The benefits of implementing high throughput sequencing in the clinic are quickly becoming apparent. However, few freely available bioinformatics pipelines have been built from the ground up with clinical genomics in mind. Here we present... more
The benefits of implementing high throughput sequencing in the clinic are quickly becoming apparent. However, few freely available bioinformatics pipelines have been built from the ground up with clinical genomics in mind. Here we present Cpipe, a pipeline designed specifically for clinical genetic disease diagnostics. Cpipe was developed by the Melbourne Genomics Health Alliance, an Australian initiative to promote common approaches to genomics across healthcare institutions. As such, Cpipe has been designed to provide fast, effective and reproducible analysis, while also being highly flexible and customisable to meet the individual needs of diverse clinical settings. Cpipe is being shared with the clinical sequencing community as an open source project and is available at http://cpipeline.org.
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Background Genomic technologies can be subject to significant batch-effects which are known to reduce experimental power and to potentially create false positive results. The Illumina Infinium Methylation BeadChip is a popular technology... more
Background Genomic technologies can be subject to significant batch-effects which are known to reduce experimental power and to potentially create false positive results. The Illumina Infinium Methylation BeadChip is a popular technology choice for epigenome-wide association studies (EWAS), but presently, little is known about the nature of batch-effects on these designs. Given the subtlety of biological phenotypes in many EWAS, control for batch-effects should be a consideration. Results Using the batch-effect removal approaches in the ComBat and Harman software, we examined two in-house datasets and compared results with three large publicly available datasets, (1214 HumanMethylation450 and 1094 MethylationEPIC BeadChips in total), and find that despite various forms of preprocessing, some batch-effects persist. This residual batch-effect is associated with the day of processing, the individual glass slide and the position of the array on the slide. Consistently across all dataset...
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Background DNA methylation has been shown to be associated with adiposity in adulthood. However, whether similar DNA methylation patterns are associated with childhood and adolescent body mass index (BMI) is largely unknown. More insight... more
Background DNA methylation has been shown to be associated with adiposity in adulthood. However, whether similar DNA methylation patterns are associated with childhood and adolescent body mass index (BMI) is largely unknown. More insight into this relationship at younger ages may have implications for future prevention of obesity and its related traits. Methods We examined whether DNA methylation in cord blood and whole blood in childhood and adolescence was associated with BMI in the age range from 2 to 18 years using both cross-sectional and longitudinal models. We performed meta-analyses of epigenome-wide association studies including up to 4133 children from 23 studies. We examined the overlap of findings reported in previous studies in children and adults with those in our analyses and calculated enrichment. Results DNA methylation at three CpGs (cg05937453, cg25212453, and cg10040131), each in a different age range, was associated with BMI at Bonferroni significance, P < 1....
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ABSTRACTBackgroundEpigenetic aging is associated with higher risk of cardiovascular disease, cancer, and all-cause mortality and may be a mechanistic link between early-life exposures, such as maternal dietary characteristics during... more
ABSTRACTBackgroundEpigenetic aging is associated with higher risk of cardiovascular disease, cancer, and all-cause mortality and may be a mechanistic link between early-life exposures, such as maternal dietary characteristics during pregnancy, and risk of adult disease.ObjectivesWe sought to determine the early-life risk factors for newborn epigenetic aging, specifically maternal dietary macronutrient intake, and whether epigenetic aging is associated with cardiovascular health markers in the newborn.MethodsEpigenetic age acceleration of 169 newborns was measured from saliva using the Horvath age calculator. Maternal diet during pregnancy was assessed using food-frequency questionnaires.ResultsNewborns with positive age acceleration were more likely to be female and have greater body fatness. Maternal intakes of saturated fat [6.2 wk epigenetic age acceleration (95% CI: 1.0, 11.3) per 5% of energy; P = 0.02] and monounsaturated fat [12.4 wk (95% CI: 4.2, 20.5) per 5% of energy; P = ...
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Significance First, our work provides critical biological interpretation of intermediate DNA methylation readouts at the nc886 differentially methylated region (DMR). nc886 was identified in multiple large-scale epigenome-wide association... more
Significance First, our work provides critical biological interpretation of intermediate DNA methylation readouts at the nc886 differentially methylated region (DMR). nc886 was identified in multiple large-scale epigenome-wide association studies (EWAS) that did not recognize that this region acts as a contiguous DMR imposed by genomic imprinting, highlighting the need to reexamine several 450k data sets. Second, strict control of genomic imprinting was thought to be required for organismal viability. Reports of polymorphic imprinting are limited to specific tissue types such as placenta and brain. In blood and somatic tissues, we show nc886 imprinting is mosaic in the population and influenced by maternal environment.
