RUNX1 regulates promoter activity in the absence of cognate DNA binding motifs.

Runt-related transcription factor 1 (RUNX1) plays an important role in normal haematopoietic cell development and function, and its function is frequently disrupted in leukaemia. RUNX1 is widely recognised as a sequence-specific DNA binding factor that recognises the motif 5'-TG(T/C)GGT-3' in promoter and enhancer regions of its target genes. Moreover, RUNX1 fusion proteins, such as RUNX1-ETO formed by the t(8;21) translocation, retain the ability to recognise and bind to this sequence to elicit atypical gene regulatory effects on bona fide RUNX1 targets. However, our analysis of publicly available RUNX1 chromatin immunoprecipitation sequencing (ChIP-Seq) data has provided evidence challenging this dogma, revealing that this motif-specific model of RUNX1 recruitment and function is incomplete. Our analyses revealed that the majority of RUNX1 genomic localisation occurs outside of promoters, that 20% of RUNX1 binding sites lack consensus RUNX motifs, and that binding in the absence of a cognate binding site is more common in promoter regions compared to distal sites. Reporter assays demonstrate that RUNX1 can drive promoter activity in the absence of a recognised DNA binding motif, in contrast to RUNX1-ETO. RUNX1-ETO supresses activity when it is recruited to promoters containing a sequence specific motif, while interestingly, it binds but does not repress promoters devoid of a RUNX1 recognition site. These data suggest that RUNX1 regulation of target genes occurs through multiple mechanisms depending on genomic location, the type of regulatory element and mode of recruitment.

Decrypting seasonal patterns of key pollen taxa in cool temperate Australia: A multi-barcode metabarcoding analysis.

Pollen allergies pose a considerable global public health concern. Allergy risk can vary significantly within plant families, yet some key pollen allergens can only be identified to family level by current optical methods. Pollen information with greater taxonomic resolution is therefore required to best support allergy prevention and self-management. We used environmental DNA (eDNA) metabarcoding to deepen taxonomic insights into the seasonal composition of airborne pollen in cool temperate Australia, a region with high rates of allergic respiratory disease. In Hobart, Tasmania, we collected routine weekly air samples from December 2018 until October 2020 and sequenced the internal transcribed spacer 2 (ITS2) and chloroplastic tRNA-Leucine tRNA-Phenylalanine intergenic spacer (trnL-trnF) regions in order to address the following questions: a) What is the genus-level diversity of known and potential aeroallergens in Hobart, in particular, in the families Poaceae, Cupressaceae and Myrtaceae? b) How do the atmospheric concentrations of these taxa change over time, and c) Does trnL-trnF enhance resolution of biodiversity when used in addition to ITS2? Our results suggest that individuals in the region are exposed to temperate grasses including Poa and Bromus in the peak grass pollen season, however low levels of exposure to the subtropical grass Cynodon may occur in autumn and winter. Within Cupressaceae, both metabarcodes showed that exposure is predominantly to pollen from the introduced genera Cupressus and Juniperus. Only ITS2 detected the native genus, Callitris. Both metabarcodes detected Eucalyptus as the major Myrtaceae genus, with trnL-trnF exhibiting primer bias for this family. These findings help refine our understanding of allergy triggers in Tasmania and highlight the utility of multiple metabarcodes in aerobiome studies.

Acquired copy number variation in prostate tumours: a review of common somatic copy number alterations, how they are formed and their clinical utility.

Prostate cancer is one of the most commonly diagnosed cancers in men and unfortunately, disease will progress in up to a third of patients despite primary treatment. Currently, there is a significant lack of prognostic tests that accurately predict disease course; however, the acquisition of somatic chromosomal variation in the form of DNA copy number variants may help understand disease progression. Notably, studies have found that a higher burden of somatic copy number alterations (SCNA) correlates with more aggressive disease, recurrence after surgery and metastasis. Here we will review the literature surrounding SCNA formation, including the roles of key tumour suppressors and oncogenes (PTEN, BRCA2, NKX3.1, ERG and AR), and their potential to inform diagnostic and prognostic clinical testing to improve predictive value. Ultimately, SCNAs, or inherited germline alterations that predispose to SCNAs, could have significant clinical utility in diagnostic and prognostic tests, in addition to guiding therapeutic selection.

