A hierarchical model to detect differential gene expression distributions, and their investigation as a reflection of dysregulation in cancer

University of Technology Sydney. Faculty of Engineering and Information Technology.Data from genome-wide gene expression studies provides a wealth of information on diseases such as cancer, which can lead to insights into disease mechanisms and advances in diagnosis and treatment. Analysis of expression data is most commonly aimed at identifying genes whose mean expression levels are increased or decreased in disease compared to normal tissue, or between disease subtypes - differential expression analysis. However, there is strong evidence that changes in the variability of gene expression, without a difference in mean, can also be relevant. Genes related to cancer have been shown to have changes in the variability of their expression between normal and tumour tissue, and these differentially variable genes have also been found to be informative for diagnostic and prognostic cancer classification. This thesis addresses several aspects of research on differential gene expression variability, and the broader concept of differential distribution, defined as any difference in the distribution of expression values between groups. This work makes three contributions to knowledge, relating to cancer classification, identification of differentially variable or distributed genes, and the biology of differential variability and distribution in cancer. Contribution 1 extends previous work by demonstrating that genes identified by differential variability or distribution can be used to classify closely related cancer subtypes, rather than purely diagnostic or prognostic classification. Contribution 2 is a Bayesian hierarchical model for RNA-seq data that provides tests for differential expression, variability and distribution. The performance of each test is compared with existing methods on simulated data and on real RNA-seq datasets modified to artificially introduce changes in expression between groups. The differential expression test is competitive with state-of-the-art methods, and the differential variability test improves on existing methods, particularly for small sample sizes. The differential distribution test is the first such test available for RNA-seq data. Contribution 3 builds on previous work by providing the first clear demonstration that differential variability and differential distribution analyses can identify cancer-related genes, and that differential expression and differential variability identify distinct sets of cancer-related genes, each with different biological functions. Overall, this research confirms and extends previous findings showing that changes in expression variability and distribution in cancer are both of biological significance and informative for classification. As well as further demonstrating the need to look beyond differential expression to a comprehensive assessment of changes in gene expression distributions, this work provides a method that enables the identification of these differentially distributed genes

Linkage to chromosome 2q32.2-q33.3 in familial serrated neoplasia (Jass syndrome)

Causative genetic variants have to date been identified for only a small proportion of familial colorectal cancer (CRC). While conditions such as Familial Adenomatous Polyposis and Lynch syndrome have well defined genetic causes, the search for variants underlying the remainder of familial CRC is plagued by genetic heterogeneity. The recent identification of families with a heritable predisposition to malignancies arising through the serrated pathway (familial serrated neoplasia or Jass syndrome) provides an opportunity to study a subset of familial CRC in which heterogeneity may be greatly reduced. A genome-wide linkage screen was performed on a large family displaying a dominantly-inherited predisposition to serrated neoplasia genotyped using the Affymetrix GeneChip Human Mapping 10 K SNP Array. Parametric and nonparametric analyses were performed and resulting regions of interest, as well as previously reported CRC susceptibility loci at 3q22, 7q31 and 9q22, were followed up by finemapping in 10 serrated neoplasia families. Genome-wide linkage analysis revealed regions of interest at 2p25.2-p25.1, 2q24.3-q37.1 and 8p21.2-q12.1. Finemapping linkage and haplotype analyses identified 2q32.2-q33.3 as the region most likely to harbour linkage, with heterogeneity logarithm of the odds (HLOD) 2.09 and nonparametric linkage (NPL) score 2.36 (P = 0.004). Five primary candidate genes (CFLAR, CASP10, CASP8, FZD7 and BMPR2) were sequenced and no segregating variants identified. There was no evidence of linkage to previously reported loci on chromosomes 3, 7 and 9

Risk Factors for Colorectal Cancer in Patients with Multiple Serrated Polyps: A Cross-Sectional Case Series from Genetics Clinics

Patients with multiple serrated polyps are at an increased risk for developing colorectal cancer (CRC). Recent reports have linked cigarette smoking with the subset of CRC that develops from serrated polyps. The aim of this work therefore was to investigate the association between smoking and the risk of CRC in high-risk genetics clinic patients presenting with multiple serrated polyps. Methods and Findings We identified 151 Caucasian individuals with multiple serrated polyps including at least 5 outside the rectum, and classified patients into non-smokers, current or former smokers at the time of initial diagnosis of polyposis. Cases were individuals with multiple serrated polyps who presented with CRC. Controls were individuals with multiple serrated polyps and no CRC. Multivariate logistic regression was performed to estimate associations between smoking and CRC with adjustment for age at first presentation, sex and co-existing traditional adenomas, a feature that has been consistently linked with CRC risk in patients with multiple serrated polyps. CRC was present in 56 (37%) individuals at presentation. Patients with at least one adenoma were 4 times more likely to present with CRC compared with patients without adenomas (OR = 4.09; 95%CI 1.27 to 13.14; P = 0.02). For females, the odds of CRC decreased by 90% in current smokers as compared to never smokers (OR = 0.10; 95%CI 0.02 to 0.47; P = 0.004) after adjusting for age and adenomas. For males, there was no relationship between current smoking and CRC. There was no statistical evidence of an association between former smoking and CRC for both sexes. Conclusion A decreased odds for CRC was identified in females with multiple serrated polyps who currently smoke, independent of age and the presence of a traditional adenoma. Investigations into the biological basis for these observations could lead to non-smoking-related therapies being developed to decrease the risk of CRC and colectomy in these patients.Daniel D. Buchanan, Kevin Sweet, Musa Drini, Mark A. Jenkins, Aung Ko Win, Dallas R. English, Michael D. Walsh, Mark Clendenning, Diane M. McKeone, Rhiannon J. Walters, Aedan Roberts, Sally-Ann Pearson, Erika Pavluk, John L. Hopper, Michael R. Gattas, Jack Goldblatt, Jill George, Graeme K. Suthers, Kerry D. Phillips, Sonja Woodal, Julie Arnold, Kathy Tucker, Amanda Muir, Michael Field, Sian Greening, Steven Gallinger, Renee Perrier, John A. Baron, John D. Potter, Robert Haile, Wendy Franke, Albert de la Chapelle, Finlay Macrae, Christophe Rosty, Neal I. Walker, Susan Parry and Joanne P. Youn

