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Angiotensin-converting enzyme 2 (ACE2) is the primary entry receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), but ACE2-independent entry has been observed in vitro for strains with the spike-E484D substitution. Here, we conduct a whole-genome CRISPR-Cas9 knockout screen using SARS-CoV-2 mouse adapted 1 (SARS-CoV-2 MA1 ), which carries spike-E484D, to identify the ACE2-independent entry mechanisms. SARS-CoV-2 MA1 infection in HEK293T cells relies on heparan sulfate and endocytic pathways, with TMEM106B, a transmembrane lysosomal protein, the most significant contributor. While SARS-CoV-2 MA1 productively infects human brain organoids and K18-hACE2 mouse brains, it does not infect C57BL/6J or Ifnar -/- mouse brains. This suggests that ACE2-independent entry via TMEM106B, which is predominantly expressed in the brain, does not overtly increase the risk of SARS-CoV-2 neuroinvasiveness in mice with endogenous Ace2 expression. Importantly, SARS-CoV-2 MA1 does not replicate in the Ace2 -/- mouse respiratory tract. Overall, this suggests that robust ACE2-independent infection by SARS-CoV-2 MA1 is likely an in vitro phenomenon with no apparent implications for infection in vivo., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Self-amplifying mRNA (SAM) vaccines can be rapidly deployed in the event of disease outbreaks. A legitimate safety concern is the potential for recombination between alphavirus-based SAM vaccines and circulating viruses. This theoretical risk needs to be assessed in the regulatory process for SAM vaccine approval. Herein, we undertake extensive in vitro and in vivo assessments to explore recombination between SAM vaccine and a wide selection of alphaviruses and a coronavirus. SAM vaccines were found to effectively limit alphavirus co-infection through superinfection exclusion, although some co-replication was still possible. Using sensitive cell-based assays, replication-competent alphavirus chimeras were generated in vitro as a result of rare, but reproducible, RNA recombination events. The chimeras displayed no increased fitness in cell culture. Viable alphavirus chimeras were not detected in vivo in C57BL/6J, Rag1 -/- and Ifnar -/- mice, in which high levels of SAM vaccine and alphavirus co-replicated in the same tissue. Furthermore, recombination between a SAM-spike vaccine and a swine coronavirus was not observed. In conclusion we state that although the ability of SAM vaccines to recombine with alphaviruses might be viewed as an environmental safety concern, several key factors substantially mitigate against in vivo emergence of chimeric viruses from SAM vaccine recipients., Competing Interests: Declaration of interests The user committee for the RepliSAFE project included members from MSD Animal Health. MSD Animal Health provided TC-83 replicons and antibodies but otherwise provided no other resources or funding, had no input into the manuscript nor the decision to publish., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The reduced pathogenicity of the omicron BA.1 sub-lineage compared to earlier variants is well described, although whether such attenuation is retained for later variants like BA.5 and XBB remains controversial. We show that BA.5 and XBB isolates were significantly more pathogenic in K18-hACE2 mice than a BA.1 isolate, showing increased neurotropic potential, resulting in fulminant brain infection and mortality, similar to that seen for original ancestral isolates. BA.5 also infected human cortical brain organoids to a greater extent than the BA.1 and original ancestral isolates. In the brains of mice, neurons were the main target of infection, and in human organoids neuronal progenitor cells and immature neurons were infected. The results herein suggest that evolving omicron variants may have increasing neurotropic potential., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Stewart, Yan, Ellis, Bishop, Dumenil, Tang, Nguyen, Larcher, Parry, Sng, Khromykh, Sullivan, Lor, Meunier, Rawle and Suhrbier.)

Mayaro virus (MAYV) is a mosquito-transmitted alphavirus that causes often debilitating rheumatic disease in tropical Central and South America. There are currently no licensed vaccines or antiviral drugs available for MAYV disease. Here, we generated Mayaro virus-like particles (VLPs) using the scalable baculovirus-insect cell expression system. High-level secretion of MAYV VLPs in the culture fluid of Sf9 insect cells was achieved, and particles with a diameter of 64 to 70 nm were obtained after purification. We characterize a C57BL/6J adult wild-type mouse model of MAYV infection and disease and used this model to compare the immunogenicity of VLPs from insect cells with that of VLPs produced in mammalian cells. Mice received two intramuscular immunizations with 1 μg of nonadjuvanted MAYV VLPs. Potent neutralizing antibody responses were generated against the vaccine strain, BeH407, with comparable activity seen against a contemporary 2018 isolate from Brazil (BR-18), whereas neutralizing activity against chikungunya virus was marginal. Sequencing of BR-18 illustrated that this virus segregates with genotype D isolates, whereas MAYV BeH407 belongs to genotype L. The mammalian cell-derived VLPs induced higher mean neutralizing antibody titers than those produced in insect cells. Both VLP vaccines completely protected adult wild-type mice against viremia, myositis, tendonitis, and joint inflammation after MAYV challenge. IMPORTANCE Mayaro virus (MAYV) is associated with acute rheumatic disease that can be debilitating and can evolve into months of chronic arthralgia. MAYV is believed to have the potential to emerge as a tropical public health threat, especially if it develops the ability to be efficiently transmitted by urban mosquito vectors, such as Aedes aegypti and/or Aedes albopictus. Here, we describe a scalable virus-like particle vaccine against MAYV that induced neutralizing antibodies against a historical and a contemporary isolate of MAYV and protected mice against infection and disease, providing a potential new intervention for MAYV epidemic preparedness.

