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A Genetic Association Study of Chromosome 11q22-24 in Two Different Samples Implicates the FXYD6 Gene, Encoding Phosphohippolin, in Susceptibility to Schizophrenia

2007; Elsevier BV; Volume: 80; Issue: 4 Linguagem: Inglês

10.1086/513475

1537-6605

Khalid Choudhury, Andrew McQuillin, Vinay Puri, Jonathan Pimm, Susmita Datta, Srinivasa Thirumalai, Robert Krasucki, Jacob Lawrence, Nicholas Bass, Digby Quested, Caroline Crombie, Gillian Fraser, Nicholas Walker, Haitham Nadeem, Sophie L. Johnson, David Curtis, David St Clair, Hugh Gurling,

14-3-3 protein interactions

Previous linkage analyses of families with multiple cases of schizophrenia by us and others have confirmed the involvement of the chromosome 11q22-24 region in the etiology of schizophrenia, with LOD scores of 3.4 and 3.1. We now report fine mapping of a susceptibility gene in the 11q22-24 region, determined on the basis of a University College London (UCL) sample of 496 cases and 488 supernormal controls. Confirmation was then performed by the study of an Aberdeen sample consisting of 858 cases and 591 controls (for a total of 2,433 individuals: 1,354 with schizophrenia and 1,079 controls). Seven microsatellite or single-nucleotide polymorphism (SNP) markers localized within or near the FXYD6 gene showed empirically significant allelic associations with schizophrenia in the UCL sample (for D11S1998, P=.021; for rs3168238, P=.009; for TTTC20.2, P=.048; for rs1815774, P=.049; for rs4938445, P=.010; for rs4938446, P=.025; for rs497768, P=.023). Several haplotypes were also found to be associated with schizophrenia; for example, haplotype Hap-F21 comprising markers rs10790212-rs4938445-rs497768 was found to be associated with schizophrenia, by a global permutation test (P=.002). Positive markers in the UCL sample were then genotyped in the Aberdeen sample. Two of these SNPs were found to be associated with schizophrenia in the Scottish sample (for rs4938445, P=.044; for rs497768, P=.037). The Hap-F21 haplotype also showed significant association with schizophrenia in the Aberdeen sample, with the same alleles being associated (P=.013). The FXYD6 gene encodes a protein called “phosphohippolin” that is highly expressed in regions of the brain thought to be involved in schizophrenia. The protein functions by modulating the kinetic properties of Na,K-ATPase to the specific physiological requirements of the tissue. Etiological base-pair changes in FXYD6 or in associated promoter/control regions are likely to cause abnormal function or expression of phosphohippolin and to increase genetic susceptibility to schizophrenia. Previous linkage analyses of families with multiple cases of schizophrenia by us and others have confirmed the involvement of the chromosome 11q22-24 region in the etiology of schizophrenia, with LOD scores of 3.4 and 3.1. We now report fine mapping of a susceptibility gene in the 11q22-24 region, determined on the basis of a University College London (UCL) sample of 496 cases and 488 supernormal controls. Confirmation was then performed by the study of an Aberdeen sample consisting of 858 cases and 591 controls (for a total of 2,433 individuals: 1,354 with schizophrenia and 1,079 controls). Seven microsatellite or single-nucleotide polymorphism (SNP) markers localized within or near the FXYD6 gene showed empirically significant allelic associations with schizophrenia in the UCL sample (for D11S1998, P=.021; for rs3168238, P=.009; for TTTC20.2, P=.048; for rs1815774, P=.049; for rs4938445, P=.010; for rs4938446, P=.025; for rs497768, P=.023). Several haplotypes were also found to be associated with schizophrenia; for example, haplotype Hap-F21 comprising markers rs10790212-rs4938445-rs497768 was found to