Konsta Duesing

A panel of genes methylated with high frequency in colorectal cancer

BackgroundThe development of colorectal cancer (CRC) is accompanied by extensive epigenetic changes, including frequent regional hypermethylation particularly of gene promoter regions. Specific genes, including SEPT9, VIM1 and TMEFF2 become methylated in a high fraction of cancers and diagnostic assays for detection of cancer-derived methylated DNA sequences in blood and/or fecal samples are being developed. There is considerable potential for the development of new DNA methylation biomarkers or panels to improve the sensitivity and specificity of current cancer detection tests.MethodsCombined epigenomic methods – activation of gene expression in CRC cell lines following DNA demethylating treatment, and two novel methods of genome-wide methylation assessment – were used to identify candidate genes methylated in a high fraction of CRCs. Multiplexed amplicon sequencing of PCR products from bisulfite-treated DNA of matched CRC and non-neoplastic tissue as well as healthy donor peripheral blood was performed using Roche 454 sequencing. Levels of DNA methylation in colorectal tissues and blood were determined by quantitative methylation specific PCR (qMSP).ResultsCombined analyses identified 42 candidate genes for evaluation as DNA methylation biomarkers. DNA methylation profiles of 24 of these genes were characterised by multiplexed bisulfite-sequencing in ten matched tumor/normal tissue samples; differential methylation in CRC was confirmed for 23 of these genes. qMSP assays were developed for 32 genes, including 15 of the sequenced genes, and used to quantify methylation in tumor, adenoma and non-neoplastic colorectal tissue and from healthy donor peripheral blood. 24 of the 32 genes were methylated in >50% of neoplastic samples, including 11 genes that were methylated in 80% or more CRCs and a similar fraction of adenomas.ConclusionsThis study has characterised a panel of 23 genes that show elevated DNA methylation in >50% of CRC tissue relative to non-neoplastic tissue. Six of these genes (SOX21, SLC6A15, NPY, GRASP, ST8SIA1 and ZSCAN18) show very low methylation in non-neoplastic colorectal tissue and are candidate biomarkers for stool-based assays, while 11 genes (BCAT1, COL4A2, DLX5, FGF5, FOXF1, FOXI2, GRASP, IKZF1, IRF4, SDC2 and SOX21) have very low methylation in peripheral blood DNA and are suitable for further evaluation as blood-based diagnostic markers.

Mechanism of fat taste perception: Association with diet and obesity

Energy homeostasis plays a significant role in food consumption and body weight regulation with fat intake being an area of particular interest due to its palatability and high energy density. Increasing evidence from humans and animal studies indicate the existence of a taste modality responsive to fat via its breakdown product fatty acids. These studies implicate multiple candidate receptors and ion channels for fatty acid taste detection, indicating a complex peripheral physiology that is currently not well understood. Additionally, a limited number of studies suggest a reduced ability to detect fatty acids is associated with obesity and a diet high in fat reduces an individuals ability to detect fatty acids. To support this, genetic variants within candidate fatty acid receptors are also associated with obesity reduced ability to detect fatty acids. Understanding oral peripheral fatty acid transduction mechanisms and the association with fat consumption may provide the basis of novel approaches to control development of obesity.

The role of vitamins and minerals in modulating the expression of microRNA

A growing number of studies in recent years have highlighted the importance of molecular nutrition as a potential determinant of health and disease. In particular, the ability of micronutrients to regulate the final expression of gene products via modulation of transcription and translation is now being recognised. Modulation of microRNA (miRNA) by nutrients is one pathway by which nutrition may mediate gene expression. miRNA, a class of non-coding RNA, can directly regulate gene expression post-transcriptionally. In addition, miRNA are able to indirectly influence gene expression potential at the transcriptional level via modulation of the function of components of the epigenetic machinery (DNA methylation and histone modifications). These mechanisms interact to form a complex, bi-directional regulatory circuit modulating gene expression. Disease-specific miRNA profiles have been identified in multiple disease states, including those with known dietary risk factors. Therefore, the role that nutritional components, in particular, vitamins and minerals, play in the modulation of miRNA profiles, and consequently health and disease, is increasingly being investigated, and as such is a timely subject for review. The recently posited potential for viable exogenous miRNA to enter human blood circulation from food sources adds another interesting dimension to the potential for dietary miRNA to contribute to gene modulation.

