[1]
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There are several lines of evidence indicating that schizophrenia and bipolar affective disorder are unlikely to be caused by single major genes but rather by multifactorial inheritance. 1,2 The two disorders may share genetic and nongenetic risk factors. 3 Significant linkage of markers to 4p16 has been found in several families segregating for bipolar disorder or schizophrenia, [4][5][6][7][8][9] which makes this a candidate region for susceptibility gene(s). Perhaps the most interesting candidate gene in this region is the gene coding for the dopamine type 5 receptor (DRD5), 10 as malfunction of the dopamine system has been implicated in psychotic disorders. 11 The present study focuses on obtaining further evidence to map susceptibility genes for schizophre-nia and bipolar affective disorder on chromosome 4, in samples of distantly related patients from the genetically isolated population of the Faroe Islands. The present study identifies segments in chromosome regions 4p16 and 4p15 that may be involved in the susceptibility to schizophrenia and bipolar affective disorder. In searching for a joint risk locus for the two disorders, a number of overlapping segments were supported by increased sharing of one or more haplotypes.
[2]
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Linkage-disequilibrium (LD)-based approaches for mapping of complex neuropsychiatric disease genes may be useful for locating susceptibility loci. 12,13 The recent founding, isolation and relatively rapid expansion of the Faroese population 14,15 is reflected in the population structure and the extensive background LD that has been observed in this population, 16,17 making this population suitable for LD-mapping of complex diseases. The affected individuals in this study share common ancestry, and it is therefore likely that they also share disease genes and alleles of surrounding markers. In LD-mapping, one searches for markers that have different allele or haplotype frequencies when comparing cases and controls, suggesting that markers where allele frequencies differ are in LD and therefore presumably lie close to a disease gene.
[3]
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Allele and haplotype frequencies of 34 microsatellite-markers were compared among cases and controls using Monte Carlo-based tests as implemented in the computer program CLUMP. 18 Association tests were performed for single markers, two-and three-marker segments (Table 1). Table 2 lists P-values for Fisher's exact tests performed for single alleles or haplotypes of markers that revealed an interesting level of association (Table 1). For a given marker or segment, Fisher's exact tests were only performed for specific alleles or haplotypes with large deviation in frequency between cases and controls, in order to evaluate whether low P-values obtained by CLUMP were preferentially caused by deviation in frequency of a single allele or haplotype. Each separate Fisher's exact test, therefore, only evaluates differences in frequencies of a specific allele or haplotype. If the P-value obtained by running CLUMP is lower, this indicates that the overall difference between cases and controls is not caused by divergence in frequency of a single allele or haplotype, but must be caused by divergence in frequencies of more than one allele or haplotype. Affected individuals who are related may share segments that are identical by decent (IBD) from a common ancestor who does not necessarily contain shared disease genes, and therefore represent false positives. The goal is to identify haplotypes shared more frequently by cases than would be expected by chance given the genealogical relation of cases. The probability that affected individuals share specific haplotypes that are IBD by chance from a known ancestor is presented in Table 2. IBD probability calculations are performed for a single haplotype, and are therefore too conservative for evaluating situations where low CLUMP P-values are caused by differences in frequencies of more than one haplotype.
[4]
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The gene for the DRD5 maps to chromosome 4p16.1 10 and dysfunction of dopamine synthesis has been implied in the pathophysiology of psychotic disorders for decades. 11 Linkage between D4S394 (4p16.1) and bipolar affective disorder was initially reported by Blackwood et al. 4 Other surveys have provided additional evidence for linkage of bipolar affective disorder or schizophrenia to markers in this region and other regions on chromosome 4. [5][6][7][8][9] There are, however, also negative reports of linkage of bipolar affective disorder or schizophrenia to chromosome 4. 9, [19][20][21] When considering bipolar patients, two segments (D4S394-D4S2983 and D4S2923-D4S2928-D4S1582) at 4p16.1 received some support (Table 1). For patients with schizophrenia, there was reasonable support for 4p16.1, as marker D4S2281, a two-marker segment (D4S2281-D4S1605) and a three-marker segment (D4S2923-D4S2928-D4S1582) appeared to be associated with schizophrenia. When combining both psychiatric disorders, chromosome 4p16.1 received further support from six partially overlapping two-and three-marker segments (D4S394-D4S2983, D4S2923-D4S2928, D4S2281-D4S1605 and D4S394-D4S2983-D4S2923; D4S2923-D4S2928-D4S1582, D4S1582-D4S1599-D4S2281). For most of these markers and segments, Fisher's exact tests revealed P-values that were either nonsignificant, an order of magnitude higher than the one obtained by running CLUMP or several alleles/haplotypes produced significant P-values.