be associated with schizophrenia, by a global permutation test (P=.002). Positive markers in the UCL sample were then genotyped in the Aberdeen sample. Two of these SNPs were found to be associated with schizophrenia in the Scottish sample (for rs4938445, P=.044; for rs497768, P=.037). The Hap-F21 haplotype also showed significant association with schizophrenia in the Aberdeen sample, with the same alleles being associated (P=.013). The FXYD6 gene encodes a protein called “phosphohippolin” that is highly expressed in regions of the brain thought to be involved in schizophrenia. The protein functions by modulating the kinetic properties of Na,K-ATPase to the specific physiological requirements of the tissue. Etiological base-pair changes in FXYD6 or in associated promoter/control regions are likely to cause abnormal function or expression of phosphohippolin and to increase genetic susceptibility to schizophrenia. Schizophrenia (SCZD [MIM #181500]) is a disease characterized by self-neglect, thought disorder, auditory hallucinations, delusions, and bizarre behavior.1Andreasen NC Symptoms, signs, and diagnosis of schizophrenia.Lancet. 1995; 346: 477-481PubMed Scopus (255) Google Scholar It has a lifetime prevalence of 0.85% in the United Kingdom. Family, twin, and adoption studies have confirmed that the disorder is highly heritable. More than 20 whole-genome linkage scans of SCZD have demonstrated that there is heterogeneity of linkage, involving multiple loci on different chromosomal regions.2Lewis CM Levinson DF Wise LH DeLisi LE Straub RE Hovatta I Williams NM Schwab SG Pulver AE Faraone SV et al.Genome scan meta-analysis of schizophrenia and bipolar disorder, part II: schizophrenia.Am J Hum Genet. 2003; 73: 34-48Abstract Full Text Full Text PDF PubMed Scopus (925) Google Scholar It is not yet known whether these loci interact. The linkage studies are interpretable only if it is accepted that at least some of these loci act independently in different families. Chromosome 11q22 was first implicated in SCZD in a linkage study of two Japanese pedigrees in which positive LOD scores between 1.0 and 1.5 were reported with marker D11S35.3Nanko S Gill M Owen M Takazawa N Moridaira J Kazamatsuri H Linkage study of schizophrenia with markers on chromosome 11 in two Japanese pedigrees.Jpn J Psychiatry Neurol. 1992; 46: 155-159PubMed Google Scholar This was confirmed in a separate study of a large Canadian pedigree, in which a maximum LOD score of 3.4 was obtained using the same genetic marker, D11S35.4Maziade M Raymond V Cliche D Fournier JP Caron C Garneau Y Nicole L Marcotte P Couture C Simard C et al.Linkage results on 11Q21-22 in eastern Quebec pedigrees densely affected by schizophrenia.Am J Med Genet. 1995; 60: 522-528Crossref PubMed Scopus (47) Google Scholar Later, we published further evidence of linkage to SCZD at 11q23.3 in a genomewide scan of very large multiply affected families.5Gurling HMD Kalsi G Brynjolfson J Sigmundsson T Sherrington R Mankoo BS Read T Murphy P Blaveri E McQuillin A et al.Genomewide genetic linkage analysis confirms the presence of susceptibility loci for schizophrenia, on chromosomes 1q32.2, 5q33.2, and 8p21-22 and provides support for linkage to schizophrenia, on chromosomes 11q23.3-24 and 20q12.1-11.23.Am J Hum Genet. 2001; 68: 661-673Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar Overall, we found a 3-point LOD score of 3.1 with markers D11S925 and D11S934. A LOD score of 3.2 was found in a single large Icelandic pedigree, again supporting the existence of locus heterogeneity. The chromosome 11q22-24 region has been shown to be one of the most well-established linkages to SCZD by a meta-analysis of 20 genome scans that employed nonparametric rank-order statistics to show that evidence of linkage at this region was nonrandom.2Lewis CM Levinson DF Wise LH DeLisi LE Straub RE Hovatta I Williams NM Schwab SG Pulver AE Faraone SV et al.Genome scan meta-analysis of schizophrenia and bipolar disorder, part II: schizophrenia.Am J Hum Genet. 