Original Article

Molecular Psychiatry (2010) 15, 1101–1111; doi:10.1038/mp.2009.96; published online 29 September 2009

Evidence for rare and common genetic risk variants for schizophrenia at protein kinase C, alpha

L S Carroll1, N M Williams1, V Moskvina1, E Russell1, N Norton1, H J Williams1, T Peirce1, L Georgieva1, S Dwyer1, D Grozeva1, E Greene1, A Farmer2, P McGuffin2, D W Morris3, A Corvin3, M Gill3, D Rujescu4, P Sham5, P Holmans1, I Jones1, G Kirov1, N Craddock1, M C O'Donovan1 and M J Owen1

  1. 1Department of Psychological Medicine, School of Medicine, Cardiff University, Henry Wellcome Building for Biomedical Research in Wales, Cardiff, UK
  2. 2King's College London, Department of Psychological Medicine, Institute of Psychiatry, London, UK
  3. 3Neuropsychiatric Genetics Group, Institute of Molecular Medicine, Trinity College Dublin, St James Hospital, Dublin, Ireland
  4. 4Department of Psychiatry, Ludwig-Maximilians-University, Munich, Germany
  5. 5Department of Psychiatry and State Key Lab for Cognitive and Brain Sciences, The University of Hong Kong, Pokfulam, Hong Kong SAR, China

Correspondence: Professor MJ Owen or Professor MC O’Donovan, Department of Psychological Medicine, Cardiff University, Henry Wellcome Building, Heath Park, Cardiff CF14 4XN, UK. E-mails: OwenMJ@cf.ac.uk or ODonovanMC@cf.ac.uk

Received 11 February 2009; Revised 28 July 2009; Accepted 18 August 2009; Published online 29 September 2009.

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Abstract

We earlier reported a genome-wide significant linkage to schizophrenia at chromosome 17 that was identified in a single pedigree (C702) consisting of six affected, male siblings with DSM-IV schizophrenia and prominent mood symptoms. In this study, we adopted several approaches in an attempt to map the putative disease locus. First, mapping the source of linkage to chromosome 17 in pedigree C702. We refined the linkage region in family C702 to a 21-marker segment spanning 11.7Mb at 17q23–q24 by genotyping a total of 50 microsatellites across chromosome 17 in the pedigree. Analysis of data from 1028 single nucleotide polymorphisms (SNPs) across the refined linkage region identified a single region of homozygosity present in pedigree C702 but not in 2938 UK controls. This spanned ~432kb of the gene encoding protein kinase C, alpha (PRKCA), the encoded protein of which has been implicated in the pathogenesis of psychiatric disorders. Analysis of pedigree C702 by oligonucleotide-array comparative genome hybridization excluded the possibility that this region of homozygosity was because of a deletion. Mutation screening of PRKCA identified a rare, four-marker haplotype (C-HAP) in the 3′ untranslated region of the gene, which was present in the homozygous state in all six affected members of pedigree C702. No other homozygotes were observed in genotype data for a total of 6597 unrelated Europeans (case N=1755, control N=3580 and parents of probands N=1262). Second, association analysis of C702 alleles at PRKCA. The low-frequency haplotype (C-HAP) showed a trend for association in a study of unrelated schizophrenia cases and controls from the UK (661 cases, 2824 controls, P=0.078 and odd ratio (OR)=1.9) and significant evidence for association when the sample was expanded to include cases with bipolar (N=710) and schizoaffective disorder (N=50) (psychosis sample: 1421 cases, 2824 controls, P=0.037 and OR=1.9). Given that all the affected members of C702 are male, we also undertook sex-specific analyses. This revealed that the association was strongest in males for both schizophrenia (446 male cases, 1421 male controls, P=0.008 and OR=3.9) and in the broader psychosis group (730 male cases, 1421 male controls, P=0.008 and OR=3.6). Analysis of C-HAP in follow-up samples from Ireland and Bulgaria revealed no evidence for association in either the whole sample or in males alone, and meta-analysis of all male psychosis samples yielded no significant evidence of association (969 male cases, 1939 male controls, 311 male probands P=0.304 and OR=1.4). Third, association mapping of the pedigree C702 linkage region. Independent of pedigree C702, genotype data from the Affymetrix 500k GeneChip set were available for 476 patients with schizophrenia and 2938 controls from the United Kingdom. SNPs in PRKCA showed evidence for association with schizophrenia that achieved gene-wide significance (P=0.027). Moreover, the same SNP was the most significantly associated marker out of the 1028 SNPs genotyped across the linkage region (rs873417, allelic P=0.0004). Follow-up genotyping in samples from Ireland, Bulgaria and Germany did not show consistent replication, but meta-analysis of all samples (4116 cases and 6491 controls) remained nominally significant (meta-analysis P=0.026, OR=1.1). We conclude that, although we have obtained convergent lines of evidence implicating both rare and common schizophrenia risk variants at PRKCA, none of these is individually compelling. However, the evidence across all approaches suggests that further study of this locus is warranted.

