Distinct chromatin structures at the monoamine oxidase-A promoter correlate with allele-specific expression in SH-SY5Y cells.

Monoamine oxidase-A (MAOA) metabolises monoamines and is implicated in the pathophysiology of psychiatric disorders. A polymorphic repetitive DNA domain, termed the uVNTR (upstream variable number tandem repeat), located at the promoter of the MAOA gene is a risk factor for many of these disorders. MAOA is on the X chromosome suggesting gender could play a role in regulation. We analysed MAOA regulation in the human female cell line, SH-SY5Y, which is polymorphic for the uVNTR. This heterozygosity allowed us to correlate allele-specific gene expression with allele-specific transcription factor binding and epigenetic marks for MAOA. Gene regulation was analysed under basal conditions and in response to the mood stabiliser sodium valproate. Both alleles were transcriptionally active under basal growth conditions; however, the alleles showed distinct transcription factor binding and epigenetic marks at their respective promoters. Exposure of the cells to sodium valproate resulted in differential allelic expression which correlated with allele-specific changes in distinct transcription factor binding and epigenetic marks at the region encompassing the uVNTR. Biochemically our model for MAOA promoter function has implications for gender differences in gene × environment responses in which the uVNTR has been implicated as a genetic risk.

A TOMM40 poly-T variant modulates gene expression and is associated with vocabulary ability and decline in nonpathologic aging.

The Translocase of Outer Mitochondrial Membrane 40 Homolog and Apolipoprotein E (TOMM40-APOE) locus has been associated with a number of age-related phenotypes in humans including nonpathologic cognitive aging, late-onset Alzheimer's disease, and longevity. Here, we investigate the influence of the TOMM40 intron 6 poly-T variant (rs10524523) on TOMM40 gene expression and cognitive abilities and decline in a cohort of 1613 community-dwelling elderly volunteers who had been followed for changes in cognitive functioning over a period of 14 years (range = 12-18 years). We showed that the shorter length poly-T variants were found to act as a repressor of luciferase gene expression in reporter gene constructs. Expression was reduced to approximately half of that observed for the very long variant. We further observed that the shorter poly-T variant was significantly associated with reduced vocabulary ability and a slower rate of vocabulary decline with age compared to the very long poly-T variants. No significant associations were observed for memory, fluid intelligence or processing speed, although the direction of effect, where the short variant was correlated with reduced ability and slower rate of decline was observed for all tests. Our results indicate that the poly-T variant has the ability to interact with transcription machinery and differentially modulate reporter gene expression and influence vocabulary ability and decline with age.

Behavioural genetics of the serotonin transporter.

The serotonin transporter is a key regulator of the bioavailability of serotonin and therefore any modulation in the expression or action of the transporter would be expected to have consequences on behaviour. The transporter has therefore become a target for pharmaceutical intervention in behavioural and mood disorders. The search for polymorphic variants in the transporter that would associate with neurological disorders has been extensive but has become focused on two domains which are both termed variable number tandem repeat (VNTR)polymorphisms. Both of these VNTRs are in non-coding DNA and therefore proposed to be mechanistically involved in a disorder through their ability to modulate transcriptional or post-transcriptional regulation of the transporter. The most extensively studied is in the promoter and is a bi-allelic insertion/deletion found in the 50 promoter region of the gene 1.2 kb upstream of the transcriptional start site. This VNTR, termed, 5-HTTLPR was initially identified as two variants containing either, 14 (short/deletion) or 16 (long/insertion) copies of a 22 bp repeat. A second widely studied VNTR found in the non-coding region of the transporter is located within intron 2 and comprises 9, 10 or 12 copies of a16­17 bp repeat termed, STin2.9, STin2.10 and STin2.12, respectively. These VNTR polymorphisms have been associated with a range of behavioural and psychiatric disorders including depression, OCD, anxiety and schizophrenia, however often the lack of reproducibility in different cohorts has led to debate on the actual association of the polymorphisms with this extensive range of neurological conditions. Here we review these two polymorphic VNTRs in depth and relate that to pharmaceutical response, their ability to regulate differential transporter expression, their core involvement in gene-environment interaction and their genetic association with specific disorders.

