MethPanel: a parallel pipeline and interactive analysis tool for multiplex bisulphite PCR sequencing to assess DNA methylation biomarker panels for disease detection.

SUMMARY: DNA methylation patterns in a cell are associated with gene expression and the phenotype of a cell, including disease states. Bisulphite PCR sequencing is commonly used to assess the methylation profile of genomic regions between different cells. Here we have developed MethPanel, a computational pipeline with an interactive graphical interface to rapidly analyse multiplex bisulphite PCR sequencing data. MethPanel comprises a complete analysis workflow from genomic alignment to DNA methylation calling and supports an unlimited number of PCR amplicons and input samples. MethPanel offers important and unique features, such as calculation of an epipolymorphism score and bisulphite PCR bias correction capabilities, and is designed so that the methylation data from all samples can be processed in parallel. The outputs are automatically forwarded to a shinyApp for convenient display, visualization and remotely sharing data with collaborators and clinicians. AVAILABILITYAND IMPLEMENTATION: MethPanel is freely available at https://github.com/thinhong/MethPanel. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Genotype-dependent associations between serotonin transporter gene (SLC6A4) DNA methylation and late-life depression.

BACKGROUND: Disrupted serotonergic signaling is often a feature of depression and the role of the serotonin transporter gene (SLC6A4), responsible for serotonin re-uptake, has received much attention in this regard. Most studies have focused on the polymorphic 5-HTTLPR upstream repeat, or DNA methylation at the promoter CpG island. Few studies have explored the influence of genetic variation across the gene on DNA methylation, and their combined association with depression risk. The aim of this study was to determine whether genetic variation in the SLC6A4 gene influences promoter DNA methylation, and whether these are associated with depression status. METHOD: The ESPRIT study involves a community-based population of older individuals (> 65 years of age). Major depressive disorder (MDD) was diagnosed according to DSM-IV (American Psychiatric Association, 1994) criteria, and severe depressive symptoms assessed by the Centre for Epidemiological Studies Depression (CES-D) Scale. Sequenom MassARRAY was used to measure SLC6A4 methylation status (n = 302). RESULTS: Nominally significant associations were observed between SLC6A4 genetic variants (5-HTTLPR, rs140700, rs4251417, rs6354, rs25528, rs25531) and DNA methylation at several CpG sites. In multivariate regression, DNA methylation was associated with depression status, but only in the presence of specific genotypes. In individuals homozygous for the short 5-HTTLPR and 5-HTTLPR/r25531 alleles, lower methylation at two CpGs was associated with depression (ß = - 0.44 to ß = - 0.31; p = 0.001 to p = 0.038). CONCLUSION: We present evidence for genotype-dependent associations between SLC6A4 methylation and depression. Genetic variants may also play a role in influencing promoter methylation levels and its association with depression.

Comprehensive methylome sequencing reveals prognostic epigenetic biomarkers for prostate cancer mortality.

BACKGROUND: Prostate cancer is a clinically heterogeneous disease with a subset of patients rapidly progressing to lethal-metastatic prostate cancer. Current clinicopathological measures are imperfect predictors of disease progression. Epigenetic changes are amongst the earliest molecular changes in tumourigenesis. To find new prognostic biomarkers to enable earlier intervention and improved outcomes, we performed methylome sequencing of DNA from patients with localised prostate cancer and long-term clinical follow-up. METHODS: We used whole-genome bisulphite sequencing (WGBS) to comprehensively map and compare DNA methylation of radical prostatectomy tissue between patients with lethal disease (n = 7) and non-lethal (n = 8) disease (median follow-up 19.5 years). Validation of differentially methylated regions (DMRs) was performed in an independent cohort (n = 185, median follow-up 15 years) using targeted multiplex bisulphite sequencing of candidate regions. Survival was assessed via univariable and multivariable analyses including clinicopathological measures (log-rank and Cox regression models). RESULTS: WGBS data analysis identified cancer-specific methylation patterns including CpG island hypermethylation, and hypomethylation of repetitive elements, with increasing disease risk. We identified 1420 DMRs associated with prostate cancer-specific mortality (PCSM), which showed enrichment for gene sets downregulated in prostate cancer and de novo methylated in cancer. Through comparison with public prostate cancer datasets, we refined the DMRs to develop an 18-gene prognostic panel. Applying this panel to an independent cohort, we found significant associations between PCSM and hypermethylation at EPHB3, PARP6, TBX1, MARCH6 and a regulatory element within CACNA2D4. Strikingly in a multivariable model, inclusion of CACNA2D4 methylation was a better predictor of PCSM versus grade alone (Harrell's C-index: 0.779 vs. 0.684). CONCLUSIONS: Our study provides detailed methylome maps of non-lethal and lethal prostate cancer and identifies novel genic regions that distinguish these patient groups. Inclusion of our DNA methylation biomarkers with existing clinicopathological measures improves prognostic models of prostate cancer mortality, and holds promise for clinical application.

