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. Author manuscript; available in PMC: 2014 May 6.
Published in final edited form as: Am J Med Genet B Neuropsychiatr Genet. 2010 Jan 5;0(1):323–331. doi: 10.1002/ajmg.b.30982

Association analysis of the PIP4K2A gene on chromosome 10p12 and schizophrenia in the Irish study of high density schizophrenia families (ISHDSF) and the Irish case-control study of schizophrenia (ICCSS)

DL Thiselton 1,*, BS Maher 1, BT Webb 2, TB Bigdeli 3, FA O’Neill 4, D Walsh 5, KS Kendler 1, BP Riley 1,3
PMCID: PMC4011176  NIHMSID: NIHMS142247  PMID: 19475563

Abstract

Molecular studies support pharmacological evidence that phosphoinositide signaling is perturbed in schizophrenia and bipolar disorder. The phosphatidylinositol-4-phosphate-5-kinase type-II alpha (PIP4K2A) gene is located on chromosome 10p12. This region has been implicated in both diseases by linkage, and PIP4K2A directly by association. Given linkage evidence in the Irish Study of High Density Schizophrenia Families (ISHDSF) to a region including 10p12, we performed an association study between genetic variants at PIP4K2A and disease. No association was detected through single-marker or haplotype analysis of the whole sample. However, stratification into families positive and negative for the ISHDSF schizophrenia high-risk haplotype (HRH) in the DTNBP1 gene and re-analysis for linkage showed reduced amplitude of the 10p12 linkage peak in the DTNBP1 HRH positive families. Association analysis of the stratified sample showed a trend toward association of PIP4K2A SNPs rs1417374 and rs1409395 with schizophrenia in the DTNBP1 HRH positive families. Despite this apparent paradox, our data may therefore suggest involvement of PIP4K2A in schizophrenia in those families for whom genetic variation in DTNBP1 appears also to be a risk factor. This trend appears to arise from under-transmission of common alleles to female cases. Follow-up association analysis in a large Irish schizophrenia case-control sample (ICCSS) showed significant association with disease of a haplotype comprising these same SNPs rs1417374-rs1409395, again more so in affected females, and in cases with negative family history of the disease. This study supports a minor role for PIP4K2A in schizophrenia etiology in the Irish population.

Keywords: PIP4K2A, schizophrenia, association, family, case-control

Schizophrenia is a complex, debilitating psychiatric disorder with lifetime prevalence ~1% [Jablensky et al., [1992]]. Genetic factors impact substantially upon risk for developing the disease, with heritability ~80% [Sullivan et al., [2003]]. Indeed, >20 genome-wide linkage scans have pinpointed genomic regions harboring schizophrenia susceptibility variants. Association studies in multiply reported linked regions have revealed several candidate genes for the disorder [Riley and Kendler, [2006]].

In the ISHDSF, a genome linkage scan of 265 families with schizophrenia gave strongest signals in 3 regions: 5q21-31, 6p24-22, 8p22-21, all since replicated [Straub et al., [2002]; Pimm et al., [2005]; Riley and Kendler, [2006]]. Elsewhere, D10S674 (10p13) gave one of the highest pairwise heterogeneity lod (H-LOD) scores, 3.2 (P = 0.0004), on an 88-family subset [Straub et al., [1998]]. Across all 265 families, strongest linkage was to D10S2443 (10p12.1) with intermediate phenotypic definition (maximum pairwise H-LOD 1.95, P = 0.005). Multipoint H-LODS gave a broad peak (maximum 1.91, P = 0.006) extending 11 cM from D10S674(10p13)-D10S1426(10p11.23). This locus has been linked to schizophrenia in other genome scans [Faraone et al., [1998]; Schwab et al., [1998], [2000]; Levinson et al., [2000]; DeLisi et al., [2002]; Lewis et al., [2003]; Holliday et al., [2005]].

One gene within the 10p-linked interval became a positional and functional candidate for schizophrenia: phosphatidylinositol-4-phosphate-5-kinase type-II alpha. Formerly PIP5K2A, its new gene symbol PIP4K2A will be used here (http://www.genenames.org). PIP4K2A is involved in the synthesis of phosphoinositide-4,5-biphosphate (PIP2), precursor to second messengers in the phosphoinositide signal transduction cascade [Doughman et al., [2003]; Weernink et al., [2004]]. Inositol phosphate metabolism is a target of mood stabilizer drugs [e.g., lithium, valproate, carbamazepine; Harwood, [2005]] and there is growing evidence for dysfunction of this pathway in schizophrenia [Kalkman, [2006]]. Linkage of schizophrenia to D10S245, <1 Mb from PIP4K2A, was shown by Maziade et al. [2001].

