Proteomic and transcriptomic profiling reveal different aspects of aging in the kidney.

Little is known about the molecular changes that take place in the kidney during the aging process. In order to better understand these changes, we measured mRNA and protein levels in genetically diverse mice at different ages. We observed distinctive change in mRNA and protein levels as a function of age. Changes in both mRNA and protein are associated with increased immune infiltration and decreases in mitochondrial function. Proteins show a greater extent of change and reveal changes in a wide array of biological processes including unique, organ-specific features of aging in kidney. Most importantly, we observed functionally important age-related changes in protein that occur in the absence of corresponding changes in mRNA. Our findings suggest that mRNA profiling alone provides an incomplete picture of molecular aging in the kidney and that examination of changes in proteins is essential to understand aging processes that are not transcriptionally regulated

Mapping in silico genetic networks of tumour suppressor genes to uncover novel gene functions and predict cancer cell vulnerabilities

Sequencing technologies have advanced the discovery of cancer-related genes. However, the functional impact of mutations in these genes and the effect of their mutated forms on a cell’s ability to withstand additional perturbations is unclear. Tumour suppressor genes are cancer-associated genes frequently acquiring loss-of-function (LOF) alterations, rendering them ineffective drug targets. Therefore, alternative methods for identifying druggable targets in cancer cells harbouring LOF alterations in tumour suppressor genes are needed. Genetic networks, namely essentiality and genetic interaction (GI) networks, have been used to attribute novel biological functions to genes and identify genotype-specific vulnerabilities, respectively. However, the low-throughput and laborious nature of in vitro genetic screens have hampered efforts to characterise cancer-associated genetic networks. In this thesis, I explored the use of in silico genetic network mapping to characterise biological functions of tumour suppressor genes and reveal possible vulnerabilities in cells harbouring LOF mutations in these genes. First, I developed a computational tool for generating genetic network maps and provided proof-of-concept using a case study on ARID1A, whose GI and protein complex interactions have been well characterised. Next, I mapped the in silico genetic networks of two tumour suppressor genes, CIC and KMT2D. To characterise CIC’s biological functions, I collaborated to perform multi-omic analyses and revealed new interactions with SWItch/Sucrose Non-Fermentable (SWI/SNF) complex members and a novel potential role in maintaining mitotic integrity. I also characterised KMT2D’s genetic and proteomic interaction networks, identifying potential roles associated with mitotic processes, metabolism, DNA repair, and immune response and predicting several synthetic lethal genetic interactors that are targets of approved or preclinical drugs, including WRN, MDM2, NDUFB5, and TUBA1B. Additionally, I found that markers for immune checkpoint inhibitor (ICI) response, including TUBA1B expression, were elevated in KMT2D-LOF cases with microsatellite instability (MSI) but not in KMT2D wildtype MSI cases in two cancer patient cohorts, indicating that KMT2D-LOF could be a potential biomarker to stratify patients to ICI treatment. The research presented in this thesis thus shows the value of interrogating genetic networks of cancer-associated genes and contributes to our understanding of tumour suppressor gene functions and cancer cell vulnerabilities.Science, Faculty ofGraduat

Whole-Genome Sequence of the C57L/J Mouse Inbred Strain.

We sequenced the complete genome of the widely used C57L/J mouse inbred strain. With 40× average coverage, we compared the C57L/J sequence with that of the C57BL/6J and identified many known as well as novel private variants. This genome sequence adds another strain to the growing number of mouse inbred strains with complete genome sequences and is a valuable resource to the scientific community. G3 (Bethesda) 2014 Sep; 4(9):1689-92

Proteomic and transcriptomic profiling reveal different aspects of aging in the kidney

Little is known about the molecular changes that take place in the kidney during the aging process. In order to better understand these changes, we measured mRNA and protein levels in genetically diverse mice at different ages. We observed distinctive change in mRNA and protein levels as a function of age. Changes in both mRNA and protein are associated with increased immune infiltration and decreases in mitochondrial function. Proteins show a greater extent of change and reveal changes in a wide array of biological processes including unique, organ-specific features of aging in kidney. Most importantly, we observed functionally important age-related changes in protein that occur in the absence of corresponding changes in mRNA. Our findings suggest that mRNA profiling alone provides an incomplete picture of molecular aging in the kidney and that examination of changes in proteins is essential to understand aging processes that are not transcriptionally regulated

FAR2 is Associated with Kidney Disease in Mice and Humans.

Mesangial matrix expansion is an important process in the initiation of chronic kidney disease, yet the genetic factors driving its development are unknown. Our previous studies have implicated Far2 as a candidate gene associated with differences in mesangial matrix expansion between mouse inbred strains. Consistent with the hypothesis that increased expression of Far2 leads to mesangial matrix expansion through increased production of platelet-activating factor precursors, we show that FAR2 is capable of mediating de novo platelet-activating factor synthesis in vitro and driven by the transcription factor NKX3.2. We demonstrate that knockdown of Far2 in mice delays the progression of mesangial matrix expansion with at least six months (equivalent to approximately 15 years in human). Furthermore, we show that increased FAR2 expression in human patients is associated with diabetic nephropathy, lupus nephritis, and IgA nephropathy. Taken together, these results highlight FAR2\u27s role in the development of mesangial matrix expansion and chronic kidney disease

Uncovering Modifier Genes of X-Linked Alport Syndrome Using a Novel Multiparent Mouse Model.