A long AAAG repeat allele in the 5' UTR of the ERR-γ gene is correlated with breast cancer predisposition and drives promoter activity in MCF-7 breast cancer cells.

We sequenced the 5' UTR of the estrogen-related receptor gamma gene (ERR-γ) in ~500 patient and volunteer samples and found that longer alleles of the (AAAG)(n) microsatellite were statistically and significantly more likely to exist in the germlines of breast cancer patients when compared to healthy volunteers. This microsatellite region contains multiple binding sites for a number of transcription factors, and we hypothesized that the polymorphic AAAG-containing sequence in the 5' UTR region of ERR-γ might modulate expression of ERR-γ. We found that the 369 bp PCR product containing the AAAG repeat drove expression of a reporter gene in estrogen receptor positive breast cancer cells. Our results support a role for the 5' UTR region in ERR-γ expression, which is potentially mediated via binding to the variable tandem AAAG repeat, the length of which correlates with breast cancer pre-disposition. Our study indicates that the AAAG tetranucleotide repeat polymorphism in ERR-γ gene 5' UTR region may be a new biomarker for genetic susceptibility to breast cancer.

The human neurokinin B gene, TAC3, and its promoter are regulated by Neuron Restrictive Silencing Factor (NRSF) transcription factor family.

We have previously shown that one of the major determinants directing the expression of the preprotachykinin-A (TAC1) gene, which encodes the neuropeptide substance P, is the transcription factor Neuronal Restrictive Silencer Factor (NSRF), which is also termed Repressor Element-1 Silencing Factor (REST). In rodent models of epilepsy, NRSF and its truncated isoform short NRSF (sNRSF), also termed REST4, are increased as an immediate response to seizure. In similar models the neurokinin B (NKB) gene (TAC3) is also induced and NKB has also been shown to be proconvulsant. In this communication we have demonstrated that both the TAC3 endogenous gene and its promoter are regulated, directly or indirectly, by the NRSF transcription factors resulting in both the increased expression of the endogenous gene and increased reporter gene activity. We demonstrate by chromatin immunoprecipitation analysis that NRSF and sNRSF will bind to the NKB promoter in vivo. Consistent with a model in which NRSF modulation of TAC3 gene expression is a mechanism that operates during epilepsy, the observed increases in both the level of the endogenous gene and the activity of the NKB promoter by these NRSF variants, were diminished by the action of the anticonvulsant drug, carbamazepine.

Expression of activity-dependent neuroprotective protein in the brain of adult rats.

Activity-dependent neuroprotective protein (ADNP) is a VIP-regulated gene, which is essential for brain development. A synthetic peptide (NAP) derived from the ADNP sequence is highly neuroprotective, therefore it has been hypothesised that ADNP has a similar role. ADNP contains classical transcription factor motifs and nuclear localisation domains, but it has also been reported to be secreted and to co-localise with microtubules, indicating that ADNP may have multiple functions. We investigated the pattern of ADNP expression by immunohistology in normal rat brain, in order to generate a framework for future studies examining changes in ADNP expression in response to noxious stimuli or in models of disease. We found widespread ADNP-like immunoreactivity in neurons throughout the rat brain, with the highest expression in the cerebellum, and strong expression in the thalamus, mesencephalon, pons and medulla oblongata. ADNP-like immunoreactivity was mainly observed in the cytoplasm of neurons, and fibre tracts were often strongly positive as well. In addition, positive neuronal nuclei were occasionally observed. ADNP-like immunoreactivity was lost in degenerating "dark" neurons, whereas it appeared to locate to the nucleus in some of the morphologically unaltered adjacent cells. Occasional astrocyte and microglial cells were also positive. We suggest that the widespread expression of ADNP may correlate with the wide-ranging protective effects of NAP, and that the cytoplasmic and axonal localisation of ADNP-like immunoreactivity suggests additional, non-transcriptional functions of ADNP.

