Gene copy number variation in schizophrenia.

Recent reports have highlighted the possibility that gene copy number variations play a role in the development of complex disorders and have suggested that some variations are very common in schizophrenic patients. We have carried out a comparative genomic hybridization screen using oligonucleotide probes of 891 candidate genes to look for very common copy number variance in schizophrenic patients. In addition we have developed a new approach for the detection and validation of putative copy number variation based upon established methods of allele quantification by DNA pooling and have used it to study 15 major candidates including dysbindin (DTNBP1), neuregulin (NRG1), RGS4 and DISC1. With the exception of positive control sequences, no copy number variations were found for any of the genes in any samples by the use of either technique. Our data for the genes studied are in line with the known existence and frequency of CNVs as reported by recent large scale studies and suggest that gene copy number variations are not more common in schizophrenics than controls, although large ethnic differences cannot be excluded.

The importance and identification of regulatory polymorphisms and their mechanisms of action.

The search for the genetic variations underlying all human phenotypes is in its infancy but must be one of the long term goals of the scientific community. There is evidence that most, if not all human phenotypes, including illnesses are influenced by the genetic makeup of the individual. There are an estimated 11 million human genetic polymorphisms with a minor allele frequency >1% and possibly many times that number of rare sequence variants. The proportion of these sequence variants which have any functional effect is unknown but it is likely that the majority of those which influence illness lie outside of the amino acid coding regions of genes, and affect the regulation of gene expression--these are called rSNPs. Recent research suggests that about 50% of genes have one or more common rSNPs associated with them and probably most if not all genes have an rSNP within the human population. In the long term, determining which polymorphisms are potentially functional must be done bio-informatically using algorithms based upon experimental data. However, at the current time, the limited data that has been obtained does not allow the creation of such an algorithm. In vitro studies suggest that a large proportion of rSNPs lie within the core and proximal promoter regions of genes but it is not clear how the majority of these influence transcription, as they do not appear to be within any known transcription factor binding sites. However, promoter regions possess a number of sequence-dependent characteristics which make them distinct from the rest of the genome, namely stability, curvature and flexibility. Subtle changes to these features may underlie the mechanisms by which many polymorphisms exert their function.

Gene copy number variation in schizophrenia.

The possibility that gene copy number variations play a role in the development of complex disorders is a topic of considerable interest. Recent reports have highlighted the large number of such variations that exist and that their occurrence varies considerably between populations. A recent report has suggested that copy number variations in four genes (GRIK3, EFNA5, AKAP5 and CACNG2) may be associated with schizophrenia. One problem with this area of study is the validation of high throughput methods such as comparative genomic hybridisation, as the latter inevitably generates false positives. We have used two contrasting methodologies to determine the validity of the findings reported above which if true would have major implications for the pathogenesis of schizophrenia. Samples from a UK population were tested using a method of allele quantification by DNA pooling and samples from Belgium and northern Sweden were tested using Multiplex Amplicon Quantification (MAQ). Both methods were used to test DNA samples used in the original investigation. No copy number variations were found for any of the genes in any samples. Our data suggests that more reliable methods need to be used to validate the existence of CNVs before full scale association studies are carried out.

Endosomal location of dopamine receptors in neuronal cell cytoplasm.

Five subtypes of dopamine receptor exist in two subfamilies: two D(1)-like (D(1) and D(5)) and three D(2)-like (D(2), D(3) and D(4)). We produced novel monoclonal antibodies against all three D(2)-like receptors and used them to localize receptors in Ntera-2 (NT-2) cells, the human neuronal precursor cell line. Most of the immunostaining for all three receptors colocalized with mannose-6-phosphate receptor, a marker for late endosomes formed by internalization of the plasma membrane. This result was obtained with antibodies against three different epitopes on the D(3) receptor, to rule out the possibility of cross-reaction with another protein, and controls without primary antibody or in the presence of competitor antigen were completely negative. In rat cerebral cortex and hippocampus, some of the dopamine receptor staining was found in similar structures in neuronal cell cytoplasm. Only some of the neurons were positive for dopamine receptors and the pattern was consistent with previously-reported patterns of innervation by dopamine-producing neurons. Endosomal dopamine receptors may provide a useful method for identifying cell bodies of dopamine-responsive neurons to complement methods that detect only active receptors in the neuronal cell membrane.

Strong bias in the location of functional promoter polymorphisms.

