Early life trauma, depression and the glucocorticoid receptor gene--an epigenetic perspective.

BACKGROUND: Hopes to identify genetic susceptibility loci accounting for the heritability seen in unipolar depression have not been fully realized. Family history remains the 'gold standard' for both risk stratification and prognosis in complex phenotypes such as depression. Meanwhile, the physiological mechanisms underlying life-event triggers for depression remain opaque. Epigenetics, comprising heritable changes in gene expression other than alterations of the nucleotide sequence, may offer a way to deepen our understanding of the aetiology and pathophysiology of unipolar depression and optimize treatments. A heuristic target for exploring the relevance of epigenetic changes in unipolar depression is the hypothalamic-pituitary-adrenal (HPA) axis. The glucocorticoid receptor (GR) gene (NR3C1) has been found to be susceptible to epigenetic modification, specifically DNA methylation, in the context of environmental stress such as early life trauma, which is an established risk for depression later in life. METHOD: In this paper we discuss the progress that has been made by studies that have investigated the relationship between depression, early trauma, the HPA axis and the NR3C1 gene. Difficulties with the design of these studies are also explored. RESULTS: Future efforts will need to comprehensively address epigenetic natural histories at the population, tissue, cell and gene levels. The complex interactions between the epigenome, genome and environment, as well as ongoing nosological difficulties, also pose significant challenges. CONCLUSIONS: The work that has been done so far is nevertheless encouraging and suggests potential mechanistic and biomarker roles for differential DNA methylation patterns in NR3C1 as well as novel therapeutic targets.

Control of gene expression by CpG island methylation in normal cells.

The role of DNA methylation in the control of mammalian gene expression has been the subject of intensive research in recent years, partly due to the critical role of CpG island methylation in the inactivation of tumour suppressor genes during the development of cancer. However, this research has also helped elucidate the role that DNA methylation plays in normal cells. At present, it is also clear that DNA methylation forms an important part of the normal cell-regulatory processes that govern gene transcription. Methylation, targeted at CpG islands, is an important part of the mechanisms that govern X-chromosome inactivation; it is also essential for the maintenance of imprinted genes and, at least in some cases, is critical in determining the cell-type-specific expression patterns of genes. Study of these examples will be important in identifying the mechanisms that control targeting of DNA methylation and how these processes are disrupted during disease pathogenesis.

CpG-island methylation and epigenetic control of resistance to chemotherapy.

Aberrant methylation of CpG islands (CpG-rich regions of DNA associated with the promoters of many genes) is associated with transcriptional inactivation of genes involved in tumour development. Genes involved in key DNA damage response pathways, such as cell-cycle control, apoptosis signalling and DNA repair can frequently become epigenetically silenced and methylated in tumours. This may lead to differences in intrinsic sensitivity of tumours to chemotherapy, depending on the specific function of the gene inactivated. Furthermore, chemotherapy itself may exert a selective pressure on epigenetically silenced drug sensitivity genes present in subpopulations of cells, leading to acquired chemoresistance. Clinical trials of epigenetic therapies are now in progress, and epigenetic profiling using DNA methylation will provide guidance on optimization of the use of these therapies with conventional chemotherapy, as well as helping to identify patient populations who may particularly benefit from such approaches.

Drug deaths in police custody: is dual diagnosis a significant factor?

This study examined cases for possible mental health issues based on a study of 43 deaths in custody that had been supervised by the PCA between 1998 and 2002 involving the use of drugs. In 18 of the 43 cases, there was evidence of one of three groups of mental health symptoms--in five cases, there was evidence of psychosis, in five of previous self-harm or suicidal attempts, and in a further eight, there were indications of anxiety or depression. This constitutes a total of 42% of the cases studied. Those with mental health factors were more likely to have swallowed the drugs used, were more likely to have used prescription drugs and were more likely to have been believed to be faking their symptoms by the officers involved in these cases. While it is recognised that mental health problems are widespread in the criminal justice system, the diversity of conditions and the marked overlap with the use of alcohol and illicit drugs has not been sufficiently recognised, either in the training of officers or in the procedures for intervention in the custody suite.

Cell type-specific methylation of an intronic CpG island controls expression of the MCJ gene.