Identification of DNA methylation biomarkers with potential to predict response to neoadjuvant chemotherapy in triple-negative breast cancer.

Neoadjuvant chemotherapy (NAC) is used to treat triple-negative breast cancer (TNBC) prior to resection. Biomarkers that accurately predict a patient's response to NAC are needed to individualise therapy and avoid chemotoxicity from unnecessary chemotherapy. We performed whole-genome DNA methylation profiling on diagnostic TNBC biopsy samples from the Sequential Evaluation of Tumours Undergoing Preoperative (SETUP) NAC study. We found 9 significantly differentially methylated regions (DMRs) at diagnosis which were associated with response to NAC. We show that 4 of these DMRs are associated with TNBC overall survival (P < 0.05). Our results highlight the potential of DNA methylation biomarkers for predicting NAC response in TNBC.

Advances in Prognostic Methylation Biomarkers for Prostate Cancer.

There is a major clinical need for accurate biomarkers for prostate cancer prognosis, to better inform treatment strategies and disease monitoring. Current clinically recognised prognostic factors, including prostate-specific antigen (PSA) levels, lack sensitivity and specificity in distinguishing aggressive from indolent disease, particularly in patients with localised intermediate grade prostate cancer. There has therefore been a major focus on identifying molecular biomarkers that can add prognostic value to existing markers, including investigation of DNA methylation, which has a known role in tumorigenesis. In this review, we will provide a comprehensive overview of the current state of DNA methylation biomarker studies in prostate cancer prognosis, and highlight the advances that have been made in this field. We cover the numerous studies into well-established candidate genes, and explore the technological transition that has enabled hypothesis-free genome-wide studies and the subsequent discovery of novel prognostic genes.

Comprehensive evaluation of targeted multiplex bisulphite PCR sequencing for validation of DNA methylation biomarker panels.

BACKGROUND: DNA methylation is a well-studied epigenetic mark that is frequently altered in diseases such as cancer, where specific changes are known to reflect the type and severity of the disease. Therefore, there is a growing interest in assessing the clinical utility of DNA methylation as a biomarker for diagnosing disease and guiding treatment. The development of an accurate loci-specific methylation assay, suitable for use on low-input clinical material, is crucial for advancing DNA methylation biomarkers into a clinical setting. A targeted multiplex bisulphite PCR sequencing approach meets these needs by allowing multiple DNA methylated regions to be interrogated simultaneously in one experiment on limited clinical material. RESULTS: Here, we provide an updated protocol and recommendations for multiplex bisulphite PCR sequencing (MBPS) assays for target DNA methylation analysis. We describe additional steps to improve performance and reliability: (1) pre-sequencing PCR optimisation which includes assessing the optimal PCR cycling temperature and primer concentration and (2) post-sequencing PCR optimisation to achieve uniform coverage of each amplicon. We use a gradient of methylated controls to demonstrate how PCR bias can be assessed and corrected. Methylated controls also allow assessment of the sensitivity of methylation detection for each amplicon. Here, we show that the MBPS assay can amplify as little as 0.625 ng starting DNA and can detect methylation differences of 1% with a sequencing coverage of 1000 reads. Furthermore, the multiplex bisulphite PCR assay can comprehensively interrogate multiple regions on 1-5 ng of formalin-fixed paraffin-embedded DNA or circulating cell-free DNA. CONCLUSIONS: The MBPS assay is a valuable approach for assessing methylated DNA regions in clinical samples with limited material. The optimisation and additional quality control steps described here improve the performance and reliability of this method, advancing it towards potential clinical applications in biomarker studies.

