The interpretation of the level of statistical significance in the allelic association studies should consider both multiple alleles and multiple markers. In the study of brain morphology, the number of statistical tests carried out should be considered. In the single marker tests of allelic association, we used empirical tests of significance, which do not need further correction for multiple alleles. For the global tests of haplotypic association with schizophrenia, further correction for both multiple alleles and multiple markers are not required because the permutation test significance takes both into account. The finding of extended transmission disequilibrium between D8S261 with schizophrenia in the family linkage sample was the reason to select this and other markers within the PCM1 gene for genotyping in the case-control sample. In addition, the marker D8S26 1 had been independently studied and implicated in schizophrenia in the National Institute of Mental Health trio sample and not as a result of the positive ETDT in the UK family sample or positive association in the UK case-control sample. The other markers showing association in the UK case-control sample were selected for being in linkage disequilibrium with each other. Taking into account that D8S261 had already been implicated in schizophrenia in prior studies and the fact that the additional markers genotyped were not independent tests of association, it can be argued that further corrections of significance values for testing association with multiple markers in the UK case-control sample are not needed.
Our results demonstrate that a chromosomal locus showing replicated evidence of linkage with schizophrenia in several family studies can be identified by observing linkage disequilibrium between markers and schizophrenia in a case-control association sample in which the cases have not been selected for a positive family history of schizophrenia. The fact that we were able to detect linkage disequilibrium in a sample of 450 cases of schizophrenia and find evidence in support of this in an independent sample of 100 US trio cases suggests that the chromosome 8p22 schizophrenia susceptibility alleles may be present in most European populations. As with other reported genetic associations with schizophrenia, it is clear that only a small fraction of research subjects with schizophrenia have a genetic susceptibility from the PCM1
gene locus. This confirms the general findings of linkage studies, which are explicable only by assuming the presence of extensive locus heterogeneity for genes involved in schizophrenia. We attribute the failure to find association in the Scottish sample as being due possiblybecauseonlyalimitedsetof3 markers could be genotyped in this sample. The UK case-control sample employed for this study association to the epsin 4 gene on chromosome 5, which encodes enthoprotin, a clathrin-associated protein involved in vesicle endocytosis in the brain and elsewhere.20
The gene PCM1
is involved in the maintenance of centrosome integrity and the regulation of the microtubule cytoskeleton. Its protein structure bears similarities to the structural myosin proteins, which are microtubule-associated proteins involved in axon guidance, synaptogenesis, functioning of the synapse, and intracellular transport along axons and dendrites.28
It is of note that a different gene disrupted in schizophrenia (DISC1
), known to straddle a translocation breakpoint on chromosome 1 that cosegregates with schizophrenia and other types of psychiatric disorder in a single large Scottish pedigree,29
also has similarities to structural proteins such as myosins. It seems likely that PCM1
has a role in the development of the nervous system and neuronal activity. For example, it is known that PCM1
interacts with the brain-specific protein huntingtin-associated protein 1 (HAP1)
that binds variably to huntingtin in relation to the number of glutamine repeat lengths present. PCM1
also interacts with cytoskeletal, vesicular, and motor proteins to mediate interactions among these different molecules.28
Our neuroimaging findings further validate the genetic data in so far as we demonstrate distinct differences in regional gray matter abnormalities in patients with chromosome 8p22 PCM1–associated schizophrenia (SZ8) compared with non-chromosome 8 PCMI–associated cases (SZ0). The significance levels reported were corrected for multiple tests and are therefore conservative. A larger sample size, which would reduce the standard error and increase confidence in the results, would require another 450 cases of schizophrenia to be sampled and genotyped at the PCM1 locus to test for association. Therefore, replication in an independent sample is desirable.
