Brain-specific epigenetic markers of schizophrenia.

Epigenetics plays a crucial role in schizophrenia susceptibility. In a previous study, we identified over 4500 differentially methylated sites in prefrontal cortex (PFC) samples from schizophrenia patients. We believe this was the first genome-wide methylation study performed on human brain tissue using the Illumina Infinium HumanMethylation450 Bead Chip. To understand the biological significance of these results, we sought to identify a smaller number of differentially methylated regions (DMRs) of more functional relevance compared with individual differentially methylated sites. Since our schizophrenia whole genome methylation study was performed, another study analysing two separate data sets of post-mortem tissue in the PFC from schizophrenia patients has been published. We analysed all three data sets using the bumphunter function found in the Bioconductor package minfi to identify regions that are consistently differentially methylated across distinct cohorts. We identified seven regions that are consistently differentially methylated in schizophrenia, despite considerable heterogeneity in the methylation profiles of patients with schizophrenia. The regions were near CERS3, DPPA5, PRDM9, DDX43, REC8, LY6G5C and a region on chromosome 10. Of particular interest is PRDM9 which encodes a histone methyltransferase that is essential for meiotic recombination and is known to tag genes for epigenetic transcriptional activation. These seven DMRs are likely to be key epigenetic factors in the aetiology of schizophrenia and normal brain neurodevelopment.

Isocitrate dehydrogenase 1 R132C mutation occurs exclusively in microsatellite stable colorectal cancers with the CpG island methylator phenotype.

The CpG Island Methylator Phenotype (CIMP) is fundamental to an important subset of colorectal cancer; however, its cause is unknown. CIMP is associated with microsatellite instability but is also found in BRAF mutant microsatellite stable cancers that are associated with poor prognosis. The isocitrate dehydrogenase 1 (IDH1) gene causes CIMP in glioma due to an activating mutation that produces the 2-hydroxyglutarate oncometabolite. We therefore examined IDH1 alteration as a potential cause of CIMP in colorectal cancer. The IDH1 mutational hotspot was screened in 86 CIMP-positive and 80 CIMP-negative cancers. The entire coding sequence was examined in 81 CIMP-positive colorectal cancers. Forty-seven cancers varying by CIMP-status and IDH1 mutation status were examined using Illumina 450K DNA methylation microarrays. The R132C IDH1 mutation was detected in 4/166 cancers. All IDH1 mutations were in CIMP cancers that were BRAF mutant and microsatellite stable (4/45, 8.9%). Unsupervised hierarchical cluster analysis identified an IDH1 mutation-like methylation signature in approximately half of the CIMP-positive cancers. IDH1 mutation appears to cause CIMP in a small proportion of BRAF mutant, microsatellite stable colorectal cancers. This study provides a precedent that a single gene mutation may cause CIMP in colorectal cancer, and that this will be associated with a specific epigenetic signature and clinicopathological features.

Genome-wide DNA methylation analysis of human brain tissue from schizophrenia patients.

Recent studies suggest that genetic and environmental factors do not account for all the schizophrenia risk, and epigenetics also has a role in disease susceptibility. DNA methylation is a heritable epigenetic modification that can regulate gene expression. Genome-wide DNA methylation analysis was performed on post-mortem human brain tissue from 24 patients with schizophrenia and 24 unaffected controls. DNA methylation was assessed at over 485,000 CpG sites using the Illumina Infinium HumanMethylation450 Bead Chip. After adjusting for age and post-mortem interval, 4641 probes corresponding to 2929 unique genes were found to be differentially methylated. Of those genes, 1291 were located in a CpG island and 817 were in a promoter region. These include NOS1, AKT1, DTNBP1, DNMT1, PPP3CC and SOX10, which have previously been associated with schizophrenia. More than 100 of these genes overlap with a previous DNA methylation study of peripheral blood from schizophrenia patients in which 27,000 CpG sites were analysed. Unsupervised clustering analysis of the top 3000 most variable probes revealed two distinct groups with significantly more people with schizophrenia in cluster one compared with controls (P=1.74 × 10(-4)). The first cluster composed of 88% of patients with schizophrenia and only 12% controls, whereas the second cluster composed of 27% of patients with schizophrenia and 73% controls. These results strongly suggest that differential DNA methylation is important in schizophrenia etiology and add support for the use of DNA methylation profiles as a future prognostic indicator of schizophrenia.

DNA methylation within the normal colorectal mucosa is associated with pathway-specific predisposition to cancer.

There are two major molecular pathways to sporadic colorectal cancer, the chromosomal instability (CIN) and the CpG island methylator phenotype (CIMP) pathways. This study recruited 166 patients undergoing colonoscopy. Biopsy samples were collected from the cecum, transverse colon, sigmoid colon and rectum. DNA methylation was quantified at 'type A' (ESR1, GATA5, HIC1, HPP1, SFRP1) and 'type C' markers (MGMT, MLH1, CDKN2A, MINT2, MINT31, IGF2, CACNA1G, NEUROG1, SOCS1, RUNX3), and LINE-1. 'Type A' genes are frequently methylated in normal and neoplastic tissues, proportional to tissue age. 'Type C' methylation is more specific for neoplasia. The last five 'type C' markers comprise a CIMP panel. The mean 'type A' and CIMP-panel methylation Z-scores were calculated. In all, 88 patients had adenomatous lesions, 32 had proximal serrated polyps (PSPs) and 50 were normal. Most 'type A' genes showed direct correlations between methylation and age (ESR1, rho=0.66, P<0.0001), with higher methylation distally (ESR1, P<0.0001). On multivariate analysis, 'type A' methylation was inversely associated with colorectal adenomas (odds ratio=0.23, P<0.001), the precursor to CIN cancers. CIMP-panel methylation was significantly associated with advanced PSPs (odds ratio=5.1, P=0.009), the precursor to CIMP cancers. DNA methylation in normal mucosa varied with age and region and was associated with pathway-specific pathology. In the future, the colorectal field could yield important information and potentially inform clinical practice.