A considerable proportion of heritable human phenotypic variation is thought to result from altered gene expression. Unfortunately, it is currently impossible to use bioinformatic analysis to discriminate between DNA sequence variants that are likely to influence gene expression and those that are not. In an attempt to define some of the characteristics of promoter polymorphisms with functional effects on gene expression, we examined 674 haplotypes representing 247 unique gene promoters using a standardized reporter gene assay system. Sequence variants that altered gene expression by 1.5-fold or more were strongly biased toward a location in the core and proximal promoter regions, 50% being within the first 100 bases 5' to the transcription start site. No bias was seen in the allele frequencies of functional and nonfunctional sequence variants. Only 33% of the functional variants were found in known consensus transcription factor binding sequences or motifs, which suggests that either there are many unknown transcription factor binding motifs or other, unknown mechanisms are involved. The genes with functional polymorphisms that are reported here for the first time include AGTRL2, CAT, CHRNA5, CTSG, CYP2D6, DLD, ERCC1, GABRA1, GABRP, HNRPH3, HIP1, IGKV1-9, KCNJ15, KCNK6, KLK1, MSMB, MYOC, NPY2R, NOTCH4, ORM2, PEDF, PTPRCAP, ST16 (IL24), SULT1A1, and TSHR.

A high proportion of polymorphisms in the promoters of brain expressed genes influences transcriptional activity.

There is increasing interest in the possibility that polymorphisms affecting gene expression are responsible for a significant proportion of heritable human phenotypic variation, including human disease. We have sought to determine if polymorphisms in the promoters of brain expressed genes are commonly functional. We screened for polymorphism 56 genes previously reported to be differentially expressed in the brains of schizophrenics [Y. Hakak, J.R. Walker, C. Li, W.H. Wong, K.L. Davis, J.D. Buxbaum, V. Haroutunian, A.A. Fienberg, Genome-wide expression analysis reveals dysregulation of myelination-related genes in chronic schizophrenia. Proc. Natl. Acad. Sci. 98 (2001) 4746-4751.]. We found 60 variants distributed across 31 of the genes. A total of 77 haplotypes representing 28 different putative promoters were analyzed in a reporter gene assay in two cell lines. Of a total of 54 sequence variants represented in the haplotypes, 12 (or around 22%) were functional according to a highly conservative definition. These were found in the promoters of eight genes: NPY, PCSK1, NEFL, KIAA0513, LMO4, HSPA1B, TF and MDH1. We therefore estimate that around 20-25% of promoter polymorphisms in brain expressed genes are functional, and this is likely to be an underestimate. Our data therefore provide for the first time empirical evidence that promoter element polymorphisms, at least in brain expressed genes, should be afforded a high priority for molecular genetic studies.

Low gene expression conferred by association of an allele of the 5-HT2C receptor gene with antipsychotic-induced weight gain.

OBJECTIVE: Association has been reported between the C allele of a -759C/T polymorphism in the promoter of the 5-HT2C receptor gene (HTR2C) and antipsychotic-induced weight gain, suggesting that polymorphic HTR2C expression influences this phenotype. The authors tested this polymorphism, and other promoter variants, for effects on HTR2C transcription. METHOD: Six HTR2C promoter haplotypes constructed from four polymorphisms were cloned into a luciferase reporter gene plasmid. Their transcriptional activities were then compared in two human cell lines. RESULTS: All haplotypes containing the -759C allele showed less transcriptional activity than haplotypes containing the -759T allele. The A allele of a -997G/A polymorphism was also associated with reduced expression. CONCLUSIONS: These findings suggest that the -759C allele is functional and results in relative underexpression of HTR2C. Reduced expression of HTR2C mRNA may underlie vulnerability to weight gain following antipsychotic treatment.

Functional analysis of polymorphisms in the promoter regions of genes on 22q11.

Segmental aneusomy, which includes chromosome 22 deletion syndrome (del(22)(q11.2q11.2)), has been associated with DiGeorge syndrome (DGS), velocardiofacial syndrome (VCFS), conotruncal anomaly face (CAF) syndrome, cat-eye syndrome (CES), der(22) syndrome, and duplication of the del(22)(q11.2q11.2) syndrome's typically deleted region. Adults with del(22)(q11.2q11.2) may develop psychiatric illnesses, including schizophrenia, schizoaffective disorder, and bipolar disorder, suggesting that lower gene dosage leads to a predisposition to these illnesses. In a bid to identify important regulatory polymorphisms (SNPs) that may emulate changes in gene dosage of the genes within the common deletion, we have analyzed the promoter region of 47 genes (44 of which encode a protein with known function) encoding proteins in and around 22q11 for sequence variants. A total of 33 of the promoters contained polymorphisms. Of those, 25 were cloned into a reporter gene vector, pGL3. The relative ability of each promoter haplotype to promote transcription of the luciferase gene was tested in each of two human cell lines (HEK293t and TE671), using a cotransfected CMV-SPAP plasmid as an internal control. Five genes (PRODH, DGCR14, GSTT2, SERPIND1, and a gene tentatively called DKFZP434P211) showed activity differences between haplotypes of greater than 1.5-fold. Of those, PRODH, which encodes proline dehydrogenase, has previously been highlighted in relation to schizophrenia, and the functional promoter polymorphism reported here may be involved in pathogenic mechanisms.