Over 50% of human genes are associated with CpG islands and DNA methylation within such CpG islands has been clearly correlated with inhibition of expression. Whereas changes in DNA methylation play a key role in a number of human diseases, in particular cancer, in normal DNA CpG islands are nearly always methylation free, regardless of the expression status of the associated gene. Only limited evidence supports a role for DNA methylation in controlling tissue-specific expression in adult somatic tissue. Loss of expression of the MCJ gene has previously been linked to increased chemotherapeutic drug resistance in ovarian cancer. We report that loss of expression of MCJ in drug-resistant ovarian cancer cell lines depends on methylation of a CpG island within its first exon, but is independent of methylation within the promoter region. Furthermore, cell type-specific expression of the MCJ gene in normal cells also depends on the methylation status of the CpG island within its first exon. The MCJ CpG island is methylated and the gene is not expressed in cells of epithelial origin, but unmethylated and expressed in cells of lymphocyte or fibroblast origin. Chromatin immunoprecipitation assays determined that MCJ CpG island methylation was associated with loss of histone acetylation in ovarian epithelial cells compared with unmethylated fibroblast cells. Reduced acetylation was observed not only within the CpG island, but also within the promoter region, suggesting that CpG island methylation may direct alterations in chromatin structure within the promoter region, leading to gene inactivation.

Threshold 2: the reliability, validity and sensitivity to change of the Threshold Assessment Grid (TAG).

OBJECTIVE: This study investigated the psychometric properties of the Threshold Assessment Grid (TAG), a new assessment of the severity of mental health problems. METHOD: A total of 605 patients were recruited from 10 mental health adult and elderly services in London, UK. TAG ratings and other standardized definitions of severe mental illness were completed by referrers. TAG, Global Assessment of Functioning (GAF), Camberwell Assessment of Need Short Appraisal Schedule (CANSAS) and Health of the Nation Outcome Scale (HoNOS) ratings were completed by mental health service staff. Construct validation on extreme groups was investigated. RESULTS: Construct and concurrent validity were good. Referrer TAG scores predicted mental health team view of referral suitability, but not whether assessments were offered. Test-retest reliability was good, interrater reliability ranged from good to poor in different domains (but adequate for total TAG score), internal consistency was appropriate. Sensitivity to change requires further investigation. CONCLUSION: The TAG can be recommended for use by all agencies when making referrals to mental health services.

Aberrant DNA methylation in cancer: potential clinical interventions.

DNA methylation, the addition of a methyl group to the carbon-5 position of cytosine residues, is the only common covalent modification of human DNA and occurs almost exclusively at cytosines that are followed immediately by a guanine (so-called CpG dinucleotides). The bulk of the genome displays a clear depletion of CpG dinucleotides, and those that are present are nearly always methylated. By contrast, small stretches of DNA, known as CpG islands, are comparatively rich in CpG nucleotides and are nearly always free of methylation. These CpG islands are frequently located within the promoter regions of human genes, and methylation within the islands has been shown to be associated with transcriptional inactivation of the corresponding gene. Alterations in DNA methylation might be pivotal in the development of most cancers. In recent years, it has become apparent that the pattern of DNA methylation observed in cancer generally shows a dramatic shift compared with that of normal tissue. Although cancers often exhibit clear reductions throughout their genomes in the levels of DNA methylation, this goes hand-in-hand with increased methylation at the CpG islands. Such changes in methylation have a central role in tumourigenesis; in particular, methylation of CpG islands has been shown to be important in transcriptional repression of numerous genes that function to prevent tumour growth or development. Studies of DNA methylation in cancer have thus opened up new opportunities for diagnosis, prognosis and ultimately treatment of human tumours.

A role for mismatch repair in control of DNA ploidy following DNA damage.