Promoter hypermethylation frequency and BRAF mutations distinguish hereditary non-polyposis colon cancer from sporadic MSI-H colon cancer.

BACKGROUND: Colorectal cancers resulting from defective DNA mismatch repair can occur in both hereditary non-polyposis colon cancer (HNPCC) and in the sporadic setting. They are characterised by a high level of microsatellite instability (MSI-H) and superficially resemble each other in that they are frequently located in the proximal colon and share features such as circumscribed tumour margins and tumour-infiltrating lymphocytes. However, significant differences can be demonstrated at the molecular level including widespread promoter hypermethylation and BRAF -activating mutations which occur significantly less often in HNPCC. AIMS: In this study, we sought to determine whether the presence of widespread promoter hypermethylation and BRAF mutations would exclude HNPCC. MATERIALS AND METHODS: We investigated the methylation status of four methylated in tumour markers (MINTs 1,2,12 and 31), and the promoter regions of 5 genes hMLH1, HPP1, MGMT, p16INK4A and p14ARF, in 21 sporadic MSI-H colorectal cancers and compared these with 18 cancers from HNPCC patients. The methylation status of CpG islands were determined by either methylation specific PCR (MSP) or combined bisulfite restricton analysis (COBRA). In addition we considered the BRAF mutation status of 18 HNPCC tumours and 19 sporadic MSI-H cancers which had been previously determined by RFLP analysis and confirmatory sequencing. RESULTS: Methylation of the promoter regions in target genes occurred less frequently within the HNPCC tumours (27% of analyses), compared with the sporadic MSI-H tumours (59% of analyses) (P < 0.001). Methylation of MINTs 1, 2, 12 and 31 occurred in 4% of analyses for HNPCC tumours contrasted with 73% for sporadic MSI-H tumours (P < 0.001). BRAF mutations were detected in 74% of sporadic tumours but none of the HNPCC cancers tested. CONCLUSIONS: The total number of genes and MINTs methylated in HNPCC was lower than in MSI-H colorectal tumours. No HNPCC tumour showed evidence of widespread promoter hypermethylation or BRAF mutation suggesting this feature could be used as a discriminator between familial and sporadic cases.

BRAF mutation is associated with DNA methylation in serrated polyps and cancers of the colorectum.

BACKGROUND AND AIMS: Mutations in BRAF have been linked with colorectal cancers (CRC) showing high level microsatellite instability (MSI-H). However, the distribution of BRAF mutations in MSI-H cancers remains to be clarified with respect to precursor lesions and the CpG island methylator phenotype (CIMP). METHODS: Forty three hyperplastic polyps (HP), nine mixed polyps (MP), five serrated adenomas (SA), 28 conventional adenomas (AD), 18 hereditary non-polyposis colorectal cancers (HNPCC), and 127 sporadic CRC (46 MSI-H and 81 non-MSI-H) were collected from patients undergoing colectomy for either CRC or hyperplastic polyposis. Twenty five of 57 serrated lesions were derived from four patients with hyperplastic polyposis. HP were further subdivided according to recently documented morphological criteria into 27 classical HP and 16 variant lesions described as "sessile serrated adenoma" (SSA). All tumours were screened for BRAF activating mutations. RESULTS: The BRAF mutation was more frequent in SSA (75%) and MP (89%) than in classical HP (19%), SA (20%), and AD (0%) (p<0.0001), and also in sporadic MSI-H cancers (76%) compared with HNPCC (0%) and sporadic non-MSI-H cancers (9%) (p<0.0001). The BRAF mutation was identified more often in CIMP-high serrated polyps (72%) and CIMP-high CRC (77%) than in CIMP-low (30%) and CIMP-negative (13%) polyps (p = 0.002) as well as CIMP-low (18%) and CIMP-negative (0%) CRC (p<0.0001). CONCLUSIONS: The BRAF mutation was frequently seen in SSA and in sporadic MSI-H CRC, both of which were associated with DNA methylation. Sporadic MSI-H cancers may originate in SSA and not adenomas, and BRAF mutation and DNA methylation are early events in this "serrated" pathway.

APC mutation and tumour budding in colorectal cancer.

AIM: To determine the frequency of tumour budding and somatic APC mutation in a series of colorectal cancers stratified according to DNA microsatellite instability (MSI) status. MATERIAL/METHODS: Ninety five colorectal cancers were genotyped for APC mutation in the mutation cluster region (exon 15) and scored for the presence of tumour budding at the invasive margin in haematoxylin and eosin stained sections. A subset was immunostained for beta catenin and p16. RESULTS: The frequency of both somatic APC mutation and tumour budding increased pari passu in cancers stratified as sporadic MSI high (MSI-H), hereditary non-polyposis colorectal cancer (HNPCC), MSI low (MSI-L), and microsatellite stable (MSS). Both budding and APC mutation were significantly less frequent in sporadic MSI-H cancers than in MSI-L or MSS cancers. Tumour buds were characterised by increased immunostaining for both beta catenin and p16. CONCLUSION: Tumour budding is associated with an adverse prognosis. The lack of budding in MSI-H colorectal cancer may account for the improved prognosis of this subset and may be explained by an intact WNT signalling pathway and/or inactivated p16(INK4a).