Idiopathic Pulmonary Fibrosis Is Associated with Common Genetic Variants and Limited Rare Variants.

Rationale: Idiopathic pulmonary fibrosis (IPF) is a rare, irreversible, and progressive disease of the lungs. Common genetic variants, in addition to nongenetic factors, have been consistently associated with IPF. Rare variants identified by candidate gene, family-based, and exome studies have also been reported to associate with IPF. However, the extent to which rare variants, genome-wide, may contribute to the risk of IPF remains unknown. Objectives: We used whole-genome sequencing to investigate the role of rare variants, genome-wide, on IPF risk. Methods: As part of the Trans-Omics for Precision Medicine Program, we sequenced 2,180 cases of IPF. Association testing focused on the aggregated effect of rare variants (minor allele frequency ⩽0.01) within genes or regions. We also identified individual rare variants that are influential within genes and estimated the heritability of IPF on the basis of rare and common variants. Measurements and Main Results: Rare variants in both TERT and RTEL1 were significantly associated with IPF. A single rare variant in each of the TERT and RTEL1 genes was found to consistently influence the aggregated test statistics. There was no significant evidence of association with other previously reported rare variants. The SNP heritability of IPF was estimated to be 32% (SE = 3%). Conclusions: Rare variants within the TERT and RTEL1 genes and well-established common variants have the largest contribution to IPF risk overall. Efforts in risk profiling or the development of therapies for IPF that focus on TERT, RTEL1, common variants, and environmental risk factors are likely to have the largest impact on this complex disease.

Identification of a novel recurrent EEF2 gene amplification in familial prostate tumors.

Recurrent tumor copy number variations (CNVs) in prostate cancer (PrCa) have predominantly been discovered in sporadic tumor cohorts. Here, we examined familial prostate tumors for novel CNVs as prior studies suggest these harbor distinct CNVs. Array comparative genomic hybridization of 12 tumors from an Australian PrCa family, PcTas9, highlighted multiple recurrent CNVs, including amplification of EEF2 (19p13.3) in 100% of tumors. The EEF2 CNV was examined in a further 26 familial and seven sporadic tumors from the Australian cohort and in 494 tumors unselected for family history from The Cancer Genome Atlas (TCGA). EEF2 overexpression was observed in seven PcTas9 tumors, in addition to seven other predominantly familial tumors (ntotal  = 34%). EEF2 amplification was only observed in 1.4% of TCGA tumors, however 7.5% harbored an EEF2 deletion. Analysis of genes co-expressed with EEF2 revealed significant upregulation of two genes, ZNF74 and ADSL, and downregulation of PLSCR1 in both EEF2 amplified familial tumors and EEF2 deleted TCGA tumors. Furthermore, in TCGA tumors, EEF2 amplification and deletion were significantly associated with a higher Gleason score. In summary, we identified a novel PrCa CNV that was predominantly amplified in familial tumors and deleted in unselected tumors. Our results provide further evidence that familial tumors harbor distinct CNVs, potentially due to an inherited predisposition, but also suggest that regardless of how EEF2 is dysregulated, a similar set of genes involved in key cancer pathways are impacted. Given the current lack of gene-based biomarkers and clinical targets in PrCa, further investigation of EEF2 is warranted.

Non-coding RNA regulation of integrins and their potential as therapeutic targets in cancer.