Molecular genetics of monoamine transporters: relevance to brain disorders.

We have demonstrated in both the human serotonin transporter gene (5HTT) and the dopamine transporter gene (DAT1) that specific polymorphic variants termed Variable Number Tandem Repeats (VNTRs), which correlate with predisposition to a number of neurological and psychiatric disorders, act as transcriptional regulatory domains. We have demonstrated that these domains can act as both tissue-specific and stimulus-inducible regulators of gene expression. As such they can act to be mechanistically associated with the progression or initiation of a behavioural disorder by altering the level of transporter mRNA, which in turn regulates the concentration of transporter in specific cells or in response to a challenge; chemical, environmental or physiological. The synergistic actions of such transcriptional domains will modulate gene expression. Our hypothesis is that these VNTR variants are one mechanism by which nurture can modify concentrations of neurotransmitters in a differential manner.

Generation of a transgenic model to address regulation and function of the human neurokinin 1 receptor (NK1R).

We have generated mouse transgenic lines using yeast artificial chromosome (YAC) technology which demonstrate expression from the human NK1 receptor (NK1R) locus. We introduced a 380 kb fragment encompassing the human NK1R gene and flanking regions which we hoped would recapitulate the expected endogenous expression of the human gene. To visualise this expression the NK1 locus co-expresses the green fluorescence protein gene (GFP) under the control of an internal ribosome entry site (IRES) sequence. We have generated five mouse lines that express the human NK1 receptor gene with and without the marker gene. All the lines incorporating the marker gene appear to exhibit the same expression pattern in analysis of selected anatomical regions throughout the mouse. The lack of a human specific NK1R antibody determined that we could not distinguish between expression of the transgene and endogenous NK1R. Our analysis has shown transgene expression in brain areas known to express NK1R in human such as the hippocampus and caudate putamen. The majority of these cells were also positive for GFP fluorescence. These transgenic lines may prove a good pre-clinical model as drugs can be addressed against both the human receptor and modulators of its expression in vivo.

The serotonin transporter intronic VNTR enhancer correlated with a predisposition to affective disorders has distinct regulatory elements within the domain based on the primary DNA sequence of the repeat unit.

We have demonstrated that a variable number tandem repeat domain (VNTR) within intron 2 of the serotonin transporter gene is a transcriptional regulatory domain which is potentially correlated with a predisposition to affective disorders and other behavioural conditions. This correlation based on copy number of the VNTR alone (nine, 10 or 12 copies of 16/17 base-pair element) has been controversial and not reproduced in all studies. We demonstrate that individual repeat elements within the VNTR domain differ in their enhancer activity in an embryonic stem cell model. This has implications for both the mechanism by which these VNTRs are correlated with the progression of the disease and suggests that clinical analysis should now be extended to correlate sequence variation within the VNTR with the disorder. The latter may resolve some of the conflicting data published to date.

Neuron restrictive silencer factor as a modulator of neuropeptide gene expression.

We hypothesize that the transcription factor neuron restrictive silencer factor (NRSF) is an important determinant of the expression of the preprotachykinin (PPTA) gene (encoding substance P and Neurokinin A) and arginine vasopressin (AVP) both in neuronal and nonneuronal cells. NRSF, a zinc finger repressor protein, binds the NRSE motif found in many neuronal specific genes at a variety of promoter locations. However, it is found in a similar location at the major transcriptional start site, within both PPTA and AVP peptide promoters. We have correlated modulation of NRSF activity with expression of AVP and PPTA in a variety of cell types, indicating the general mechanism by which this protein may regulate expression. Specifically, they are as follows:(1). Expression of NRSF dramatically represses PPTA promoter activity in reporter gene constructs in primary cultures of DRG neurons.(2). The PPTA promoter activity is regulated differentially in osteoarthritic compared to normal chondrocytes. This regulation correlates with the region containing the NRSE site.(3). We have correlated a splice variant of NRSF with the establishment and progression of small cell lung carcinoma (SCLC) and demonstrated that NRSF variants can directly affect the activity of the AVP promoter in reporter gene constructs. If the deregulated expression of peptides in these diseases point to the mechanism determining the pathology, then perhaps targeting protocols that correct this deregulation may also reverse the specific disease phenotypes. Our data would indicate that modulation of NRSF activity would be a target for such intervention.