Stopkova et al. [2003] first reported association between PIP4K2A and both schizophrenia and bipolar disorder (BPD). Schwab et al. [2006] investigated PIP4K2A following significant association with D10S211 (900 kb from PIP4K2A) in their 10p-linked sample. After initial results [Sewekow et al., [2003]] 31 polymorphisms were tested covering PIP4K2A plus ~100 kb flanking regions. Fifteen variants spanning 73.6 kb (PIP4K2A intron 4 → 3′UTR) were significantly associated with schizophrenia, in a region of high intermarker linkage disequilibrium (LD). Major alleles were over-transmitted to affected individuals and as a 12-SNP haplotype over 43 kb (intron 6 → 3′UTR, P = 0.037 after correction). For non-synonymous SNP rs10828317 (Ser251Asp; exon 7) the associated major allele encoding Ser (P = 0.001) was associated with schizophrenia in a case-control study [Bakker et al., [2007]; P = 0.0004]. Schwab et al. [2006] also detected association with rs1417374, ~125 kb upstream of PIP4K2A (P = 0.0009).

Jamra et al. [2006] attempted to replicate Sewekow et al. [2003], analyzing 5 PIP4K2A SNPs in a sample of 268 schizophrenia patients, 260 BPD patients and 325 controls. Despite including rs1417374 and one SNP from the high-LD region, they found no association to either disease. Because of its positional candidacy, and association with schizophrenia in another 10p-linked Caucasian sample [which, like the ISHDSF, showed association with DTNBP1; Schwab et al., [2006]], we decided to investigate association with PIP4K2A in the ISHDSF. Follow-up analysis was performed on The Irish Case-Control Study Sample (ICCSS).

The ISHDSF comprises 265 families with 1,408 individuals for genotyping, divided into four concentric diagnostic categories: core schizophrenia (d2), narrow spectrum (“intermediate phenotype” d5), broad (d8) and very broad spectrum psychiatric disease [d9; Kendler et al., [1996]]. The ICCSS has 1021 cases with schizophrenia or poor-outcome schizoaffective disorder (DSM-III-R) from psychiatric facilities in the Republic of Ireland and Northern Ireland. For the 627 controls inclusion criteria was no history of psychotic illness. Family history (FH) was assessed by clinical interview using FH-RDC criteria [Endicott et al., [1975]]. Inclusion in the study required all grandparents to be born in Ireland/UK and appropriate informed consent.

We assessed five PIP4K2A SNPs in the ISHDSF, all significantly associated in Schwab et al. [2006] (rs1417374, rs1409395, rs10828317, rs746203, rs2364115) and one microsatellite [Stopkova et al., [2003]], named PIK53_54 [Schwab et al., [2006]] or D10S0969i [Tamiya et al., [2005]]. PCR and single-base extension primers were designed manually for rs1417374 and rs2364115 (available on request). Other primer sequences were from Schwab et al. [2006]. SNP genotyping was performed by template-directed dye-terminator incorporation with fluorescence polarization detection (FP-TDI) using AcycloPrime FP SNP detection kits (PerkinElmer Life Sciences, Boston, MA) and manufacturer’s instructions, with an automated allele scoring platform [Van den Oord et al., [2003]]. PIK53_54 was typed via standard PCR using one fluorescent dye-labeled primer (6-FAM), electrophoresis on an SCE7610 capillary sequencer (Spectrumedix LLC, State College, PA) and allele-calling using Spectrumedix genotyping software. Genotypes were examined for incompatibilities within families by PEDCHECK v1.1 [O’Connell et al., [1993]], unlikely recombinations by MERLIN [Abecasis et al., [2002]], and corrected as necessary. For unambiguously resolvable errors, respective genotypes were deleted for the whole family. For individuals with >50% missing genotypes all data were deleted (n = 94). Average genotyping rate was 95% (max 98%, min 89%).