BACKGROUND: Integrins are integral to cell signalling and management of the extracellular matrix, and exquisite regulation of their expression is essential for a variety of cell signalling pathways, whilst disordered regulation is a key driver of tumour progression and metastasis. Most recently non-coding RNAs in the form of micro-RNA (miRNA) and long non-coding RNA (lncRNA) have emerged as a key mechanism by which tissue dependent gene expression is controlled. Whilst historically these molecules have been poorly understood, advances in 'omic' technologies and a greater understanding of non-coding regions of the genome have revealed that non-coding RNAs make up a large proportion of the transcriptome. CONCLUSIONS AND PERSPECTIVES: This review examines the regulation of integrin genes by ncRNAs, provides and overview of their mechanism of action and highlights how exploitation of these discoveries is informing the development of novel chemotherapeutic agents in the treatment of cancer. MiRNA molecules have been the most extensively characterised and negatively regulate most integrin genes, classically regulating genes through binding to recognition sequences in the mRNA 3'-untranslated regions of gene transcripts. LncRNA mechanisms of action are now being elucidated and appear to be more varied and complex, and may counter miRNA molecules, directly engage integrin mRNA transcripts, and guide or block both transcription factors and epigenetic machinery at integrin promoters or at other points in integrin regulation. Integrins as therapeutic targets are of enormous interest given their roles as oncogenes in a variety of tumours, and emerging therapeutics mimicking ncRNA mechanisms of action are already being trialled.

Functional genomics for curation of variants in telomere biology disorder associated genes: A systematic review.

PURPOSE: Patients with an underlying telomere biology disorder (TBD) have variable clinical presentations, and they can be challenging to diagnose clinically. A genomic diagnosis for patients presenting with TBD is vital for optimal treatment. Unfortunately, many variants identified during diagnostic testing are variants of uncertain significance. This complicates management decisions, delays treatment, and risks nonuptake of potentially curative therapies. Improved application of functional genomic evidence may reduce variants of uncertain significance classifications. METHODS: We systematically searched the literature for published functional assays interrogating TBD gene variants. When possible, established likely benign/benign and likely pathogenic/pathogenic variants were used to estimate the assay sensitivity, specificity, positive predictive value, negative predictive value, and odds of pathogenicity. RESULTS: In total, 3131 articles were screened and 151 met inclusion criteria. Sufficient data to enable a PS3/BS3 recommendation were available for TERT variants only. We recommend that PS3 and BS3 can be applied at a moderate and supportive level, respectively. PS3/BS3 application was limited by a lack of assay standardization and limited inclusion of benign variants. CONCLUSION: Further assay standardization and assessment of benign variants are required for optimal use of the PS3/BS3 criterion for TBD gene variant classification.

Remote primary care during the COVID-19 pandemic for people experiencing homelessness: a qualitative study.

BACKGROUND: The COVID-19 pandemic has caused unprecedented disruption and change to the organisation of primary care, including for people experiencing homelessness who may not have access to a phone. Little is known about whether the recent changes required to deliver services to people experiencing homelessness will help to address or compound inequality in accessing care. AIM: To explore the experience and impact of organisational and technology changes in response to COVID-19 on access to health care for people experiencing homelessness. DESIGN AND SETTING: An action-led and participatory research methodology was employed in three case study sites made up of primary care services delivering care for people experiencing homelessness. METHOD: Individual semi-structured interviews were conducted with 21 people experiencing homelessness and 22 clinicians and support workers. Interviews were analysed using a framework approach. RESULTS: The move to remote telephone consultations highlighted the difficulties experienced by participants in accessing health care. These barriers included problems at the practice level associated with remote triage as participants did not always have access to a phone or the means to pay for a phone call. This fostered increased reliance on support workers and clinicians working in the community to provide or facilitate a primary care appointment. CONCLUSION: The findings have emphasised the importance of addressing practical and technology barriers as well as supporting communication and choice for mode of consultation. The authors argue that consultations should not be remote 'by default' and instead take into consideration both the clinical and social factors underpinning health.

Urban-rural prostate cancer disparities in a regional state of Australia.