Adenomatous polyposis coli (APC), beta-catenin, and cadherin are expressed in human bone and cartilage.

AIMS: Members of the cadherin and catenin families are involved in chondrogenesis and catenin gene mutations have been detected in malignant tumours of bone. This study was undertaken to assess in detail expression of cadherin, beta-catenin and the associated tumour suppressor gene product APC in bone and cartilage at different stages of human skeletal maturity and in non-neoplastic and neoplastic osteoarticular disease. METHODS AND RESULTS: Immunohistochemical staining of formalin-fixed paraffin-embedded normal and osteoarthritic adult articular cartilage, fetal growth plate and a series of tumours of bone and cartilage was undertaken with a panel of antibodies against APC, beta-catenin, and pan-cadherin. This study demonstrated expression of APC, beta-catenin and cadherin in normal and diseased bone and cartilage. APC was present both in osteoblasts and osteoclasts but not in osteocytes. Although only weak APC staining of occasional growth plate hypertrophic chondrocytes and normal articular chondrocytes was seen, APC staining was increased in osteoarthritic articular cartilage. beta-catenin and pan-cadherin staining was strongly positive in osteoclasts and osteoblasts, with expression being lost when bone cells differentiated into osteocytes. Expression of APC, beta-catenin and pan-cadherin in bone tumours was similar to that of non-neoplastic adult tissues. CONCLUSIONS: These findings suggest previously unrecognized roles for APC in regulation of function of chondrocytes, osteoblasts and osteoclasts and support the view that catenin-cadherin interactions are important in regulation of bone cell activity. Abnormalities of expression or function of these molecules may be important in formation of bone tumours and their clinical behaviour.

Familial adenomatous polyposis coli in South Africa--molecular basis and diagnosis.

OBJECTIVE: To determine the molecular basis and establish a routine molecular diagnostic service for familial adenomatous polyposis coli (FAP) families in South Africa. DESIGN: The coding region of the adenomatous polyposis coli (APC) gene in affected FAP kindreds was screened using heteroduplex analysis, single-strand conformation polymorphism analysis and the protein truncation test. SETTING: Department of Human Genetics, University of Stellenbosch, and the Cancer Research Campaign Laboratories, Department of Pathology, University of Edinburgh and Molecular Medicine Centre, Western General Hospital, Edinburgh, Scotland (academic visit of 6 months). SUBJECTS: FAP-affected individuals and at-risk family members in 28 apparently unrelated South African families. RESULTS: A total of nine different APC mutations was identified, allowing DNA-based diagnosis in 20 families. Three of these mutations have not been described previously in other populations. CONCLUSION: Pre-symptomatic diagnosis using direct mutation detection is cost-effective and surgical intervention has the potential to prevent cancer in at-risk individuals from FAP families.

Dysregulated expression of beta-catenin marks early neoplastic change in Apc mutant mice, but not all lesions arising in Msh2 deficient mice.

We have analysed the pattern of beta-catenin expression by immunohistochemistry in mice singly or multiply mutant for Apc, p53 and Msh2. We observed increased expression of beta-catenin in all intestinal lesions arising on an ApcMin+/- background. In all categories of lesion studied mosaic patterns of beta-catenin expression were observed, with the proportion of cells showing enhanced expression decreasing with increasing lesion size. p53 status did not alter these patterns. We also show that beta-catenin dysregulation marks pancreatic abnormalities occurring in ApcMin+/- and (ApcMin+/-, p53-/-) mice. In these mice both adenomas and adenocarcinomas of the pancreas arose and were characterized by increased expression of beta-catenin. We have extended these analyses to intestinal lesions arising in mice mutant for the mismatch repair gene Msh2. In these mice, increased expression of beta-catenin was again observed. However, in contrast with ApcMin+/- mice, a subset of lesions retained normal expression. Taken together, these findings show that increased expression of beta-catenin is an efficient marker of early neoplastic change in both murine intestine and pancreas in Apc mutant mice. However, we also show that dysregulation of beta-catenin is not an obligate step in the development of intestinal lesions, and therefore that genetic events other than the loss of Apc function may initiate the transition from normal to neoplastic epithelium.