We assessed 5 PIP4K2A SNPs in the ICCSS, using Taqman SNP genotyping assays from ABI (www.appliedbiosystems.com): rs1417374, rs1409395, rs10828317, rs746203, and rs8341, as proxy for rs2364115 (no assay, complete LD). Manufacturer’s instructions were followed, with slight modifications (available on request). For individuals with >50% missing genotypes all data were deleted (n = 66). Average genotype rate was 98% (max 99%, min 96%). Genotyping error rate was estimated from 205 duplicate genotypes. Of these, 94% were successfully collected and none were discordant, yielding an error rate <0.005%.

Genotype deviation from Hardy-Weinberg equilibrium (HWE) and pairwise intermarker LD were calculated using Haploview v4.0 [Barrett et al., [2005]] from unrelated founders (ISHDSF) or controls (ICCSS). We used the Tagger function of Haploview to estimate the proportion of common regional variation (MAF > 0.20) identified in HapMap Phase II data that was tagged at r2 > 0.8 by the SNPs (including proxies for PIK53_54) genotyped in this study.

We performed association analysis using PDTPHASE v4.0 [Martin et al., [2000]; Dudbridge, [2003]] for the ISHDSF and COCAPHASE [Dudbridge, [2003]] for the ICCSS. For haplotype analyses, we included only those with frequency >2% to avoid statistical problems from rare alleles/haplotypes. To investigate epistatis between known susceptibility variants in DTNBP1 and other linked loci in the ISHDSF, we stratified the sample on the DTNBP1 high-risk haplotype (HRH; up to and including d8 affection), re-analyzing for linkage to schizophrenia using GENEHUNTER [v1.3; Kruglyak et al., [2006]].

For FH-conditioned analyses (ICCSS), all FH- cases were set to unknown affection status. For sex-stratified analysis, only male or female subjects (ICCSS) or affected offspring (ISHDSF) were used. We further explored influence of subject sex on association between PIP4K2A and schizophrenia in the ICCSS via logistic regression, parameterizing each genotype (assuming an additive model), including sex and testing for their interaction. Gender-specific LD contrast analysis between cases and controls was also performed, using correlation and D measures of LD.

Accounting for all comparisons, including DTNBP1 HRH-stratified and sex-specific analyses, we used false discovery rate (FDR) methodology to control false positive rate. The procedure applied controls FDR for correlated data [“nested” diagnostic categories d2-d9 in the ISHDSF and SNPs in LD; Benjamini and Hochberg, [1995]; Storey, [2003]]. We also used weighted FDR to allow inclusion of prior information [Genovese et al., [2006]; Roeder et al., [2006]] via the R package wFDR (http://wpicr.wpic.pitt.edu/WPICCompgen/fdr/) that is, P-values of published single marker tests of PIP4K2A association with schizophrenia as prior weights. For multiply reported tests we combined P-values using the approach of Fisher [1932]. Previously untested marker haplotype combinations were assigned a prior P = 0.5. For the ICCSS, permutation testing was performed in Haploview [Barrett et al., [2005]].

All SNP genotype frequencies conformed to HWE (data not shown). Regional LD structure in the ISHDSF and ICCSS is consistent with prior reports [HapMap CEU release 21, Schwab et al., [2006]] that is, a high-LD block spanning PIP4K2A exon 7 → 3′UTR in modest LD with rs1409395 (intron 1; Table I). 5′ Upstream SNP rs1417374 shows modest LD with rs1409395 (D′ ≃ 0.5), lower with the high-LD block (D′ ≃ 0.4; Table I). SNP allele frequencies were similar in the ISHDSF, ICCSS, and dbSNP (CEU) (data not shown).

Table I.

Allele Frequencies and Intermarker Pairwise LD (D′ and r2 Values) for PIP4K2A Gene SNPs Used in This Study

