Presentación de Resultados

Dopamine receptors, in particular dopamine D2-like receptors, have long been implicated in the pathogenesis o f schizophrenia (see section 1.8). Apart from the dopamine receptors, many other genes fitting several candidate gene models have also been tested for association with schizophrenia: models o f serotonergic dysfunction, viral/autoimmune theories and neurodevelopmental disturbances. These have not been addressed in this thesis. The next few sections provide a summary o f some o f the linkage and association studies o f the five dopamine receptors with schizophrenia.

1.12.2.1 Linkage analysis

Several studies have used the five dopamine receptor loci as candidate genes to test for genetic linkage to schizophrenia and have failed to find an association (see review by Kendler and Diehl, 1993). One such study was performed by Coon et al. (1993). The five loci were tested in nine multigeneration families. The results provided evidence for the lack o f a mutation at any o f the receptor gene sites that could be a major contributor to schizophrenia. Another recent study excluded DRD2 as a candidate gene (Grassi et a l,

1996). Wiese et al. (1993) failed to show linkage between schizophrenia and the DRD3 locus in four Icelandic pedigrees. Nanko et al. (1994) and Asherson et al. (1996) also failed to show linkage between schizophrenia and the DRD3 locus in two Japanese pedigrees for dominant and recessive models and in 24 families (non-parametric linkage study o f 90 affected sibling pairs also failed to show linkage) respectively.

Shaikh et al. (1994) used the polymorphic 48bp repeat sequence o f the DRD4 (discussed in the next section) as a marker to perform a linkage study involving 24 families. Two- point lod score analyses with a number o f single gene models ranging fi*om near dominant

Introduction to near recessive revealed no evidence for linkage. Also the data were examined with non- parametric sib-pair analysis and no excess sharing o f alleles between affected sib-pairs was observed. This study failed to show linkage to the DRD4 locus. Kalsi et al. (1996) investigated the association o f the DRD5 with schizophrenia. 23 Icelandic and English pedigrees containing multiple cases o f schizophrenia were genotyped using a highly informative microsatellite for the DRD5. Negative lod scores were obtained under assumptions o f autosomal dominant and recessive inheritance. The authors concluded that mutations o f the DRD5 are not a major cause o f schizophrenia in these pedigrees but due to the probable existence o f locus heterogeneity, the D RD5 may be o f aetiologic importance in other families with schizophrenia.

Despite the fact that no linkage was found between dopamine receptors and schizophrenia, it is possible that a certain polymorphic form o f the dopamine receptors may increase disease susceptibility or contribute to the severity o f symptoms. Based on this hypothesis association studies were initiated, as discussed below.

1.12.2.2 Association studies

A large number o f case/ control association studies have been performed, where the distribution o f a specific allele o f a polymorphic locus is tested in unrelated affected individuals compared to unrelated controls. This method o f study is powerful for detecting susceptibility loci o f small to moderate effects. The controls would be matched by ethnicity, sex, age and would have preferably been psychiatrically evaluated. The only problem with this design is stratification effects, where a sample o f the population contains a disease and a marker allele more commonly than expected without a causal relationship between them. This problem can be avoided by careful selection o f unrelated patients and controls from a specific population.

Four polymorphic sites have been detected in the human D R D l but none has been shown to be associated with schizophrenia (Ohara et a l, 1993; Cichon et a l, 1994a and b). Sobell et al. (1995) examined the entire gene in 78 unrelated schizophrenic individuals using the dideoxy finger-printing (ddF) screening method. Nine different sequence changes were identified. Five o f these generate protein alterations but none showed statistically significant association with schizophrenia or other neuropsychiatrie diseases. A novel polymorphism causing an amino acid change from serine to cysteine at codon

Introduction

311 o f the DRD2 was originally reported by Itokawa et al. (1993). This substitution was the first sequence variation described in the coding sequence o f the human DRD2. Tw o studies have shown a significant association between the cysteine 311 allele and schizophrenia (Arinami et al., 1994; Shaikh et a i, 1994). Thereafter several studies have tried to replicate these findings and so far none has shown a positive association. One o f the recent studies by Verga et al. (1997) also investigated this polymorphism but failed to show any association. This paper provides a summary o f the studies investigating this polym orphism until the present study.

The DRD4 and DRD3 are the two o f the five dopamine receptor genes that have been investigated in a large number o f studies (discussed in the next few sections), mainly because o f the 10-fold affinity for clozapine by the DRD4 and the implication o f the DRD3 in the limbic hypothesis o f schizophrenia (see sections 1.8.1 and 1.8.2).

(i) Polvmorphims in the DRD4

An interesting feature o f DRD4 is the existence of several polymorphic variants o f this receptor which could conceivably lead to biochemical or physiological differences. This observation has prompted thorough investigations at structural, pharmacological, biochemical, genetic and clinical levels to detennine a possible role o f this receptor in

schizophrenia and its treatment. Figure 1.21 illustrates the currently known

polymorphisms in the DRD4.