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Over the last few years a number of restriction enzymes that cut DNA only if cytosines within their recognition sequences are methylated have been characterized and become commercially available. Cleavage with these enzymes to release DNA... more
Over the last few years a number of restriction enzymes that cut DNA only if cytosines within their recognition sequences are methylated have been characterized and become commercially available. Cleavage with these enzymes to release DNA fragments in a methylation-dependent manner can be combined with a novel method of amplification, Helper Dependent Chain Reaction (HDCR), to selectively amplify these fragments. HDCR uses "Helper" oligonucleotides as templates for extension of the free 3' end of target fragments to incorporate tag sequences at the ends of fragments. These tag sequences are then used for priming of amplification of target fragments. Modifications to the amplification primers (Drivers) and the Helpers ensure that there is selection for the sequences within target fragments with each cycle of amplification. The combination of methylation-dependent enzymes and HDCR allows the sensitive and selective amplification of methylated DNA sequences without the ne...
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Solid tumors shed DNA into circulation, and there is growing evidence that the detection of circulating tumor DNA (ctDNA) has broad clinical utility, including monitoring of disease, prognosis, response to chemotherapy and tracking tumor... more
Solid tumors shed DNA into circulation, and there is growing evidence that the detection of circulating tumor DNA (ctDNA) has broad clinical utility, including monitoring of disease, prognosis, response to chemotherapy and tracking tumor heterogeneity. The appearance of ctDNA in the circulating cell-free DNA (ccfDNA) isolated from plasma or serum is commonly detected by identifying tumor-specific features such as insertions, deletions, mutations and/or aberrant methylation. Methylation is a normal cell regulatory event, and since the majority of ccfDNA is derived from white blood cells (WBC), it is important that tumour-specific DNA methylation markers show rare to no methylation events in WBC DNA. We have used a novel approach for assessment of low levels of DNA methylation in WBC DNA. DNA methylation in 29 previously identified regions (residing in 17 genes) was analyzed in WBC DNA and eight differentially-methylated regions (DMRs) were taken through to testing in clinical samples...
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Combined Bisulfite Restriction Analysis (COBRA) quantifies DNA methylation at a specific locus. It does so via digestion of PCR amplicons produced from bisulfite-treated DNA, using a restriction enzyme that contains a cytosine within its... more
Combined Bisulfite Restriction Analysis (COBRA) quantifies DNA methylation at a specific locus. It does so via digestion of PCR amplicons produced from bisulfite-treated DNA, using a restriction enzyme that contains a cytosine within its recognition sequence, such as TaqI. Here, we introduce COBRA-seq, a genome wide reduced methylome method that requires minimal DNA input (0.1-1.0 mg) and can either use PCR or linear amplification to amplify the sequencing library. Variants of COBRA-seq can be used to explore CpG-depleted as well as CpG-rich regions in vertebrate DNA. The choice of enzyme influences enrichment for specific genomic features, such as CpG-rich promoters and CpG islands, or enrichment for less CpG dense regions such as enhancers. COBRA-seq coupled with linear amplification has the additional advantage of reduced PCR bias by producing full length fragments at high abundance. Unlike other reduced representative methylome methods, COBRA-seq has great flexibility in the cho...
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Genome-wide linkage studies have identified the 9q22 chromosomal region as linked with colorectal cancer (CRC) predisposition. A candidate gene in this region is transforming growth factor β receptor 1 (TGFBR1). Investigation of TGFBR1... more
Genome-wide linkage studies have identified the 9q22 chromosomal region as linked with colorectal cancer (CRC) predisposition. A candidate gene in this region is transforming growth factor β receptor 1 (TGFBR1). Investigation of TGFBR1 has focused on the common genetic variant rs11466445, a short exonic deletion of nine base pairs which results in truncation of a stretch of nine alanine residues to six alanine residues in the gene product. While the six alanine (*6A) allele has been reported to be associated with increased risk of CRC in some population based study groups this association remains the subject of robust debate. To date, reports have been limited to population-based case-control association studies, or case-control studies of CRC families selecting one affected individual per family. No study has yet taken advantage of all the genetic information provided by multiplex CRC families. We have tested for an association between rs11466445 and risk of CRC using several famil...