dbSNP name SNP alias Genome location Distance (Hg17) Inter SNP (kb) MAF PIP4K2A location rs1417374 rs1409395 rs10828317 rs746203 rs2364115
(a) ISHDSF sample
rs1417374 TSC615653 23168481 0.31 Intergenic 5′ - 0.32 0.18 0.14 0.18
rs1409395 TSC599587 22972497 196 0.32 Intron 1 0.56 - 0.48 0.45 0.6
rs10828317 PIK153_154 22879634 93 0.31 Exon 7 0.42 0.7 - 0.62 0.65
rs746203 TSC123902 22870547 9 0.35 Intron 8 0.31 0.65 0.87 - 0.71
rs2364115 TSC1513749 22857725 11.2 0.34 Intergenic 3′ 0.4 0.76 0.86 0.86 -
dbSNP name SNP alias Genome location (Hg17) Inter SNP Distance (kb) MAF PIP4K2A location rs1417374 rs1409395 rs10828317 rs746203 rs8341
(b) ICCSS sample
rs1417374 TSC615653 23168481 0.31 Intergenic 5′ - 0.21 0.52 0.52 0.09
rs1409395 TSC599587 22972497 196 0.34 Intron 1 0.49 - 0.14 0.49 0.52
rs10828317 PIK153_154 22879634 93 0.34 Exon 7 0.4 0.73 - 0.7 0.69
rs746203 TSC123902 22870547 9 0.39 Intron 8 0.36 0.77 0.94 - 0.91
rs8341 TSC1513749 22857725 11.2 0.37 Intergenic 3′ 0.36 0.77 0.9 0.99 -
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N.B. D′ below the diagonal, r2 above the diagonal. Shading indicates degree of LD.

For the ISHDSF, we saw no association with single markers in any diagnostic group, in the whole sample (Table SI) or restricted to the 10p-linked subset (data not shown). For susceptibility loci of small effect, positive association may only be detected with haplotypes [Akey et al., [2001]]. However, key haplotypes from Schwab et al. [2006] showed no greater evidence for association here, perhaps owing to high regional LD (data not shown).

Re-analysis of the ISHDSF genome scan data [Straub et al., [2002]] conditioned on the DTNBP1 HRH (B.T. Webb, unpublished work) showed an increased 10p linkage signal in the HRH− families (n = 176) and negative linkage in the HRH+ families (n = 73; Fig. 1) that is, DTNBP1 HRH+ families do not appear to harbor the 10p risk locus (by linkage), or, they attenuate the 10p linkage signal from DTNBP1 HRH− families when analyzed altogether. Somewhat counter intuitively, we saw uncorrected evidence for association to PIP4K2A SNPs rs1417374 and rs1409395 in the DTNBP1 HRH+ families (Table II). We also saw nominal association of haplotypes comprising rs1417374 with PIP4K2A LD-block SNPs (Table II) in this subset, a significant under-transmission of the common alleles (P = 0.012; Table II; Table SII). We saw no association between PIP4K2A and schizophrenia in DTNBP1 HRH− families (data not shown).

Figure 1.

Figure 1

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Linkage signal on chromosome 10p in the ISHDSF, in the whole sample at the d5 (intermediate phenotype) diagnostic category, and stratified on the presence/absence of DTNBP1 high-risk haplotype. [Normal View 29K | Magnified View 51K]

Table II.

Uncorrected P-Values From Association Analysis With PDTPHASE of PIP4K2A Markers in DTNBP1 High-Risk Haplotype Positive Families of the ISHDSF Sample

Markers (see haplotype*) d2 d5 d8 d9
rs1417374 (1) 0.068 0.040 0.041 0.028
rs1409375 (2) 0.049 0.090 0.140 0.104
rs10828317 (3) 0.281 0.301 0.309 0.256
rs746203 (4) 0.963 0.943 0.677 0.490
PIK53_54 (5) 0.983 0.801 0.565 0.594
rs2364115 (6) 0.185 0.228 0.286 0.345
*Haplotype d2 d5 d8 d9
1–2 0.055 0.046 0.069 0.026
1–3 0.144 0.071 0.087 0.038
1–4 0.217 0.065 0.041 0.040
1–6 0.077 0.033 0.047 0.045
1–2–6 0.120 0.098 0.151 0.090
1–3–4 0.243 0.049 0.074 0.030
1–4–6 0.134 0.034 0.031 0.038
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Significant values are highlighted in bold.

Sex-stratified association analysis revealed the positive trend for markers rs1417374-rs1409395 to derive from over- (or under)transmission of the minor (or major) alleles to affected females (Table IIIA). This reached nominal significance for rs1409395 at d8 (P = 0.042) and in DTNBP1 HRH+ families, for rs1417374 and rs1409395 at d2-d9 (Table IIIB). We found no significant difference in the transmission counts according to sex alone (analyzed as 2 × 2 tables), refuting the notion of transmission ratio distortion on 10p [data not shown; Paterson and Petronis, [1999]]. However, only one single marker test (rs1409395 in female DTNBP1 HRH+ d8 cases) was significant at FDR = 20% under either weighting scheme.