Next-generation sequencing: a challenge to meet the increasing demand for training workshops in Australia

The widespread adoption of high-throughput next-generation sequencing (NGS) technology among the Australian life science research community is highlighting an urgent need to up-skill biologists in tools required for handling and analysing their NGS data. There is currently a shortage of cutting-edge bioinformatics training courses in Australia as a consequence of a scarcity of skilled trainers with time and funding to develop and deliver training courses. To address this, a consortium of Australian research organizations, including Bioplatforms Australia, the Commonwealth Scientific and Industrial Research Organisation and the Australian Bioinformatics Network, have been collaborating with EMBL-EBI training team. A group of Australian bioinformaticians attended the train-the-trainer workshop to improve training skills in developing and delivering bioinformatics workshop curriculum. A 2-day NGS workshop was jointly developed to provide hands-on knowledge and understanding of typical NGS data analysis workflows. The road show–style workshop was successfully delivered at five geographically distant venues in Australia using the newly established Australian NeCTAR Research Cloud. We highlight the challenges we had to overcome at different stages from design to delivery, including the establishment of an Australian bioinformatics training network and the computing infrastructure and resource development. A virtual machine image, workshop materials and scripts for configuring a machine with workshop contents have all been made available under a Creative Commons Attribution 3.0 Unported License. This means participants continue to have convenient access to an environment they had become familiar and bioinformatics trainers are able to access and reuse these resources.

Vitamin D Receptor Genotype Modulates the Correlation between Vitamin D and Circulating Levels of let-7a/b and Vitamin D Intake in an Elderly Cohort

Background and Aims: Circulating microRNAs (miRNAs) are linked to disease and are potential biomarkers. Vitamin D may modulate miRNA profiles, and vitamin D status has been linked to risk of disease, including cardiovascular disease and cancers. We hypothesise that genotypic variance influences these relationships. We examined the correlations between vitamin D intake and circulating levels of the miRNAs let-7a/b, and the involvement of two common vitamin D receptor (VDR) polymorphisms, BsmI and ApaI. Methods: Two hundred participants completed food frequency and supplement questionnaires, and were assayed for circulating let-7b expression by qPCR. Polymorphisms were detected using restriction fragment length polymorphism-PCR. Results: let-7b expression negatively correlated with vitamin D intake (rs = -0.20, p = 0.005). The magnitude and direction of correlation were maintained in the presence of the BsmI restriction site (rs = -0.27, p = 0.0005). However, in the absence of BsmI restriction site, the direction of the correlation was reversed (rs = +0.319, p = 0.0497). These correlations were significantly different (z-score = 2.64, p = 0.0085). The correlation between vitamin D intake and let-7a was only significant in those without the ApaI restriction site. Conclusions: The correlation between vitamin D intake and let-7a/b expression in this cohort varies with VDR genotype. This study highlights the importance of considering underlying genotypic variance in miRNA expression studies and in nutritional epigenetics generally.

Risk-conscious correction of batch effects: maximising information extraction from high-throughput genomic datasets

BackgroundBatch effects are a persistent and pervasive form of measurement noise which undermine the scientific utility of high-throughput genomic datasets. At their most benign, they reduce the power of statistical tests resulting in actual effects going unidentified. At their worst, they constitute confounds and render datasets useless. Attempting to remove batch effects will result in some of the biologically meaningful component of the measurement (i.e. signal) being lost. We present and benchmark a novel technique, called Harman. Harman maximises the removal of batch noise with the constraint that the risk of also losing biologically meaningful component of the measurement is kept to a fraction which is set by the user.ResultsAnalyses of three independent publically available datasets reveal that Harman removes more batch noise and preserves more signal at the same time, than the current leading technique. Results also show that Harman is able to identify and remove batch effects no matter what their relative size compared to other sources of variation in the dataset. Of particular advantage for meta-analyses and data integration is Harman’s superior consistency in achieving comparable noise suppression - signal preservation trade-offs across multiple datasets, with differing number of treatments, replicates and processing batches.ConclusionHarman’s ability to better remove batch noise, and better preserve biologically meaningful signal simultaneously within a single study, and maintain the user-set trade-off between batch noise rejection and signal preservation across different studies makes it an effective alternative method to deal with batch effects in high-throughput genomic datasets. Harman is flexible in terms of the data types it can process. It is available publically as an R package (https://bioconductor.org/packages/release/bioc/html/Harman.html), as well as a compiled Matlab package (http://www.bioinformatics.csiro.au/harman/) which does not require a Matlab license to run.

A Comparison of Collection Techniques for Gene Expression Analysis of Human Oral Taste Tissue.