The human hyaluronan synthase genes: genomic structures, proximal promoters and polymorphic microsatellite markers.

The glycosaminoglycan (GAG) hyaluronan (HA) is a key component of the vertebrate extracellular matrix (ECM) and is synthesised by the HA synthase (HAS) enzymes HAS1, HAS2 and HAS3 at the plasma membrane. Accumulating evidence emphasises the relevance of HA metabolism in an increasing number of processes of clinical interest including renal fibrosis and peritoneal mesothelial wound healing. In the present study, the genomic sequences and organisation of the genes encoding the human HAS isoforms were deduced, in silico, from reference cDNA and genomic sequence data. These data were confirmed in vitro by sequencing of PCR-amplified HAS exons and flanking genomic sequences, comparison with sequence data for the corresponding murine Has orthologues, rapid amplification of 5' cDNA ends analysis and luciferase reporter assays on putative proximal promoter sequences. The HAS1 gene comprised five exons, with the translation start site situated 9bp from the 3' end of exon 1. In contrast, the genomic structures for HAS2 and both HAS3 variants spanned four exons, exon 1 forming a discrete 5'-untranslated region (5'-UTR) and the translation start site lying at nucleotide 1 of exon 2. Dinucleotide microsatellite loci were identified in intron 1 of HAS1 and HAS2, and immediately upstream of the HAS3 gene and their utility as linkage markers demonstrated in genomic DNA (gDNA) studies. We thus present a comprehensive resource for mutation detection screening of all HAS exons and/or linkage analysis of each HAS gene in a variety of disorders for which they are attractive candidates.

Promoter polymorphisms in glutathione-S-transferase genes affect transcription.

The glutathione-S-transferases are a group of enzymes that play a major role in detoxification and defence against toxic, carcinogenic and other compounds. We analysed the proximal promoters of 14 genes encoding glutathione-S-transferase for polymorphism. Ten of the promoters contained sequence variants, nine of which we were able to clone into a reporter gene vector, pGL3. The relative ability of each haplotype to promote transcription of the luciferase gene was tested in each of two human cell lines (HEK293t and TE671) using a cotransfected CMV-SEAP plasmid as a control. Four genes (GSTA1, GSTA2, GSTM4 and GSTT2) showed activity differences greater than 1.5-fold between haplotypes, and a fifth gene (MGST1) showed a 1.4-fold difference. The promoter sequence variants in these genes may therefore play a role in human variation, susceptibility to diseases and the effects of drugs.

A high proportion of chromosome 21 promoter polymorphisms influence transcriptional activity.

We have sought to obtain an unbiased estimate of the proportion of polymorphisms in promoters of human genes that have functional effects. We carried out polymorphism discovery on a randomly selected group of 51 gene promoters mapping to human chromosome 21 and successfully analyzed the effect on transcription of 38 of the sequence variants. To achieve this, a total of 53 different haplotypes from 20 promoters were cloned into a modified pGL3 luciferase reporter gene vector and were tested for their abilities to promote transcription in HEK293t and JEG-3 cells. Up to seven (18%) of the 38 tested variants altered transcription by 1.5-fold, confirming that a surprisingly high proportion of promoter region polymorphisms are likely to be functionally important. The functional variants were distributed across the promoters of CRYAA, IFNAR1, KCNJ15, NCAM2, IGSF5, and B3GALT5. Three of the genes (NCAM2, IFNAR1, and CRYAA) have been previously associated with human phenotypes and the polymorphisms we describe here may therefore play a role in those phenotypes.

Will we ever find the genes for addiction?