Table IIIA.

Association Analysis by PDTPHASE Between PIP4K2A Markers and Schizophrenia According to Offspring Sex in the ISHDSF Sample

P-values (male affecteds)
P-values (female affecteds)
Single markers d2 d5 d8 d9 d2 d5 d8 d9
rs1417374 0.641 0.640 0.721 0.883 0.140 0.066 0.114 0.098
rs1409375 0.677 0.738 0.721 0.795 0.076 0.060 0.042 0.061
rs10828317 0.965 0.677 0.816 0.505 0.978 0.634 0.650 0.710
rs746203 0.808 0.670 0.605 0.398 0.634 0.619 0.340 0.884
PIK53_54 0.689 0.864 0.743 0.874 0.788 0.519 0.352 0.311
rs2364115 0.957 0.409 0.441 0.438 0.927 0.509 0.409 0.617
Haplotype
rs1417374-rs1409375 0.316 0.440 0.509 0.713 0.164 0.120 0.137 0.148
Detailed output for rs1409375 to show under-transmission of common allele to affected females
d8
All offspring
Allele Z P Trio-T Trio-NT AffSib UnafSib Freq
 1 −0.9715 0.3313 857 859.7 943 956 0.6798
 2 0.9715 0.3313 411 408.3 393 380 0.3202
 Global test: chi-sq 0.9438, P 0.3313
d8
Male affected offspring
Allele Z P Trio-T Trio-NT AffSib UnafSib Freq
 1 0.3569 0.7212 566 548.8 301 293 0.6867
 2 −0.3569 0.7212 246 263.2 109 117 0.3133
 Global test: chi-sq 0.1274, P 0.7212
d8
Female affected offspring
Allele Z P Trio-T Trio-NT AffSib UnafSib Freq
 1 −2.038 0.04154 291 314 155 173 0.6767
 2 2.038 0.04154 165 142 99 81 0.3233
 Global test: chi-sq 4.154, P 0.04154
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Significant (uncorrected) P-values are shown in bold, those showing a trend in gray.

Table IIIB.

Association Analysis by PDTPHASE Between PIP4K2A Markers and Schizophrenia According to Offspring Sex in the DTNBP1 HRH+ Families

P-values (male cases)
P-values (female cases)
Single markers d2 d5 d8 d9 d2 d5 d8 d9
rs1417374 0.693 0.483 0.379 0.287 0.004 0.013 0.017 0.012
rs1409375 0.370 0.460 0.417 0.578 0.001 0.006 0.012 0.005
rs10828317 0.842 0.805 0.644 0.841 0.084 0.065 0.088 0.030
rs746203 0.843 0.833 0.762 0.405 0.647 0.186 0.250 0.581
PIK53_54 0.684 0.956 0.945 0.995 0.297 0.039 0.140 0.075
rs2364115 0.667 0.853 0.678 0.701 0.282 0.123 0.141 0.166
*Haplotype (see below)
rs1417374-rs1409375 0.518 0.61 0.529 0.469 0.0009 0.005 0.011 0.004
d2 diagnostic category (female cases)
Haplotype Z P Trio-T Trio-NT AffSib UnafSib Freq
1-1 −3.376 0.0007 49.86 67.72 51.11 59.45 0.61
1–2 0.7885 0.4304 14.5 11.93 13.63 14.83 0.13
2-1 0.9808 0.3267 8.147 6.747 5.577 2.552 0.09
2-2 3.003 0.0027 27.49 13.61 27.68 21.17 0.17
Global test: chi-sq 16.5, P 0.0009
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Significant (uncorrected) global P-values are in bold.

Follow-up analysis in the ICCSS showed significant yet modest association between rs1417374 (P = 0.023), rs1409395 (P = 0.026), and the rs1417374-rs1409395 common allele haplotype (P = 0.003) with schizophrenia (Table IV). This results from under-representation of common alleles in cases, echoing the sex-specific association in the ISHDSF DTNBP1 HRH+ families (Table IIIB). Although the associations with rs1417374 and rs1409395 in the ICCSS did not survive 100,000 permutations (perm P = 0.121 and P = 0.139), their common haplotype remained significant (perm P = 0.015).

Table IV.