Keywords:

schizophrenia; gene; association; bipolar disorder; schizoaffective disorder; PRKCA

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Introduction

Schizophrenia is a severe psychiatric disorder with a lifetime morbid risk of ~1% worldwide.1 Fundamental characteristics of the disorder include psychotic symptoms, such as delusions and hallucinations, and also negative symptoms, such as reduced motivation and social withdrawal.2 Family, twin and adoption studies have unequivocally shown that the population variance in the risk of developing schizophrenia is largely attributable to genetic factors, with heritability estimates of about 80%.1 Although the number of risk loci, their effect size and mode-of-transmission remain unknown, the decrement in risk of developing the disorder as genetic distance increases is consistent with schizophrenia being a complex, multifactorial disorder,3, 4 with multiple alleles acting to alter the risk.5 There are two main models of the allelic architecture of schizophrenia: the common-disease/common-variant and common-disease/rare-variant hypotheses. However, current findings would indicate that the two hypotheses are not mutually exclusive, with strong support for common but low-risk alleles6, 7 and also highly penetrant but rare alleles.8, 9, 10

In the course of a whole-genome linkage study of schizophrenia, we earlier identified a single pedigree (family C702) consisting of six affected male siblings, which showed genome-wide significant evidence for linkage to chromosome 17p11.2–q25.1.11 All affected siblings shared both haplotypes across most of chromosome 17, consistent with the segregation of a recessive, high-penetrant risk allele. In this study, having first refined the linkage region to a 21-marker segment spanning 11.7Mb at 17q23–q24, we followed several parallel lines of enquiry.

First, we identified the gene encoding protein kinase C, alpha (PRKCA) as the most plausible biological candidate gene in the region. Several direct and indirect lines of evidence have implicated this kinase in the pathogenesis of psychiatric disorders.12, 13, 14, 15, 16, 17 Directed mutation screening of PRKCA identified a rare, four-marker haplotype (C-HAP) in the 3′ untranslated region (UTR) of the gene that was present in the homozygous state in all six affected members of pedigree C702. No other homozygotes were observed in unrelated Europeans (N=6597; case N=1755, control N=3580 and parents of probands N=1262).

Having identified a rare haplotype as a possible explanation for the linkage to chromosome 17 in pedigree C702, we sought to determine whether heterozygosity at C-HAP confers susceptibility to schizophrenia and related psychotic disorders. Analysis in a case–control sample from the United Kingdom showed some evidence for association and a male-specific effect, which was of interest given that all the affected siblings in pedigree C702 are male. Follow-up analysis in samples from Ireland and Bulgaria did not replicate this finding, although as the haplotype is rare much larger sample sizes may be required to confirm or refute any claim of this as a risk allele for schizophrenia and related psychotic disorders.

Concurrently with the mutation screening and association analysis, we sought to identify regions of consecutive homozygous single nucleotide polymorphisms (SNPs) that could indicate transmission of a founder allele in pedigree C702.18, 19 We analyzed Affymetrix 500k GeneChip data from 1028 SNPs across the refined linkage region in a single member of pedigree C702. This identified a single region of homozygosity that was present in pedigree C702 but not in 2938 UK controls. This spanned ~432kb of the PRKCA locus including the 3′UTR and the rare variants forming C-HAP. Analysis of pedigree C702 by oligonucleotide-array comparative genome hybridization (CGH) excluded the possibility that this region of apparent homozygosity was due to a deletion.

Finally, we tested the hypothesis that the linkage region, and PRKCA in particular, might contain common alleles that confer risk of schizophrenia. Thus, we undertook association mapping based on Affymetrix 500k GeneChip data, which were available for 476 patients with schizophrenia and 2938 controls from the United Kingdom. SNPs in PRKCA showed evidence for association with schizophrenia that achieved gene-wide significance. Moreover, one of these SNPs was the most significantly associated marker out of the 1028 SNPs genotyped across the linkage region. Follow-up genotyping in samples from Ireland, Bulgaria and Germany did not show replication, but meta-analysis of all genotyped samples remained nominally significant.

