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Fluoropyrimidines (FP; 5-fluorouracil, capecitabine, and tegafur) are a commonly prescribed class of antimetabolite chemotherapies, used for various solid organ malignancies in over 2 million patients globally per annum. Dihydropyrimidine dehydrogenase (DPD), encoded by the DPYD gene, is the critical enzyme implicated in FP metabolism. DPYD variant genotypes can result in decreased DPD production, leading to the development of severe toxicities resulting in hospitalization, intensive care admission, and even death. Management of toxicity incurs financial burden on both patients and healthcare systems alike. Upfront DPYD genotyping to identify variant carriers allows an opportunity to identify patients who are at high risk to suffer from serious toxicities and allow prospective dose adjustment of FP treatment. This approach has been shown to reduce patient morbidity, as well as improve the cost-effectiveness of managing FP treatment. Upfront DPYD genotyping has been recently endorsed by several countries in Europe and the United Kingdom. This review summarizes current knowledge about DPD deficiency and upfront DPYD genotyping, including clinical and cost-effectiveness outcomes, with the intent of supporting implementation of an upfront DPYD genotyping service with individualized dose-personalization.

Sharing genomic variant interpretations across laboratories promotes consistency in variant assertions. A landscape analysis of Australian clinical genetic-testing laboratories in 2017 identified that, despite the national-accreditation-body recommendations encouraging laboratories to submit genotypic data to clinical databases, fewer than 300 variants had been shared to the ClinVar public database. Consultations with Australian laboratories identified resource constraints limiting routine application of manual processes, consent issues, and differences in interpretation systems as barriers to sharing. This information was used to define key needs and solutions required to enable national sharing of variant interpretations. The Shariant platform, using both the GRCh37 and GRCh38 genome builds, was developed to enable ongoing sharing of variant interpretations and associated evidence between Australian clinical genetic-testing laboratories. Where possible, two-way automated sharing was implemented so that disruption to laboratory workflows would be minimized. Terms of use were developed through consultation and currently restrict access to Australian clinical genetic-testing laboratories. Shariant was designed to store and compare structured evidence, to promote and record resolution of inter-laboratory classification discrepancies, and to streamline the submission of variant assertions to ClinVar. As of December 2021, more than 14,000 largely prospectively curated variant records from 11 participating laboratories have been shared. Discrepant classifications have been identified for 11% (28/260) of variants submitted by more than one laboratory. We have demonstrated that co-design with clinical laboratories is vital to developing and implementing a national variant-interpretation sharing effort. This approach has improved inter-laboratory concordance and enabled opportunities to standardize interpretation practices.

Background Accumulating evidence suggests that breastfeeding exclusivity and duration are positively associated with child cognition. This study investigated whether DNA methylation, an epigenetic mechanism modified by nutrient intake, may contribute to the link between breastfeeding and child cognition. The aim was to quantify the relationship between global DNA methylation and cognition and behavior at 4 years of age. Methods Child behavior and cognition were measured at age 4 years using the Wechsler Preschool and Primary Scale of Intelligence, third version (WPPSI‐III), and Child Behavior Checklist (CBC). Global DNA methylation (%5‐methylcytosines (%5mC)) was measured in buccal cells at age 4 years, using an enzyme‐linked immunosorbent assay (ELISA) commercial kit. Linear regression models were used to quantify the statistical relationships. Results Data were collected from 73 children recruited from the Women and Their Children's Health (WATCH) study. No statistically significant associations were found between global DNA methylation levels and child cognition or behavior (p > .05), though the estimates of effect were consistently negative. Global DNA methylation levels in males were significantly higher than in females (median %5mC: 1.82 vs. 1.03, males and females, respectively, (p, This study quantified the relationship between global DNA methylation and cognition and behavior at 4 years of age. Data were collected from 73 children indicated no statistically significant associations were found between global DNA methylation levels and child cognition or behavior (p > .05). Global DNA methylation levels in males were significantly higher than in females (median %5mC: 1.82 vs. 1.03, males and females, respectively, (p