Association of PIP4K2A SNPs With Schizophrenia in the ICCSS

SNPs Case freq Control freq Chi-sq P Perm P Odds ratio
rs1417374 0.68 0.72 5.195 0.023 0.121 0.94
rs1409395 0.64 0.68 4.943 0.026 0.139 0.94
rs10828317 0.66 0.68 1.411 0.235 0.784 0.97
rs746203 0.60 0.63 3.965 0.046 0.236 0.94
rs8341 0.62 0.64 2.529 0.112 0.490 0.98
rs1417374-rs1409395
Haplotype
1-1 0.54 0.59 8.869 0.003 0.015 0.91
1–2 0.11 0.09 1.497 0.221 0.761 1.13
2-1 0.14 0.13 1.413 0.235 0.784 1.12
2-2 0.22 0.19 3.019 0.082 0.385 1.14
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Significant (uncorrected) values are shown in bold.

Like the ISHDSF, association with rs1417374 in the ICCSS stems primarily from allele frequency differences in female cases versus controls (P = 0.028; Table V) but does not survive 100,000 permutations (perm P = 0.145). Significant association with common allele haplotype rs1417374-rs1409395 in female cases (P = 0.009, global P = 0.049; Table II) survived permutation (perm P = 0.045). After accounting for gender in the logistic regression model, the association between rs1417374 and schizophrenia (unadjusted P = 0.031, OR (95% CI) = 1.197 (1.017–1.408)) and between rs1409395 and schizophrenia (unadjusted P = 0.022, OR (95% CI) = 1.207 (1.027–1.418)) were not significant after correction (data not shown). No other markers were significant in the model and we found no significant differences in LD pattern between cases and controls for correlation or D′-based composite LD comparisons (data not shown).

Table V.

Association of PIP4K2A SNPs With Schizophrenia in the Sex-Stratified ICCSS

SNPs Female cases (major allele freq) Female controls (major allele freq) Chi-sq P Perm P Odds ratio Male cases (major allele freq) Male controls (major allele freq) Chi-sq P Odds ratio
rs1417374 0.68 0.74 4.804 0.028 0.143 0.92 0.68 0.70 0.896 0.343 0.97
rs1409395 0.64 0.68 1.986 0.158 0.623 0.94 0.64 0.68 3.301 0.068 0.96
rs10828317 0.66 0.70 2.021 0.155 0.615 0.94 0.66 0.66 0.035 0.851 0.99
rs746203 0.60 0.65 3.430 0.063 0.306 0.92 0.60 0.62 0.884 0.347 0.94
rs8341 0.62 0.65 1.449 0.228 0.774 0.95 0.62 0.64 0.946 0.330 0.97
rs1417374-rs1409395
Haplotype
1-1 0.54 0.62 6.890 0.009 0.043 0.87 0.53 0.57 2.38 0.123 0.93
1–2 0.10 0.07 3.682 0.055 0.268 1.48 0.11 0.11 0.07 0.790 0.96
2-1 0.14 0.12 0.834 0.361 0.926 1.15 0.15 0.13 0.75 0.387 1.11
2-2 0.22 0.19 1.269 0.260 0.820 1.14 0.22 0.19 1.85 0.173 1.14
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Significant (uncorrected) P-values are in bold.

There was no greater evidence in the ICCSS for association in FH+ cases (n = 256) compared to controls, or significant difference in allele frequencies between FH+ and FH− (n = 467) cases (data not shown). However, nominally significant associations were observed between several PIP4K2A SNPs and schizophrenia in FH− cases versus controls (Table VI). Only rs1417374 (P = 0.004) and haplotype rs1417374-rs1409395 (P = 0.0008) remained significant after 100,000 permutations (perm P = 0.023, perm P = 0.004 respectively).

Table VI.

Association Analysis of PIP4K2A SNPs in the FH− Cases Compared to Controls

SNP Case freq Control freq Chi-sq P Perm P Odds ratio
rs1417374 0.343 0.282 8.54 0.004 0.024 1.22
rs1409395 0.367 0.317 5.43 0.020 0.158 1.16
rs10828317 0.351 0.322 1.97 0.161 0.807 1.09
rs746203 0.414 0.368 4.68 0.031 0.230 1.13
rs8341 0.401 0.355 4.51 0.034 0.247 1.13
rs1417374-rs1409395
Haplotype
1-1 0.54 0.59 8.869 0.0008 0.004 0.88
1–2 0.11 0.09 1.497 0.033 0.590 1.20
2-1 0.14 0.13 1.413 0.366 0.985 1.10
2-2 0.22 0.19 3.019 0.032 0.242 1.24
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Significant (uncorrected) P-values are in bold.

