Biology subjects

Ward, J. C.

Publications and source records attributed to Ward, J. C..

3 recordsLinked to original sources

bioRxiv2026

DNA methylation signatures of mismatch repair-deficient colorectal cancer

BackgroundColorectal cancer (CRC) is a molecularly heterogeneous disease shaped by both genetic and epigenetic alterations. Approximately 15% of CRCs display widespread CpG island hypermethylation, known as the CpG Island Methylator Phenotype (CIMP). CIMP-high (CIMP-H) tumours frequently exhibit MLH1 promoter hypermethylation, leading to mismatch repair deficiency (MMRd) and microsatellite instability (MSI). However, DNA methylation patterns associated with MSI, independent of CIMP and MLH1 silencing, and the influence of clinical variables such as anatomical location and patient age on the CRC methylome remain poorly characterised. MethodsWe performed epigenome-wide DNA methylation profiling of 259 primary CRC tissue samples using the Illumina EPICv2 array, comparing differential methylation between MSI and microsatellite stable (MSS) CRC, adjusting for tumour purity, MLH1 promoter methylation, CIMP status, and anatomical location, to account for known confounders. We further evaluated the independent effects of anatomical location and patient age on global methylation patterns. ResultsEpigenome-wide differential methylation between MSS and MSI CRC was dominated by MLH1 promoter hypermethylation. After adjusting for MLH1 hypermethylation and CIMP status, we identified a distinct set of 656 CpG sites associated with MMRd independent of MLH1 silencing. These included hypermethylation at LRP6, GSK3{beta}, and CDK12, implicating altered WNT signalling and transcriptional regulation pathways. Comparison of MSI subgroups revealed the co-occurrence of MLH1 hypermethylation with promoter hypermethylation at TXNRD1. Anatomical location showed a strong independent effect on methylation patterns, while we observed only modest effects of patient age on the CRC methylome after adjustment for confounders. ConclusionsWe identified a distinct methylation profile distinguishing MSS and MSI CRC, including MLH1-independent markers of MMRd, as well as novel differentially methylated loci within MSI subgroups. We further showed that anatomical location has a strong independent impact on the CRC methylome. Together, these findings refine the molecular characterisation of CRC and highlight potential epigenetic markers that could inform patient stratification and precision oncology.

cancer biology↗

bioRxiv2023

Genetic evidence of a functional linkage between the RNA-dependent RNA polymerase and the highly structured S fragment located at the 5' end of the genome of foot-and-mouth disease virus.

Secondary and tertiary RNA structures play key roles in genome replication of single stranded positive sense RNA viruses. Complex, functional structures are particularly abundant in the untranslated regions of picornaviruses, where they are involved in initiation of translation, priming of new strand synthesis and genome circularisation. The 5' UTR of foot-and-mouth disease virus (FMDV) is predicted to include a c. 360 nucleotide-long stem-loop, termed the short (S) fragment. This structure is highly conserved and essential for viral replication, but the precise function(s) are unclear. Here, we used selective 2' hydroxyl acetylation analysed by primer extension (SHAPE) to experimentally-determine aspects of the structure, alongside comparative genomic analyses to confirm structure conservation from a wide range of field isolates. To examine its role in virus replication, we introduced a series of deletions to the distal and proximal regions of the stem loop. These truncations affected genome replication in a size-dependent and, in some cases, host cell-dependent manner. Furthermore, during passage of viruses incorporating the largest tolerated deletion from the proximal region of the S fragment stem loop, an additional mutation was selected in the viral RNA-dependent RNA polymerase, 3Dpol.These data suggest that the S fragment and 3Dpol interact in the formation of the FMDV replication complex.

molecular biology↗

bioRxiv2022

Processing of the hepatitis E virus polyprotein can be mediated by a cellular protease

The genomes of positive-sense RNA viruses encode polyproteins that are essential for controlling viral replication. These viral polyproteins must undergo proteolysis (also termed polyprotein processing) to generate functional protein units. This proteolysis can be performed by virally-encoded proteases as well as host cellular proteases, and is generally believed to be a key step in regulating viral replication. Hepatitis E virus (HEV), a leading cause of acute viral hepatitis, translates its positive-sense RNA genome to generate a polyprotein, termed pORF1, which is necessary and sufficient for viral genome replication. However, the mechanism of polyprotein processing in HEV remains to be determined. In this study, we aimed to understand processing of this polyprotein and its role in viral replication using a combination of in vitro translation experiments and HEV sub-genomic replicons. Our data suggest no evidence for a virally-encoded protease or auto-proteolytic activity as in vitro translation predominantly generates unprocessed viral polyprotein precursors. However, seven cleavage sites within the polyprotein (suggested by bioinformatic analysis) are susceptible to the host cellular protease, thrombin. Using a sub-genomic replicon system, we demonstrate that mutagenesis of these sites prevents replication, as does pharmacological inhibition of serine proteases. Overall, our data supports a model where HEV uses host proteases to support its replication and could have uniquely evolved not to rely on a virally-encoded protease for replication. Author summaryPositive-strand RNA viruses produce polyproteins that are cleaved by proteases that control viral replication. The polyproteins of all well studied positive-strand viruses undergo proteolysis in a highly controlled manner to generate functional proteins and regulate the transition from translation to RNA replication. Proteolysis of viral polyproteins is generally performed by virally-encoded proteases, although host cell proteases are used by some viruses. In this report, we provide evidence that suggests that hepatitis E virus, a medically important human pathogen, does not encode a protease and unlike other viral polyproteins cannot undergo auto-catalytic processing. Instead, we provide evidence that the polyprotein is susceptible to proteolysis by host cell proteases and that this is essential for viral replication. Our data contradict the previous dogma of positive-sense viral replication and suggests a model where this virus has evolved to use a host protease to control viral replication and tropism.

microbiology↗