Background: During the early postnatal period, the impact of nutrition on DNA methylation has not been well studied in humans. The aim was to quantify the relationship between one-carbon metabolism nutrient intake during the first three years of life and global DNA methylation levels at four years. Design: Childhood dietary intake was assessed using infant feeding questionnaires, food frequency questionnaires, 4-day weighed food records and 24-h food records. The dietary records were used to estimate the intake of methionine, folate, vitamins B2, B6 and B12 and choline. The accumulative nutrient intake specific rank from three months to three years of age was used for analysis. Global DNA methylation (%5-methyl cytosines (%5-mC)) was measured in buccal cells at four years of age, using an enzyme-linked immunosorbent assay (ELISA) commercial kit. Linear regression models were used to quantify the statistical relationships. Results: Data were collected from 73 children recruited from the Women and their Children’s Health (WATCH) study. No association was found between one-carbon metabolism nutrient intake and global DNA methylation levels (P > 0.05). Global DNA methylation levels in males were significantly higher than in females (median %5-mC: 1.82 vs. 1.03, males and females respectively, (P < 0.05)). Conclusion: No association was found between the intake of one-carbon metabolism nutrients during the early postnatal period and global DNA methylation levels at age four years. Higher global DNA methylation levels in males warrants further investigation.

The pyrimidine analog, 5-aza-2’-deoxycytidine (5-aza-dC) is a DNA methyltransferase inhibitor that triggers DNA demethylation leading to the reactivation of epigenetically silenced tumor suppressor genes. To understand the shift in gene expression which mediates the beneficial 5-aza-dC effects in leukemia, we have treated human myeloid derived leukemic cells with 5-aza-dC. Target genes were identified first in MV4-11 cells using a genome-wide gene expression profiling assay to detect differences in treated and untreated cells. From this analysis six genes were identified (HOXA4, HOXD4, HOXA8, HOXD12, CD9 and RGS2) as being significantly different expressed after treatment. To validate microarray data, we performed quantitative PCR on these genes from multiple leukemic cells. The results suggest that these genes are epigenetically regulated indicating that dysregulation of HOXA4, HOXD4, HOXA8, HOXD12, CD9 and RGS2 expression may play an important role in establishing the malignant phenotype in AML.

Epigenetic aberrations such as global hypomethylation and gene-specific hypermethylation are key events that underlie tumour development. Such scenarios are brought about by the loss of control of methylation patterns which typically are reversed in neoplasia in comparison to normal states. Despite the methylation process being termed epigenetic, suggesting that it is not a heritable condition, there is strong evidence in mouse models suggesting that epimutations within the germline may provide a mechanism through which methylation variations can be transmissible to offspring. The first half of the review will focus on the nature of methylation-induced gene silencing and transmission of this information through the germline. The latter half will focus on the cause of aberrant DNA methylation.

From the *Immunology and Infectious Diseases Unit, John Hunter Hospital, Newcastle, Australia; School of Biomedical Sciences, The University of Newcastle, Callaghan, Australia; Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, Australia; and Department of Immunology and Microbiology, Rush University Medical Center, Chicago, Illinois.

Schizophrenia has a strong genetic basis, and genome-wide association studies (GWAS) have shown that effect sizes for individual genetic variants which increase disease risk are small, making detection and validation of true disease-associated risk variants extremely challenging. Specifically, we first identify genes with exons showing differential expression between cases and controls, indicating a splicing mechanism that may contribute to variation in disease risk and focus on those showing consistent dif- ferential expression between blood and brain tissue. We then perform a genome-wide screen for SNPs associated with both normalised exon intensity of these genes (so called splicing QTLs) as well as as- sociation with schizophrenia. We identified a number of significantly associated loci with a biologically plausible role in schizophrenia, including MCPH1, DLG3, ZC3H13, and BICD2, and additional loci that influence splicing of these genes, including YWHAH. Our approach of integrating genome-wide exon intensity with genome-wide polymorphism data has identified a number of plausible SZ associated loci.

Mutations in the recognized breast cancer susceptibility genes BRCA1, BRCA2, TP53, ATM, and CHEK2 account for approximately 20% of hereditary breast cancer. This raises the possibility that mutations in other biologically relevant genes may be involved in genetic predisposition to breast cancer. In this study, BRIP1, PALB2, and RAD51C were sequenced for mutations as a result of previously being associated with breast cancer risk due to their role in the double-strand break repair pathway and their close association with BRCA1 and BRCA2. Two truncating mutations in PALB2 (Q66X and W1038X), one of which is has not been reported before, were detected in an independent Australian cohort of 70 individuals with breast or ovarian cancer, and have strong family histories of breast or breast/ovarian cancer. In addition, six missense variants predicted to be causative were detected, one in BRIP1 and five in PALB2. No causative variants were identified in RAD51C. This study supports recent observations that although rare, PALB2 mutations are present in a small but substantial proportion of inherited breast cancer cases, and indicates that RAD51C at a population level does not account for a substantial number of familial breast cancer cases.

