NRG1, and schizophrenia. Yang et al., in a study of 246 Han Chinese trios, reported a significant association between this polymorphism and schizophrenia (Yang et al . 2003). In an attempt to replicate this finding, Hong and co-workers conducted case-control and family-based association studies of NRG1-Arg38Gln in 228 schizophrenic patients and 269 control individuals (Hong et al., 2004). The family-based analysis showed that the 38Gln allele was transmitted in excess to affected offspring, however no association was detected in their case-control analysis. The discrepancy between results from family-based and case-control studies can be explained in various ways. Firstly, the NRG1 polymorphism may be a disease locus of small effect and, therefore, the failure of these investigators to detect any association in their case-control

There have also been reports of association studies between a non-synonymous NRG1 polymorphism (Arg38Gln), located in exon 2 of NRG1, and schizophrenia. Yang et al., in a study of 246 Han Chinese trios, reported a significant association between this polymorphism and schizophrenia (Yang et al . 2003). In an attempt to replicate this finding, Hong and co-workers conducted case-control and family-based association studies of NRG1-Arg38Gln in 228 schizophrenic patients and 269 control individuals (Hong et al., 2004). The family-based analysis showed that the 38Gln allele was transmitted in excess to affected offspring, however no association was detected in their case-control analysis. The discrepancy between results from family-based and case-control studies can be explained in various ways. Firstly, the NRG1 polymorphism may be a disease locus of small effect and, therefore, the failure of these investigators to detect any association in their case-control