12bp 13bp 48bp

ins/de del repeats

5’ --- ^ ---■ ■ ---3’

I II I III IV

V al-194-gly

Fig ure 1.21; S ch em atic rep resen tatio n o f p o ly m o rp h ism s in the h um an D R D 4 (adapted from Sanya! and V an Toi, 1997). T he solid black line rep resen ts introns, the black boxes rep resen ts exons.

The I2bp insertion/deletion polymorphism located in exon 1 encodes a sequence o f four amino acids in the extracellular N-terminal part of the receptor, that borders the first putative TMD (Catalano et al., 1993). The polymorphism occurs as a 2-fold repeat in the more common variant (allele 1) and is represented only once in the rarer variant (allele

Introduction 2). Catalano et al. (1993) observed a significant difference in genotype frequencies o f this polymorphism between patients with delusional disorder and controls. 27% o f patients suffering from delusional disorder carried the allele 2 compared with only 8% o f the controls. Nevertheless, no significant differences were observed between schizophrenics and controls. In a recent publication by Ohara et al. (1996), an association between the D2-like receptors and schizophrenia was investigated. Once again, no statistically significant association with schizophrenia was observed, except a trend with positive symptoms. Schizophrenics heterozygous for the 12bp repeat in exon 1 had a lower total positive symptom score before medication compared with schizophrenics homozygous for allele 1 (two 1 2bp repeats).

A second polymorphism, a 13bp deletion located in exon 1, which occurs with a frequency o f 2% in the German population, was reported by NOthen et al. (1994). This deletion alters the reading frame from amino acid 79 and thus generates a new stop codon downstream at amino acid 99. This deletion is situated in the second TMD and includes Asp 80 which is highly conserved within the family o f catecholaminergic receptors and is postulated to act as the counterion in catecholamine binding (Strader et a l, 1988, see section 1.9.3.3, table 1.2). The mRNA is predicted to code for an aberrant protein o f 98 amino acids with a calculated molecular mass o f 1 IKDa. As a result the protein encoded is non-fimctional and is therefore a complete loss-of-function mutation. The frequency o f this mutation was examined in a total o f 277 control subjects, 163 schizophrenic patients and 144 patients with bipolar affective disorder and 134 patients with Tourette’s syndrome (NOthen et a l, 1994). The mutation occurred in similar frequencies in all the samples, indicating that heterozygosity for this mutation is not causally related to these major psychiatric diseases. A later study carried out on 196 schizophrenic patients and 162 healthy individuals o f Italian descent, also confirmed the previous negative findings (Di Bella et a l, 1996). This 13bp deletion is the first example o f a naturally occurring mutation leading to a non-functional dopamine receptor protein in humans.

A mononucleotide G repeat variant was identified in the first intron by Petronis et al. (1994). A nucleotide change from T to G, causing a valine to glycine substitution at codon 194 in exon 3 was identified by Seeman et al. (1994). This substitution was shown to result in a non-functional protein. This polymorphism has been shown not to

Introduction be associated with schizophrenia (Seeman et a l, 1994).

Polymorphic forms o f the DRD4 containing the 48bp repeat have been shown to exist in the general population. The 48bp repeat is situated in the third exon o f the gene (Van Tol et a l, 1992). Screening human genomic libraries has revealed 19 different repeat units located at variable positions within the repeat region (Van Toi et a l, 1992; Litcher et a l,

1993; Asghari et a l, 1994). Further analysis o f the repeat region has shown that the alleles vary not only in the number o f repeats but also in the sequence o f the repeats and the order in which they appear (Litchter et a l, 1993). Due to these differences, a total o f 27 different haplotypes, coding for 20 different receptor forms have been found (Litcher et a l, 1993; Asghari et a l, 1994, see figure 1.22). The protein encoded varies b y multiples o f 16 amino acids in the putative third cytoplasmic loop region, the region thought to be involved in G-protein coupling (see section 1.9.3.3).

The different forms o f DRD4 have been shown to display pharmacological differences when expressed in COS cells. For example, the seven-repeat form was observed to be less sensitive to changes in agonist or antagonist (clozapine) affinity induced by addition o f sodium chloride. The two and four repeat forms exhibited six to eight fold higher dissociation constants for clozapine in the presence o f sodium chloride than in its absence (Van Toi et a l, 1992). Whether these pharmacological differences observed in vitro translate into meaningful differences in dmg sensitivity in the intact brain remains to be seen. On the other hand, a study conducted by Asghari et a l (1994), which involved the transient expression o f full length cDNAs encoding two, three, four, five, six, seven and nine repeat sequence in COS-7 cells, provided evidence for the lack o f influence o f the polymorphic repeat on the DRD4 binding profiles to clozapine and dopamine. However several studies have already disproved any association between this polymorphic repeat and schizophrenia (Tanaka cr a/., 1995; Ohara a/., 1996). One such study investigated 80 schizophrenic and 81 control subjects, and examined the allelic association between the patient groups and the controls, with respect to clinical characteristics, but no association was found. The frequency o f individual alleles by repeat number (2-7) was similar in the two groups (Nanko et a l, 1993). In another study performed by Petronis et a l (1995), no genetic association was showed with schizophrenia, although a trend towards an excess o f the allele with seven repeats was observed. Sommer et a l (1993) investigated the 48bp repeat polymorphism in 115

Introduction