schizophrenia is a severe , particularly devastating , psychiatric disorder affecting approximately 1% of the general population .
even though schizophrenia is highly heritable , the research for chromosomal loci and candidate genes has not provided any consistent results .
combinations of genetic , epigenetic , and environmental factors participate in the development of the disease .
since these factors have not been identified , the diagnosis of schizophrenia is based on phenotypic symptoms only .
therefore , the identification of susceptibility genes is likely to provide valuable insights into the etiology and pathogenesis of the disease , consequently leading to the development of more effective treatments .
catechol - o - methyltransferase ( comt ) is an enzyme , which catalyses the o - methylation of catecholamine neurotransmitters such as dopamine , adrenaline , and noradrenaline .
a  reformulated  hypothesis of the dopamine 's role in the disease states that hyperdopaminergic functioning in subcortical structures is associated with positive symptoms such as hallucinations and delusions , whereas hypodopaminergic functioning in prefrontal cortical regions is associated with negative and cognitive symptoms .
dopamine is inactivated either by reuptake into the neurons that release dopamine into the synapse or through metabolism by monoamine oxidase or comt . in most areas of the brain ,
reuptake inactivation predominates , so that comt does not markedly influence dopamine levels . by contrast , in the prefrontal cortex , that mediates the cognitive functions , which are impaired in schizophrenia , dopamine levels are sensitive to comt levels .   velocardiofacial syndrome ( vcfs )
patients with vcfs display an extremely high incidence of schizophrenia about 25% to 30% , and 22q11 deletion occurs in 2% of diagnosed schizophrenics .
comt gene maps to the vcfs region of chromosome 22 [ 7 , 8 ] .   due to its involvement in the catabolic clearance of dopamine and its location in the 22q11 microdeletion area
the best - studied variant is a functional single - nucleotide polymorphism , which results in a valine to methionine mutation at position 158 ( commonly referred to as val158met or rs4680 ) .
the val variant has higher enzymatic comt activity and thermostability than its methionine counterpart , leading to more efficient degradation of dopamine .
therefore , the val variant is related to poor performance on certain tests of working memory and to insufficient brain activation [ 11 , 12 ] .  
there is weak and inconsistent evidence that the val variant may be associated with increased risk of schizophrenia .
most case control studies and meta - analysis [ 14 , 15 ] do not support association , whereas studies using a family design actually do . other variants , which have been studied for association with schizophrenia , are rs737865 and rs165599 .
the present study examines evidence for association of the three snps ( rs737865 , rs4680 , and rs165599 ) and their haplotypes with schizophrenia in a greek population .
  the study sample consisted of 108 cases with verified schizophrenia and 97 healthy individuals .
blood samples of schizophrenia patients were collected from hospitalized patients in the 10th clinical department of the attica 's psychiatric hospital  dafni ,  whereas control blood samples were obtained from healthy individuals , who had free anamnesis of schizophrenia and volunteered to participate in this study .  
table 1 shows the distribution of cases and controls by sociodemographic characteristics . gender distribution and median age were similar in the two groups of participants .
the inclusion criteria specified that all patients fulfilled the diagnostic criteria of schizophrenia according to the diagnostic and statistical manual of mental disorder ( dsm - iv ) .
all participants were of greek ethnic origin and had signed the informed - consent form .
the study was approved by hospital 's ethic committee . according to the exclusion criteria , subjects who denied providing informed consent or those
whose psychosis was judged secondary to substance use or a known neurological disorder , such as epilepsy , based on the consensus diagnostic procedure , were not eligible to participate .
the three snps ( rs737865 , rs4680 , and rs165599 ) were genotyped using pcr real - time analysis on the lightcycler 480 ( roche ) .
lightmix kit ( tib - molbiol ) , which contained specific primers and probes for 96 pcr - rt chain reactions , and lightcycler genotyping 480 dna master of roche diagnostics , which contains tag dna polymerase , reaction buffer , mixture of dntp ( with datp , dctp , dgtp , and dutp ) and 15  mm mgcl2 were used .
briefly 20  l of reaction master mix for comt gene was performed with h2o 10.4  l , reagent mix 1.0  l , lightcycler genotyping 480 dna master 2.0  l , mgcl2 1.6  l , dna 5.0  l ( ~50  ng ) .
thermal cycling conditions for pcr were as follows : denaturation at 95c for 10 minutes , 1 cycle , followed by 45 cycles of 95c for 10  sec , 60c for 10  sec and 72c for 15  sec , followed by melting at 95c for 30  sec , 40c for 2  min and 75c for 1  sec , 1 cycle and cooling at 40c for 30  sec , 1 cycle .
deviations from hardy - weinberg equilibrium ( hwe ) were assessed using the chi - square test at each snp locus separately in cases and controls .
to assess the differences in demographic characteristics , we used chi - square for categorical data and t test for continuous data .