2003; 73: 34-48Abstract Full Text Full Text PDF PubMed Scopus (925) Google Scholar Chromosome 11q22.3-q24.1 was ranked third of all regions, and, when the different sample sizes of the 20 SCZD genome scans were taken into account, it was ranked fourth. This provided considerable confidence that an SCZD susceptibility locus was likely to exist in the 11q22-24 region. Allelic association studies were performed to fine map an 11q22-24 SCZD susceptibility gene by detecting linkage disequilibrium (LD). The University College London (UCL) case-control sample consisted of 496 unrelated cases and 488 ancestrally matched controls. Research subjects were selected only if both parents were English, Scottish, or Welsh, with at least three grandparents having the same origins. Subjects were also included if the fourth grandparent was of another white European origin but were excluded if one grandparent was of Jewish or non–European Union (EU) (before the enlargement of the EU in 2004) ancestry. These data were recorded in an ancestry questionnaire, with confirmation from family histories noted on medical records. U.K. National Health Service (NHS) multicenter and local research ethics–committee approval was obtained, and all participating subjects signed an approved consent form after reading an information sheet. Each schizophrenic research subject had received a diagnosis and assessment by NHS psychiatrists as part of routine clinical diagnosis and treatment. Those with short-term drug-induced psychoses, psychoses with either learning disability or head injury, and other symptomatic psychoses were excluded. Schizophrenic subjects were recruited on the basis of having an International Classification of Diseases version 10 (ICD10) diagnosis of SCZD recorded in medical case-history notes after clinical interview by NHS psychiatrists. The diagnoses were confirmed by a senior psychiatrist, usually within 1 wk. Schedule for Affective Disorders and Schizophrenia–Lifetime Version (SADS-L) interview was completed for all cases and controls by a research psychiatrist.6Spitzer RL Endicott J The Schedule for Affective Disorders and Schizophrenia, Lifetime Version. 3rd ed. New York State Psychiatric Institute, New York1977Google Scholar Schizophrenic subjects were then chosen on the basis of having received a diagnosis at the “probable level” of the Research Diagnostic Criteria (RDC).7Spitzer RL Endicott J Robins E Research Diagnostic Criteria for a selected group of functional disorders. 3rd ed. New York State Psychiatric Institute, New York1978Google Scholar Patients with schizoaffective bipolar disorder or schizomania were not included. All screened control individuals were also interviewed by a psychiatrist, specifically for the study, and were selected for having no family history of SCZD, alcoholism, or bipolar disorder, according to self-report by the research subject. They were then interviewed with the SADS-L schedule and were included only if they were found to have no present or lifetime history of any RDC-defined mental disorder. The UCL case-control sample has been used in studies of other chromosomal regions and has implicated the chromosome 5q33 epsin 4 (CLINT1) gene encoding enthoprotin, the chromosome 8p22 PCM1 gene, and the chromosome 1q23 UHMK1 gene as the cause of SCZD.8Pimm J McQuillin A Thirumalai S Lawrence J Quested D Bass N Lamb G Moorey H Datta SR Kalsi G et al.The Epsin 4 gene on chromosome 5q, which encodes the clathrin-associated protein enthoprotin, is involved in the genetic susceptibility to schizophrenia.Am J Hum Genet. 