We conclude that convergent evidence suggests the PRKCA locus may be responsible for the linkage observed in pedigree C702 and that both rare and common genetic variation at this locus may influence susceptibility to schizophrenia and related disorders. Although the data do not unequivocally confirm any of these findings, further work at this locus may be fruitful.

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Materials and methods

All genomic positions and regions are given in Human March 2006 assembly (Hg18. National Center for Biotechnology Information (NCBI) Build 36.1) as found on the University of California, Santa Cruz (UCSC) genome browser (http://genome.ucsc.edu/).

Diagnosis and consanguinity testing of pedigree C702

Pedigree C70211 consists of six affected male siblings and their parents. DNA was available from all individuals except the mother. All affected individuals had a consensus best-estimate diagnosis of Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM-IV) schizophrenia based on a semistructured lifetime psychiatric interview, Schedules for Clinical Assessment in Neuropsychiatry20 and review of case notes. Age at first hospitalization ranged from 17 to 22 years, and each had a chronic course of illness with multiple admissions. It is notable that both manic and depressive symptoms occurred in all siblings. Mood disturbance was a prominent feature of illness in three of the siblings, who met the Research Diagnostic Criteria21 for schizoaffective disorder, depressed type in two and schizoaffective disorder, bipolar type in the other. In the other siblings multiple mood symptoms were present but they did not meet the criteria required for a manic or depressive syndrome.

Analysis of 372 autosomal microsatellite markers revealed no evidence of consanguinity in family C702 (see Supplementary information).

Samples for association studies

All samples used in this study have been described earlier; however, a detailed description is given in the Supplementary material. All cases had a DSM-IV diagnosis of schizophrenia, bipolar I disorder or schizoaffective disorder (bipolar type). The UK case samples consisted of a total of 696 individuals with schizophrenia,6, 22 1736 patients with bipolar I disorder and 62 patients with schizoaffective disorder.23, 24 The UK control sample used for rare allele analysis consisted of 2824 Caucasians resident in the British Isles. Affymetrix 500k GeneChip data from a further control sample of 2938 individuals were also available and used for homozygosity mapping, association mapping and analysis of rs873417.6, 24

Replication samples were obtained from Irish, Bulgarian and German populations. The Irish case–control sample consisted of 312 individuals with schizophrenia, 89 patients with schizoaffective disorder and a total of 1804 unrelated controls (two independent control samples; N=806 and N=998).6, 25 A Bulgarian case–control sample consisted of 611 patients with schizophrenia, 93 patients with schizoaffective disorder and 658 unrelated controls.6 A Bulgarian parent-proband trios sample included 431 probands with schizophrenia, 156 probands with bipolar I disorder and 49 probands with schizoaffective disorder. These samples were included in the Bulgarian case–control sample.26 The German case–control sample consisted of 758 individuals with schizophrenia and 1897 controls.6 Further details of all association samples are given in the Supplementary information.

Genotyping

Microsatellite marker genotyping
 

A total of 50 microsatellites were analyzed in pedigree C702, which included 14 microsatellites reported earlier.11 The average heterozygosity was 0.69 (0.19–0.9) and the average inter-marker genetic distance was ~2.5cM (sex-averaged). The deCODE genetic map27 was used as the spatial template, but was supplemented by a later map.28 All markers were genotyped as described earlier.11 Marker names and genomic positions are presented in Supplementary Figure SF1.

Single nucleotide polymorphism genotyping
 

In house SNP genotype data were generated primarily using either the MassARRAY iPlex platform (Sequenom, San Diego, CA, USA) or the Amplifluor UniPrimer (Millipore, Billerica, MA, USA) systems according to the manufacturers’ recommendations. Genotypes were called in duplicate, blind to sample identity and to the calls of the other rater. All carriers of the specific haplotype identified in C702 (C-HAP) or individuals that failed genotyping for these alleles were called by an additional independent genotyping method, either SNaPshot (Applied Biosystems, Foster City, CA, USA) or direct sequencing. Primers and thermocycling conditions are available on request. The details of genotyping using the GeneChip 500K Mapping Array (Affymetrix) are described elsewhere6, 24 and quality control metrics for SNPs analyzed are given in the relevant results sections.