Warmer climatic conditions have been associated with fewer COVID-19 cases. Herein we infected K18-hACE2 mice housed at the standard animal house temperature of ∼22 °C, or at ∼31 °C, which is considered to be thermoneutral for mice. On day 2 post infection, RNA-Seq analyses showed no significant differential gene expression lung in lungs of mice housed at the two temperatures, with almost identical viral loads and type I interferon responses. There was also no significant difference in viral loads in lungs on day 5, but RNA-Seq and histology analyses showed clearly elevated inflammatory signatures and infiltrates. Thermoneutrality thus promoted lung inflammation. On day 2 post infection mice housed at 31 °C showed reduced viral loads in nasal turbinates, consistent with increased mucociliary clearance at the warmer ambient temperature. These mice also had reduced virus levels in the brain, and an ensuing amelioration of weight loss and a delay in mortality. Warmer air temperatures may thus reduce infection of the upper respiratory track and the olfactory epithelium, resulting in reduced brain infection. Potential relevance for anosmia and neurological sequelae in COVID-19 patients is discussed., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

How well mouse models recapitulate the transcriptional profiles seen in humans remains debatable, with both conservation and diversity identified in various settings. Herein we use RNA-Seq data and bioinformatics approaches to analyze the transcriptional responses in SARS-CoV-2 infected lungs, comparing 4 human studies with the widely used K18-hACE2 mouse model, a model where hACE2 is expressed from the mouse ACE2 promoter, and a model that uses a mouse adapted virus and wild-type mice. Overlap of single copy orthologue differentially expressed genes (scoDEGs) between human and mouse studies was generally poor (≈15-35%). Rather than being associated with batch, sample treatment, viral load, lung damage or mouse model, the poor overlaps were primarily due to scoDEG expression differences between species. Importantly, analyses of immune signatures and inflammatory pathways illustrated highly significant concordances between species. As immunity and immunopathology are the focus of most studies, these mouse models can thus be viewed as representative and relevant models of COVID-19., Competing Interests: The authors have declared that no competing interests exist.

Human ACE2 Human angiotensin converting enzyme 2 (hACE2) is the key cell attachment and entry receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), with the original SARS-CoV-2 isolates unable to use mouse ACE2 (mACE2). Herein we describe the emergence of a SARS-CoV-2 strain capable of ACE2 -independent infection and the evolution of mouse-adapted (MA) SARS-CoV-2 by in vitro serial passaging of virus in co-cultures of cell lines expressing hACE2 and mACE2. MA viruses evolved with up to five amino acid changes in the spike protein, all of which have been seen in human isolates. MA viruses replicated to high titers in C57BL/6J mouse lungs and nasal turbinates and caused characteristic lung histopathology. One MA virus also evolved to replicate efficiently in several ACE2 -negative cell lines across several species, including clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) ACE2 knockout cells. An E484D substitution is likely involved in ACE2 -independent entry and has appeared in only ≈0.003 per cent of human isolates globally, suggesting that it provided no significant selection advantage in humans. ACE2 -independent entry reveals a SARS-CoV-2 infection mechanism that has potential implications for disease pathogenesis, evolution, tropism, and perhaps also intervention development., (© The Author(s) 2022. Published by Oxford University Press.)