Apoptosis and carcinogenesis.

Apoptosis has long been known to be effected through a common sequence of structural changes, despite the wide variety of initiating stimuli. These common structural events appear to depend upon activation of a set of enzymes (caspases) which direct a strongly conserved, terminal effector pathway. The regulation of this pathway, and in particular its coupling to DNA damage, appears to be critical in maintaining at low levels the number of mutated cells within tissues. The frequency with which tumours (experimental and human) bear deficiency in p53 or MSH-2 repair function may indicate the importance of these proteins in coupling DNA damage to apoptosis.

Caspase-mediated cleavage of APC results in an amino-terminal fragment with an intact armadillo repeat domain.

During the effector phase of apoptosis, caspase activation appears to be responsible for the distinctive structural changes of apoptosis and perhaps for some of the changes in function of the doomed cells. There is therefore interest in identifying caspase substrates and the details of the cleavage events. Here we define precisely the event responsible for generation of a stable 90 kDa fragment from the oncosuppressor protein adenomatous polyposis coli (APC). Using synthetic radiolabeled APC peptides as substrate, we demonstrate cleavage by cytosolic extracts from preapoptotic cells. This cleavage was reproduced by recombinant caspase-3 and blocked by a tetrapeptide inhibitor Ac-DEVD-CHO, which is specific for caspase-3 family members. Inhibitors specific for caspase-1 and -8 however, were less effective in blocking APC cleavage. Mutation of a candidate DNID caspase-3 target site completely abolished cleavage. This cleavage may be of biological importance since the 90 kDa fragment consists of a sequence that is highly conserved in the human, rat, mouse, Xenopus, and Drosophila APC, although wide sequence divergence is observed in Drosophila immediately carboxy-terminal to the DNID site. Furthermore, cleavage at this site separates two significant functional domains: an amino-terminal armadillo repeat and an adjacent series of beta-catenin binding sites. Further circumstantial evidence for the significance of APC-related pathways in apoptosis is provided by the observation that apoptosis also induces cleavage of beta-catenin itself, a protein known to accumulate in cells depleted in functional APC and that appears to link cell-cell signaling to changes in transcription and cell movement.

The pattern of K-ras mutation in pulmonary adenocarcinoma defines a new pathway of tumour development in the human lung.

Codon 12 of the K-ras oncogene was screened for mutations in 65 surgically-resected primary pulmonary adenocarcinomas and in 32 tissue foci of alveolar atypical hyperplasia (AAH) by a polymerase chain reaction (PCR)-based method. Mutations in either position 1 or position 2 of codon 12 were detected in 16 tumours (25 per cent). When analysed by site of origin, mutations were seen in 9/26 (35 per cent) parenchymal and in 0/12 bronchial adenocarcinomas (P < 0-02), K-ras mutations were seen in five AAH lesions from four patients. DNA sequencing showed that the great majority of mutations in both adenocarcinomas and AAH were G-T transversions. These findings provide support for the classification of pulmonary adenocarcinomas into bronchial and parenchymal subtypes and also provide molecular evidence to support the importance of AAH in the development of parenchymal cancers.

APC expression in normal human tissues.

The tumour suppressor gene APC codes for a 2843-amino acid protein whose precise functions are still poorly understood. This paper describes the development of two new antisera to APC (to amino- and carboxy-terminal epitopes) which permit localization of the protein by immunohistochemistry in archival paraffin sections. The protein is expressed in a wide variety of normal epithelial tissues. Its distribution frequently coincides with the location of post-replicative cells within tissues. Staining patterns demonstrate that the APC protein, although often diffusely cytoplasmic in distribution, may also accumulate in the apical and immediately subapical regions, or along the lateral margins of certain cells. These results indicate that APC is significant in many tissues in addition to the colorectal epithelium. They are compatible with a function related to signalling at the adherens junction and possibly with other more complex roles in cells committed to terminal differentiation.