AIM: To assess the likelihood of finding genes which predispose to addiction and to present this information in a form accessible to the general readership of Addiction. METHODS: Review of the evidence that genetic factors play a significant role in the process of addiction and the proximity of the identification of these factors. RESULTS: The search for the genetic susceptibility variants for many complex illnesses has been ongoing for decades, with increased pace in the last 20 years. However, until very recently only a small number of such variants have been found. Recent studies have used several thousand samples in genome-wide association studies and the latest genotyping technology and have reported a growing number of successes, but have highlighted the need for even larger samples and new statistical methods or new experimental approaches to identify fully the genes involved in the disease process. The phenotype for addiction to drugs is not well defined, and the heritability of addiction to drugs of abuse is far from clear and may be small compared to that of many other complex disorders. The absolute requirement for the administration of drugs before addiction can occur, and other environmental factors known to have a major effect, makes the selection of both probands and controls challenging for genetic studies. Many candidate genes put forward so far as susceptibility genes may be unrelated to the underlying process referred to as addiction but, rather, are related to the propensity to take drugs in the first place. CONCLUSIONS: It is the underlying biological process which changes to an alternative state following addiction, which is the target of investigation, and it is not clear that even genome-wide association studies with sample sizes a magnitude greater than those reported so far would identify the genes involved which have the largest effect. Ultimately, modern neurobiological approaches may identify this process and the genes involved, and even at this stage identifying the susceptibility variants will require both biological as well as genetic analysis.

In silico discrimination of single nucleotide polymorphisms and pathological mutations in human gene promoter regions by means of local DNA sequence context and regularity.

DNA sequence features were sought that could be used for the in silico ascertainment of the likely functional consequences of single nucleotide changes in human gene promoter regions. To identify relevant features of the local DNA sequence context, we transformed into consensus tables the nucleotide composition of sequences flanking 101 promoter SNPs of type C<-->T or A<-->G, defined empirically as being either 'functional' or 'non-functional' on the basis of a standardised reporter gene assay. The similarity of a given sequence to these consensus tables was then measured by means of the Shapiro-Senapathy score. A decision rule with the potential to discriminate between empirically ascertained functional and non-functional SNPs was proposed that potentiated discrimination between functional and non-functional SNPs with a sensitivity of 80% and a specificity of 20%. Two further datasets (viz. disease-associated SNPs of types A<-->G and C<-->T (N = 75) and pathological promoter mutations (transitions, N = 114)) were retrieved from the Human Gene Mutation Database (HGMD; http://www.hgmd.org/) and analyzed using consensus tables derived from the functional and non-functional promoter SNPs; approximately 70% were correctly recognized as being of probable functional significance. Complexity analysis was also used to quantify the regularity of the local DNA sequence environment. Functional SNPs/mutations of type C<-->T were found to occur in DNA regions characterized by lower average sequence complexity as measured with respect to symmetric elements; complexity values increased gradually from functional SNPs and pathological mutations to functional disease-associated SNPs and non-functional SNPs. This may reflect the internal axial symmetry that frequently characterizes transcription factor binding sites.

Haplotypes at the dystrobrevin binding protein 1 (DTNBP1) gene locus mediate risk for schizophrenia through reduced DTNBP1 expression.

The DTNBP1 gene, encoding dysbindin, is now generally considered to be a susceptibility gene for schizophrenia. However, the confidence with which this hypothesis can be held has to be tempered by the poor reproducibility between studies in terms of the exact nature of the associated haplotypes, by the failure so far to identify any specific susceptibility variants and by the absence of any demonstrated function associated with any of the risk haplotypes. In the present study, we show that a defined schizophrenia risk haplotype tags one or more cis-acting variants that results in a relative reduction in DTNBP1 mRNA expression in human cerebral cortex. Subsidiary analyses suggest that risk haplotypes identified in other sample groups of white European ancestry also index lower DTNBP1 expression, whereas putative 'protective' haplotypes index high DTNBP1 expression. Our data indicate that variation in the DTNBP1 gene confers susceptibility to schizophrenia through reduced expression, and that this, therefore, represents a primary aetiological mechanism in the disorder.

Allele-specific gene expression differences in humans.

In the last decade, the search for the genetic origins of phenotypic variation has expanded beyond the non-synonymous variants which alter the amino acid sequence of the encoded protein, and many examples of sequence variants which alter gene expression have been found. Recently, using both traditional and novel technologies, a number of surveys have been carried out to examine the frequency with which cis-acting sequence variants or other cis-acting effects, alter gene expression either in vitro or in vivo. Microarray data have shown that the expression of many genes varies markedly between individuals and allele-specific expression studies have shown that the source of much of this variation appears to be cis-acting effects. A significant proportion of the variation may originate in gene promoter regions and a large number of sequence variants which have functional effect in vitro have been found. The evidence suggests that given a large enough population, most, if not all genes may have allele-specific expression differences in at least some individuals and finding the genetic origins of each of these and linking the former to a possible phenotype must be a major long term goal of the biomedical community.

Polymorphically duplicated genes: their relevance to phenotypic variation in humans.