Global microplastic (MP) contamination and the effects on the environment are well described. However, the potential for MP consumption to affect human health remains controversial. Mice consuming ≈80 μg/kg/day of 1 μm polystyrene MPs via their drinking water showed no weight loss, nor were MPs detected in internal organs. The microbiome was also not significantly changed. MP consumption did lead to small transcriptional changes in the colon suggesting plasma membrane perturbations and mild inflammation. Mice were challenged with the arthritogenic chikungunya virus, with MP consumption leading to a significantly prolonged arthritic foot swelling that was associated with elevated Th1, NK cell and neutrophil signatures. Immunohistochemistry also showed a significant increase in the ratio of neutrophils to monocyte/macrophages. The picture that emerges is reminiscent of enteropathic arthritis, whereby perturbations in the colon are thought to activate innate lymphoid cells that can inter alia migrate to joint tissues to promote inflammation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Granzyme A (GZMA) is a serine protease secreted by cytotoxic lymphocytes, with Gzma -/- mouse studies having informed our understanding of GZMA's physiological function. We show herein that Gzma -/- mice have a mixed C57BL/6J and C57BL/6N genetic background and retain the full-length nicotinamide nucleotide transhydrogenase ( Nnt ) gene, whereas Nnt is truncated in C57BL/6J mice. Chikungunya viral arthritis was substantially ameliorated in Gzma -/- mice; however, the presence of Nnt and the C57BL/6N background, rather than loss of GZMA expression, was responsible for this phenotype. A new CRISPR active site mutant C57BL/6J Gzma S211A mouse provided the first insights into GZMA's bioactivity free of background issues, with circulating proteolytically active GZMA promoting immune-stimulating and pro-inflammatory signatures. Remarkably, k-mer mining of the Sequence Read Archive illustrated that ≈27% of Run Accessions and ≈38% of BioProjects listing C57BL/6J as the mouse strain had Nnt sequencing reads inconsistent with a C57BL/6J genetic background. Nnt and C57BL/6N background issues have clearly complicated our understanding of GZMA and may similarly have influenced studies across a broad range of fields., Competing Interests: DR, TL, TD, CB, KY, EN, PB, AS No competing interests declared, (© 2022, Rawle et al.)

Purpose: Branchpoint elements are required for intron removal, and variants at these elements can result in aberrant splicing. We aimed to assess the value of branchpoint annotations generated from recent large-scale studies to select branchpoint-abrogating variants, using hereditary cancer genes as model., Methods: We identified branchpoint elements in 119 genes associated with hereditary cancer from 3 genome-wide experimentally-inferred and 2 predicted branchpoint data sets. We then identified variants that occur within branchpoint elements from public databases. We compared conservation, unique variant observations, and population frequencies at different nucleotides within branchpoint motifs. Finally, selected minigene assays were performed to assess the splicing effect of variants at branchpoint elements within mismatch repair genes., Results: There was poor overlap between predicted and experimentally-inferred branchpoints. Our analysis of cancer genes suggested that variants at -2 nucleotide, -1 nucleotide, and branchpoint positions in experimentally-inferred canonical motifs are more likely to be clinically relevant. Minigene assay data showed the -2 nucleotide to be more important to branchpoint motif integrity but also showed fluidity in branchpoint usage., Conclusion: Data from cancer gene analysis suggest that there are few high-risk alleles that severely impact function via branchpoint abrogation. Results of this study inform a general scheme to prioritize branchpoint motif variants for further study., Competing Interests: Conflict of Interest The authors declare no conflicts of interest., (Copyright © 2021 American College of Medical Genetics and Genomics. Published by Elsevier Inc. All rights reserved.)

SARS-CoV-2 uses the human ACE2 (hACE2) receptor for cell attachment and entry, with mouse ACE2 (mACE2) unable to support infection. Herein we describe an ACE2-lentivirus system and illustrate its utility for in vitro and in vivo SARS-CoV-2 infection models. Transduction of non-permissive cell lines with hACE2 imparted replication competence, and transduction with mACE2 containing N30D, N31K, F83Y and H353K substitutions, to match hACE2, rescued SARS-CoV-2 replication. Intrapulmonary hACE2-lentivirus transduction of C57BL/6J mice permitted significant virus replication in lung epithelium. RNA-Seq and histological analyses illustrated that this model involved an acute inflammatory disease followed by resolution and tissue repair, with a transcriptomic profile similar to that seen in COVID-19 patients. hACE2-lentivirus transduction of IFNAR-/- and IL-28RA-/- mouse lungs was used to illustrate that loss of type I or III interferon responses have no significant effect on virus replication. However, their importance in driving inflammatory responses was illustrated by RNA-Seq analyses. We also demonstrate the utility of the hACE2-lentivirus transduction system for vaccine evaluation in C57BL/6J mice. The ACE2-lentivirus system thus has broad application in SARS-CoV-2 research, providing a tool for both mutagenesis studies and mouse model development., Competing Interests: The authors have declared that no competing interests exist.