2005; 76: 902-907Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 9Gurling HM Critchley H Datta SR McQuillin A Blaveri E Thirumalai S Pimm J Krasucki R Kalsi G Quested D et al.Genetic association and brain morphology studies and the chromosome 8p22 pericentriolar material 1 (PCM1) gene in susceptibility to schizophrenia.Arch Gen Psychiatry. 2006; 63: 844-854Crossref PubMed Scopus (75) Google Scholar, 10Puri V McQuillin A Choudhury K Datta S Pimm J Thirumalai S Krasucki R Lawrence J Quested D Bass N et al.Fine mapping by genetic association implicates the chromosome 1q23.3 gene UHMK1, encoding a serine/threonine protein kinase, as a novel schizophrenia susceptibility gene.Biol Psychiatry. 2006; (electronically published September 13, 2006; accessed February 19, 2007)(http://www.journals.elsevierhealth.com/periodicals/bps/article/PIIS0006322306008067/abstract)Google Scholar The sample has also been used to exclude association between SCZD and markers at the chromosome 1q23 RGS4 and CAPON (NOS1AP) genes.11Puri V McQuillin A Thirumalai S Lawrence J Krasucki R Choudhury K Datta S Kerwin S Quested D Bass N et al.Failure to confirm allelic association between markers at the CAPON gene locus and schizophrenia in a British sample.Biol Psychiatry. 2006; 59: 195-197Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 12Rizig MA McQuillin A Puri V Choudhury K Datta S Thirumalai S Lawrence J Quested D Pimm J Bass N et al.Failure to confirm genetic association between schizophrenia and markers on chromosome 1q23.3 in the region of the gene encoding the regulator of G-protein signaling 4 protein (RGS4).Am J Med Genet B Neuropsychiatr Genet. 2006; 141: 296-300Crossref Scopus (28) Google Scholar Genomic DNA for the UCL case-control sample was extracted from whole-blood samples by use of standard cell lysis, proteinase K digestion, and phenol/chloroform ethanol precipitation method, as described elsewhere.8Pimm J McQuillin A Thirumalai S Lawrence J Quested D Bass N Lamb G Moorey H Datta SR Kalsi G et al.The Epsin 4 gene on chromosome 5q, which encodes the clathrin-associated protein enthoprotin, is involved in the genetic susceptibility to schizophrenia.Am J Hum Genet. 2005; 76: 902-907Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar All DNA samples were quantified with Picogreen (Molecular Probes) by use of fluorimetry. Fifteen reference microsatellite markers at chromosomal loci not thought to be involved in SCZD were genotyped in a subset of samples (200 cases and 300 controls). This was done to detect genetic heterogeneity between the cases and controls and to confirm that the samples were genetically well matched. In addition, a statistical test with use of CHECKHET was employed to detect subjects with an atypical genetic background.13Curtis D North BV Gurling HM Blaveri E Sham PC A quick and simple method for detecting subjects with abnormal genetic background in case-control samples.Ann Hum Genet. 2002; 66: 235-244Crossref PubMed Google Scholar No evidence of genetic heterogeneity between cases and controls was found with use of the reference markers. The CHECKHET test detected two schizophrenic subjects with abnormal genotypes. These were xcluded from the sample before any chromosome 11 markers were genotyped. The Aberdeen sample consisted of 858 subjects with SCZD and 591 controls. The cases were recruited through Scottish psychiatric hospitals and met DSM-III-R or DSM-IV criteria of SCZD, with use of an operational criteria checklist (OPCRIT). Diagnosis of SCZD was confirmed through agreement by two independent senior psychiatrists on the basis of structured clinical interviews for DSM-III-R/DSM-IV (SCID) and inspection of psychiatric case notes. All controls were volunteers