Mutation screening of protein kinase C, alpha

The genomic structure of PRKCA was determined by overlaying RefSeq NM_002737.2 and transcripts BC053321, AB209475 and X52479 (GenBank human messenger RNAs (mRNAs)). PCR amplimeres were designed to span all exons, 1kb 5′ to the start of transcription (maximum fragment size 600bp) and an additional nine putative regulatory motifs (total of 1061bp) predicted in silico (http://zlab.bu.edu/cluster-buster/). Details of the primers and PCR conditions are available on request. All assays were amplified using standard or touchdown protocols as described earlier29 in 14 unrelated, white subjects from the UK meeting DSM-IV criteria for schizophrenia (seven males and seven females) and also a C702-affected sibling. After PCR, each amplimere was screened for DNA variants by bi-directional sequencing using Big-Dye (v3.1) terminator chemistry and an ABI3100 sequencer according to the manufacturer's instructions (Applied Biosystems).

Screen for homozygous segments in the pedigree C702 linkage region

One affected sibling from pedigree C702 (702.06) and 2938 unrelated UK controls were genotyped as part of an earlier Genome-Wide Association Study.6, 24 The genotypes of 702.06 for the 1028 SNPs located within the refined IBD2 (Identical-By-Descent 2) linkage region were combined with data for an additional 23 microsatellite markers spanning the same region and were manually interrogated for genomic segments containing greater than or equal to2 consecutive homozygous markers (SNP or microsatellite). The frequency of each homozygous segment identified in sibling 702.06 was then determined using the same SNP data in the sample of 2938 controls.24

Oligonucleotide-array comparative genome hybridization

A custom tiling-path oligonucleotide CGH array of 387218 oligonucleotide features was designed by Roche NimbleGen, Inc. (Madison, WI, USA) to span the repeat-masked C702 IBD2 region (chr17:52996269–64651294) at an average resolution of 3.67bp. High-resolution oligonucleotide-array comparative genome hybridization was performed using two affected C702 siblings who were each separately hybridized to two unrelated controls from the United Kingdom. All sample labelling, hybridization, raw data generation and analysis were performed by NimbleGen Systems Inc.

Statistical analyses

Assessment of marker Hardy–Weinberg equilibrium and tests of single-locus association in the association samples were performed by χ2-test, Fisher's exact test or Cochran–Mantel–Haenszel test using PLINK 1.00.30 Calculation of linkage disequilibrium between SNPs was performed using Haploview.31 Meta-analysis of rare alleles identified in pedigree C702 was performed using an exact conditional Cochran–Mantel–Haenszel test.

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Results

Mapping the source of linkage to chromosome 17 in pedigree C702

Refinement of the C702 IBD linkage region
 

The originally reported chromosome 17 IBD2 linkage region in pedigree C702 spanned a total of 69.2cM.11 Typing an additional 36 microsatellite markers allowed us to refine this region to 12.57cM (11.7Mb at chr17:52996269–64651294), spanning 21 markers from D17S1604 to D17S940 (Supplementary Figure SF1).

Screen for homozygous segments in the pedigree C702 linkage region
 

Pedigree C702 may be segregating a highly penetrant, rare, recessive acting allele as identified by the linkage to 11.7Mb at 17q23–q24. Rare segments of consecutive homozygous genotypes within the linked region in a C702 sibling may identify the source of the linkage signal. To identify these rare homozygous regions we analyzed a total of 1028 SNPs from within the C702 IBD2 region that had been genotyped in C702.06 and 2938 controls using the Affymetrix 500k GeneChip.6 The SNPs had an overall genotyping call rate of 0.997 (min=97.5%) and all had a Hardy–Weinberg equilibrium P-value of >0.001. High-quality genotypes were available for 1023 SNPs for the C702 affected sibling, which when combined with the genotypes of 23 microsatellite markers revealed 83 regions that harbored greater than or equal to2 consecutive homozygous markers. No regions contained greater than or equal to2 consecutive markers with missing genotypes. Using the SNP data only, a single homozygous region was absent in 2938 controls. This was composed of 82 SNPs (and one microsatellite marker in pedigree C702; D17S942), and was located at chr17:61851033–62282771 that spanned the 3′UTR of PRKCA and included the rare variants in pedigree C702 identified in parallel analyses (see below and Figure 1).

Figure 1.
Figure 1 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

The UCSC Genome Browser 2004 freeze at the protein kinase C, alpha (PRKCA) locus with added custom tracks. Shown in descending order is the chromosomal base position, chromosomal band, RefSeq and GenBank Human mRNAs focused on in this study, microsatellite markers, regions of homozygosity (numbers indicate rank from rarest to commonest diplotype in 2938 UK controls. Therefore ‘1’ indicates the rarest C702 diplotype), areas re-sequenced in 14 UK individuals with schizophrenia and a C702 sibling, novel variants discovered, the four rare variants identified as homozygous in pedigree C702, the 99 GWAS single nucleotide polymorphisms (SNPs) analyzed in the candidate gene study of PRKCA, and the associated SNP rs873417. GWAS, Genome-Wide Association Study.