Cardiac injury and dysfunction occur in COVID-19 patients and increase the risk of mortality. Causes are ill defined but could be through direct cardiac infection and/or inflammation-induced dysfunction. To identify mechanisms and cardio-protective drugs, we use a state-of-the-art pipeline combining human cardiac organoids with phosphoproteomics and single nuclei RNA sequencing. We identify an inflammatory "cytokine-storm", a cocktail of interferon gamma, interleukin 1β, and poly(I:C), induced diastolic dysfunction. Bromodomain-containing protein 4 is activated along with a viral response that is consistent in both human cardiac organoids (hCOs) and hearts of SARS-CoV-2-infected K18-hACE2 mice. Bromodomain and extraterminal family inhibitors (BETi) recover dysfunction in hCOs and completely prevent cardiac dysfunction and death in a mouse cytokine-storm model. Additionally, BETi decreases transcription of genes in the viral response, decreases ACE2 expression, and reduces SARS-CoV-2 infection of cardiomyocytes. Together, BETi, including the Food and Drug Administration (FDA) breakthrough designated drug, apabetalone, are promising candidates to prevent COVID-19 mediated cardiac damage., Competing Interests: Declaration of interests R.J.M., J.E.H., G.A.Q.-R., D.M.T., and E.R.P. are co-inventors on patents relating to cardiac organoid maturation and cardiac therapeutics. J.E.H. is co-inventor on licensed patents for engineered heart muscle. R.J.M., E.R.P., D.M.T., B.G., and J.E.H. are co-founders, scientific advisors, and stockholders in Dynomics. D.M.T. and B.G. are employees of Dynomics. C.H., D.G., L.F., J.J., M.S., N.C.W.W., and E.K. are employees of Resverlogix. S.J.N. received honoraria and research support from Resverlogix. QIMR Berghofer Medical Research Institute filed a patent on the use of BETi., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Poxvirus systems have been extensively used as vaccine vectors. Herein a RNA-Seq analysis of intramuscular injection sites provided detailed insights into host innate immune responses, as well as expression of vector and recombinant immunogen genes, after vaccination with a new multiplication defective, vaccinia-based vector, Sementis Copenhagen Vector. Chikungunya and Zika virus immunogen mRNA and protein expression was associated with necrosing skeletal muscle cells surrounded by mixed cellular infiltrates. The multiple adjuvant signatures at 12 hours post-vaccination were dominated by TLR3, 4 and 9, STING, MAVS, PKR and the inflammasome. Th1 cytokine signatures were dominated by IFNγ, TNF and IL1β, and chemokine signatures by CCL5 and CXCL12. Multiple signatures associated with dendritic cell stimulation were evident. By day seven, vaccine transcripts were absent, and cell death, neutrophil, macrophage and inflammation annotations had abated. No compelling arthritis signatures were identified. Such injection site vaccinology approaches should inform refinements in poxvirus-based vector design., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: NAP, LL and JDH own Sementis shares. JDH is the current CSO of Sementis. AS was a consultant for Sementis. PMH was the previous CEO/CSO of Sementis. LL and NAP have had, and/or currently have, salary and/or project support from funds provided, whole or in part, via Sementis. Sementis had no role in the design and interpretation of the study, or in preparation of the manuscript.

Getah virus (GETV) is a mosquito-transmitted alphavirus primarily associated with disease in horses and pigs in Asia. GETV was also reported to have been isolated from mosquitoes in Australia in 1961; however, retrieval and sequencing of the original isolates (N544 and N554), illustrated that these viruses were virtually identical to the 1955 GETV MM2021 isolate from Malaysia. K-mer mining of the >40,000 terabases of sequence data in the Sequence Read Archive followed by BLASTn confirmation identified multiple GETV sequences in biosamples from Asia (often as contaminants), but not in biosamples from Australia. In contrast, sequence reads aligning to the Australian Ross River virus (RRV) were readily identified in Australian biosamples. To explore the serological relationship between GETV and other alphaviruses, an adult wild-type mouse model of GETV was established. High levels of cross-reactivity and cross-protection were evident for convalescent sera from mice infected with GETV or RRV, highlighting the difficulties associated with the interpretation of early serosurveys reporting GETV antibodies in Australian cattle and pigs. The evidence that GETV circulates in Australia is thus not compelling., Competing Interests: The authors declare no conflicts of interests.

