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The exact etiology of schizophrenia remainsundetermined but accumulating evidence sug-gests that disturbances in neurodevelopment mayrepresent one contributory factor. Netrin G1, arecently cloned gene from the mouse, has beenshown to play a potential role in the formation ofneural circuitry. To determine whether this gene isinvolved in the development of psychosis, we per-formed a genetic association study of humannetrin G1 gene in schizophrenia. First, we deter-mined the human genomic structure of netrin G1by direct comparisons between cDNA andgenome sequences, and by database searches. Forthe subsequent examination of heterozygosity, weselected 10 single nucleotide polymorphisms(SNPs) for an association test in case (n = 180) andcontrol (n = 180) samples. Among these SNPs,IVS8-1467C＞T showed significant allelic associa-tion (nominal P = 0.020) with disease. This SNP islocated in a haplotype block of ~40 kb and haplo-types in this block also displayed significantassociation (most significant P = 0.017). Thesefindings suggest that netrin G1 or a nearby genemay contribute to the overall genetic risk forschizophrenia.

Key words: netrin family, laminet 1, axon guid-ance, haplotype, linkage disequilibrium

Introduction

Schizophrenia is a common and devastating mentaldisorder of unknown etiology. Multiple factors includingrisk-conferring genes and undefined environmentalvariables are thought to contribute to overall suscepti-bility.1 One etiological hypothesis is that neurodevel-opmental abnormalities are at least partially involved inthe manifestation of schizophrenia. This assertion issupported by a range of epidemiological, clinical andneurobiological evidence.2

The developing nervous system is dependent on theactions of various secreted factors and membraneproteins that allow neuronal axons to find their correcttargets. The proteins that provide these cues includenetrins, ephrins, semaphorins and slits.3 Classicalnetrins identified as laminin like molecules that directmigration in Caenorhabditis elegans are solublesecreted proteins that provide bifunctional axon guid-ance signals that can mediate either attraction orrepulsion.4 Three classical netrin molecules (1, 2 and3) have been characterized in vertebrates.5 The genefamily is structurally related to the short arms of thelaminin Î chain, comprising a laminin VI domain, threeLE repeats, similar to the laminin V domain and a pos-itively charged heparin-binding carboxyl domain.6

Recently, netrin G1 (also called laminet 1) has beenidentified in the mouse.7 Its predicted domain structureresembles that of the laminin Ç chain and the protein is

Original Article

Case-control association study of human netrin G1 gene in Japanese schizophrenia

Masayuki Fukasawa1,2, Mika Aoki1,2, Kazuo Yamada1, Yoshimi Iwayama-Shigeno1, Hitomi Takao1,Joanne Meerabux1, Tomoko Toyota1,2, Toru Nishikawa2 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 2004; 51: 121–128

Correspondence to: Takeo Yoshikawa, M.D., Ph.D.Laboratory for Molecular PsychiatryRIKEN Brain Science Institute2-1 Hirosawa, Wako-city, Saitama 351-0198, JapanTel: +81(Japan)-48-467-5968Fax: +81(Japan)-48-467-7462E-mail: [email protected] February 6; Accepted March 19, 2004

linked to the plasma membrane by a glycosyl phos-phatidyl-inositol (GPI) lipid anchor, an important featurethat distinguishes them from classical netrins.7,8

Additionally, netrin G1 does not bind to receptors forclassical netrins, nor does it attract circumferentiallygrowing axons from the cerebellar plate in explantextracts.7 No orthologues for netrin G1 gene have beenfound in the C. elegans or D. melanogaster. Theseobservations suggest that netrin G1 may play an as yet,undetermined role in cell architecture that is unique tovertebrates.

Based on the potential relevance of netrin G1 to neu-rodevelopment, we performed a genetic analysis of thisgene in schizophrenia.