Many reports have shown a link between mismatch repair (MMR) deficiency and loss of normal cell cycle control, particularly loss of G2 arrest. However almost all of these studies utilized transformed cell lines, and thus the involvement of other genes in this phenotype cannot be excluded. We have examined the effects of cisplatin treatment on primary embryo fibroblasts (MEFs) derived from mice in which the MMR gene Msh2 had been inactivated (Msh2(-/-)). This analysis determined that both primary Msh2(-/-) and wild type (WT) fibroblasts exhibited an essentially identical G2 arrest following cisplatin treatment. Similarly, we observed a cisplatin-induced G2 arrest in immortalized MMR deficient (Mlh1(-/-) and Pms2(-/-)) and WT MEFs. p53 deficient primary MEFs (p53(-/-)) exhibited both a clear G2 arrest and an increase in cells with a DNA content of 8N in response to cisplatin. When the Msh2 and p53 defects were combined (p53(-/-)/Msh2(-/-)) the G2 arrest was essentially identical to the p53(-/-) fibroblasts. However, the p53(-/-)/Msh2(-/-) fibroblasts demonstrated a further increase in cells with an 8N DNA content, above that seen in the p53(-/-) fibroblasts. These results suggest that loss of MMR on its own is not enough to overcome G2 arrest following exposure to cisplatin but does play a role in preventing polyploidization, or aberrant DNA reduplication, in the absence of functional p53.

Primary ovarian carcinomas display multiple methylator phenotypes involving known tumor suppressor genes.

Mounting evidence suggests that aberrant methylation of CpG islands is a major pathway leading to the inactivation of tumor suppressor genes and the development of cancer. Recent studies on colorectal and gastric cancer have defined a CpG island methylator phenotype (CIMP), which involves the targeting of multiple genes by promoter hypermethylation. To determine the role of methylation in ovarian cancer, we have investigated the methylation status of 93 primary ovarian tumors at ten loci using methylation-specific polymerase chain reaction (MSP). Seven of the loci (BRCA1, HIC1, MINT25, MINT31, MLH1, p73 and hTR) were found to be methylated in a significant proportion of the ovarian tumors, and methylation of at least one of these was found in the majority (71%) of samples. Although concurrent methylation of multiple genes was commonly seen, this did not seem to be due to a single CIMP phenotype. Instead the results suggest the presence of at least three groups of tumors, two CIMP-positive groups, each susceptible to methylation of a different subset of genes, and a further group of tumors not susceptible to CpG island methylation, at least at the loci studied.

Threshold 3: the feasibility of the Threshold Assessment Grid (TAG) for routine assessment of the severity of mental health problems.

BACKGROUND: Evidence-based practice requires the development of measures which are suitable for everyday clinical use ('feasible'). There is no consensus as to how to establish feasibility. METHOD: The feasibility of a new assessment - the Threshold Assessment Grid (TAG) - for use when making referrals to mental health services was tested by training mental health teams in using the TAG and other standardised assessments, asking referrers to ten mental health services in London also to complete a TAG, surveying TAG users, and evaluating a feedback meeting at which TAG data were presented. RESULTS: One hundred and one (61%) mental health staff received training, and 445 (74%) referrers of 600 patients completed TAGs. Sixty-five (65%) questionnaires from TAG users were completed, and 24 (80%) people attending feedback meetings evaluated the TAG. These allowed the extent to which the TAG is brief, simple, relevant, acceptable, available and valuable to be investigated. CONCLUSION: The TAG exhibited good feasibility when used by mental health staff, and moderate feasibility when used by referrers. This approach can be used to investigate the feasibility of other standardised assessments.

Reversal of drug resistance in human tumor xenografts by 2'-deoxy-5-azacytidine-induced demethylation of the hMLH1 gene promoter.

Loss of DNA mismatch repair because of hypermethylation of the hMLH1 gene promoter occurs at a high frequency in a number of human tumors. A role for loss of mismatch repair (MMR) in resistance to a number of clinically important anticancer drugs has been shown. We have investigated whether the demethylating agent 2'-deoxy-5-azacytidine (DAC) can be used in vivo to sensitize MMR-deficient, drug-resistant ovarian (A2780/cp70) and colon (SW48) tumor xenografts that are MLH1 negative because of gene promoter hypermethylation. Treatment of tumor-bearing mice with the demethylating agent DAC at a nontoxic dose induces MLH1 expression. Re-expression of MLH1 is associated with a decrease in hMLH1 gene promoter methylation. DAC treatment alone has no effect on the growth rate of the tumors. However, DAC treatment sensitizes the xenografts to cisplatin, carboplatin, temozolomide, and epirubicin. Sensitization is comparable with that obtained by reintroduction of the hMLH1 gene by chromosome 3 transfer. Consistent with loss of MMR having no effect on sensitivity in vitro to Taxol, DAC treatment has no effect on the Taxol sensitivity of the xenografts. DAC treatment does not sensitize xenografts of HCT116, which lacks MMR because of hMLH1 mutation. Because there is emerging data on the role of loss of MMR in clinical drug resistance, DAC could have a role in increasing the efficacy of chemotherapy for patients whose tumors lack MLH1 expression because of hMLH1 promoter methylation.