Full figure and legend (205K)

Oligonucleotide-array comparative genome hybridization in pedigree C702
 

Given the unstable nature of chromosome 17,32 the presence of a hemizygous deletion could potentially account for both the linkage and the region of apparent homozygosity observed in pedigree C702. We therefore used oligonucleotide-array comparative genome hybridization to analyze the C702 linkage region using two affected C702 siblings and two unrelated controls. The inter-hybridization quality across all four hybridization experiments was good, giving Pearson's correlations of 0.37–0.46. The C702 IBD2 region is thought to harbor a number of polymorphic copy number variations (UCSC March 2004 and 2006; Structural Variation track); however, probes within these regions showed no evidence of a mean log2 ratio that approached ±0.5 (range of −0.01 to −0.15) (Supplementary Table ST3). Specific analysis of the region of homozygosity at the PRKCA locus failed to identify any evidence for a deletion at this locus (24766 probes; mean log2 ratio range of −0.03 to −0.01; Supplementary Figure SF2 and Supplementary Table ST3).

Mutation screening of protein kinase C, alpha
 

Re-sequencing all PRKCA exons and putative regulatory regions in individual 702.06 and 14 unrelated individuals with schizophrenia identified a total of 44 sequence variants, of which 27 had not been reported earlier in the single nucleotide polymorphism database (Supplementary Table ST2); 29 were exonic (2 coding, 27 3′UTR), 11 were located in the flanking intronic sequence and 4 were located 5′ to the PRKCA transcriptional start site.

In all, 4 of the 44 identified DNA variants were homozygous in the C702 affected sibling (702.06) for an allele that was not observed in the other 14 unrelated individuals with schizophrenia (rs62621678, rs62621679, rs62621677 and rs62621676) and these were prioritized for further analysis. The location of all four DNA variants relative to the multiple PRKCA isoforms is shown in Figure 1. Three of these SNPs (rs62621678, rs62621679 and rs62621677) were located within a 267bp region of the 3′UTR of PRKCA isoform AB209475. Sequencing this region in the 90 HapMap CEU individuals revealed that all three variants were in perfect linkage disequilibrium (r2=1); therefore, for simplicity, the haplotype (rs62621678(G)–rs62621679(G)–rs62621677(A)) that carries the rare alleles that segregate in family C702 is termed C-1 (Table 1). The fourth SNP (rs62621676) was not polymorphic in the 90 HapMap CEU individuals and is located within the 3′UTR of the PRKCA isoform X52479 (Table 1). For simplicity, the haplotype that segregates in family C702 and is composed of the rare alleles of all four markers (rs62621678(G)–rs62621679(G)–rs62621677(A)–rs62621676(T)) is termed C-HAP (Table 1).


Homozygosity for the C702 haplotype (C-HAP) in European samples
 

To identify individuals homozygous for the rare diplotype observed at PRKCA in pedigree C702, C-HAP was assayed in case and control samples from Europe (UK: schizophrenia N=661, schizoaffective disorder N=50, bipolar I disorder N=710 and control N=2824; Irish: schizophrenia N=296, schizoaffective disorder N=76 and control N=806; Bulgarian (probands and both parents): schizophrenia N=431, schizoaffective disorder N=49 and bipolar I disorder N=156). We did not observe the C-HAP homozygous diplotype carried by affected members of pedigree C702 in any of the 6597 unrelated individuals from Europe with full genotype data (case N=1755, control N=3580 and parents of probands N=1262).

Association analysis of C702 alleles at protein kinase C, alpha

UK schizophrenia and psychosis case–control samples
 

We initially genotyped C-1, rs62621676 and C-HAP in a UK schizophrenia case–control sample of 661 cases and 716 controls. There was weak but significant linkage disequilibrium between C-1 and rs62621676 (D=0.24 (CI 0.05–0.58; r2=0.01)). Nevertheless, the phase probabilities were such that carriers of the minor alleles at both C-1 and rs62621676 nearly always carried the C-HAP haplotype (probability 0.94). We also later observed in the Bulgarian trios (in which phase can be more accurately defined) that in all cases, in probands with rare alleles at both C-1 and rs62621676, these were in the same haplotype (Supplementary Table ST4). Given this, and that all transmissions of C-HAP were in phase, we classed all individuals carrying the minor alleles at both C-1 and rs62621676 as carriers of the C-HAP haplotype.