Material and Methods

SubjectsSchizophrenic samples were composed of 90

males (mean age, 40.3±8.6 years) and 90 females(mean age, 47.1±13.0 years). All patients were diag-nosed according to the criteria of the Diagnostic andStatistical Manual of Mental Disorders, Fourth Edition(DSM-IV) for schizophrenia, to give a best-estimate life-time diagnosis with consensus from at least two expe-

rienced psychiatrists (Yamada K, Toyota T andYoshikawa T). The interview parameters includedthose described in the Structured Clinical Interview ForDSM-IV Axis I Disorders. All available medicalrecords and family informant reports were also takeninto consideration. Controls comprising 90 males(mean age, 39.3±11.5 years) and 90 females (meanage, 46.9±11.9 years), were recruited from hospitalstaff and company employees documented to be freefrom psychoses. All of our samples were collected fromcentral Japan.

The present study was approved by the EthicsCommittees of RIKEN and Tokyo Medical and DentalUniversity, and all participants provided writteninformed consent.

Determination of genomic organization of netrin G1gene

A mouse netrin G1 cDNA sequence NM_030699 anda human EST (expressed sequence tag) cloneKIAA0976 sequence (NM_014917) were compared tohuman BAC clones forming the contig NT_029860using BLAST, to determine the intron/exon structure ofthe human netrin G1 gene. This led to the identificationof 10 exons, with translation starting within exon 2 (Fig.1). The UCSC April 2003 draft assembly of the human

M. FUKASAWA et al. J Med Dent Sci122

Fig. 1. Genomic structure and locations of polymorphic sites for the human netrin G1 gene. Exons are denoted by boxes, with untranslatedregions in open boxes and translated regions in closed boxes. The sizes of exons (bp) and introns (kb) are also shown. The rs number of eachSNP is the NCBI SNP cluster ID from the dbSNP database (http://www.ncbi.nlm.nih.gov/SNP/).

genome (UCSC Genome Bioinformatics web site,http://genome.ucsc.edu/) included only exons 1 to 5 inits gene prediction program. “A” from the ATG initiationcodon was considered at +1.

Single nucleotide polymorphisms (SNPs) searchand genotyping

We consulted the JSNP database (http://snp.ims.u-tokyo.ac.jp/) and The SNP Consortium Ltd database(http://snp.cshl.org/) to find polymorphisms within thenetrin G1 gene, and identified a total of 16 SNPs (Fig.1). We first genotyped these SNPs using 40 randomlychosen schizophrenic samples, and direct sequencingof PCR products (the SNPs located in exons andnearby introns) or the TaqMan method9 (AppliedBiosystems, Foster City, California, US) (the SNPslocated in deep introns). The primers used for PCRamplification are shown in Table 1. Sequencing of PCRproducts was performed using the BigDye TerminatorCycle Sequencing FS Ready Reaction kit (AppliedBiosystems) and the ABI 3700 Genetic Analyzer(Applied Biosystems). The SNPs that were not poly-

morphic in the 40 samples were excluded from furthergenetic analyses. These included IVS2-66691A＞T(rs4471296, http://www.ncbi.nlm.nih.gov/SNP/), IVS2-48075A＞G (rs4550083), IVS2-6059G＞A (rs4463701),IVS3-9907G＞ T (rs1899775), IVS6+1905G＞ A(rs556157) and 1746+5572C＞A (rs2166017) (Fig. 1).The remaining 10 variants were genotyped in all sam-ples using the TAQman method, that utilizes the 5’-exonuclease activity of the Taq polymerase in combi-nation of PCR and competitive hybridization.9 Probesand primers were designed using the Assays-by-Design File Builder v2.0 software and the PrimerExpress software (Applied Biosystems) (Table 2).PCR reactions were performed using an ABI 9700 ther-mocycler, and fluorescence-based genotyping wasconducted using an ABI 7900 sequence detectionsystem and SDS v2.0 software (Applied Biosystems).The samples with ambiguous genotypes were notused in statistical analyses.

Statistical analysisDeviation from Hardy-Weinberg equilibrium was

123NETRIN G1 AND SCHIZOPHRENIA

Table 1. PCR Primers used to examine nucleotide variants in the NTNG1

examined using the Á2 test. Differences in genotypeand allele frequency were evaluated using Fisher’sexact test. Linkage disequilibrium (LD) statistics werecalculated using COCAPHASE10 (http://www.hgmp.mrc.ac.uk/~fdudbrid/software/). Estimation and com-parison of haplotype frequencies were also madeusing COCAPHASE. Graphical overview of pair-wiseLD strength between markers was made using GOLDsoftware11 (http://www.well.ox.ac.uk/asthma/GOLD/).Power calculations were performed using the PowerCalculator (http://calculators.stat.ucla.edu/powercalc/).