Functional assays that assess mRNA splicing can be used in interpretation of the clinical significance of sequence variants, including the Lynch syndrome-associated mismatch repair (MMR) genes. The purpose of this study was to investigate the contribution of splicing assay data to the classification of MMR gene sequence variants. We assayed mRNA splicing for 24 sequence variants in MLH1 , MSH2 , and MSH6 , including 12 missense variants that were also assessed using a cell-free in vitro MMR activity (CIMRA) assay. Multifactorial likelihood analysis was conducted for each variant, combining CIMRA outputs and clinical data where available. We collated these results with existing public data to provide a dataset of splicing assay results for a total of 671 MMR gene sequence variants (328 missense/in-frame indel), and published and unpublished repair activity measurements for 154 of these variants. There were 241 variants for which a splicing aberration was detected: 92 complete impact, 33 incomplete impact, and 116 where it was not possible to determine complete versus incomplete splicing impact. Splicing results mostly aided in the interpretation of intronic (72%) and silent (92%) variants and were the least useful for missense substitutions/in-frame indels (10%). MMR protein functional activity assays were more useful in the analysis of these exonic variants but by design they were not able to detect clinically important splicing aberrations identified by parallel mRNA assays. The development of high throughput assays that can quantitatively assess impact on mRNA transcript expression and protein function in parallel will streamline classification of MMR gene sequence variants., (Copyright © 2020 Thompson, Walters, Parsons, Dumenil, Drost, Tiersma, Lindor, Tavtigian, de Wind, Spurdle and the InSiGHT Variant Interpretation Committee.)

Background: WNT activation is a hallmark of colorectal cancer. BRAF mutation is present in 15% of colorectal cancers, and the role of mutations in WNT signaling regulators in this context is unclear. Here, we evaluate the mutational landscape of WNT signaling regulators in BRAF mutant cancers., Methods: we performed exome-sequencing on 24 BRAF mutant colorectal cancers and analyzed these data in combination with 175 publicly available BRAF mutant colorectal cancer exomes. We assessed the somatic mutational landscape of WNT signaling regulators, and performed hotspot and driver mutation analyses to identify potential drivers of WNT signaling. The effects of Apc and Braf mutation were modelled, in vivo, using the Apc min/+ and Braf V637 /Villin-Cre ERT2/+ mouse, respectively., Results: RNF43 was the most frequently mutated WNT signaling regulator (41%). Mutations in the beta-catenin destruction complex occurred in 48% of cancers. Hotspot analyses identified potential cancer driver genes in the WNT signaling cascade, including MEN1, GNG12 and WNT16. Truncating APC mutation was identified in 20.8% of cancers. Truncating APC mutation was associated with early age at diagnosis (p < 2 × 10 -5 ), advanced stage (p < 0.01), and poor survival (p = 0.026). Apc min/+ /Braf V637 animals had more numerous and larger SI and colonic lesions (p < 0.0001 and p < 0.05, respectively), and a markedly reduced survival (median survival: 3.2 months, p = 8.8 × 10 -21 ), compared to animals with Apc or Braf mutation alone., Conclusions: the WNT signaling axis is frequently mutated in BRAF mutant colorectal cancers. WNT16 and MEN1 may be novel drivers of aberrant WNT signaling in colorectal cancer. Co-mutation of BRAF and APC generates an extremely aggressive neoplastic phenotype that is associated with poor patient outcome., Competing Interests: The authors declare no conflict of interest.