We did not detect any evidence for allelic association to the minor allele at C-1 (P=0.49, odd ratio (OR)=0.9) although there was a trend for association at rs62621676 (P=0.088, OR=1.9) (Table 2). However, we did detect nominally significant evidence for association to C-HAP (P=0.048, OR=3.8). All tests of association reported are one-tailed for all odd ratios >1, given the a priori hypothesis that the minor alleles identified in family C702 are risk alleles.


Given that all affected individuals in family C702 are male, we additionally performed a gender-specific association analysis (Table 2). This revealed evidence suggesting a male-specific effect at both rs62621676 (446 male cases, 482 male controls, Pmales=0.045, ORmales=2.4; Pfemales=1, ORfemales=0.5) and also at C-HAP (Pmales=0.049, ORmales=6.6; Pfemales=1, ORfemales=1.1).

Given the prominence of affective symptoms in pedigree C702 as well as the reported evidence for linkage of these related disorders to 17q,33, 34, 35, 36, 37, 38, 39, 40 we broadened the diagnostic criteria to also include an additional 50 cases with schizoaffective disorder and 710 with bipolar I disorder, and also included a further 2108 controls. This also resulted in evidence for association in the full UK sample at C-HAP (1421 cases, 2824 controls; P=0.037, OR=1.9) as well as in the male subjects only (730 male cases and 1421 male controls) at both rs62621676 (Pmales=0.015, ORmales=2.1; Pfemales=0.706, ORfemales=0.8) and more strongly at C-HAP (Pmales=0.008, ORmales=3.6; Pfemales=0.800, ORfemales=1.1) (Table 2).

To test for an effect that differed by gender, we undertook a log-likelihood test (1 df) performed on the UK case–control data (1421 cases and 2824 controls) in which two models were compared, with and without the gender × SNP interaction term. This resulted in suggestive evidence for an interaction between gender and rs62621676 in predicting affection status that fell short of conventional levels of statistical significance (P=0.060) and weaker evidence at the rare C-HAP (P=0.116).

Follow-up samples and meta-analysis
 

Analysis of rs62621676 and C-HAP in follow-up samples from Ireland and Bulgaria revealed no evidence for association in either the whole sample or in males alone (Supplementary Table ST4). In a meta-analysis of all male psychosis samples combined (969 male cases, 1939 male controls and 354 male-proband trios), no significant evidence for association remained at either rs62621676 (Pmales=0.290, ORmales=1.3) or C-HAP (Pmales=0.304, ORmales=1.4) (Table 2). We did not observe the C-HAP homozygous diplotype carried by family C702 in any of the 6597 unrelated individuals studied (case N=1755, control N=3580 and parents of probands N=1262).

Association mapping of the pedigree C702 linkage region

It is a reasonable assumption that some susceptibility loci for complex diseases may include both rare highly penetrant alleles as well as more common risk alleles with lower penetrance.41, 42 We, therefore, analyzed genotype data for the 1028 SNPs included on the Affymetrix 500K Mapping Array used for homozygosity mapping of the C702 IBD2 linkage region (chr17:52996269–64651294), in 476 cases and 2938 controls from our schizophrenia Genome-Wide Association Study.6 A SNP 14500bp 3′ to PRKCA was the most significantly associated SNP in the C702 linkage region (rs873417; allelic P=0.0004, OR=1.4 and genotypic P=0.002; see Supplementary Figure SF3).

We also tested for association specifically at PRKCA. A total of 99 SNPs spanned the PRKCA locus (plus 20kb 5′ and 3′) at chr17:61709388–62257324. Each SNP had a call rate >97% and Hardy–Weinberg equilibrium P-values for each assay were >0.01 in controls, cases and combined. These 99 SNPs capture 57% of the common variation present in the HapMap CEU sample (HapMap Release 22, Phase II, April 2007) at an r2>0.8 (MAF>0.01; Hardy–Weinberg equilibrium P-value >0.001). Six SNPs yielded nominally significant evidence for allelic association with the strongest evidence at rs873417, which was experiment-wide significant (10000 permutations, P=0.027) (Supplementary Table ST5). No evidence for a significant gender-specific association survived permutation.

rs873417 was next genotyped in an additional 163 schizophrenia cases with schizophrenia and 62 cases with schizoaffective disorder,24 and again this expanded data set provided significant evidence for allelic association (P=0.001, OR=1.3) (Table 3). However, combined analysis of independent schizophrenia-schizoaffective disorder case–control samples from the UK, Ireland, Germany and Bulgaria by meta-analysis resulted in only a trend for association with rs873417 (P=0.079, OR=1.1; Table 3).