Our evidence suggests that brain regions showing the greatest differences between the schizophrenic groups are subsumed within a wider matrix of regional brain abnormalities that span other genetic susceptibilities to schizophrenia. Nevertheless, the peak locations of reduced gray matter volume in SZ8 and SZ0 groups predict important patterns of neuropsychological deficits or vulnerability that can perhaps inform etiopathological mechanisms. Selective gray matter deficit in orbitofrontal cortex, observed in the SZ8 group, is likely to preferentially compromise neural mechanisms supporting reward-related processing and motivational behaviors. In such cases, symptomatology of the patient may be biased toward affective and behavioral features. In contrast, patients with non–chromosome 8 PCMI–associated schizophrenia may exhibit more mnemonic and perceptual features.12
Relevant to this is the recent research showing that families with evidence for linkage to 8p21-22 had significantly more affective deterioration, poorer outcome, more thought disorder, and fewer depressive symptoms than affected individuals from non– 8p21-22–linked families.30
There is evidence of a second schizophrenia susceptibility locus called neuregulin 1 (NRG1)
on chromosome 8p21-2231
and 2 replications were successful.32-34
This locus is about 20 million bases toward the centromere from the PCM1
8p22 locus (). Gerber et al35
also found evidence of allelic association with schizophrenia on chromosome 8p21 at or near the calcineurin (PPP3CC)
gene locus. TwoJapanese research groups now report36,37
association with schizophrenia at the frizzled (FZD3)
gene locus suggesting that there may be 4 loci increasing susceptibility on the short arm of chromosome 8. This is compatible with the multiple lod score peaks along the short arm of chromosome 8 in our previous genome linkage scan of schizophrenia.4
In the case-control sample in which we have implicated the PCM1
gene, we have also found weak but significant evidence for association between schizophrenia and the NRG1
gene (Susmitta Datta, PhD, unpublished data, January 2006) but no evidence for association between markers at the PPP3CC
genes and schizophrenia (Susmitta Datta, PhD, unpublished data, January 2006). In addition to the epsin 4 gene association, 2 further allelic associations with markers at the FXYD6
(phosphohippolin) gene on chromosome 1 1q23.3 (Khalid Choudhury, BSc, unpublished data, January 2006) and the KIST serine threonine kinase (UHMK1)
gene on chromosome 1q23.338
have been found in the same sample. All of these associations have been found by following up localizations originally made in our family linkage analyses.
Further collections of genotyped patients are now needed to confirm the PCM1 8p22 allelic association with schizophrenia and to replicate the finding of brain morphology changes associated with PCM1 -associated marker alleles. Modest but statistically significant levels for PCM1 marker association with schizophrenia have been found in this study. This is compatible with the fact that linkage studies have already proven heterogeneity of linkage in schizophrenia. The linkage studies can only be explicable if one assumes that no single schizophrenia susceptibility gene has an effect in more than 5% to 10% of families. By inference, this would also apply to case-control samples as well. High levels of significance really would not be expected from a case-control sample if only 5% to 10% of cases shared a common genetic susceptibility.
Because some of the single nucleotide polymorphism marker alleles associated with schizophrenia are at both low and high frequencies, sufficient numbers of cases and controls are needed to obtain a good chance of replication. Assuming complete linkage disequilibrium between markers and disease alleles for a significance of P<.05 and a power of 80% of replicating the result (ie, 1 in 4) with a low-frequency allele present in 5% of controls and 10% of cases, then 600 cases and 600 controls should be enough. For a high-frequency disease-associated allele present in 50% of cases and in 45% of controls, then 1300 cases and 1300 controls are needed. We can now sequence DNA from cases who have inherited PCM1 gene marker alleles associated with schizophrenia to define the precise etiological base pair changes or mutations increasing susceptibility to schizophrenia. If these can be found, then exactly how PCM1 shapes regional brain structure and function can be understood. The alternative splicing of PCM1 messenger RNA, interaction of PCM1 with other proteins, posttranslational modification, phosphorylation, and glycosylation may all play a role in mediating genetic effects on the disease.