BACKGROUND: Mutations in METHODS: We created a cohort of genetically diverse XLAS male and female mice using the Diversity Outbred mouse resource and measured albuminuria, GFR, and gene expression. Using a quantitative trait locus approach, we mapped modifier genes that can best explain the underlying phenotypic variation measured in our diverse population. RESULTS: Genetic analysis identified several loci associated with the variation in albuminuria and GFR, including a locus on the X chromosome associated with X inactivation and a locus on chromosome 2 containing CONCLUSION: With this novel approach, we emulated the variability in the severity of kidney phenotypes found in human patients with Alport Syndrome through albuminuria and GFR measurements. This approach can identify modifier genes in kidney disease that can be used as novel therapeutic targets

Uncovering Modifier Genes of X-linked Alport Syndrome Using a Novel Multi-parent Mouse Model

BACKGROUND: Mutations in METHODS: We created a cohort of genetically diverse XLAS male and female mice using the Diversity Outbred mouse resource and measured albuminuria, GFR, and gene expression. Using a quantitative trait locus approach, we mapped modifier genes that can best explain the underlying phenotypic variation measured in our diverse population. RESULTS: Genetic analysis identified several loci associated with the variation in albuminuria and GFR, including a locus on the X chromosome associated with X inactivation and a locus on chromosome 2 containing CONCLUSION: With this novel approach, we emulated the variability in the severity of kidney phenotypes found in human patients with Alport Syndrome through albuminuria and GFR measurements. This approach can identify modifier genes in kidney disease that can be used as novel therapeutic targets

Linked-read Sequencing Analysis Reveals Tumor-specific Genome Variation Landscapes in Neurofibromatosis Type 2 (NF2) Patients.

HYPOTHESIS: We hypothesize that genomic variants including deletions, insertions, inversions, and tandem duplications beyond the changes in tumor suppressor NF2 gene affect gene expression of tumor-specific pathways in vestibular schwannomas (VS) patients with Neurofibromatosis type 2 (NF2), thus contributing to their clinical behavior. BACKGROUND: Genomic variation could reconfigure transcription in NF2 transformation process. Therefore, genome-wide high-resolution characterization of structural variants (SV) landscapes in NF2 tumors can expand our understanding of the genes regulating the clinical phenotypes in NF2-associated VS. METHODS: We performed whole-genome haplotype-specific structural variation analysis using synthetic linked reads generated through microfluidics-based barcoding of high molecular weight DNA followed by high-coverage Illumina paired-end whole-genome sequencing from 10 patients\u27 tumors of different growth rates and their matching blood samples. RESULTS: NF2 tumor-specific deletions and large SVs were detected and can be classified based on their association with tumor growth rates. Through detailed annotation of these mutations, we uncover common alleles affected by these deletions and large SVs that can be associated with signaling pathways implicated in cell proliferation and tumorigenesis. CONCLUSION: The genomic variation landscape of NF2-related VS was investigated through whole-genome linked-read sequencing. Large SVs, in addition to deletions, were identified and may serve as modulators of clinical behavior

Multi-Omic Analysis of CIC’s Functional Networks Reveals Novel Interaction Partners and a Potential Role in Mitotic Fidelity

CIC encodes a transcriptional repressor and MAPK signalling effector that is inactivated by loss-of-function mutations in several cancer types, consistent with a role as a tumour suppressor. Here, we used bioinformatic, genomic, and proteomic approaches to investigate CIC’s interaction networks. We observed both previously identified and novel candidate interactions between CIC and SWI/SNF complex members, as well as novel interactions between CIC and cell cycle regulators and RNA processing factors. We found that CIC loss is associated with an increased frequency of mitotic defects in human cell lines and an in vivo mouse model and with dysregulated expression of mitotic regulators. We also observed aberrant splicing in CIC-deficient cell lines, predominantly at 3′ and 5′ untranslated regions of genes, including genes involved in MAPK signalling, DNA repair, and cell cycle regulation. Our study thus characterises the complexity of CIC’s functional network and describes the effect of its loss on cell cycle regulation, mitotic integrity, and transcriptional splicing, thereby expanding our understanding of CIC’s potential roles in cancer. In addition, our work exemplifies how multi-omic, network-based analyses can be used to uncover novel insights into the interconnected functions of pleiotropic genes/proteins across cellular contexts.Medicine, Faculty ofOther UBCNon UBCMedical Genetics, Department ofPathology and Laboratory Medicine, Department ofReviewedFacultyResearcherGraduat