recruited through general practices from the same region of Scotland and were ethnically matched. The control samples were screened for absence of psychiatric illness. Informed consent was obtained from all patients and control individuals. A proportion of the Aberdeen sample was employed elsewhere in tests for genetic association between NRG1 and SCZD.14Stefansson H Sarginson J Kong A Yates P Steinthorsdottir V Gudfinnsson E Gunnarsdottir S Walker N Petursson H Crombie C et al.Association of neuregulin 1 with schizophrenia confirmed in a Scottish population.Am J Hum Genet. 2003; 72: 83-87Abstract Full Text Full Text PDF PubMed Scopus (479) Google Scholar Random markers on chromosome 11q22-24, close to the maximum LOD score position in the UCL SCZD family-linkage study, were initially selected for fine mapping.5Gurling HMD Kalsi G Brynjolfson J Sigmundsson T Sherrington R Mankoo BS Read T Murphy P Blaveri E McQuillin A et al.Genomewide genetic linkage analysis confirms the presence of susceptibility loci for schizophrenia, on chromosomes 1q32.2, 5q33.2, and 8p21-22 and provides support for linkage to schizophrenia, on chromosomes 11q23.3-24 and 20q12.1-11.23.Am J Hum Genet. 2001; 68: 661-673Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar Five of these random microsatellite markers were genotyped before positive association with D11S1998 was found. Further nonrandom microsatellite and SNP markers close to D11S1998 were then chosen for genotyping. Microsatellite marker TTTC20.2 was identified from a simple repeat found in the UCSC Genome Browser database May 2004 assembly. The forward (5′-AGGACCACACTCAGCCTCAC-3′) and reverse (5′-GGGAAGGAAGGAGAGAGAGAG-3′) primers were designed using Primer3.15Rozen S Skaletsky H Primer3 on the WWW for general users and for biologist programmers.Methods Mol Biol. 2000; 132: 365-386Crossref PubMed Google Scholar The primer sequences for D11S29, D11S4127, D11S1998, D11S939, D11S976, D11S4195, D11S925, D11S964, D11S836, and D11S934 were obtained from the GDB Human Genome Database and Ensembl. Microsatellite markers were genotyped in an initial sample of 443 controls and 480 schizophrenic patients in the UCL sample. PCR amplification of these markers was performed using an M13-tailed primer and a second nontailed primer. A third universal M13 sequence primer labeled with infrared dye IRD700 or IRD800 was used to hybridize against the M13-tailed locus-specific primer. Microsatellite-marker fragment sizes were separated and visualized with either of the two infrared dyes on LiCor 4200L sequencers. Genotyping was accomplished using SAGA-GT genotyping software (LiCor). The genotypes were then checked by eye, with all allele calls verified by a second independent person. Any discrepant genotypes were repeated. SNPs rs11999, rs869789, rs529623, rs479991, rs2019655,rs10790212, rs3087563, rs1815774, rs876798, rs876797, r1s4938445, rs4938446, rs11216598, and rs631898 were chosen from Ensembl. Further SNPs—rs4579962, rs678776, rs11216567, rs10892181, rs3168238, rs564989, rs12363888, rs11608153, rs512481, rs476130, rs7121573, rs873713, rs10790218, rs11605223, rs3809043, rs3809042, and rs497768—were then selected from the International HapMap Project with use of the Haploview tagger function.16Barrett JC Fry B Maller J Daly MJ Haploview: analysis and visualization of LD and haplotype maps.Bioinformatics. 2005; 21: 263-265Crossref PubMed Scopus (11421) Google Scholar, 17de Bakker PI Yelensky R Pe’er I Gabriel SB Daly MJ Altshuler D Efficiency and power in genetic association studies.Nat Genet. 