A number of disorders are known to be caused by duplication of genes, but these are all rare events. However, there is evidence that polymorphic gene duplication may be common and a growing number of genes are known to be duplicated in a polymorphic manner although phenotypes cannot be associated with most of these. Gene duplication occurring due to cytogenetic abnormalities such as Down syndrome predisposes the patients to a variety of complex disorders. It is possible therefore that many complex disorders and variable phenotypes are associated with duplication of genes.

Cis-acting variation in the expression of a high proportion of genes in human brain.

Much of the genetic component of human phenotypic diversity, including susceptibility to disease, is proposed to be the result of cis-acting influences on gene expression. If this hypothesis is correct, it implies that cis-acting regulatory variation is a common phenomenon. However, direct evidence in support of this view is scarce. We have applied highly quantitative measures of allele-specific expression in order to screen an average of 19 informative subjects (range 8-26) for the presence of common cis-acting influences on the expression of 15 genes by using RNA derived from human brain. We found that, in seven of the 15 assayed genes, at least one individual exhibited relative differences in allelic expression of 20% or more and, in one gene (DTNBP1), allelic expression differences exceeded 50%. These results suggest that cis-acting variation in gene expression commonly occurs in native tissue and hence provide empirical support for the hypothesis that this is potentially an important mechanism underlying human phenotypic diversity.

Experimental analysis of the annotation of promoters in the public database.

The ability to identify and examine promoter elements is important to researchers who wish to understand how gene expression is regulated in normal and pathological states. Unfortunately, the number of human promoters that have been directly experimentally defined is small. In order to determine if promoter sequences can be identified by simply aligning mRNA and genomic sequences, we have used a reporter gene assay to assess the promoter activity of the immediate 5' region flanking 38 mRNAs mapping to chromosome 21. For comparison, we have measured the activities of 19 sequences not thought to be promoters and 39 sequences taken from the Eukaryotic Promoter Database. Our results suggest that alignment of reference mRNAs to genomic sequence allows promoters to be identified for at least 75% of genes. These data provide the first empirical evidence that the current state of annotation of the genome is sufficient to allow molecular geneticists to correctly identify promoter sequences for most genes for which reference mRNA and genomic sequences are available.

Functional analysis of human promoter polymorphisms.

The potential importance of gene regulation in disease susceptibility and other inherited phenotypes has been underlined by the observation that the human genome contains fewer protein coding genes than expected. Promoter sequences are potential sources of polymorphism affecting gene expression, although to date there are no large-scale systematic studies that have determined how frequently such variants occur. We have used denaturing high performance liquid chromatography to screen the first 500 bp of the 5' flanking region of 170 opportunistically selected genes identified from the Eukaryotic Promoter Database (EPD) for common polymorphisms. Using a screening set of 16 chromosomes, single-nucleotide polymorphisms were found in approximately 35% of genes. It was attempted to clone each of these promoters into a T-vector constructed from the reporter gene vector pGL3. The relative ability of each promoter haplotype to promote transcription of the luciferase gene was tested in each of three human cell lines (HEK293, JEG and TE671) using a co-transfected SEAP-CMV plasmid as a control. The findings suggest that around a third of promoter variants may alter gene expression to a functionally relevant extent.

A haplotype implicated in schizophrenia susceptibility is associated with reduced COMT expression in human brain.

The gene encoding catechol-O-methyltransferase (COMT) is a strong candidate for schizophrenia susceptibility, owing to the role of COMT in dopamine metabolism, and the location of the gene within the deleted region in velocardiofacial syndrome, a disorder associated with high rates of schizophrenia. Recently, a highly significant association was reported between schizophrenia and a COMT haplotype in a large case-control sample (Shifman et al. 2002). In addition to a functional valine-->methionine (Val/Met) polymorphism, this haplotype included two noncoding single-nucleotide polymorphisms (SNPs) at either end of the COMT gene. Given the role of COMT in dopamine catabolism and that deletion of 22q11 (containing COMT) is associated with schizophrenia, we postulated that the susceptibility COMT haplotype is associated with low COMT expression. To test this hypothesis, we have applied quantitative measures of allele-specific expression using mRNA from human brain. We demonstrate that COMT is subject to allelic differences in expression in human brain and that the COMT haplotype implicated in schizophrenia (Shifman et al. 2002) is associated with lower expression of COMT mRNA. We also show that the 3' flanking region SNP that gave greatest evidence for association with schizophrenia in that study is transcribed in human brain and exhibits significant differences in allelic expression, with lower relative expression of the associated allele. Our results indicate that COMT variants other than the Val/Met change are of functional importance in human brain and that the haplotype implicated in schizophrenia susceptibility is likely to exert its effect, directly or indirectly, by down-regulating COMT expression.