neuregulin 1 ( nrg1 ) is a ligand for the neu / erbb2 protooncogene and closely related to cell - cell signal interactions required for the growth and development of multiple organ systems .
nrg1 exerts its effect on the epithelium , cardiovascular system , and central nervous system ( cns ) . in early embryogenesis
, nrg1 is expressed on neural tissue , respiratory epithelium , and endocardium , and in later stage predominantly expressed in neural tissue .
genetic factors which promote neuronal development and modulate synaptic plasticity may influence the development and symptoms of schizophrenia .
nrg1 plays a role in antipsychotic treatment of schizophrenia   and may affect dopamine receptors ( d2 and d3 ) .
nrg1 is involved in the abnormal gamma - aminobutyric acid ( gaba ) neurotransmission in schizophrenia , together with erbb4 , which is synaptic receptor of nrg1 and regulates synaptic maturation .
furthermore , n - methyl - d - aspartate ( nmda ) receptor functional change is related to schizophrenia .
nrg1 polymorphisms were reported to be associated with susceptibility to schizophrenia in iceland population , and replication studies of scottish   and chinese   populations . in another scottish population study ,
, we investigated the relationship between nrg1 polymorphisms and the development of schizophrenia in korean population .
a total of 221 schizophrenia patients and 359 control subjects ( 44.26.3 years ) were recruited .
the schizophrenia group consisted of 122 males and 99 females , and the control group was comprised of 180 males and 179 females .
schizophrenia patients were selected among participants who visited at the departments of neuropsychiatry in the east - west neomedical center and kyung hee medical center , seoul , korea .
patients were diagnosed with schizophrenia by two psychiatrists according to the diagnostic and statistical manual of mental disorders ( dsm - iv ) .
subjects with other psychiatric disorders , neurological diseases , and any severe diseases were excluded .
controls subjects were recruited among participants who assessed as mentally healthy through a general health examination program .
this study was conducted in accordance with the guidelines of the helsinki declaration and approved by the ethics review committee of medical research institute , kyung hee university medical center , seoul , korea .
we searched the promoter and coding regions of the nrg1 gene in the snp database of the national center for biotechnology information ( http://www.ncbi.nlm.nih.gov/snp , build 135 ) , and selected two promoter snps ( rs7014762 , -1174 a / t and rs11998176 , -788 a / t ) and one missense snp ( rs3924999 , arg253gln ) among the nrg1 snps . peripheral blood sample of each subject was collected in heparin or edta tubes .
dna isolation kit for cells and tissues ( roche , indianapolis , in , usa ) was used for extracting genomic dna .
polymerase chain reactions ( pcrs ) were performed as the following condition : 35 cycles at 94 for 30 sec , 58 for 30 sec , 72 for 30 sec , and 1 cycle at 72 for 5 min for the final reaction .
the primer sequences for pcrs were as following : rs7014762 ( sense , tgccaacttgcagaatcttggg ; anti - sense , aatgggcgatagatccacactg ) , rs11998176 ( sense , cagtgtggatctatcgcccatt ; anti - sense , aacgctctctctccttgcagcg ) , and rs3924999 ( sense , gatccattttcgctcatccattt ; anti - sense , cccaaagagctgggattacagtt ) ( table 1 ) .
the pcr products were processed through direct sequencing ( macrogen , seoul , korea ) , and genotypes of each snp were analyzed with seqmanii software ( dnastar , madison , wi , usa ) .
we applied multiple logistic regression models in analysis of genotype data : codominant1 ( major allele homozygotes vs. heterozygotes ) , codominant2 ( major allele homozygotes vs. minor allele homozygotes ) , dominant ( major allele homozygotes vs. heterozygotes+minor allele homozygotes ) , recessive ( major allele homozygotes+heterozygotes vs. minor allele homozygotes ) , and log - additive ( major allele homozygotes vs. heterozygotes vs. minor allele homozygotes ) models .
odd ratios ( or ) , 95% confidence intervals ( ci ) , and p - values were calculated with snpstats ( http://bioinfo.iconcologia.net/index.php?module=snpstats ) and spss 18.0 ( spss inc . ,
haploview version 4.2 ( daly lab , cambridge , ma , usa ) was used in determining linkage disequilibrium ( ld ) block and haplotypes .
a total of 221 schizophrenia patients and 359 control subjects ( 44.26.3 years ) were recruited .
the schizophrenia group consisted of 122 males and 99 females , and the control group was comprised of 180 males and 179 females .
schizophrenia patients were selected among participants who visited at the departments of neuropsychiatry in the east - west neomedical center and kyung hee medical center , seoul , korea .
patients were diagnosed with schizophrenia by two psychiatrists according to the diagnostic and statistical manual of mental disorders ( dsm - iv ) .
subjects with other psychiatric disorders , neurological diseases , and any severe diseases were excluded .
controls subjects were recruited among participants who assessed as mentally healthy through a general health examination program .