Threshold Assessment Grid (TAG): the development of a valid and brief scale to assess the severity of mental illness.

BACKGROUND: Lack of consensus about the meaning of severe mental illness makes it difficult to prioritise the severely mentally ill for specialist mental health care. The goal of this study was to develop a valid and brief assessment of severity of mental illness. METHOD: Six search workshops (n = 57) using consensus techniques developed a draft assessment acceptable to users, carers, practitioners and policy makers. A two-round Delphi consultation (n = 58) was held to identify consensus on this instrument. RESULTS: Search workshops agreed seven domains relevant to identifying the severely mentally ill: intentional and unintentional self-harm, risk from and to others, and survival, psychological, and social needs and disabilities. The Delphi consultation indicated at least agreement with all aspects in both rounds. CONCLUSIONS: The Threshold Assessment Grid (TAG) is a brief method of identifying the severely mentally ill, which has adequate face, concurrent, construct and content validity.

A role for methylation of the hMLH1 promoter in loss of hMLH1 expression and drug resistance in ovarian cancer.

Experimental evidence from several sources has identified a link between mismatch repair deficiency and cytotoxic drug resistance. Selection for cisplatin resistance in the human ovarian cancer cell line A2780, results in loss of expression of the mismatch repair protein hMLH1 in most (90%) of the resultant cisplatin-resistant cell lines. Here we demonstrate that the cisplatin sensitive parental cell line displays methylation of the promoter of only one hMLH1 allele, but that the resistant cell lines all exhibit hyper-methylation of the promoters of both hMLH1 alleles. Full methylation of all sites tested was found to be invariably associated with loss of hMLH1 expression, whereas a partial increase in methylation appears compatible with either loss or maintenance of expression. In addition treatment of two of the resistant cell lines with 5-azacytidine, a known inhibitor of methylation, results in re-expression of hMLH1. Clonogenic assays demonstrate that the 5-azacytidine treated cells show increased sensitivity to cisplatin. Furthermore, 12.5% (3/ 24) of ovarian tumours show hypermethylation of the hMLH1 promoter. Expression of hMLH1 is absent in the tumours that are hypermethylated, while all the unmethylated tumours still express the protein. This analysis suggests that methylation of the hMLH1 promoter may be a common mechanism for loss of hMLH1 expression, and possibly for cisplatin-resistance, in ovarian cancer.

Analysis of the CAVEOLIN-1 gene at human chromosome 7q31.1 in primary tumours and tumour-derived cell lines.

We identified CAVEOLIN-1 as a candidate for a tumour suppressor gene mapping to human chromosome 7q31.1. A number of studies suggest that caveolin could function as a tumour suppressor. Expression of caveolin, and in turn the number of caveolae within a cell, are inversely correlated with the transforming ability of numerous oncoproteins, including H-ras, v-abl, and bcr-abl, and caveolin is a major transformation-dependent substrate of v-src. Heterologous expression of caveolin has been shown to abrogate anchorage-independent growth and induce apoptosis in transformed fibroblasts and also to suppress anchorage-independent growth in human mammary carcinoma cells. We have analysed the status and expression of the human CAVEOLIN-1 gene in primary tumours and tumour-derived cell lines. We found no evidence for mutation of CAVEOLIN-1 in human cancers. Additionally, we found that while the first two exons of CAVEOLIN-1 are associated with a CpG island, this is not methylated in either primary tumours or in tumour-derived cell lines in which Caveolin-1 expression is low or undetectable. The level of expression of Caveolin-1 does not correlate with loss of heterozygosity at the CAVEOLIN-1 locus in these same cell lines. Contrary to other published studies, we have shown that CAVEOLIN-1 is not expressed in normal breast ductal epithelial cells in vivo. CAVEOLIN-1 is however highly expressed in breast myoepithelial cells and its expression is retained in tumours derived from breast myoepithelium. Together our data refute a role for CAVEOLIN-1 as a breast tumour suppressor gene in vivo.

