The pericentromeric region of chromosome 18,especially 18p11.2, is described as a schizophreniasusceptibility locus. We had previously clonedtwo novel brain-derived transcripts from thisregion: the gene for a second human myo-inositolmonophosphatase (IMPA2) and a gene ofunknown function, C18orf1. Recently, we reporteda distortion of transmission of the tandem repeatmarker D18S852, embedded in the 3’-untranslatedregion of C18orf1, in schizophrenia, using a family-based association test. A subsequent case-controlstudy also revealed a significant associationbetween the haplotype constructed from D18S852and the 6409T>C polymorphism located inC18orf1 and schizophrenia. In the present study,we screened the C18orf1 gene for mutations andidentified a novel single nucleotide polymorphism(SNP), -96T>C in exon 2. This SNP showed signifi-cant genotypic (P = 0.048) and allelic association (P= 0.005) with schizophrenia in a case-controlstudy. The distributions of haplotypes defined byD18S852 and -96T>C were different between con-trol and schizophrenia groups (P = 0.021). Thesefindings suggest that C18orf1 or a gene nearby

may contribute to the overall genetic risk forschizophrenia.

Key words: association, chromosome 18, haplotype,linkage disequilibrium

Introduction

Schizophrenia is a common and devastating mentaldisorder of unknown etiology. Multiple factors includingrisk-conferring genes and undefined environmentalvariables may contribute to overall susceptibility.1

Several chromosomal loci have been reported toshow linked with schizophrenia,2 one of which is thepericentromeric region of chromosome 18. Schwab etal.3 demonstrated linkage and association of schizo-phrenia to genetic markers within and near the G-olfα(GNAL) gene on 18p11.2. Williams et al.4 detected 18pas a locus exerting a nominal significance in their first-stage genome scan of schizophrenic sibling pairs.Cytogenetic studies have also identified schizo-phrenic patients who carried a pericentric inversion ofchromosome 185 and translocations between 2p11.2and 18p11.2,6 and between 18p11.1 and 21p11.1,7 sup-porting the notion that the short arm/pericentromericregion of chromosome 18 is involved in the manifesta-tion of schizophrenia.

To identify susceptibility gene(s) for functional psy-choses on chromosome 18, we isolated 25 novelbrain-derived transcripts by employing a cDNA selec-

Original Article

C18orf1 located on chromosome 18p11.2 may confer susceptibility to schizophrenia

Mika Kikuchi1,2, Kazuo Yamada1, Tomoko Toyota1,2 and Takeo Yoshikawa1

1) Laboratory for Molecular Psychiatry, RIKEN Brain Science Institute, Saitama, Japan2) Section of Psychiatry and Behavioral Sciences, Tokyo Medical and Dental University Graduate School,Tokyo, Japan

J Med Dent Sci 2003; 50: 225–229

Corresponding Author: Takeo Yoshikawa, M.D., Ph.D.Laboratory for Molecular PsychiatryRIKEN Brain Science Institute2-1 Hirosawa, Wako-city, Saitama 351-0198JapanTel: +81 (Japan) - 48 - 467 - 5968Fax: +81 (Japan) - 48 - 467 - 7462E-mail: takeo@brain.riken.go.jpReceived May 7; Accepted June 13, 2003

tion strategy.8 Among these, was the gene for a secondhuman myo-inositol monophosphatase (IMPA2)9,10

and a gene of unknown function, C18orf1,11 bothmapping to 18p11.2. Our genetic analysis of C18orf1showed evidence for a distortion of transmission of theD18S852 triplet repeat, present in the 3’-untranslatedregion of C18orf1, in schizophrenic trios (patients andtheir parents). A subsequent case-control study con-firmed a significant association between the haplotypeconstructed from D18S852 and 6409T>C polymor-phism located in C18orf1, and schizophrenia.12 In thisstudy, we performed mutation screening of theC18orf1 gene, and conducted association tests usingthe newly detected polymorphism in a case-control par-adigm.