Data for rs873417 were also available for 1566 samples with bipolar I disorder.24 A significant association was again observed with the C allele at rs873417 (P=0.031, OR=1.1; Table 3); however, as the controls are the same as for the UK schizophrenia association sample this does not constitute fully independent support. A combined analysis of all phenotypes, when all UK and non-UK samples were considered in a meta-analysis, was nominally significant (P=0.026, OR=1.1; Table 3).

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Discussion

We earlier reported genome-wide significant evidence for linkage at chromosome 17p11.2–q25.1 in a single pedigree.11 Linkage to 17q has also been reported by others for both schizophrenia and bipolar disorder (see Supplementary Figure SF4),33, 34, 35, 36, 37, 38, 39, 40, 43 suggesting that a locus or loci for psychiatric illness is likely to exist in this region.

The linkage region we reported by in our earlier study11 encompassed much of chromosome 17, in which the affected C702 siblings were apparently IBD2. This is compatible with a recessive model, as were many of the other linkages reported to this region.34, 36, 37, 40 In this study, the use of additional microsatellites refined the maximum IBD2 region to 11.7Mb at 17q23.2–q24.3.

We subsequently followed several complementary lines of investigation. First, we identified the gene encoding PRKCA as the most plausible biological candidate gene in the region. Several direct and indirect lines of evidence have implicated this kinase in the pathogenesis of psychiatric disorders. Protein kinase C, alpha (PKCα), is an isoform of the PKC family of kinases, and an enzyme with a multitude of interacting partners44 and cellular roles.45, 46, 47 PKCα has important roles in a plethora of functions potentially relevant to the pathogenesis of psychiatric disorders, including synaptic signalling and long-term potentiation/depression,48, 49 neurite growth and neuronal development,50, 51 and possibly in myelination.52 PKCα is involved in certain types of memory12, 13 including working memory,12 which has been suggested as an endophenotype for schizophrenia.53

More direct evidence implicating PKCα in major psychiatric disorder comes from the observation that it is present at lower levels in the anterior cingulate cortex of patients with schizophrenia15 and in peripheral tissue of bipolar subjects54, 55 as well as in the prefrontal cortex and hippocampus of teenage suicide victims.16 Furthermore, recent evidence implicates PKCα as a therapeutic target in the treatment of mania.17

Mutation screening of PRKCA identified homozygosity for a number of low-frequency, exonic (3′UTR) alleles in affected members of pedigree C702. These were subsequently shown to be absent in homozygous form in 6597 unrelated European individuals. The alleles have population frequencies in UK and Irish controls of 0.008 (rs62621676) and 0.004 for the four-marker haplotype (C-HAP). Given this estimate, and assuming Hardy–Weinberg equilibrium, only one person in ~62500 individuals would be expected to carry this rare diplotype. Our observation then of such a rare finding in our family showing linkage to the same region suggests the hypothesis that one or more of these alleles directly confers risk of illness or that the risk haplotype is tagging variation elsewhere that confers risk. As the alleles are located in the 3′UTR, this confers the possibility that they act to alter the secondary structure of the pre-mRNA or mRNA, which could result in altered pre-mRNA splicing or disrupted translation. Such a role is supported by the observation in silico that rs62621676 alters the secondary structure of transcript X52479 (http://www.genebee.msu.su/services/rna2_reduced.html; data not shown). However, this hypothesis requires experimental validation.

Concurrently, a purely genetic analysis of the family C702 linkage region also pointed to PRKCA as the locus explaining the linkage signal observed in the pedigree. We sought to identify a rare recessive locus by systematically scanning the IBD2 linkage region for regions of homozygosity present in the affected siblings but absent in controls (N=2938). Across the whole linkage region, we found only a single segment compatible with the rare, highly penetrant recessive model. This encompassed the 3′ of PRKCA.