statistical inference for single , univariate snp associations in the comt gene was derived from fisher 's exact test .
the odds ratios for dominant , recessive , and additive genetic models for each individual snp were calculated .
the relevant p values derived from wald test for dominant and recessive models and armitage test for trend for additive model .
subsequently , all possible combinations of the three snps were constructed , and thus , differences in haplotype frequencies between schizophrenia patients and control individuals were calculated using chi - square test .
a logistic regression analysis was performed to estimate the risk for schizophrenia of each one of the eight haplotypes .
snp rs737865 carries the alleles t and c and the possible genotypes of that polymorphism are t / t , t / c , or c / c .
snp rs4680 carries the alleles a and g , and its genotypes are a / a , g / a , or g / g . a and g are the two alleles of snp rs165599 , and the possible genotypes are a / a , g / a , or g / g ( table 2 ) . to further explore the combination of genotype is from which arise the t / a / a and t / g / g haplotypes , multivariate logistic regression analysis was performed estimating the risk of schizophrenia in each combination .
the sas statistical package ( version 9.1 , sas institute inc , cary , nc ) was used to analyze the data .
polymorphism rs4680 was in accordance with hardy - weinberg equilibrium ( hwe ) , whereas borderline evidence for deviation from hwe was identified in polymorphism rs165599 among controls ( x = 5.11 ; p = 0.03 ) and in rs737865 among cases ( x = 4.15 ; p = 0.04 ) .
all the three genetic models , recessive , dominant , and additive , in the three individual snps failed to investigate any evidence for a possible genetic contribution of the snps to schizophrenia susceptibility ( table 3 ) .
further , a haplotype analysis was performed by constructing all the three - site haplotypes that consisted of the alleles of snps rs4680 , rs165599 , and rs737865 ( table 4 ) .
the most frequent haplotypes were t / a / a and t / g / g .
significant differences in the frequencies were found between patients and controls regarding t / a / a and t / g / g .
specifically , cases had higher frequency of t / a / a ( p = 0.010 ) and lower frequency of t / g / g ( p = 0.088 ) as compared to the controls .
when a logistic regression analysis was performed , with schizophrenia as outcome , wild type as the baseline category , and the eight haplotypes as explanatory variables , a significant adverse effect of t / a / a was revealed .
therefore , compared to the baseline expression of t / g / g , participants with t / a / a were found to be associated with 52% higher risk of schizophrenia ( or = 1.52 ; 95% ci : 1.122.08 ; p = 0.008 ) , whereas there was no evidence for association with the psychiatric illness regarding all the other haplotypes . to evaluate further the supporting evidence for the increased risk of t / a / a and for protective effect of t / g / g
, a more detailed analysis was performed examining all the possible combinations of genotypes from which the t / a / a and t / g / g haplotypes were derived ( tables 5 and 6 ) .
such analysis showed that after taking the most frequent expression , t / t - a / a - a / a , as baseline , participants with t / c - a / a - a / a were at increased risk for developing the disease ( or = 2.13 ; 95% ci : 1.024.47 ; p = 0.045 ) .
similarly , participants with t / t - a / a - g / a were more likely to develop the disease ( or = 3.20 ; 95% ci : 1.0210.05 ; p = 0.046 ) as compared to those with t / t - a / a - a / a .
regarding t / g / g , we found a protective effect of t / t - g / g - g / g ( or = 0.22 ; 95% ci : 0.090.56 ; p = 0.001 ) and t / t - g / a - g / g ( or = 0.33 ; 95% ci : 0.120.87 ; p = 0.025 ) both compared to the baseline , most frequent , category t / t - g / a - g / a . in this multivariate model , two combinations , namely , t / t - g / g - g / a and t / c - g / a - g / g , were excluded given their extremely low frequencies .
previously , shifman et al .   reported a large study of comt in schizophrenia , including over 700 patients and 4000 controls .
the researchers used israeli ashkenazi jewish population , a well - characterized homogeneous population , in order to reduce genetic variance , and they tested the same snps examined in our study , firstly each snp independently and further the combination of the two snps haplotype as well as the three snps haplotype .
the study showed that in that specific population the association of the val / met polymorphism ( rs4680 ) with schizophrenia was modest ( p = 0.024 ) , whereas the two other polymorphisms rs737865 and rs165599 ( located in intron 1 and 3 flanking region , resp . ) were highly significantly associated , as was the haplotype of all three markers g - g - g of rs737865 , rs4680 , and rs165599 ( p = 9.6  10 ) .
two independent studies , namely the study of chen et al .   and the study of handoko et el .   have reported a significant association of comt with schizophrenia . but both of them reported different alleles of the three snps consisting the risk haplotype , than the alleles described by shifman et al . .
specifically , chen et al .   studied the association of comt with schizophrenia in irish families with high density of schizophrenia .
they concluded that val allele might carry a small increase in disease susceptibility , but they did not confirm any association of rs737865 and rs165599 . in our study none of the three snps were found to be independently associated with schizophrenia .