Material and Methods

SubjectsSchizophrenic samples were composed of 49

females (mean age, 50±12 years) and 59 males(mean age, 48±12 years). The controls were 48females (mean age, 45±10 years) and 58 males(mean 42±10 years). All the samples were derivedfrom the same region of central Japan. Best-estimatelifetime diagnoses of schizophrenia were madeaccording to criteria of the Diagnostic and StatisticalManual of Medical Disorders, 4th edition (DSM-IV) andby the consensus of at least two experienced psychia-trists. In addition to direct interviews, all availablemedical records and information from relatives and hos-

pital staff were considered. The present protocol wasapproved by the ethics committees of both RIKEN andTokyo Medical and Dental University. Writteninformed consent was obtained from all participantsafter receiving an explanation of the protocol and pur-pose of the study. All samples were taken in accor-dance with the Helsinki declaration.

Mutation screening and genotyping of the C18orf1gene

We screened for polymorphisms in the protein codingregions and exon-intron junctions of the C18orf1 α andβ isoforms by PCR followed by sequencing. Theprimers used for amplification are listed in Table 1. Allexons except 2 and 5 were amplified using rTaq(Takara, Tokyo, Japan). To amplify exons 2 and 5,MasterAmp DN buffer (Epicentre, Madison, WI, USA)and rTaq were used. The PCR reactions were per-formed starting at 96 °C for 3 min, followed by 35 cycleof 95 °C for 50 s, 59 °C for 50 s, 72 °C for 1 min, and afinal extension period at 72 °C for 10 min. Sequencingof the PCR product was conducted using the dGTPBigDye Terminator Ready Reaction Kit (PE AppliedBiosystems, Foster City, CA, USA) on an ABI 377 DNAsequencer (PE Applied Biosystems).

Statistics analysisDeviations from Hardy-Weinberg equilibrium, linkage

disequilibrium statistics and estimated haplotype fre-quencies were computed using Arlequin software13

(http://lgb.unige.ch/arlequin/). Genotypic, allelic andhaplotypic associations were assessed using the

M. KIKUCHI et al. J Med Dent Sci226

Table 1. PCR primers and conditions used to screen for nucleotide variants in the C18orf1 gene

CLUMP program14 (http://www.mds.qmw.ac.uk/stat-gen/dcurtis/software.html) with 10,000 simulations,which calculates empirical P-values using a MonteCarlo permutation procedure.

Results

Polymorphism identifiedThe initiation ATG codon of the C18orf1 α variant is

located in exon 2, while that of the β form is in exon 4a(Fig. 1).11 The stop codon for both isoforms is encodedby exon 6. To find functional polymorphisms in the pro-tein-coding sequences of the C18orf1 gene, theregion spanning from the translation start site to thestop codon including splice boundaries weresequenced from 30 schizophrenic patients. No unsyn-onymous mutation was detected, but we found anovel T>C SNP located 96bp upstream from the ATGcodon in exon 2 (Fig. 1).

Genetic analysis of the -96T>C polymorphismWe typed the -96T>C SNP in 108 schizophrenic

patients and 106 age- and sex-matched controls. ThisSNP showed a modest genotypic association (P =0.048), and significant allelic association (P = 0.005)with schizophrenia (Table 2). The T allele was overlyrepresented in the disease group [odds ratio and 95%C.I. = 1.99 (1.19 – 3.30)]. The genotypic frequenciesconformed to Hardy-Weinberg equilibrium in both thecontrol (P =0.314) and schizophrenia (P = 0.248)samples.

Linkage disequilibrium (LD) analysis among poly-morphisms in the IMPA2 and C18orf1 genes, andD18S40

In prior genetic studies of the chromosome 18 shortarm, we detected evidence of associations in schizo-phrenia with the following genes/markers: D18S852and 6409T>C within the C18orf1 gene,12 IVS1-15G>Aand 800C>T within IMPA2,15 and D18S40.12

Therefore, we examined LD among the markersincluding the newly identified -96T>C polymorphism ofthe C18orf1 gene (Table 3). The -96T>C was in signif-

227C18orf1 LOCUS CONFERS SUSCEPTIBILITY TO SCHIZOPHRENIA

Fig. 1. Genomic structure and cDNA isoforms (α and β ) of the C18orf1 gene. The numbers in the exon boxes denote nucleotide posi-tions (A of the initiation codon ATG is counted as +1). The locations of three polymorphisms, -96T>C, D18S852 and 6409T>C are alsoshown.