UnlabelledEpigenetic regulation of genes involves the coordination of DNA methylation and histone modifications to maintain transcriptional status. These two features are frequently disrupted in malignancy such that critical genes succumb to inactivation. 5-aza-2'-deoxycytidine (5-aza-dC) is an agent which inhibits DNA methyltransferase, and holds great potential as a treatment for cancer, yet the extent of its effectiveness varies greatly between tumour types. Previous evidence suggests expression status after 5-aza-dC exposure cannot be explained by the DNA methylation status alone.AimWe sought to identify chromatin changes involved with short and long term gene reactivation following 5-aza-dC exposure. Two colorectal cancer cell lines, HCT116 and SW480, were treated with 5-aza-dC and then grown in drug-free media to allow DNA re-methylation. DNA methylation and chromatin modifications were assessed with bisulfite sequencing and Chromatin Immuno-Precipitation analysis.ResultsIncreased H3 acetylation, H3K4 tri-methylation and loss of H3K27 tri-methylation were associated with reactivation. Hypermethylated genes that did not show increased acetylation were transiently expressed with 5-aza-dC treatment before reverting to an inactive state. Three reactivated genes, CDO1, HSPC105 and MAGEA3, were still expressed 10 days post 5-aza-dC treatment and displayed localised hypomethylation at the transcriptional start site, and also an increased enrichment of histone H3 acetylation.ConclusionsThese observations suggest that hypomethylation alone is insufficient to reactivate silenced genes and that increased Histone H3 acetylation in unison with localised hypomethylation allows long term reversion of these epigenetically silenced genes. This study suggests that combined DNA methyltransferase and histone deacetylase inhibitors may aid long term reactivation of silenced genes.

Background DNA methylation and histone acetylation are epigenetic modifications that act as regulators of gene expression. Aberrant epigenetic gene silencing in tumours is a frequent event, yet the factors which dictate which genes are targeted for inactivation are unknown. DNA methylation and histone acetylation can be modified with the chemical agents 5-aza-2'-deoxycytidine (5-aza-dC) and Trichostatin A (TSA) respectively. The aim of this study was to analyse de-methylation and re-methylation and its affect on gene expression in colorectal cancer cell lines treated with 5-aza-dC alone and in combination with TSA. We also sought to identify methylation patterns associated with long term reactivation of previously silenced genes. Method Colorectal cancer cell lines were treated with 5-aza-dC, with and without TSA, to analyse global methylation decreases by High Performance Liquid Chromatography (HPLC). Re-methylation was observed with removal of drug treatments. Expression arrays identified silenced genes with differing patterns of expression after treatment, such as short term reactivation or long term reactivation. Sodium bisulfite sequencing was performed on the CpG island associated with these genes and expression was verified with real time PCR. Results Treatment with 5-aza-dC was found to affect genomic methylation and to a lesser extent gene specific methylation. Reactivated genes which remained expressed 10 days post 5-aza-dC treatment featured hypomethylated CpG sites adjacent to the transcription start site (TSS). In contrast, genes with uniformly hypermethylated CpG islands were only temporarily reactivated. Conclusion These results imply that 5-aza-dC induces strong de-methylation of the genome and initiates reactivation of transcriptionally inactive genes, but this does not require gene associated CpG island de-methylation to occur. In addition, for three of our selected genes, hypomethylation at the TSS of an epigenetically silenced gene is associated with the long term reversion of gene expression level brought about by alterations in the epigenetic status following 5-aza-dC treatment.

Hereditary non-polyposis colorectal cancer (HNPCC) is an autosomal dominantly inherited syndrome caused by germline mutations in mismatch repair (MMR) genes. HNPCC patients have a lifetime risk of 80% of developing colorectal cancer (CRC); however the likely age of onset is difficult to predict. A single C>T polymorphism located within the promoter region of the DeltaDNMT3B gene has recently been reported to be associated with a significant increase to the risk of early onset CRC. In this study we determined the DeltaDNMT3B genotype in 404 confirmed HNPCC participants (total of 194 CRC cases) from Australia (203) and Poland (201). From the total number of participants there were 194 diagnosed cases of CRC and 210 healthy MMR gene mutation carriers. The study was undertaken to assess whether the reported effect observed in a previous study of 146 HNPCC patients is consistent in a larger separate and unrelated participant cohort. Through the statistical tests of Kaplan-Meier survival analysis and Cox hazard regression models we did not observe any significant association between the DeltaDNMT3B C>T SNP and early onset CRC in HNPCC patients.