Ethnicity and risk for colorectal cancers showing somatic BRAF V600E mutation or CpG island methylator phenotype

Colorectal cancers arising from serrated polyps are characterized by the CpG island methylator phenotype (CIMP) and somatic mutation (V600E) in the BRAF proto-oncogene. Few epidemiologic studies have investigated risk factors for these tumors. We conducted a cohort study of 41,328 residents of Melbourne, Australia that included 9,939 participants of southern European origin and 31,389 of Anglo-Celtic origin. Colorectal adenocarcinomas were identified from population-based cancer registries. BRAF V600E mutation in tumors was determined using a PCR-based allelic discrimination method. Tumors were classified as CIMP positive when at least three of five markers (RUNX3, CACNA1G, SOCS1, NEUROG1, and IGF2) were methylated according to MethyLight analysis. Hazard ratios (HR) and 95% confidence intervals (95% CI) were estimated by Cox regression with adjustment for risk factors for colorectal cancer. During follow-up, 718 participants were diagnosed with colorectal cancer. CIMP assays were done for 579 and BRAF V600E mutation testing for 582. After adjustment for other risk factors, when compared with people of Anglo-Celtic origin, those of southern European origin had lower incidence of colorectal cancer that had CIMP (HR, 0.32; 95% CI, 0.16-0.67) or BRAF mutations (HR, 0.30; 95% CI, 0.16-0.58) but similar incidence of colorectal cancer without CIMP (HR, 0.86; 95% CI, 0.70-1.05) or BRAF (HR, 0.90; 95% CI, 0.74-1.11). People of southern European origin had lower risk of colorectal cancers with CIMP and BRAF mutation than people of Anglo-Celtic origin, which may in part be due to genetic factors that are less common in people of southern European origin

Germline mutations and somatic inactivation of <i>TRIM28</i> in Wilms tumour

<div><p>Wilms tumour is a childhood tumour that arises as a consequence of somatic and rare germline mutations, the characterisation of which has refined our understanding of nephrogenesis and carcinogenesis. Here we report that germline loss of function mutations in <i>TRIM28</i> predispose children to Wilms tumour. Loss of function of this transcriptional co-repressor, which has a role in nephrogenesis, has not previously been associated with cancer. Inactivation of <i>TRIM28</i>, either germline or somatic, occurred through inactivating mutations, loss of heterozygosity or epigenetic silencing. <i>TRIM28</i>-mutated tumours had a monomorphic epithelial histology that is uncommon for Wilms tumour. Critically, these tumours were negative for TRIM28 immunohistochemical staining whereas the epithelial component in normal tissue and other Wilms tumours stained positively. These data, together with a characteristic gene expression profile, suggest that inactivation of <i>TRIM28</i> provides the molecular basis for defining a previously described subtype of Wilms tumour, that has early age of onset and excellent prognosis.</p></div

DNA sequence and methylation of <i>TRIM28</i>.

<p>(<b>A</b>) Family 1. 2-bp deletion (c.525_526del) in the blood of case 39 (39B), the kidney of case 37 (37K) and the blood of their mother (37M). The father (37F) was unaffected. The tumours from cases 37 and 39 (37T and 39T) showed loss of heterozygosity. (<b>B</b>) Family 2. Germline deletion/insertion (c.1746_1747delinsC) in blood DNA from case 399 (399N) with loss of heterozygosity in tumours 399T and 249T. (<b>C</b>) Somatic deletion/insertion mutation (c.1935delinsGA) in W117 tumour (W117T) and reference sequence in the adjacent kidney (W117K). (<b>D</b>) The proportion of methylated CpGs in exon 1 of <i>TRIM28</i> in W117T as measured by targeted bisulfite PCR. For each CpG site the black portion of the bar shows the proportion of methylated reads.</p

Somatic genetic changes in Wilms tumours.

<p>The left side shows the number of somatic non-synonymous and truncating mutations for each tumour detected by MuTect2. The single somatic variant in W117 is the <i>TRIM28</i> mutation. The right side shows the fractional length of aberrant copy number segments as determined by ADTEx.</p

Dendrogram from unsupervised hierarchical clustering of gene expression of 17 Wilms tumours.

<p>IGF2, refers to <i>IGF2</i> status where blue = loss of imprinting, and red = loss of heterozygosity at <i>IGF2</i>. Rests refers to the presence of nephrogenic rests (NR) were blue = intralobar NR, red perilobar NR and purple NR of unknown type. For each gene, red boxes indicate the presence of mutation, whereas the grey box denotes gene deletion.</p