2005; 37: 1217-1223Crossref PubMed Scopus (1455) Google Scholar All SNP markers were determined using KASPar (KBioscience), a modified Amplifluor SNP-genotyping method. Of the samples, 17% from each microtitre plate were reduplicated to detect possible errors and to confirm the reproducibility of genotypes. The data were then analyzed to confirm Hardy-Weinberg equilibrium (HWE). Any markers lacking HWE were repeated using an alternative Taqman method. Before any association analysis, the genotype data were assessed using the SCANGROUP program in GENECOUNTING.18Zhao JH Curtis D Sham PC Model-free analysis and permutation tests for allelic associations.Hum Hered. 2000; 50: 133-139Crossref PubMed Scopus (380) Google Scholar, 19Zhao JH Lissarrague S Essioux L Sham PC GENECOUNTING: haplotype analysis with missing genotypes.Bioinformatics. 2002; 18: 1694-1695Crossref PubMed Scopus (107) Google Scholar, 20Curtis D Knight J Sham PC Program report: GENECOUNTING support programs.Ann Hum Genet. 2006; 70: 277-279Crossref PubMed Scopus (43) Google Scholar This program highlights any potential genotyping errors by identifying significant differences in haplotypic frequencies between any single 96-well microtitre plate and haplotypic frequencies in all other plates combined. It is argued that genotyping errors can show up as rare haplotypes occurring on just a single plate, whereas true haplotypic associations with disease will appear as differences in haplotype frequencies between cases and controls spread across a number of plates. Allelic association analysis with SCZD was performed using a simple χ2 test for biallelic SNPs and by using CLUMP for microsatellite markers. CLUMP employs an empirical Monte Carlo test of significance that does not require further correction for multiple alleles.21Sham PC Curtis D Monte Carlo tests for associations between disease and alleles at highly polymorphic loci.Ann Hum Genet. 1995; 59: 97-105Crossref PubMed Scopus (852) Google Scholar Subtests of the CLUMP program include T1, Pearson’s χ2 statistic of the “raw” contingency table; T2, the χ2 statistic of a table with rare alleles grouped together to prevent small expected cell counts; T3, the largest of the χ2 statistics of 2×2 tables, each of which compares one allele against the rest grouped together; and T4, the largest of the χ2 statistics of all possible 2×2 tables, which compares any combination of alleles against the rest. To select regions of interest for further investigation, we took account of the output of the most significant of the four statistics by use of CLUMP. Likewise, we considered the pointwise P values for markers individually rather than attempting to derive an overall P value for the region on the basis of combining results from multiple markers. The multilocus genotypes were then analyzed for haplotypic association with SCZD by use of GENECOUNTING, which computes maximum-likelihood estimates of haplotype frequencies from phase-unknown case-control data. The significance of any overall haplotypic association with SCZD was then obtained with a permutation test.18Zhao JH Curtis D Sham PC Model-free analysis and permutation tests for allelic associations.Hum Hered. 2000; 50: 133-139Crossref PubMed Scopus (380) Google Scholar, 19Zhao JH Lissarrague S Essioux L Sham PC GENECOUNTING: haplotype analysis with missing genotypes.Bioinformatics. 2002; 18: 1694-1695Crossref PubMed Scopus (107) Google Scholar, 20Curtis D Knight J Sham PC Program report: GENECOUNTING support programs.Ann Hum Genet. 