's hapolype analyses showed that a - g - a haplotype for snps rs737865 , rs4680 , and rs165599 was preferentially transmitted to the affected subjects .
this finding was different from the reported g - g - g haplotype found in ashkenazi jews and different from the risk haplotype of our study .
studied the above snps in a sample of australian caucasian families , containing 107 patients with schizophrenia .
the haplotype analyses showed that a - a - a combination for snps rs737865 , rs4680 , and rs165599 was the risk haplotype for the studied population .
that haplotype was in accordance with the risk haplotype found in our study in greek population .
analytically , in the present study the allele frequency of comt val / met polymorphism was typical to the european population , since in europe the two alleles are very similar in frequency .
furthermore , a three - marker haplotype analysis was performed , consisting of combinations of the putative functional snps . in this study , cases have higher frequency of the rs737865/rs4680/rs165599 t - a - a haplotype and lower frequency of t - g - g haplotype .
the c allele at rs737865 corresponds to the g allele in the study of shifman et al . , and
the t allele , which refers to the a allele , is the wild - type allele of rs737865 polymorphism .
the t - g - g haplotype , which consists of the three wild types alleles of polymorphisms rs737865 , rs4680 , and rs165599 , is present at reasonably high frequencies in controls .
furthermore , this study shows that compared to the baseline expression t - g - g , participants with t - a - a haplotype were found to be associated with 52% higher risk of schizophrenia ( table 4 ) . conclusively , among studies of european subjects , which examined the same snps described in the present study , different populations have been reported to exhibit an association between schizophrenia and the different triple snp haplotypes .
as already mentioned , examples of this include the g - g - g haplotype in ashkenazi jews , the a - g - a haplotype in irish families , the a - g - g haplotype which was significantly under transmitted in australian family - affected samples , and the protective haplotype a - a - a in a case - control study of caucasian subjects .
the high - risk haplotype in the ashkenazi sample was actually under transmitted to affected individuals in our sample .
is the first and largest population - based association study to date to find a significant association between comt haplotype , constructed by the three snps , and schizophrenia
. the ashkenazi jewish population may not be directly comparable with other european populations since this haplotype may possibly contribute risk to schizophrenia in a different pathway across multiple ethnic populations .
furthermore , the phenotypic complexity of the disorder makes it difficult to achieve a great clinical homogeneity in the sample of patients .
the diagnosis of the disease is based upon clinical phenomena , which may not correctly reflect the underlying biology that predisposes to illness and the absence of biological markers limits the homogeneity of diagnosis .
one approach that has been suggested is to subdivide schizophrenia into components of symptom complexes and to develop targets of narrower clinical features .
furthermore , all possible genotype combinations of the risk haplotype t - a - a were examined . patients with schizophrenia displayed an excess of the tc - aa - aa genotype homozygous for the two mutant alleles and the tt - aa - ga genotype , homozygous for the mutant rs4680 allele and heterozygous for rs165599 .
similarly , a protective effect of genotypes tt - gg - gg and tt - ga - gg was documented .
in addition , bray et al . , showed that shifman 's risk haplotype   g - g - g ( c - g - g ) is associated with low comt mrna expression in prefrontal cortex .
it is possible that the opposite haplotype a - a - a ( t - a - a ) , which is the risk haplotype in this study confers high comt mrna expression .
this speculation is compatible with the reformulated theory of hypofrontality in schizophrenia [ 5 , 11 ] .
the current study shows an association of comt gene and schizophrenia in a greek population .
the haplotype analysis indicates that the t - a - a haplotype had high association with schizophrenia .
 schizophrenia , a severe psychiatric condition , is characterized by disturbances of cognition , emotion , and social functioning . 
 the disease affects almost 1% of world population . 
 recent studies evaluating the role of catechol - o - methyltransferase enzyme ( comt ) polymorphisms in the pathogenesis of schizophrenia have resulted in ambiguous findings . 
 the current study examined the association of schizophrenia with three comt polymorphisms , namely , rs737865 , rs4680 , and rs165599 in a greek population . 
 there was no significant association between schizophrenia and any of the three snps examined . 
 however , haplotype analysis showed that cases have higher frequency of the t - a - a haplotype , and participants with that haplotype were at increased risk for developing schizophrenia ( or = 1.52 ; cl  :  1.122.08 ; p = 0.008 ) . 
 furthermore , patients with schizophrenia displayed an excess of tc / aa / aa and the tt / aa / ga genotypes . similarly a protective effect of tt / gg / gg and tt / ga / gg was suggested by our results . 
1. Background
2. Subjects and Methods 
3. Results and Discussion
4. Discussion
5. Conclusion 