Men living in regional and remote areas experience disparities in prostate cancer (PrCa) diagnosis, clinical characteristics and treatment modalities. We sought to determine whether such disparities exist in PrCa patients from Tasmania; a regional state of Australia with the second-highest rate of diagnosis and where over a third of residents live in outer regional and remote areas. Our study included clinicopathological data from 1526 patients enrolled in the Prostate Cancer Outcomes Registry-Tasmania. Regression analyses were undertaken to determine whether demographic, clinical and treatment variables differed between inner regional and outer regional/remote patients. Men from outer regional/remote areas were significantly more likely to reside in lower socio-economic areas, be diagnosed at a later age and with more clinically aggressive features. However, in contrast to previous studies, there were no overall differences in diagnostic or treatment method, although men from outer regional/remote areas took longer to commence active treatment and travelled further to do so. This study is the first to investigate PrCa disparities in a wholly regional Australian state and highlights the need to develop systematic interventions at the patient and healthcare level to improve outcomes in outer regional and remote populations in Australia and across the globe.

A novel long non-coding RNA regulates the integrin, ITGA2 in breast cancer.

PURPOSE: ITGA2 encodes the integrin, α2 which mediates metastatic progression, and is a predictor of poor prognosis and chemoresistance in breast cancer. Decreased ITGA2 promoter methylation is implicated as a driver of increased gene expression in aggressive prostate and pancreatic tumours, however the contribution of altered methylation to ITGA2 expression changes in breast tumours has not been examined. METHODS: ITGA2 gene methylation and gene expression was examined in publicly available breast cancer datasets, and ITGA2 promoter methylation was mapped by targeted bisulphite sequencing analysis in breast tumour cell lines. The expression of a putative regulatory long noncoding RNA (lncRNA) was examined by qPCR and its' functionality was investigated using gene knockdown (antisense oligonucleotides) and over expression in breast cancer cell lines. RESULTS: In breast tumours and breast cancer cell lines the ITGA2 promoter is largely unmethylated, with gene expression variable in tumour subtypes, irrespective of promoter methylation. A novel lncRNA (AC025180.1;ENSG00000249899), named herein I2ALR, was identified at the ITGA2 gene locus, and was variably expressed in breast tumours and breast cancer cell subtypes. I2LAR knockdown resulted in upregulation of ITGA2 gene expression, whilst over-expression of I2ALR resulted in downregulation of ITGA2 mRNA. Further, examination of two downstream targets of ITGA2 associated with breast tumor stemness and metastasis (CCND1 and ACLY), revealed concomitant gene expression changes in response to I2ALR modulation. CONCLUSION: I2ALR represents a novel regulatory molecule targeting ITGA2 expression in breast tumours; a finding of significant and topical interest to the development of therapeutics targeting this integrin.

Analysis of a large prostate cancer family identifies novel and recurrent gene fusion events providing evidence for inherited predisposition.

There is strong interest in the characterisation of gene fusions and their use to enhance clinical practices in prostate cancer (PrCa). Significantly, ~50% of prostate tumours harbour a gene fusion. Inherited factors are thought to predispose to these events but, to date, only one study has investigated gene fusions in a familial context. Here, we examined the prevalence and diversity of gene fusions in 14 tumours from a single large PrCa family, PcTas9, using the TruSight® RNA Fusion Panel and Sanger sequencing validation. These fusions were then explored in The Cancer Genome Atlas (TCGA) PrCa data set (n = 494). Overall, 64.3% of PcTas9 tumours harboured a gene fusion, including known erythroblast transformation-specific (ETS) fusions involving ERG and ETV1, and two novel gene fusions, C19orf48:ETV4 and RYBP:FOXP1. Although 3' ETS genes were overexpressed in PcTas9 and TCGA tumour samples, 3' fusion of FOXP1 did not appear to alter its expression. In addition, PcTas9 fusion carriers were more likely to have lower-grade disease than noncarriers (p = 0.02). Likewise, TCGA tumours with high-grade disease were less likely to harbour fusions (p = 0.03). Our study further implicates an inherited predisposition to PrCa gene fusion events, which are associated with less aggressive tumours. This knowledge could lead to clinical strategies to predict men at risk for fusion-positive PrCa and, thus, identify patients who are more or less at risk of aggressive disease and/or responsive to particular therapies.