2006; 70: 277-279Crossref PubMed Scopus (43) Google Scholar GENECOUNTING was also used to calculate pairwise LD between all markers with use of the LDPAIRS program; this was visualized using LocusView 2.0. One the five initial randomly selected microsatellites in the 11q22-24 region, D11S1998, showed allelic association with SCZD (P=.021; CLUMP T2) (tables Table 1, Table 2). After this, other markers to be genotyped were selected nonrandomly by virtue of being very likely to be in LD with marker D11S1998. From these nonrandom microsatellite markers, TTTC20.2 was also found to be associated with SCZD (P=.048; CLUMP T4). None of the other genotyped microsatellite markers localized to chromosome 11q22-24 showed association with SCZD (P values are for CLUMP T1 statistic: for D11S29, P=.899; for D11S4127, P=.521; for D11S939, P=.460; for D11S976, P=.393; for D11S4195, P=.287; for D11S925, P=.379; for D11S964, P=.132; for D11S836, P=.423; for D11S934, P=.561). After microsatellite analysis, SNP markers localized near positive markers D11S1998 and TTTC20.2 were selected for genotyping. SNPs rs3168238 (P=.009; odds ratio [OR] 1.64), rs1815774 (P=.049; OR 1.21), rs4938445 (P=.010; OR 1.31), rs4938446 (P=.025; OR 1.26), and rs497768 (P=.023; OR 1.24) were all found to be associated with SCZD, with six further SNPs (for rs869789, P=.097; for rs11216567, P=.072; for rs7121573, P=.076; for rs876797, P=.086; for rs873713, P=.091; and for rs10790218, P=.099) showing a trend toward association.Table 1Allelic Association Tests with SCZD at the FXYD6 Locus in the UCL SampleNo. of Observed Alleles for Allelic BaseMarker and SampleMarker Location (bp)Previous Marker Distance (bp)χ2PaTwo-tailed significance from 2×2 χ2, with 1 df.ACGTrs11999:117196128.91.340 Control542300 Case521261rs869789:1171965964682.75.097 Control107753 Case138772rs4579962:117197544948.03.860 Control274688 Case266680rs529623:117198465921.60.440 Control473395 Case475427rs678776:1172019543,489.00.975 Control285671 Case282666rs479991:1172021682141.17.280 Control665197 Case715187D11S199811720294177312.60.021bP from CLUMP Monte Carlo subtest T2.rs11216567:1172036937523.25.072 Control88070 Case82989rs10892181:1172047961,103.49.484 Control351609 Case320594rs2019655:117205088292.60.437 Control208664 Case230674rs10790212:1172079002,812.47.493 Control640240 Case666232rs3087563:1172131475,247.09.763 Control415451 Case422472rs3168238:1172148551,7086.80.009cOR 1.64. Control76874 Case47887rs564989:117214964109.30.584 Control222742 Case226712rs12363888:1172183973,433.04.843 Control90755 Case90953rs11608153:1172218733,476.48.489 Control821129 Case805115rs512481:1172240902,217.90.344 Control452494 Case425507rs476130:1172330939,003.64.423 Control89070 Case85377TTTC20.21172345501,45717.54.048dP from CLUMP Monte Carlo subtest T4.rs7121573:1172350404903.15.076 Control382564 Case331579rs1815774:1172366491,6093.87.049eOR 1.21. Control342528 Case311583rs876798:1172426506,001.45.503 Control125739 Case123797rs876797:1172429573072.94.086 Control207651 Case186712rs873713:1172451262,1692.86.091 Control230710 Case197733rs10790218:1172472052,0792.73.099 Control374576 Case334602rs4938445:1172502133,0086.68.010fOR 1.31. Control299579 Case250632rs4938446:117250259465.00.025gOR 1.26. Control296570 Case271655rs11216598:1172536623,403.07.798 Control727149 Case771153rs631898:117253688262.31.128 Control419451 Case466434rs11605223:1172540543661.41.235 Control490470 Case462494rs3809043:117254325271.57.452 Control92929 Case89834rs3809042:1172543305.21.646 Control27925 Case23899rs497768:1172559501,6205.21.023hOR 1.24. Control416500 Case371553a Two-tailed significance from 2×2 χ2, with 1 df.b P from CLUMP Monte Carlo subtest T2.c OR 1.64.d P from CLUMP Monte Carlo subtest T4.e OR 1.21.f OR 1.31.g OR 1.26.h OR 1.24. Open table in a new tab Table 2Fragment Sizes of Microsatellite Markers at the FXYD6 Locus in the UCL SampleNo. of Observed Alleles by PopulationMarker and Fragment Size (bp)ControlCaseD11S1998: 1506086 15810 16220 1663827 170405428 174275239 1787560 18276 18610TTTC20.2: 23284 236314 2402216 24235 244130130 2461110 24897137 2505447 252113104 2546577 2567569 2586062 2603151 2626057 2641214 2664632 26834 2701723 27495 27821 28210 Open table in a new tab Tests of three-marker