Results

The alignment of cDNA and EST sequences withgenomic sequence revealed that the human netrin G1gene is comprised of 10 exons (Fig. 1) located on chro-mosome 1p13.3 (http://genome.ucsc.edu/). A data-

base search for polymorphisms detected only intronicSNPs within the gene, and we selected 16 roughlyequidistant SNPs. Then we examined the heterozy-gosity of each SNP using 40 unrelated DNAs andexcluded six SNPs for further analyses based on theirlow heterozygosity (frequencies of minor alleles ＜ 1%).The remaining SNPs were designated SNP1-10 (Fig.1), and were genotyped in 180 schizophrenics and 180age- and gender-matched controls.

All genotyped polymorphisms were in Hardy-Weinberg equilibrium in both case and control samples(Table 3). Of the 10 SNPs, SNP8 (IVS8-1476C＞T)(NCBI dbSNP accession No. rs1373336,http://www.ncbi.nlm.nih.gov/SNP/) displayed a mar-ginally significantly different genotypic distributionbetween patients with schizophrenia and control sub-jects (P = 0.057; Table 3). Allelic distribution of SNP8showed a significant deviation in schizophrenics com-pared to controls: the C allele was over-represented in


Table 2. TaqMan primer and probe sequences used to examine nucleotide variants in the NTNG1

schizophrenia (nominal P = 0.020, odds ratio = 1.44,95% confidence interval = 1.06–1.96) (Table 3). AfterBonferroni correction for the multiple testing of 10SNPs, the deviation was no longer significant.

Next, we examined pair-wise linkage disequilibrium(LD) between markers. D’ (normalized D) and r2

(squared correlation coefficient) values were computedin controls. Both LD measures take values between 0(lack of LD) and 1 (complete LD). LD relationshipsbetween SNPs are shown in Table 4 and Fig. 2.SNPs5-9 were in the same LD block using the twomeasures. The polymorphism (SNP8) associated withschizophrenia was located in this LD block. We exam-ined two and three SNP-based haplotypic associationsin a sliding manner, using the 10 polymorphisms thatspanned netrin G1 gene (Fig. 3). The combinations ofSNP7-SNP8 and SNP6-SNP7-SNP8 showed signifi-

cant associations with schizophrenia (global P =0.017 and 0.021, respectively). For two SNP haplo-types, the haplotype G (SNP7)-C (SNP8) was signifi-cantly more frequent in schizophrenia (frequency =0.461) than in control group (0.362) (P = 0.007, oddsratio = 1.51, 95% C.I. = 1.12–2.03). For three SNPhaplotypes, the haplotype T (SNP6)-G (SNP7)-C(SNP8) was significantly more over-represented inschizophrenia (0.458) than in controls (0.364) (P =0.010, odds ratio = 1.46, 95% C.I. = 1.08–1.98). Theresults of these haplotypic associations were consistentwith those of gene LD structure and allelic (genotypic)association of SNP8 with schizophrenia.


Table 3. Genotypic and allelic distributions of the netrin G1 gene polymorphisms

Discussion

The mouse orthologue of netrin G1 gene was firstcloned by Nakashiba et al7 in 2000. They showed thatnetrin G1 transcripts were first detected at embryonicday 12 in mid and hindbrain regions, reaching peak lev-els at perinatal stages in various brain regions that

include the olfactory bulb mitral cells, thalamus anddeep cerebellar nuclei. Expression was primarilyrestricted to the central nervous system, with mostprominent expression in the thalamus.7 The thalamicneurons relay afferents to the cerebral cortex from var-ious sensory systems, and thus the thalamus isdeemed to modulate the motor response to sensory


Table 4. Pairwise marker-to-marker LD statistics of NTNG1

Fig. 2. LD map of the netrin G1 locus. GOLD plot of color-coded pair-wise disequilibrium statistics (r2 in left and D’ in right) is shown.Red and yellow indicate areas of strong LD. For SNP numbers, see Fig. 1.