Differential methylation pattern of xenobiotic metabolizing genes and susceptibility to Balkan endemic nephropathy, in a cohort of Romanian patients.

A severe, chronic and irreversible kidney disease affecting discrete rural populations in the Balkan Peninsula countries, Balkan endemic nephropathy (BEN) has been a scientific puzzle for more than half a century. Many environmental and other factors have been suggested as the primary cause and recent significant findings have linked BEN to aristolochic acids, phytotoxins derived from the plant Aristolochia clematitis, found in high density in the endemic areas. However, given that the incidence of BEN is less than 10% in affected villages, and it tends to have a family aggregation, as yet unidentified genetic factors may also play a role. To further explore this possibility, a pilot study was initiated to investigate the DNA methylation of CYP1A1, CYP1A2, NAT1, NQO1 and GSTT1 in blood samples from a group of Romanian BEN patients, compared to healthy controls and non-BEN chronic kidney disease (CKD) subjects. Our study revealed a more pronounced hypomethylation pattern in BEN and non-BEN CKD groups, compared to the healthy control group at specific CpGs across all five genes interrogated. Average methylation across the five regions investigated indicated significant differences only at GSTT1, in both BEN patients (p = 0.028) and non-BEN disease subjects (p = 0.015), relative to healthy individuals. Since GSTT1 active genotype appears to be a common feature of Serbian and Romanian BEN patients, GSTT1 epigenetic variation and increased gene activity could act as a predisposing (co)factor in BEN populations from the affected countries. BEN and non-BEN CKD groups show similar methylation patterns with exception of GSTT1 CpG8 (p = 0.046).

DNA methylation and genetic variation of the angiotensin converting enzyme (ACE) in depression.

BACKGROUND: Depression is one of the most prevalent psychiatric disorders, and in older persons is associated with high levels of comorbidity and under-treatment. Dysfunction of the hypothalamic-pituitary-adrenal (HPA) stress axis is consistently observed in the older population as well as depressed patients, with the angiotensin converting enzyme (ACE) a key regulator of the stress response. Epigenetic regulation of ACE may play an important role in HPA axis (dys)regulation. OBJECTIVE: To investigate ACE promoter methylation as a biomarker of late-life depression, and its association with genetic variation and cortisol secretion. METHOD: The longitudinal general population ESPRIT study is aimed at investigating psychiatric disorders in older persons (n=1863, average age=73). Depression was assessed using the Mini International Neuropsychiatric Interview according to DSM-IV criteria and the Centre for Epidemiologic Studies Depression Scale (CES-D). Genotype information for seven polymorphisms across the ACE gene was also available. Blood and saliva samples collected at baseline and used to extract DNA and measure cortisol, respectively. Sequenom MassARRAY was used to measure promoter DNA methylation of the ACE gene (n=552). RESULTS: There was no evidence of an association between ACE promoter methylation and depression. However, there was evidence that ACE genetic variants influenced methylation, and modified the association between depression and methylation (Δ at various sites; -2.05% to 1.74%; p=0.019 to 0.039). Multivariate analyses were adjusted for participants' lifestyle, health and medical history. Independent of depression status, ACE methylation was inversely correlated with cortisol levels (r=-0.336, p=0.042). CONCLUSION: This study provides evidence that associations between ACE methylation and depression are genotype-dependent, suggesting that the development of reliable depression biomarkers may need to consider methylation levels in combination with underlying genetic variation. ACE methylation may also be a suitable biomarker of cortisol and/or HPA axis activity.