haplotypic association with use of rs11216598, rs11605223, and rs497768 (Hap-F7) provided significant evidence of association with SCZD, with a global permutation significance of P=.0013. Several other two-, three-, and four-marker tests of haplotypic association were also significantly positive, as shown in table 3. Nearly all the genotyped markers that show significant association with SCZD or a positive trend are localized within the FXYD domain–containing ion transport regulator 6 (FXYD6 [MIM *606683]) gene. The exceptions to these are markers rs869789, D11S1998, and rs11216567, situated on FXYD2. These three markers show significant LD with several of the markers in FXYD6. Results of pairwise LD statistics between all markers genotyped and their respective locations are shown in figure 1.Table 3Haplotypic Association Tests with SCZD at the FXYD6 Locus in the UCL SampleEstimated Haplotype Frequency (%)No. of Markers and Haplotype ReferenceHaplotypeGlobal Empirical PaHaplotype-permutation test empirical P, based on 9,999 permutations.Alleles Increasing in CasesControlsCases2: HAP-F1rs3168238-rs497768.0028 T-G47.956.03: HAP-F2rs564989-rs476130-rs876797.0043 C-C-C11.414.9 HAP-F3rs564989-rs476130-rs497768.0048 T-C-G41.648.7 HAP-F4rs564989-rs876797-rs873713.0010 C-C-T16.923.8 HAP-F5rs476130-rs11605223-rs497768.0027 C-G-G11.317.1 HAP-F6rs1815774-rs4938445-rs4938446.0066 G-G-T58.164.0 HAP-F7rs11216598-rs11605223-rs497768.0013 A-G-G11.416.8 HAP-F8rs11605223-rs3809043-rs497768.0021 G-C-G11.517.34: HAP-F9rs3168239-rs564989-rs876797-rs873713.0013 T-C-C-T17.923.1 HAP-F10rs3168238-rs12363888-rs11605223-rs497768.0057 T-C-G-G10.817.2 HAP-F11rs3168238-rs476130-rs11605223-rs497768.0026 T-C-G-G10.716.9 HAP-F12rs3168238-rs11605223-rs3809043-rs497768.0016 T-G-C-G10.817.3 HAP-F13rs3168238-rs11605223-rs3809042-rs497768.0035 T-G-G-G10.917.2 HAP-F14rs1815774-rs10790218-rs4938445-rs11605223.0055 G-G-G-G40.546.7 HAP-F15rs1815774-rs4938445-rs4938446-rs11605223.0159 G-G-T-G40.647.2 HAP-F16rs10790218-rs4938445-rs4938446-rs11605223.0066 G-G-T-G40.846.3 HAP-F17rs11216598-rs11605223-rs3809043-rs497768.0019 A-G-C-G11.416.8a Haplotype-permutation test empirical P, based on 9,999 permutations. Open table in a new tab To verify the significance of the positive allelic and haplotypic association found in the UCL sample, replication was attempted in an Aberdeen case-control sample with use of SNPs rs869789, rs11216567, rs10790212, rs3168238, rs876797, rs4938445, and rs497768. With a one-tailed test of significance, two SNPs were found to show association with SCZD in the Aberdeen sample (for rs4938445, P=.044; for rs497768, P=.037) (table 4). The allele-frequency increase for SNP rs3168238 (P=.074), although not significantly associated with SCZD, displayed a trend in the direction opposite to that found in the UCL sample. When the data from the UCL and the Aberdeen samples were combined, three SNP markers showed significant allelic association (for rs869789, P=.023; for rs4938445, P=.002; and for rs497768, P=.005), with two further SNPs displaying a trend toward association (for rs11216567, P=.058, and for rs876797, P=.063) (table 4).Table 4Allelic Association Tests with SCZD at the FXYD6 Locus in the UCL, Aberdeen, and Combined SamplesUCLaData as shown in tables 1.AberdeenCombined (UCL and Aberdeen)No. (%) of Observed Alleles for Allelic BaseNo. (%) of Observed Alleles for Allelic BaseNo. (%) of Observed Alleles for Allelic BaseMarker and SampleMarker Location (bp)ACGTχ2PbTwo-tailed significance (P) from 2×2 χ2, with 1 df.ACGTχ2PcOne-tailed significance (P) from 2×2 χ2, with 1 df.ACGTχ2PbTwo-tailed significance (P) from 2×2 χ2, with 1 df.rs869789:1171965962.75.0971.996.0795.170.023 Control107 (.12)753 (.88)173 (.15)985 (.85)280 (.14)1,738 (.86) Case138 (.15