Background & Aims: Colorectal cancer is an epigenetically heterogeneous disease, however, the extent and spectrum of the CpG island methylator phenotype (CIMP) is not clear., Methods: Genome-scale methylation and transcript expression were measured by DNA Methylation and RNA expression microarray in 216 unselected colorectal cancers, and findings were validated using The Cancer Genome Atlas 450K and RNA sequencing data. Mutations in epigenetic regulators were assessed using CIMP-subtyped Cancer Genome Atlas exomes., Results: CIMP-high cancers dichotomized into CIMP-H1 and CIMP-H2 based on methylation profile. KRAS mutation was associated significantly with CIMP-H2 cancers, but not CIMP-H1 cancers. Congruent with increasing methylation, there was a stepwise increase in patient age from 62 years in the CIMP-negative subgroup to 75 years in the CIMP-H1 subgroup (P < .0001). CIMP-H1 predominantly comprised consensus molecular subtype 1 cancers (70%) whereas consensus molecular subtype 3 was over-represented in the CIMP-H2 subgroup (55%). Polycomb Repressive Complex-2 (PRC2)-marked loci were subjected to significant gene body methylation in CIMP cancers (P < 1.6 × 10 -78 ). We identified oncogenes susceptible to gene body methylation and Wnt pathway antagonists resistant to gene body methylation. CIMP cluster-specific mutations were observed in chromatin remodeling genes, such as in the SWItch/Sucrose Non-Fermentable and Chromodomain Helicase DNA-Binding gene families., Conclusions: There are 5 clinically and molecularly distinct subgroups of colorectal cancer. We show a striking association between CIMP and age, sex, and tumor location, and identify a role for gene body methylation in the progression of serrated neoplasia. These data support our recent findings that CIMP is uncommon in young patients and that BRAF mutant polyps in young patients may have limited potential for malignant progression., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Conventional adenomas are initiated by APC gene mutation that activates the WNT signal. Serrated neoplasia is commonly initiated by BRAF or KRAS mutation. WNT pathway activation may also occur, however, to what extent this is owing to APC mutation is unknown. We examined aberrant nuclear β-catenin immunolocalization as a surrogate for WNT pathway activation and analyzed the entire APC gene coding sequence in serrated and conventional pathway polyps and cancers. WNT pathway activation was a common event in conventional pathway lesions with aberrant nuclear immunolocalization of β-catenin and truncating APC mutations in 90% and 89% of conventional adenomas and 82% and 70% of BRAF wild-type cancers, respectively. WNT pathway activation was seen to a lesser extent in serrated pathway lesions. It occurred at the transition to dysplasia in serrated polyps with a significant increase in nuclear β-catenin labeling from sessile serrated adenomas (10%) to sessile serrated adenomas with dysplasia (55%) and traditional serrated adenomas (9%) to traditional serrated adenomas with dysplasia (39%) (P=0.0001). However, unlike the conventional pathway, truncating APC mutations were rare in the serrated pathway lesions especially sessile serrated adenomas even when dysplastic (15%) and in the BRAF mutant cancers with microsatellite instability that arise from them (8%). In contrast, APC missense mutations that were rare in conventional pathway adenomas and cancers (3% in BRAF wild-type cancers) were more frequent in BRAF mutant cancers with microsatellite instability (32%). We conclude that increased WNT signaling is important in the transition to malignancy in the serrated pathway but that APC mutation is less common and the spectrum of mutations is different than in conventional colorectal carcinogenesis. Moderate impact APC mutations and non-APC-related causes of increased WNT signaling may have a more important role in serrated neoplasia than the truncating APC mutations common in conventional adenomas.

Colorectal cancer is a major cause of cancer death and approximately 20% arises within serrated polyps, which are under-recognized and poorly understood. Human serrated colorectal polyps frequently exhibit both oncogenic BRAF mutation and widespread DNA methylation changes, which are important in silencing genes restraining neoplastic progression. Here, we investigated whether in vivo induction of mutant Braf is sufficient to result in coordinated promoter methylation changes for multiple cancer-related genes. The Braf V637E mutation was induced in murine intestine on an FVB;C57BL/6J background and assessed for morphological and DNA methylation changes at multiple time points from 10 days to 14 months. Extensive intestinal hyperplasia developed by 10 days post-induction of the mutation. By 8 months, most mice had murine serrated adenomas with dysplasia and invasive cancer developed in 40% of mice by 14 months. From 5 months onwards, Braf mutant mice showed extensive, gene-specific increases in DNA methylation even in hyperplastic mucosa without lesions. This demonstrates that persistent oncogenic Braf signaling is sufficient to induce widespread DNA methylation changes. This occurs over an extended period of time, mimicking the long latency followed by rapid progression of human serrated neoplasia. This study establishes for the first time that DNA methylation arises slowly in direct response to prolonged oncogenic Braf signaling in serrated polyps; this finding has implications both for chemoprevention and for understanding the origin of DNA hypermethylation in cancer generally.

Liver metastasis is the major cause of death following a diagnosis of colorectal cancer (CRC). In this study, we compared the copy number profiles of paired primary and liver metastatic CRC to better understand how the genomic structure of primary CRC differs from the metastasis. Paired primary and metastatic tumors from 16 patients and their adjacent normal tissue samples were analyzed using single nucleotide polymorphism arrays. Genome-wide chromosomal copy number alterations were assessed, with particular attention to 188 genes known to be somatically altered in CRC and 24 genes that are clinically actionable in CRC. These data were analyzed with respect to the timing of primary and metastatic tissue resection and with exposure to chemotherapy. The genomic differences between the tumor and paired metastases revealed an average copy number discordance of 22.0%. The pairs of tumor samples collected prior to treatment revealed significantly higher copy number differences compared to post-therapy liver metastases ( P = 0.014). Loss of heterozygosity acquired in liver metastases was significantly higher in previously treated liver metastasis samples compared to treatment naive liver metastasis samples ( P = 0.003). Amplification of the clinically actionable genes ERBB2 , FGFR1 , PIK3CA or CDK8 was observed in the metastatic tissue of 4 patients but not in the paired primary CRC. These examples highlight the intra-patient genomic discrepancies that can occur between metastases and the primary tumors from which they arose. We propose that precision medicine strategies may therefore identify different actionable targets in metastatic tissue, compared to primary tumors, due to substantial genomic differences., Competing Interests: CONFLICTS OF INTEREST The authors have nothing to declare.