This study attempted to replicate association of PIP4K2A with schizophrenia in the ISHDSF. We found no evidence for this in the whole sample. Markers rs1417374 (5′ upstream) and rs1409395 (intron 1) were nominally associated with disease in the DTNBP1 HRH+ families, via under-transmission of common alleles preferentially to affected females. In follow-up analysis of the ICCSS, rs1417374 and rs1409395 were significantly associated with schizophrenia, similarly sex-specific (under-representation in female cases of common-allele haplotype) and in FH− individuals.

Previously, major alleles of rs1417374 and rs1409395 were over-transmitted to cases regardless of gender [Schwab et al., [2006]]. We observe their under-representation in female cases from both ICCSS and DTNBP1 HRH+ families (ISHDSF). This negative association of previously positively associated alleles is one structure of the “flip-flop” phenomenon [Lin et al., [2007]]. The other structure (positive association of opposite alleles between two samples) was seen with PIP4K2A and schizophrenia by He et al. [2007]. The latter pattern is intuitively consistent with multiple risk alleles in the gene. The former is consistent with the schizophrenia family data of Hennah et al. [2005], where under-transmission to affected females of haplotype HEP3 in the DISC1 gene suggested a sex-specific protective variant. However, their sample had over-representation of HEP3, upwardly biasing the expected transmissions so that the observed under-transmission to affected females was more likely an over-transmission to affected males [Hennah et al., [2005]]. Closer inspection of our data for rs1409395 indicates a potentially similar epiphenomenon here (data not shown). Moreover, Schwab et al. [2006] detected PIP4K2A association in their 10p-linked families. The fact that most significant (albeit negative) association occurred in ISHDSF families unlinked to 10p, and FH− cases of the ICCSS, may suggest that PIP4K2A variants associated in our Irish samples are acting as protective alleles.

Two reviews have demonstrated a male/female risk ratio of 1.4 for developing schizophrenia [Aleman et al., [2003]; McGrath et al., [2004]]. A recent genome-wide association study of schizophrenia identified female-specific association to a SNP in the RELN gene (P = 2.9 × 10−5), with significant gene-sex interaction (P = 4.2 × 10−3) and replicated in additional populations [Shifman et al., [2008]]. Sex hormones were postulated to mediate this effect via modulation of RELN expression, impacting cortical structure and susceptibility to psychosis. Although we detected no straightforward genotype-sex interaction for PIP4K2A via logistic regression, sex hormones may affect PIP4K2A expression, for example, estrogen-response elements are predicted in the promoter via Matinspector (www.genomatix.de, data not shown).

Although only five markers over 315 kb were used to cover PIP4K2A and the 5′ region highlighted by Schwab et al. [2006], this effectively tagged ~50% of regional SNPs, capturing haplotypic diversity reasonably well in our study. However, for rs1417374, the most significant SNP of Schwab et al. [2006] and significantly associated in the ICCSS, the ISHDSF maybe underpowered to detect disease association. We estimated power using rs1417374 and the Genetic Power Calculator [Purcell et al., [2003]] under “TDT for discrete traits.” Reducing the ISHDSF to 265 trios, with high risk (associated) allele frequency 0.69 and relative risk set at 1.1, 1.3, and 1.5 for the heterozygous genotype and multiplicative model, the ISHDSF had power 11%, 48%, and 82% respectively to detect association at α = 0.05. This is a conservative estimate, considering the multigenerational nature of ISHDSF pedigrees. Moreover, Straub et al. [1998] noted the 10p susceptibility locus segregated in only 5–15% families, so we might not expect strong association signals in the whole sample.

In summary, this study shows that genetic variation in PIP4K2A plays a minor role in schizophrenia in the Irish population. Together with associations in these samples between AKT1 and disease [Thiselton et al., [2008]; in preparation], these data provide additional evidence that schizophrenia, at least in part, may result from defective phosphoinositide signaling.

Supplementary Material

supplemental data 1
supplemental data 2

Acknowledgments

Funded by:

NIH; Grant Number: MH041953

We are very grateful to all those patients and family members who participated in this study. Special thanks also to Dr. Sibylle Schwab and Dr. Dieter Wildenauer for sharing their data prior to publication and for helpful discussions during the course of the study.

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supplemental data 2
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