stimuli or to perform “sensory-motor gating”.12

Interestingly, netrin G1 knockout mice exhibited areduced level of prepulse inhibition (PPI) (Itohara,personal communication). PPI is demonstrated by areduction in response amplitude to a startle stimuluswhen this stimulus is immediately preceded by aweaker prestimulus.13 This sensorimotor phenomenonoccurs across-species and it has been investigated asa model to understand the pathophysiology of schizo-phrenia.14 Schizophrenic patients often display pro-found impairments in PPI, raising the possibility that adeficit in the filtering or “gating” of sensory informationmay explain some of the fundamental symptomsobserved in schizophrenia, including an overflow ofsensory stimulation and disintegration of cognitivefunctions.13 Therefore, netrin G1 is a potentiallyintriguing target for genetic studies in schizophreniafrom both its role in physiological functions relevant toschizophrenia pathology, and its molecular involvementin neuronal circuit development.

Our case-control analysis revealed that the IVS8-1467C＞T (SNP8) polymorphism of netrin G1 gene isnominally significantly associated with schizophrenia,with the IVS8-1467C allele overrepresented in schizo-phrenia (allelic P = 0.020, and ＞ 0.05 after Bonferronicorrection for multiple testing). Power calculationswere performed based on an arbitrary assumption ofrelative risk and frequency of risk allele. When a relativerisk of 2.0 was assumed, the present sample displayed89% power to detect significant association (Ï＜0.05,frequency of risk allele = 0.3, two-sided). With a relativerisk of 1.5, our samples had 45% power to detect sig-nificant association (Ï＜0.05, frequency of risk allele =0.3, two-sided). For LD structure, Abecasis et al15

suggested a D’ value of greater than 0.33 as a useful

measure of LD. Nakajima et al16 proposed r2＞0.1 as acriterion for useful LD. According to their criteria, our LDanalysis revealed that SNP8 was located in an LD blockspanning from SNP5 to SNP9 with a gap betweenSNP5 and the neighboring SNP4. Haplotype analysisconsistently showed that the haplotypes comprisingSNP7-SNP8 and SNP6-SNP7-SNP8 in this LD blockwere associated with schizophrenia. These resultssuggest that the real disease-causing variant(s), ifone exists, may reside in the 3’ half region of the gene.We examined sequence variation in the exons andflanking introns using 40 schizophrenics and theprimers shown in Table 1, but found no novel polymor-phisms. In order to search for candidate functional vari-ant(s), it is necessary to extend polymorphismscreening to unscreened genomic regions.

The mouse genomic structure of netrin G1 gene isvery similar to that of the human ortholog. Equally, wehave detected various human netrin G1 transcripts gen-erated by alternative splicing as seen in the mouse.7 Inhumans, the splicing involves exons 6, 7, 8 and 9 whichcode for an unknown domain and two laminin repeattype domains (Meerabux et al in preparation). Theseexons are within the same haplotype block. Therefore itis tempting to speculate that dysregulation of transcriptprocessing may have some role in schizophrenia sus-ceptibility. However, the SNPs5, 6, 7, 8 and 9 are allembedded in introns and distant from branch points orsplicing donor and acceptor sites, making them lesslikely to control the efficiency of alternative splicing.More thorough genomic and genetic analyses areneeded to corroborate the contribution of netrin G1gene to schizophrenia susceptibility and refine thepredisposing allele(s). The close paralogue, netrin G2gene, also identified from the mouse quite recently, also


Fig. 3. Results of two-marker and three-marker haplotype analyses in schizophrenia. For two (three)-markeranalysis, a sliding window of two (three) markers was tested, with one (two)-marker overlaps. The global P valuesshown, represent the overall significance when the observed versus expected frequencies of all of the haplotypesare considered together. The P values were calculated using the COCAPHASE program. For SNP numbers, seeFig. 1.

warrants future genetic analysis in schizophreniabased in its complimentary pattern of expression withnetrin G1.17

In conclusion, our data suggest the possibleinvolvement of human netrin G1 or a nearby gene in thevulnerability to schizophrenia.

Acknowledgements

We are grateful to all participants in this study.

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