TELO-SCOPE study: a randomised, double-blind, placebo-controlled, phase 2 trial of danazol for short telomere related pulmonary fibrosis.

INTRODUCTION: Recent discoveries have identified shortened telomeres and related mutations in people with pulmonary fibrosis (PF). There is evidence to suggest that androgens, including danazol, may be effective in lengthening telomeres in peripheral blood cells. This study aims to assess the safety and efficacy of danazol in adults and children with PF associated with telomere shortening. METHODS AND ANALYSIS: A multi-centre, double-blind, placebo-controlled, randomised trial of danazol will be conducted in subjects aged >5 years with PF associated with age-adjusted telomere length ≤10th centile measured by flow fluorescence in situ hybridisation; or in children, a diagnosis of dyskeratosis congenita. Adult participants will receive danazol 800 mg daily in two divided doses or identical placebo capsules orally for 12 months, in addition to standard of care (including pirfenidone or nintedanib). Paediatric participants will receive danazol 2 mg/kg/day orally in two divided doses or identical placebo for 6 months. If no side effects are encountered, the dose will be escalated to 4 mg/kg/day (maximum 800 mg daily) orally in two divided doses for a further 6 months. The primary outcome is change in absolute telomere length in base pairs, measured using the telomere shortest length assay (TeSLA), at 12 months in the intention to treat population. ETHICS AND DISSEMINATION: Ethics approval has been granted in Australia by the Metro South Human Research Ethics Committee (HREC/2020/QMS/66385). The study will be conducted and reported according to Standard Protocol Items: Recommendations for Interventional Trials guidelines. Results will be published in peer-reviewed journals and presented at international and national conferences. TRIAL REGISTRATION NUMBERS: NCT04638517; Australian New Zealand Clinical Trials Registry (ACTRN12620001363976p).

A rare variant in EZH2 is associated with prostate cancer risk.

Prostate cancer (PrCa) is highly heritable, and although rare variants contribute significantly to PrCa risk, few have been identified to date. Herein, whole-genome sequencing was performed in a large PrCa family featuring multiple affected relatives spanning several generations. A rare, predicted splice site EZH2 variant, rs78589034 (G > A), was identified as segregating with disease in all but two individuals in the family, one of whom was affected with lymphoma and bowel cancer and a female relative. This variant was significantly associated with disease risk in combined familial and sporadic PrCa datasets (n = 1551; odds ratio [OR] = 3.55, P = 1.20 × 10-5 ). Transcriptome analysis was performed on prostate tumour needle biopsies available for two rare variant carriers and two wild-type cases. Although no allele-dependent differences were detected in EZH2 transcripts, a distinct differential gene expression signature was observed when comparing prostate tissue from the rare variant carriers with the wild-type samples. The gene expression signature comprised known downstream targets of EZH2 and included the top-ranked genes, DUSP1, FOS, JUNB and EGR1, which were subsequently validated by qPCR. These data provide evidence that rs78589034 is associated with increased PrCa risk in Tasmanian men and further, that this variant may be associated with perturbed EZH2 function in prostate tissue. Disrupted EZH2 function is a driver of tumourigenesis in several cancers, including prostate, and is of significant interest as a therapeutic target.

Exploring the experiences of changes to support access to primary health care services and the impact on the quality and safety of care for homeless people during the COVID-19 pandemic: a study protocol for a qualitative mixed methods approach.