[5]
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The DRD5 gene is located between markers D4S2923 and D4S2928 on 4p16.1. Fisher's exact tests for the combined data set yielded P-values below 0.08 for two haplotypes of this segment (Table 2), suggesting that the difference between cases and controls (Table 1) might be explained by a combination of these haplotypes. Only one of these (132-191) showed increased frequency among cases. IBD probabilities suggest that IBD by chance is less likely considering the specified genealogy, and that this haplotype may show increased sharing among cases due to LD with a common risk allele.
[6]
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For schizophrenia and for both disorders combined, a single haplotype (132-191-124) of D4S2923-D4S2928-D4S1582 turned out to have significant differences in frequency between cases and controls (Table 1), although two other haplotypes also contributed to the very low CLUMP P-value in the combined data set (Table 2). IBD calculations for the combined data set suggest that haplotype sharing by chance is less likely, and this segment could therefore potentially contain a risk locus in association with the two-marker haplotype.
[7]
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The two-marker segment D4S403-D4S3048 (4p15) appeared to be associated with schizophrenia and the two disorders combined (Table 1). Fisher's exact tests revealed differences in frequency of two haplotypes (Table 2). IBD probabilities calculated according to Houwen et al 22 indicate that each of these differences considered separately is less likely to be IBD by chance.
[8]
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The present study of patients from the Faroe Islands provides no support for the previously suggested regions on chromosomees 4q31.21 and 4q35, 5,8 neither for each of the disorders considered separately nor for the combined data set (Table 1).
[9]
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Many of the markers and segments are in close proximity and, based on a previous study of LD among markers on chromosome 22, 17 presumably in extensive LD. The association tests performed in the present study cannot, therefore, be considered independent. There is no generally accepted method to make the appropriate correction, and this issue is further complicated by the fact that LD varies between populations and among different genomic regions. [23][24][25] The P-values presented here are, therefore, not corrected for multiple tests.
[10]
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The patients in this study are distantly related, and can therefore, in principle, not be considered as independent samples. All affected individuals are, however, related more than three generations ago and the bias introduced is considered to be relatively small. As we have detailed knowledge of the genealogy of the patients, this allows us to calculate the likelihood of IBD by chance and estimate the false-positive probability for specific haplotypes.
[11]
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We have previously shown that the essential assumption, that cases and controls represent sub-samples of the same panmictic population and share the same genetic history, seems valid for our sample. 16,17 In conclusion, our study supports chromosome region 4p16.1 for both bipolar affective disorder and schizophrenia. A single marker and two-and three-marker segments, with varying degrees of overlap within the 7.75 cM region between markers D4S394 and D4S1605, provide evidence for Fisher's exact test identifies differences in allele or haplotype frequencies between cases and controls for specific alleles or haplotypes. P 0 is the probability of all cases having the most frequent haplotype IBD by chance from a known ancestor (i.e., false-positive probability) according to Houwen et al. 22 P 1 is the same probability summed over all segments typed on chromosome 4. P 2 is the genome-wide probability of IBD according to the method by Durham and Feingold. 28 The presence or absence of the most frequent haplotype could not always be unambiguously determined due to phase uncertainties and the total number of chromosomes typed was therefore lowered accordingly. Due to the conservative way P 1 and P 2 are calculated, they may exceed a value of 1 when the observed value of the haplotype of interest are small. P 2 is considered to be the most conservative.
[12]
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association with schizophrenia and/or the two disorders combined. In some of the segments, specific haplotypes occur with increased frequency in both disorders. This supports the possibility that the locus on 4p16 may predispose to schizophrenia as well as bipolar affective disorder. Based on historical records and geography, the Faroese population is likely to be genetically related to parts of the Scottish and Irish populations, and it is thus possible that the same risk loci, and possibly even haplotypes, may be shared for complex disorders. It is therefore not surprising that chromosome 4p16 has been implied in studies of patients collected from the Scottish population 4,9 and other neighbouring populations. [5][6][7] This study is the first to support these findings, providing evidence for a possible shared risk locus on 4p16 for the two disorders on the basis of two-and three-marker haplotypes. The present study will also make the comparison with specific haplotypes in affected individuals from Scotland and Ireland possible, as standardised allele sizes have been used. This could potentially assist in further delineation of a minimum critical region that harbours the risk locus.