this study was conducted in accordance with the guidelines of the helsinki declaration and approved by the ethics review committee of medical research institute , kyung hee university medical center , seoul , korea .
we searched the promoter and coding regions of the nrg1 gene in the snp database of the national center for biotechnology information ( http://www.ncbi.nlm.nih.gov/snp , build 135 ) , and selected two promoter snps ( rs7014762 , -1174 a / t and rs11998176 , -788 a / t ) and one missense snp ( rs3924999 , arg253gln ) among the nrg1 snps . peripheral blood sample of each subject was collected in heparin or edta tubes .
dna isolation kit for cells and tissues ( roche , indianapolis , in , usa ) was used for extracting genomic dna .
polymerase chain reactions ( pcrs ) were performed as the following condition : 35 cycles at 94 for 30 sec , 58 for 30 sec , 72 for 30 sec , and 1 cycle at 72 for 5 min for the final reaction .
the primer sequences for pcrs were as following : rs7014762 ( sense , tgccaacttgcagaatcttggg ; anti - sense , aatgggcgatagatccacactg ) , rs11998176 ( sense , cagtgtggatctatcgcccatt ; anti - sense , aacgctctctctccttgcagcg ) , and rs3924999 ( sense , gatccattttcgctcatccattt ; anti - sense , cccaaagagctgggattacagtt ) ( table 1 ) .
the pcr products were processed through direct sequencing ( macrogen , seoul , korea ) , and genotypes of each snp were analyzed with seqmanii software ( dnastar , madison , wi , usa ) .
we applied multiple logistic regression models in analysis of genotype data : codominant1 ( major allele homozygotes vs. heterozygotes ) , codominant2 ( major allele homozygotes vs. minor allele homozygotes ) , dominant ( major allele homozygotes vs. heterozygotes+minor allele homozygotes ) , recessive ( major allele homozygotes+heterozygotes vs. minor allele homozygotes ) , and log - additive ( major allele homozygotes vs. heterozygotes vs. minor allele homozygotes ) models . odd ratios ( or ) , 95% confidence intervals ( ci ) , and p - values were calculated with snpstats ( http://bioinfo.iconcologia.net/index.php?module=snpstats ) and spss 18.0 ( spss inc . ,
haploview version 4.2 ( daly lab , cambridge , ma , usa ) was used in determining linkage disequilibrium ( ld ) block and haplotypes .
the age of the schizophrenia group was 42.010.6 ( mean agestandard deviation ) years and that of the control group was 44.26.3 years ( data not shown ) .
table 2 shows the genotype and allele frequencies of the three examined snps ( rs7014762 , -1174 a / t ; rs11998176 , -788 a / t ; rs3924999 , arg253gln ) of nrg1 in the control group and the schizophrenia group .
the genotype distributions of the three tested snps were consistent with the hardy - weinberg equilibrium ( hwe ) in the control group ( rs7014762 , hwe p=0.91 ; rs11998176 , hwe p=0.71 ; rs3924999 , hwe p=0.78 , data not shown ) .
the genotype ( t / t : a / t : a / a genotypes ) distributions of rs7014762 in the control and schizophrenia groups were 43.9%:44.5%:11.6% and 44.1%:45.5%:10.4% , respectively .
the allele ( t : a alleles ) distributions of rs7014762 in the control and schizophrenia groups were 66.1%:33.9% and 66.8%:33.2% .
the genotype ( a / a : a / t : t / t ) distributions of rs11998176 in the control and schizophrenia groups were 86.1%:13.4%:0.5% and 82.8%:16.7%:0.5% , respectively .
the allele ( a : t ) distributions of rs11998176 in the control and schizophrenia groups were 92.8%:7.2% and 91.2%:8.8% , respectively .
the genotype and allele distributions of two snps ( rs7014762 and rs11998176 ) were not significant between controls and schizophrenia patients ( p>0.05 , table 2 ) .
however , the genotype ( a / a : a / g : g / g ) distributions of rs3924999 in the control and schizophrenia groups were 55.2%:38.7%:6.1% and 64.7%:32.1%:3.2% , respectively .
the differences were significant association ( or=0.67 , 95% ci=0.47 - 0.95 , p=0.022 in the dominant model and or=0.69 , 95% ci=0.51 - 0.93 , p=0.013 in the log - additive model ) .
the allele ( a : g ) distributions of rs3924999 in the control and schizophrenia groups were 74.5%:25.5% and 80.8%:19.2% .
the difference of allele distributions showed significant association ( or=0.70 , 95% ci=0.52 - 0.93 , p=0.014 ) ( table 2 ) .
ld block was determined using haploview 4.2 , and one ld block between rs7014762 and rs11998176 was constructed ( fig .