Variability in human taste perception is associated with both genetic and environmental factors. The influence of taste receptor expression on this variability is unknown, in part, due to the difficulty in obtaining human oral tissue that enables quantitative expression measures of taste genes. In a comparison of six current techniques (Oragene RNeasy Kit, Isohelix swab, Livibrush cytobrush, tongue saliva, cheek saliva collection, and fungiform papillae biopsy), we identify the fungiform papillae biopsy is the optimal sampling technique to analyse human taste gene expression. The fungiform papillae biopsy resulted in the highest RNA integrity, enabling amplification of all the assessed taste receptor genes (TAS1R1, TAS1R2, TAS1R3, SCNN1A and CD36) and taste tissue marker genes (NCAM1, GNAT3 and PLCβ2). Furthermore, quantitative expression was observed in a subset of taste genes assessed from the saliva collection techniques (cheek saliva, tongue saliva and Oragene RNA kit). These saliva collection techniques may be useful as a non-invasive alternative sampling technique to the fungiform papillae biopsy. Identification of the fungiform papillae biopsy as the optimal collection method will facilitate further research into understanding the effect of gene expression on variability in human taste perception.

An association between the PTGS2 rs5275 polymorphism and colorectal cancer risk in families with inherited non-syndromic predisposition

Recently our group completed a genome-wide linkage study investigating Australian and Spanish families with inherited risk of colorectal cancer (CRC). A minor linkage peak from that study located on chromosome 1 correlates with the location of a known CRC risk-modifying gene, prostaglandin synthase (PTGS2). PTGS2 encodes the inducible prostaglandin synthase enzyme cyclooxygenase-2 (COX-2). Prostaglandins are implicated in the initiation of carcinogenesis and progression of tumours. Sequencing of PTGS2 in a small subset of affected individuals identified a high frequency of the minor C allele of single nucleotide polymorphism rs5275. We then genotyped the rs5275 polymorphism in 183 affected and 223 unaffected individuals from our CRC predisposed families. Tests for association in the presence of linkage were made using family-based association tests. The C allele was found to be significantly associated (P<0.01) with diagnosis of hereditary non-syndromic CRC (P=0.0094, dominant model) and an earlier age of diagnosis (P=0.0089, heterozygous-advantage model). Interestingly, by stratifying the age of diagnosis data, we observed a speculative gender-discordant effect. Relative to other groups, female CC carriers were diagnosed less when young, but by 60 years of age were the most at risk group. Conversely, CT carriers of both genders showed a consistently earlier diagnosis relative to TT carriers. Our results suggest potential differential age-and gender-dependent efficacies of chemopreventative COX-2 inhibitors in the context of non-syndromic colorectal cancer.

Vitamin D Receptor Polymorphisms Relate to Risk of Adenomatous Polyps in a Sex-Specific Manner

ABSTRACT Vitamin D receptor (VDR) gene polymorphisms may influence risk for adenomatous polyps (AP), a benign precursor to colon cancer, via modulation of vitamin D sensitive pathways, including cell proliferation and differentiation. However, results have been mixed and any association remains contentious. Failure to clinically exclude the presence of (AP in control cohorts may contribute to the lack of consensus. Therefore, we assessed the role of the FokI, BsmI, ApaI, and TaqI VDR polymorphisms in modifying risk for AP, adjusting for a range of dietary and lifestyle variables. Blood was collected from colonoscopy patients (n = 258) and VDR polymorphisms assessed by restriction fragment length polymorphism. Dietary habits were estimated from food frequency questionnaires. Odds ratios for AP were calculated by genotype, stratified by sex, and adjusted for age, lifestyle, and dietary factors. FokI was associated with modified risk for AP in males, whereas the BsmI/ApaI/TaqI haplotype was associated with modified risk in females. No interaction was found between VDR variants and vitamin D intake. This study offers novel insight into the potential for VDR genetics to contribute to risk for AP and is the first to demonstrate a sex-specific relationship between these polymorphisms and risk for AP.

Copy number variation in hereditary non-polyposis colorectal cancer.

Hereditary non-polyposis colorectal cancer (HNPCC) is the commonest form of inherited colorectal cancer (CRC) predisposition and by definition describes families which conform to the Amsterdam Criteria or reiterations thereof. In ~50% of patients adhering to the Amsterdam criteria germline variants are identified in one of four DNA Mismatch repair (MMR) genes MLH1, MSH2, MSH6 and PMS2. Loss of function of any one of these genes results in a failure to repair DNA errors occurring during replication which can be most easily observed as DNA microsatellite instability (MSI)—a hallmark feature of this disease. The remaining 50% of patients without a genetic diagnosis of disease may harbour more cryptic changes within or adjacent to MLH1, MSH2, MSH6 or PMS2 or elsewhere in the genome. We used a high density cytogenetic array to screen for deletions or duplications in a series of patients, all of whom adhered to the Amsterdam/Bethesda criteria, to determine if genomic re-arrangements could account for a proportion of patients that had been shown not to harbour causative mutations as assessed by standard diagnostic techniques. The study has revealed some associations between copy number variants (CNVs) and HNPCC mutation negative cases and further highlights difficulties associated with CNV analysis.