The CpG Island Methylator Phenotype (CIMP) is fundamental to an important subset of colorectal cancer; however, its cause is unknown. CIMP is associated with microsatellite instability but is also found in BRAF mutant microsatellite stable cancers that are associated with poor prognosis. The isocitrate dehydrogenase 1 (IDH1) gene causes CIMP in glioma due to an activating mutation that produces the 2-hydroxyglutarate oncometabolite. We therefore examined IDH1 alteration as a potential cause of CIMP in colorectal cancer. The IDH1 mutational hotspot was screened in 86 CIMP-positive and 80 CIMP-negative cancers. The entire coding sequence was examined in 81 CIMP-positive colorectal cancers. Forty-seven cancers varying by CIMP-status and IDH1 mutation status were examined using Illumina 450K DNA methylation microarrays. The R132C IDH1 mutation was detected in 4/166 cancers. All IDH1 mutations were in CIMP cancers that were BRAF mutant and microsatellite stable (4/45, 8.9%). Unsupervised hierarchical cluster analysis identified an IDH1 mutation-like methylation signature in approximately half of the CIMP-positive cancers. IDH1 mutation appears to cause CIMP in a small proportion of BRAF mutant, microsatellite stable colorectal cancers. This study provides a precedent that a single gene mutation may cause CIMP in colorectal cancer, and that this will be associated with a specific epigenetic signature and clinicopathological features.

There is increasing epidemiological and molecular evidence that cutaneous melanomas arise through multiple causal pathways. The purpose of this study was to explore the relationship between germline and somatic mutations in a population-based series of melanoma patients to reshape and refine the divergent pathway model for melanoma. Melanomas collected from 123 Australian patients were analyzed for melanocortin-1 receptor (MC1R) variants and mutations in the BRAF and NRAS genes. Detailed phenotypic and sun exposure data were systematically collected from all patients. We found that BRAF-mutant melanomas were significantly more likely from younger patients and those with high nevus counts, and were more likely in melanomas with adjacent neval remnants. Conversely, BRAF-mutant melanomas were significantly less likely in people with high levels of lifetime sun exposure. We observed no association between germline MC1R status and somatic BRAF mutations in melanomas from this population. BRAF-mutant melanomas have different origins from other cutaneous melanomas. These data support the divergent pathways hypothesis for melanoma, which may require a reappraisal of targeted cancer prevention activities.

Background: The length polymorphism repeat in the promoter region of the serotonin transporter gene (5HTTLPR) is one of the most studied polymorphisms for association with a range of psychiatric and personality phenotypes. However, the original 5HTTLPR assay is prone to bias toward short allele calling., Methods: We designed new assays for the 5HTTLPR suitable for large-scale genotyping projects and we genotyped 13 single nucleotide polymorphisms (SNPs) in a 38-kilobase region around the 5HTTLPR, including SNP rs25531, a polymorphism of the 5HTTLPR long allele. Association analysis was conducted for major depression and/or anxiety disorder in unrelated cases (n = 1161) and control subjects (n = 1051) identified through psychiatric interviews administered to a large population sample of Australian twin families. Participants had been scored for personality traits several years earlier (n > or = 2643 unrelated individuals)., Results: We identified a two-SNP haplotype proxy for 5HTTLPR; the CA haplotype of SNPs rs4251417 and rs2020934 is coupled with the short allele of 5HTTLPR (r(2) = .72). We found evidence for association (p = .0062, after accounting for multiple testing) for SLC6A4 SNPs rs6354 and rs2020936 (positioned in a different linkage disequilibrium [LD] block about 15.5 kb from 5HTTLPR) with anxiety and/or depression and neuroticism, with the strongest association for recurrent depression with onset in young adulthood (odds ratio = 1.55, 95% confidence interval = 1.16-2.06)., Conclusions: The associated SNPs are in the same LD block as the variable number of tandem repeats serotonin transporter intron 2 marker, for which association has previously been reported.