Second, as we did not detect homozygosity for C-HAP in other cases, we sought support for an involvement in schizophrenia and related disorders by seeking to detect an association, in heterozygous state, with disorder. We also tested the secondary hypotheses that association might particularly involve males, given that all of the affected in pedigree C702 are male. Although some evidence to support this hypothesis was obtained in the UK sample for C-HAP (Pmales=0.008, OR=3.9), we were unable to obtain evidence for replication in independent samples. Because of the reported linkage to chromosome 17 in bipolar affective disorders (Supplementary Figure SF4), the prominent affective symptoms present with affected members of pedigree C702 and recent literature implicating genetic association across traditional psychiatric diagnostic boundaries,56 diagnostic criteria were broadened to include other psychotic disorders. However, this did not greatly alter the pattern of findings (Pmales=0.008, ORmales=3.6) and when independent replication samples were again included in a meta-analysis no statistically significant association was observed (Pmales=0.304, ORmales=1.4) (Table 2).

The true risk conferred by any individual C-HAP allele or combination thereof is unknown. However, to have 80% power to detect an effect at P=0.05 in a sample of 700 cases and 700 controls (the approximate size of the UK schizophrenia discovery sample), the relative risk imparted by the rare allele at C-1 would need to be >1.8, and >3 at rs62621676. Although association is detected in the United Kingdom, this may be an example of the ‘winner's curse’, in which the effect size is overestimated. However, if one copy of the rare haplotype at C-HAP imparts a relative risk of only 1.5, then >6000 cases and controls will be required to detect any association. Therefore, to rigorously test the null hypothesis at the C-1 and rs62621676 alleles (and haplotype), extremely large association samples will be required.

Third, we tested the hypothesis that the linkage region and PRKCA in particular might contain common alleles that confer risk of schizophrenia. Thus, we undertook association studies based on Affymetrix 500k GeneChip data that were available for 476 patients with schizophrenia and 2938 controls from the United Kingdom. SNPs in PRKCA showed evidence for association with schizophrenia that achieved gene-wide significance (P=0.027). Moreover, one of these SNPs was the most significantly associated marker out of the 1028 SNPs genotyped across the linkage region (rs873417, allelic P=0.0004). Follow-up genotyping in samples from Ireland, Bulgaria and Germany did not show consistent replication but meta-analysis of all genotyped samples including cases with bipolar disorder (4116 cases and 6491 controls) remained nominally significant (meta-analysis P=0.026, OR=1.1).

The results for rs873417 remain equivocal at best. Failure to obtain replication in follow-up samples could reflect differences in linkage disequilibrium structure at this locus across Europe, as has been alluded to in earlier studies57, 58 or the result may be a false-positive. However, our finding of gene-wide significant evidence for association at PRKCA, taken together with the fact that the SNPs genotyped in this study detect 57% of even the HapMap CEU-defined common genetic variation at PRKCA, suggests that that further fine-scale association mapping of this locus is required. In a preliminary attempt to address this, we imputed genotypes at missing SNPs using the HapMap CEU data and the program IMPUTE,59 but this failed to identify any associations that were more significant than rs873417 (data not shown).

In summary, we have presented several lines of convergent evidence that PRKCA represents a susceptibility locus for schizophrenia and related psychiatric illness. If the pedigree C702 phenotype and linkage signal at chromosome 17 is because of the convergence of rare alleles acting through a highly penetrant recessive mechanism, then the evidence of rare homozygosity is supportive of PRKCA being the linked locus. Furthermore, the exonic location of the alleles forming the rare homozygous diplotype suggests they may be causative. In addition, there is some evidence to suggest the same variants impart a small risk in heterozygotes. Independent of pedigree C702, association mapping of the 11.7Mb region of linkage also suggests that PRKCA is the most likely source of common allele association in the region. Although each of these lines of evidence implicates PRKCA as a disease susceptibility locus, none unequivocally sustains the case for involvement of the gene and so further studies at this locus will be required to confirm this hypothesis.

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Acknowledgements

This study makes use of data generated by the Wellcome Trust Case Control Consortium. A full list of the investigators who contributed to the generation of the data is available from www.wtccc.org.uk. Funding for the project was provided by the Wellcome Trust under award 076113. The UK research was supported by grants from the MRC, the Wellcome Trust and by a NIMH (USA) CONTE: 2 P50MH066392-05A1. In Dublin, the research was supported by Science Foundation Ireland, the Health Research Board (Ireland), and the Wellcome Trust. We are grateful to Professor John Waddington for sample recruitment. Irish controls were supplied by Dr Joe McPartlin and the Trinity College Biobank. We also thank the Department of Psychiatry, LMU Munich for clinical characterization of the Munich subjects and the processing of the samples. Recruitment in Munich was partially supported by GlaxoSmithKline.

Supplementary Information accompanies the paper on the Molecular Psychiatry website