1 ) . in order to investigate association between the haplotypes of ld block snps and schizophrenia ,
there were three haplotypes ( ta , 0.585 ; aa , 0.336 ; tt , 0.078 in frequency ) .
the haplotypes showed no significant difference between the control group and the schizophrenia group ( table 3 ) .
nrg1 is well - known schizophrenia - related gene , and there have been association studies of nrg1 snps with schizophrenia susceptibility from a decade ago [ 10 - 13 ] .
a genome - wide association study ( gwas ) used 33 families consisted by 110 patients in iceland was performed in the year 2002 and the researchers identified nrg1 as a schizophrenia candidate gene .
they concluded that the ' at - risk ' haplotypes at the 5 ' end of the nrg1 gene is responsible for the analysis result .
replication study was conducted in scottish population , and they found that the ' at - risk ' haplotype and rs35753505 snp were still significant .
however , in another replication study conducted in han chinese populations , they did not show significance , instead , they found a new haplotype ' hap china 3 ' consisted by three groups of microsatellite markers . in another scottish population study , rs6988339 and rs3757930 snps , which were in near the 3 ' region of the nrg1 gene and
not previously studied in iceland population , were associated with bipolar disorder as well as schizophrenia .
such differences suggest that various snps of nrg1 may contribute or not to the schizophrenia development in certain populations . in our study results ,
two promoter snps ( rs7014762 , -1174 a / t and rs11998176 , -788 a / t ) selected from the nrg1 gene were not statistically associated with risk of schizophrenia , and one missense snp ( rs3924999 , arg253gln ) was associated with the development of schizophrenia .
the rs7014762 snp was once reported to be associated with bipolar disorder , however , not with schizophrenia . and
there was no prior study of rs11998176 ( promoter , -788 a / t ) .
however , the study result showed no association between rs11998176 and schizophrenia development in korean population .
the rs3924999 snp has been frequently reported in schizophrenia - related researches . in a nrg1 genetic study in chinese population
another chinese population study showed that rs3924999 was associated with schizophrenia risk , especially in the subgroup of patients with positive symptom . in the other study of chinese population ,
rs3924999 was associated with the prepulse inhibition ( ppi ) , which is a measurable schizophrenia phenotypic feature . on the contrary ,
association of rs3924999 with auditory p300 in schizophrenia was reported . in our study result ,
the g allele of rs3924999 , which is minor allele , showed less odds ratio in the schizophrenia group than that of the control group in the dominant model ( or=0.67 , 95% ci=0.47 - 0.95 , p=0.022 ) and the log - additive model ( or=0.69 , 95% ci=0.51 - 0.93 , p=0.013 ) .
allelic distribution of rs3924999 was significantly different between schizophrenia and controls ( or=0.70 , 95% ci=0.52 - 0.93 , p=0.014 ) .
these results were slight association , however , it might be suggested from the result that the variant allele g may have a dominant effect and more protective effect from schizophrenia than the majority allele a of rs3924999 , or the a allele of rs3924999 might be an allele with increased risk of schizophrenia development .
the g allele of rs3924999 changes original 253th amino acid arginine to variant amino acid glutamine , and its change might have modified the cell signaling of nrg1 . in conclusion , this study results showed that a missense snp rs3924999 ( arg253gln ) of nrg1 was weakly associated with the development of schizophrenia in korean population .
two other snps of nrg1 ( rs7014762 and rs11998176 ) did not show significant associations .
the results suggest that rs3924999 of the nrg1 gene may contribute to schizophrenia development in korean population .
 neuregulin 1 ( nrg1 ) is associated with the pathogenesis of schizophrenia through controlling activation and signaling of neurotransmitter receptors . 
 influence to schizophrenia development by the nrg1 gene may differ in individuals , and genetic polymorphism is one of the factors affecting their differences . 
 association between three single nucleotide polymorphisms ( snps ) ( rs7014762 , -1174 a / t ; rs11998176 , -788 a / t ; rs3924999 , arg253gln ) of nrg1 and the development of schizophrenia was analyzed in 221 schizophrneia and 359 control subjects . 
 polymerase chain reaction and direct sequencing were performed to obtain genotype data of nrg1 snps of the subjects . in analysis of genetic data , multiple logistic regression models ( codominant1 , codominant2 , dominant , recessive , and log - additive model ) were applied . 
 snpstats and spss 18.0 were used to calculate odds ratio ( or ) , 95% confidence interval ( ci ) , and p - value of each model . 
 the genotype distributions of rs3924999 were associated with schizophrenia development ( or=0.67 , 95% ci=0.47 - 0.95 , p=0.022 in the dominant model and or=0.69 , 95% ci=0.51 - 0.93 , p=0.013 in the log - addtive model ) and allelic distributions also showed significant association ( or=0.70 , 95% ci=0.52 - 0.93 , p=0.014 ) . 
 the results suggest that rs3924999 of the nrg1 gene may be associated with schizophrenia susceptibility . 
INTRODUCTION
MATERIALS AND METHODS
Schizophrenia and control subjects
SNP selection and genotyping
Statistical analysis
RESULTS
DISCUSSION