BACKGROUND: Despite high level of health care need amongst people experiencing homelessness, poor access is a major concern. This is sometimes due to organisational and bureaucratic barriers, but also because they often feel stigmatised and treated badly when they do seek health care. The COVID-19 pandemic and the required social distancing measures have caused unprecedented disruption and change for the organisation of primary care, particularly for people experiencing homelessness. Against this backdrop there are many questions to address regarding whether the recent changes required to deliver services to people experiencing homelessness in the context of COVID-19 will help to address or compound problems in accessing care and inequalities in health outcomes. METHODS: An action led and participatory research methodology will be employed to address the study objectives. Interviews with people experiencing homelessness were will be conducted by a researcher with lived experience of homelessness. Researchers with lived experience are able to engage with vulnerable communities in an empathetic, non-judgemental way as their shared experience promotes a sense of trust and integrity, which in turn encourages participation in research and may help people speak more openly about their experience. The experiences of health professionals and stakeholders delivering and facilitating care for people experiencing homelessness during the pandemic will also be explored. DISCUSSION: It is important to explore whether recent changes to the delivery of primary care in response to the COVID-19 pandemic compromise the safety of people experiencing homelessness and exacerbate health inequalities. This could have implications for how primary healthcare is delivered to those experiencing homelessness not only for the duration of the pandemic but in the future.

Epigenetic regulation of the ITGB4 gene in prostate cancer.

BACKGROUND: Examination of epigenetic changes at the ITGB4 gene promoter reveals altered methylation at different stages of prostate tumour progression and these changes may, in part, explain the complex patterns of gene expression of this integrin observed. Transcriptional re-programming perturbs expression of cell adhesion molecules and underpins metastatic tumour cell behaviour. Decreasing expression of the cell adhesion molecule ITGB4, which encodes the beta subunit of the integrin, alpha6 beta4 (α6ß4), has been correlated with increased tumour aggressiveness and metastasis in multiple tumour types including prostate cancer. Paradoxically, in vitro studies in tumour cell models demonstrate that ITGB4 mediates cell mobility and invasion. Herein we examined whether transcriptional re-programming by methylation influenced ITGB4 gene expression at different stages of prostate cancer progression. Bisulphite sequencing of a large CpG island in the ITGB4 gene promoter identified differentially methylated regions in prostate cancer cell lines representing a localised tumour (22Rv1), lymph node metastasis (LNCaP), and a bone metastasis (PC-3). The highest levels of methylation were observed in the CpG island surrounding the ITGB4 transcription start site in PC-3 cells, and this observation also correlated with higher gene expression of ITGB4 in these cells. Furthermore, PC-3 cells expressed two distinct transcripts, using an alternate transcription start site, which was not detected in other cell lines. In prostate tumour biopsy samples, patterns of methylation across the ITGB4 promoter were similar overall in matched primary and metastatic samples (n = 4 pairs), with a trend toward loss of methylation at specific sites in metastatic lesions.

Recurrence patterns identify aggressive form of human papillomavirus-dependent vulvar cancer.

BACKGROUND: Vulvar cancer is rare and, as a result, is understudied. Treatment is predominantly surgery, irrespective of the type of vulvar cancer, and is associated with physical, emotional and sexual complications. A cluster of human papillomavirus (HPV)-dependent vulvar cancer patients was identified in Arnhem Land Northern Territory (NT), Australia, in which young Indigenous women were diagnosed at 70 times the national incidence rate. AIMS: To assess whether women from the Arnhem Land cluster differ from women with vulvar squamous cell carcinoma (VSCC) and vulvar intraepithelial neoplasia (VIN) resident elsewhere in the NT in recurrence after treatment, disease progression and mortality. MATERIALS AND METHODS: A retrospective cohort study of NT-resident women diagnosed with VIN or invasive vulvar cancer (VSCC) between 1 January 1993 and 30 June 2015 was undertaken. Time to recurrence was assessed using cumulative incidence plots and Fine and Gray competing risk regression models. Mean cumulative count was used to estimate the burden of recurrent events. RESULTS: Indigenous women from Arnhem Land experienced more recurrences after treatment than non-Indigenous women, the cancers recurred faster, and Indigenous women have worse survival at five years. CONCLUSIONS: In characterising the epidemiological features of this cluster, we have identified a particularly aggressive form of vulvar cancer. This provides a unique opportunity for elucidating the aetiopathological pathways driving vulvar cancer development that may ultimately lead to preventive and therapeutic targets for this neglected malignancy. Further, these findings have important implications for clinical practice and HPV vaccination policy in the affected population.