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Argonautes are highly conserved proteins found in almost all eukaryotes and some bacteria and archaea. In humans, there are eight argonaute proteins evenly distributed across two clades, the Ago clade (AGO1-4) and the Piwi clade... more
Argonautes are highly conserved proteins found in almost all eukaryotes and some bacteria and archaea. In humans, there are eight argonaute proteins evenly distributed across two clades, the Ago clade (AGO1-4) and the Piwi clade (PIWIL1-4). The function of Ago proteins is best characterized by their role in RNA interference (RNAi) and cytoplasmic post-transcriptional gene silencing (PTGS) - which involves the loading of siRNA or miRNA into argonaute to direct silencing of genes at the posttranscriptional or translational level. However, nuclear-localized, as opposed to cytoplasmic, argonaute-small RNA complexes may also orchestrate the mechanistically very different process of transcriptional gene silencing, which results in prevention of transcription from a gene locus by the formation of silent chromatin domains. More recently, the role of argonaute in other aspects of epigenetic regulation of chromatin, alternative splicing and DNA repair is emerging. This review focuses on the a...
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An important feature of cancer development and progression is the change in DNA methylation patterns, characterized by the hypermethylation of specific genes concurrently with an overall decrease in the level of 5-methylcytosine.... more
An important feature of cancer development and progression is the change in DNA methylation patterns, characterized by the hypermethylation of specific genes concurrently with an overall decrease in the level of 5-methylcytosine. Hypomethylation of the genome can affect both single-copy genes, repeat DNA sequences and transposable elements, and is highly variable among and within cancer types. Here, we review our current understanding of genome hypomethylation in cancer, with a particular focus on hypomethylation of the different classes and families of repeat sequences. The emerging data provide insights into the importance of methylation of different repeat families in the maintenance of chromosome structural integrity and the fidelity of normal transcriptional regulation. We also consider the events underlying cancer-associated hypomethylation and the potential for the clinical use of characteristic DNA methylation changes in diagnosis, prognosis or classification of tumors.
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The 13th American Association of Cancer Research (AACR) Special Conference on Cancer Epigenetics was held in San Juan, Puerto Rico, on January 20–23, 2010. This event heralded insights arising from the large sequencing efforts within the... more
The 13th American Association of Cancer Research (AACR) Special Conference on Cancer Epigenetics was held in San Juan, Puerto Rico, on January 20–23, 2010. This event heralded insights arising from the large sequencing efforts within the NIH Epigenetics Roadmap Project and the Cancer Genome Atlas Project, as well as important new discoveries in the basic biology of epigenetics and results of epigenetic drug clinical trials. A summary of the recent findings is presented here, with particular emphasis on the major themes of the conference. Cancer Res; 70(19); 7372–8. ©2010 AACR.
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Background Tandem repeats (TRs) are unstable regions commonly found within genomes that have consequences for evolution and disease. In humans, polymorphic TRs are known to cause neurodegenerative and neuromuscular disorders as well as... more
Background Tandem repeats (TRs) are unstable regions commonly found within genomes that have consequences for evolution and disease. In humans, polymorphic TRs are known to cause neurodegenerative and neuromuscular disorders as well as being associated with complex diseases such as diabetes and cancer. If present in upstream regulatory regions, TRs can modify chromatin structure and affect transcription; resulting in altered gene expression and protein abundance. The most common TRs are short tandem repeats (STRs), or microsatellites. Promoter located STRs are considerably more polymorphic than coding region STRs. As such, they may be a common driver of phenotypic variation. To study STRs located in regulatory regions, we have performed genome-wide analysis to identify all STRs present in a region that is 2 kilobases upstream and 1 kilobase downstream of the transcription start sites of genes. Results The Short Tandem Repeats in Regulatory Regions Table, STaRRRT, contains the result...
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Background The 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... more
Background The 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. Methods Combined 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 perip...
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The biochemical mechanism of short RNA-induced TGS (transcriptional gene silencing) in mammals is unknown. Two competing models exist; one suggesting that the short RNA interacts with a nascent transcribed RNA strand (RNA–RNA model) and... more
The biochemical mechanism of short RNA-induced TGS (transcriptional gene silencing) in mammals is unknown. Two competing models exist; one suggesting that the short RNA interacts with a nascent transcribed RNA strand (RNA–RNA model) and the other implying that short RNA forms a heteroduplex with DNA from the unwound double helix, an R-loop structure (RNA–DNA model). Likewise, the requirement for DNA methylation to enact TGS is still controversial. In vitro assays using purified recombinant murine Dnmt (DNA methyltransferase) 1-dN (where dN indicates an N-terminal truncation), 3a and 3b enzymes and annealed oligonucleotides were designed to question whether Dnmts methylate DNA in a RNA–DNA heteroduplex context and whether a RNA–DNA heteroduplex R-loop is a good substrate for Dnmts. Specifically, model synthetic oligonucleotides were used to examine methylation of single-stranded oligonucleotides, annealed oligonucleotide duplexes, RNA–DNA heteroduplexes, DNA bubbles and R-loops. Dnmt...