Table 2. Allelic and genotypic distributions of -96T>C in C18orf1

icant LD with D18S852 (P = 0.039) (Table 3).

Haplotype analysisThe -96T>C SNP and D18S852 marker from

C18orf1 gene, were in LD with each other, allowing usto construct haplotypes and calculate haplotype fre-quencies. The distributions of haplotypes were signifi-cantly different between control and schizophreniagroups (P = 0.021) (Table 4).

Discussion

The C18orf1 gene was originally cloned from brainexpressed transcripts that had been selected throughhybridization to chromosome 18-specific cosmidclones.8,11 The gene was predicted to have at least foursplice variants and to encode 306 (α 1 form), 288 (α 2),248 (β 1) or 230 (β 2) amino-acid proteins, and a 7.1kb-long 3’-UTR (Fig. 1).11 A protein motif search sug-gested the presence of a putative type Ib transmem-brane domain in both α and β isoforms and a low-den-sity lipoprotein receptor class A domain in the α-specificN-terminus.11 We have also shown evidence of RNAediting in the 5’-UTR of β form,11 but the precise func-tion of the gene remains unknown. Recently, Xu et al.16

reported a novel androgen-induced gene calledPMEPA1 located on chromosome 20q13, of which thepredicted protein is similar in size to the β 1 isoform ofC18orf1 (67% sequence identity at the protein level).They suggested that C18orf1 and PMEPA1 belong to anovel gene family. More recently, Rae et al.17

described PMEPA1 as a solid tumor-associated geneand named it STAG1/PMEPA1. They also indicated

M. KIKUCHI et al. J Med Dent Sci228

Table 4. Haplotype frequencies of the C18orf1 gene

Table 3. Pairwise linkage disequilibrium among polymorphisms in the IMPA2 and C18orf1genes, and D18S40

that both C18orf1 and STAG1/PMEPA1 have potentialbinding sites for src homology 3 (SH3) and tryptophantryptophan (WW) domains. These genes may functionby interacting with signaling molecules.

Including the present results, the three genes(GNAL, IMPA2, C18orf1) on the short arm of chromo-some 18 show association with schizophrenia or func-tional psychoses.3,12,15 These genes are localized to alimited intervals of approximately 1.5 Mb. Association/linkage disequilibrium behavior over a relatively shortsequence stretch is extremely variable,18 partlybecause LD is determined by the heterozygosity andhistory of markers and other confounding factors. Insome cases the association of markers with a diseaseand the measure of LD between the markers, are notconsistent in the genomic vicinity of the diseaselocus.19 This situation makes the precise identificationof susceptibility variants formidable. However, evi-dence for the association of C18orf1 gene polymor-phisms with schizophrenia in addition to evidenceimplicating GNAL3 and IMPA215 provides cogent sup-port for the existence of a causal susceptibilitygene(s) at 18p11.2. Excluding C18orf1 a transcriptsearch using the UCSC Human Genome Browser v6(http://genome.ucsc.edu/goldenPath/hgTracks.html)failed to detect any other genes within the radius of 50kb from -96T>C and D18S852. In the present study, wescreened only the protein-coding region of C18orf1.The genomic interval surrounding C18orf1, especiallythe promoter region, warrants further scrutiny.

Acknowledgements

We are grateful to all participants in this study. Wethank Dr. Meerabux for her critical reading of themanuscript. The study was partly funded by a Grant-in-Aid for Scientific Research (No. 12307020) from theJSPS, Japan.

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229C18orf1 LOCUS CONFERS SUSCEPTIBILITY TO SCHIZOPHRENIA