Rationale and design. PRiSM Psychosis Study I.

BACKGROUND: This paper sets out the rationale for the PRiSM Psychosis Study, and the research design used. Nine accompanying papers present the main results. The questions addressed by the PRiSM Psychosis Study are: can the gains of experimental studies which have demonstrated benefits arising from treatment by community mental health teams be translated to routine settings? If so, are the benefits diluted in ordinary clinical practice? What are the costs? METHOD: A prospective nonrandomised controlled trial of two types of community mental health service, in two phases: case identification followed by patient interviews. For the case identification the research team conducted the complete ascertainment of all prevalent cases of psychosis in the two study catchment areas in the index year (1991-1992). From all 514 patients with psychotic disorders thus identified, 302 were randomly allocated for interview, along with a key informant clinician and a carer. Interviews were under taken at two time points, two years apart. RESULTS: This paper presents the socio-demographic, clinical and ethnic characteristics of the patients. CONCLUSIONS: The people with psychosis interviewed for the PRiSM Psychosis Study are representative of the whole epidemiologically based patient population identified.

Sexual and relationship problems amongst patients with severe chronic psychoses.

Recent studies have begun to examine the complexity and frequency of sexual and relationship problems amongst samples of community resident people with severe psychoses. For the purposes of this study, subjects with severe persistent psychoses and under the care of a single community team were interviewed using a semi-structured clinical interview and structured diagnostic interview of marital and sexual satisfaction. Functional disability was assessed by the Multnomah Community Ability Scale. Amongst those interviewed (n = 53) the prevalence of sexual difficulties was 47.5% and 30.8% for men (n = 40) and women (n = 13), respectively. The majority of men (82.5%) and some of the women (38.5%) were not in intimate relationships; 42.5% of men and 38.5% of women had never had a sexual relationship. Amongst community resident patients with severe psychoses, the level of unmet need for specific interventions (including assessment procedures, psychotherapeutic, pharmaco-therapeutic) for sexual and relationship dysfunction is high. This warrants evaluation of service structures and treatment packages tailored for this group.

Current approaches to identifying the severely mentally ill.

Mental health care is expensive to provide and resources should be targeted. Possible approaches to such a prioritization are outlined. In the United Kingdom, care is to be provided on the basis of need. The key issue is then identifying the severely mentally ill, who are most in need of mental health care. Definitions of severe mental illness used in research studies are reviewed, indicating a lack of consensus about identifying this group. Current practice in England was surveyed, by obtaining written documentation from 20 agencies on the eligibility criteria they use for deciding whether someone should receive mental health care. Government departments, user groups and professional bodies were also surveyed. The findings indicate that definitions of severe mental illness use the five dimensions of safety, informal and formal support, diagnosis, disability and duration--the SIDDD dimensions. These dimensions offer a framework for developing definitions of severe mental illness at the local level, thereby identifying the priority group for mental health care.

Molecular characterization of patients with 18q23 deletions.

The 18q- syndrome is a deletion syndrome that is characterized by mental retardation, hearing loss, midfacial hypoplasia, growth deficiency, and limb anomalies. Most patients with this syndrome have deletions from 18q21-qter. We report on three patients with deletions of 18q23. A mother and daughter with identical deletions of 18q23 have many of the typical features of the 18q- syndrome, including midfacial hypoplasia and hearing loss. In contrast, the third patient has few of the symptoms of the 18q- syndrome. A contig of the 18q23 region was generated to aid in the mapping of the breakpoints. FISH was used to map both breakpoints to the same YAC clone. Furthermore, somatic-cell hybrids from the daughter and the third patient were isolated. The mapping results of sequence-tagged sites relative to the two breakpoints were identical, suggesting that the two deletion breakpoints map very close to one another. The analyses of these patients demonstrate that the critical region for the 18q- syndrome maps to 18q23 but that a deletion of 18q23 does not always lead to the clinical features associated with the syndrome. These patients demonstrate the wide phenotypic variability associated with deletions of 18q.