Research Interests: Molecular Biology, Biology, Medicine, Gene Silencing, Biological Sciences, and 13 moreMethylation, DNA methyltransferases, DNA methylation, CpG islands, Post-Transcriptional Gene Regulation, Biochemical, CHEMICAL SCIENCES, Substrate Specificity, Recombinant Proteins, Sulfates, DNMT, Restriction Mapping, and Medical and Health Sciences
The CAHM gene (Colorectal Adenocarcinoma HyperMethylated), previously LOC100526820, is located on chromosome 6, hg19 chr6:163 834 097-163 834 982. It lacks introns, encodes a long non-coding RNA (lncRNA) and is located adjacent to the... more
The CAHM gene (Colorectal Adenocarcinoma HyperMethylated), previously LOC100526820, is located on chromosome 6, hg19 chr6:163 834 097-163 834 982. It lacks introns, encodes a long non-coding RNA (lncRNA) and is located adjacent to the gene QKI, which encodes an RNA binding protein. Deep bisulphite sequencing of ten colorectal cancer (CRC) and matched normal tissues demonstrated frequent hypermethylation within the CAHM gene in cancer. A quantitative methylation-specific PCR (qMSP) was used to characterize additional tissue samples. With a threshold of 5% methylation, the CAHM assay was positive in 2/26 normal colorectal tissues (8%), 17/21 adenomas (81%), and 56/79 CRC samples (71%). A reverse transcriptase-qPCR assay showed that CAHM RNA levels correlated negatively with CAHM % methylation, and therefore CAHM gene expression is typically decreased in CRC. The CAHM qMSP assay was applied to DNA isolated from plasma specimens from 220 colonoscopy-examined patients. Using a threshold ...
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Up to 25% of colorectal cancer (CRC) may be caused by inherited genetic variants that have yet to be identified. Previous genome-wide linkage studies (GWLSs) have identified a new loci postulated to contain novel CRC risk genes amongst... more
Up to 25% of colorectal cancer (CRC) may be caused by inherited genetic variants that have yet to be identified. Previous genome-wide linkage studies (GWLSs) have identified a new loci postulated to contain novel CRC risk genes amongst affected families carrying no identifiable mutations in any of the known susceptibility genes for familial CRC syndromes. To undertake a new GWLS, we recruited members from 54 non-syndromic families from Australia and Spain where at least two first-degree relatives were affected by CRC. We used single-nucleotide polymorphism arrays to genotype 98 concordant affected relative pairs that were informative for linkage analyses. We tested for genome-wide significance (GWS) for linkage to CRC using a quantile statistic method, and we found that GWS was achieved at the 5% level. Independently, using the PSEUDO gene-dropping algorithm, we also found that GWS for linkage to CRC was achieved (P=0.02). Merlin non-parametric linkage analysis revealed significant linkage to CRC for chromosomal region 10p15.3–p15.1 and suggestive linkage to CRC for regions on 14q and 9q. The 10p15.3–p15.1 has not been reported to be linked to hereditary CRC in previous linkage studies, but this region does harbour the Kruppel-like factor 6 (KLF6) gene that is known to be altered in common CRC. Further studies aimed at localising the responsible genes, and characterising their function will give insight into the factors responsible for susceptibility in such families, and perhaps shed further light on the mechanisms of CRC development.
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An important feature of cancer development and progression is the change in DNA methylation patterns, characterized by the hypermethylation of specific genes concurrently with an overall decrease in the level of 5-methylcytosine.... more
An important feature of cancer development and progression is the change in DNA methylation patterns, characterized by the hypermethylation of specific genes concurrently with an overall decrease in the level of 5-methylcytosine. Hypomethylation of the genome can affect both single-copy genes, repeat DNA sequences and transposable elements, and is highly variable among and within cancer types. Here, we review our current understanding of genome hypomethylation in cancer, with a particular focus on hypomethylation of the different classes and families of repeat sequences. The emerging data provide insights into the importance of methylation of different repeat families in the maintenance of chromosome structural integrity and the fidelity of normal transcriptional regulation. We also consider the events underlying cancer-associated hypomethylation and the potential for the clinical use of characteristic DNA methylation changes in diagnosis, prognosis or classification of tumors.