Extracolonic features of familial adenomatous polyposis in patients with sporadic colorectal cancer.

We have investigated the occurrence of attenuated extracolonic manifestations (AEMs) of familial adenomatous polyposis (FAP) in patients with non-polyposis colorectal cancer. In a prospective case-control study, we observed that significantly more colorectal cancer patients exhibited AEM than did age and sex-matched controls (19.5% vs 7.5%, P < 0.004). However patients with AEMs do not have occult FAP, as we found no heterozygous adenomatous polyposis coli (APC) gene mutations despite extensive analysis of constitutional DNA. Genome-wide DNA replication errors (RERs) occur in a proportion of colorectal cancers, particularly right-sided lesions and in almost all tumours from hereditary non-polyposis colorectal cancer (HNPCC) patients. As AEMs have been reported in familial colon cancer cases, we investigated the relationship of AEMs to tumour RER phenotype. There was indeed an excess of AEMs in patients with right-sided tumours (30.2% of 53 patients vs 14.7% of 116 patients, P < 0.03) and in those with RER tumours (3 out of 12 patients with RER tumours vs none out of 21 patients with non-RER tumours, P < 0.05). Two patients with AEM were from HNPCC families compared with none of those without AEM (P < 0.05). The association of AEMs with colorectal cancer is intriguing, and we speculate that it may be a manifestation of mutational mosaicism of the APC gene, perhaps associated with a constitutional defect in DNA mismatch pair.

Loss of heterozygosity at 5q21 in non-small cell lung cancer: a frequent event but without evidence of apc mutation.

Four genetic polymorphisms in the APC and MCC genes at chromosome 5q21 were analysed for loss of heterozygosity (LOH) in 97 primary squamous carcinomas and adenocarcinomas of the lung. LOH was identified in at least two polymorphic loci in 41 percent of informative cases. There was no significant difference in the frequency of LOH between squamous carcinomas and adenocarcinomas. Within the adenocarcinoma group, however, LOH appeared to be more common in tumours having a bronchial origin (5/9; 56 per cent) than in parenchymal adenocarcinoma (6/21; 29 per cent). All 32 tumours showing LOH at one or more polymorphic sites were examined for mutations in the mutation cluster region (MCR) of APC by single-strand conformational polymorphism (SSCP) analysis. Mutations were not detected in any of these cases. We therefore propose that it is likely that a tumour suppressor gene on 5q other than APC is involved in the pathogenesis of lung cancer.

Microsatellite instability and the role of hMSH2 in sporadic colorectalcancer.

Microsatellite instability (MSI) occurs in most tumours from patients with hereditary non-polyposis colorectal cancer (HNPCC) and in around 17% of sporadic colorectal cancers. Germline defects in mismatch repair (MMR) genes are responsible for the majority of large HNPCC families, with hMSH2 accounting for at least 50%. MMR gene defects also occur in a small proportion of sporadic colorectal tumours with MSI. Here we report a systematic analysis of mismatch repair deficiency in 215 Scottish patients with sporadic colorectal tumours. We found that 16.4% of tumours exhibited MSI; survival analysis by Cox proportional hazards method showed a substantial survival advantage for patients with tumours showing MSI, independent of other prognostic factors. Tumours with MSI were screened for hMSH2 mutations and although 61% were found to have alterations, of these only 1/24 was exonic. The majority of these changes were reductions in length at intronic mononucleotide tracts and we postulate that these alterations are the result of a genetic defect elsewhere, although they may compromise hMSH2 function as a second step in tumourigenesis. Our findings indicate that instability confers an improved prognosis in colorectal cancer and, despite the fact that these two groups of tumours share similar biological characteristics, the genetic basis of HNPCC and sporadic colorectal cancer with MSI is different.