The Val158Met polymorphism of the gene encoding catechol-O-methyltransferase (COMT) is one of the most widely tested variants for association with psychiatric disorders, but replication has been inconsistent including both sex limitation and heterogeneity of the associated allele. In this study we investigate the association between three SNPs from COMT and anxiety and depression disorders and neuroticism all measured within the same study sample. Participants were selected as sibling pairs (or multiples) that were either concordant or discordant for extreme neuroticism scores from a total sample of 18,742 Australian twin individuals and their siblings. All participants completed the Composite International Diagnostic Interview (CIDI) from which diagnoses of DSM-IV depression and anxiety disorders were determined. Of the participants, 674 had a diagnosis of anxiety and/or depression from 492 families. Study participants (n = 2,045 from 987 families) plus, where possible, their parents were genotyped for rs737865, rs4680 (Val158Met), and rs165599. Using family based tests we looked for association between these variants and neuroticism, depression, anxiety, panic disorder and agarophobia (PDAG) and obsessive compulsive disorder. We found no convincing evidence for association either in allelic or genotypic tests for the total sample or when the sample was stratified by sex. Haplotype T-G-G showed weak association (P = 0.042) with PDAG before correction for multiple testing; association between this haplotype and schizophrenia has been previously reported in an Australian sample.

Objectives: Association of the valine/methionine variant at codon 66 (Val66Met) of brain derived neurotrophic factor (BDNF) has been reported inconsistently across a spectrum of psychiatric disorders. Haplotypes of six tagging single nucleotide polymorphisms (SNPs) of a 37-kb region of dystrobrevin-binding protein 1 (DTNBP1) were found to be associated with schizophrenia. These haplotypes have not been studied extensively for other psychiatric disorders but are plausible candidates for anxiety and depression disorders. Here, association between variants of BDNF and DTNBP1, and multiple anxiety and depression phenotypes is explored., Methods: Study participants were selected as sibling pairs that were either concordant or discordant for extreme neuroticism scores from a total sample of 18 742 Australian twin individuals and their siblings. All participants completed detailed Composite International Diagnostic Interview from which diagnoses of Diagnostic and Statistical Manual of Mental Disorders (DSM)-IV depression and anxiety disorders were determined. Six hundred and seventy-four participants had a diagnosis of anxiety and/or depression from 492 families. The BDNF Val66Met and six DTNBP1 (rs3213207, rs1011313, rs2619528, rs760761, rs1018381, rs2619538) SNPs were genotyped on samples from study participants (n=2045 from 987 families) and, where possible, their parents (n=787). Family-based association tests were conducted between the individual SNPs and the DTNBP1 six SNP haplotypes and anxiety, depression, and neuroticism., Results: We found no convincing evidence for association between any of the variants studied and anxiety, depression, or neuroticism., Conclusion: This study sample is relatively large but our results do not support an association between BDNF Val66Met and anxiety, depression, or neuroticisim. DTNBP1 haplotypes, which have been found to be risk factors for schizophrenia, are unlikely to be risk factors for anxiety and depression.

Somatic mutations of the BRAF gene are common in melanomas and nevi but the contribution of polymorphisms in this gene to melanoma or nevus susceptibility remains unclear. An Australian melanoma case-control sample was typed for 16 single nucleotide polymorphisms (SNP) within the BRAF gene, and five SNP in three neighboring genes. The sample comprised 755 melanoma cases from 740 families stratified by family history of melanoma and controls from 635 unselected twin families (2,239 individuals). Ancestry of the cases and controls was recorded, and the twins had undergone skin examination to assess total body nevus count, degree of freckling, and pigmentation phenotype. Genotyping was carried out via primer extension followed by matrix-assisted laser desorption ionization-time of flight mass spectrometry. SNP in the BRAF gene were found to be weakly associated with melanoma status but not with development of nevi or freckles. The estimated proportion of attributable risk of melanoma due to variants in BRAF is 1.6%. This study shows that BRAF polymorphisms predispose to melanoma but the causal variant has yet to be determined. The burden of disease associated with this variant is greater than that associated with the major melanoma susceptibility locus CDKN2A, which has an estimated attributable risk of 0.2%.

A common single nucleotide polymorphism (SNP) in the 5' untranslated region (5'UTR) of the epidermal growth factor (EGF) gene modulates the level of transcription of this gene and hence is associated with serum levels of EGF. This variant may be associated with melanoma risk, but conflicting findings have been reported. An Australian melanoma case-control sample was typed for the EGF+61A>G transversion (rs4444903). The sample comprised 753 melanoma cases from 738 families stratified by family history of melanoma and 2387 controls from 645 unselected twin families. Ancestry of the cases and controls was recorded, and the twins had undergone skin examination to assess total body nevus count, degree of freckling and pigmentation phenotype. SNP genotyping was carried out via primer extension followed by matrix-assisted laser desorption time of flight (MALDI-TOF) mass spectroscopy. The EGF+61 SNP was not found to be significantly associated with melanoma status or with development of nevi or freckles. Among melanoma cases, however, G homozygotes had thicker tumors (p=0.05), in keeping with two previous studies. The EGF polymorphism does not appear to predispose to melanoma or nevus development, but its significant association with tumor thickness implies that it may be a useful marker of prognosis.