Doctor of Philosophy in Medical Genetics (PhD)
Studying the Pharmacogenomics of serious adverse drug reactions in patients with pediatric cancers
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Dissertations completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest dissertations.
Chronic primary systemic vasculitis (PSV) describes a diverse group of debilitating and potentially life-threatening diseases, characterized by inflammation of blood vessels within various organs such as the kidneys, lungs, brain, eyes, and skin. Subtypes of the small- to medium-sized vessel vasculitides are particularly challenging to classify due to many overlapping clinical symptoms. Their differentiation is important, however, as there is evidence that different subtypes benefit from different treatment approaches. The rarity of vasculitis in children has limited pediatric-specific PSV studies and the clinical approach to pediatric PSV is adapted primarily from adult studies. Not surprisingly, adult-derived classification criteria are imperfect for children and consequently, fail to classify up to two thirds of children with small- to medium-vessel PSV. The objective of this dissertation is to identify genetic markers and select, circulating biomarkers to better our understanding and clinical approach to managing the disease and to improve outcomes of children with chronic PSV. Using biological samples and clinical metadata from a worldwide cohort of children with chronic PSV, this dissertation reports (1) genetic associations specific to pediatric autoimmune vasculitis through employing a genome-wide association study; (2) a high prevalence of autoantibodies to lysosome associated membrane protein-2 in pediatric PSV that correlate to vasculitis-associated kidney dysfunction; and (3) the identification of nine patients, originally diagnosed with chronic PSV, harbouring novel and/or rare variants in adenosine deaminase 2 (ADA2) – these genetic data alongside having abrogated ADA2 enzyme activity have led to their reclassification as having a new monogenic form of vasculitis, deficiency of adenosine deaminase 2. This dissertation reports the first large-scale genotype and biomarker study of primary vasculitis focusing solely on pediatric cases. Improved classification is critical for timely therapeutic intervention, for identification of appropriately classified children for clinical trials, and for research, all of which will improve our understanding of the disease and the quality of life of children suffering from chronic PSV.
Theses completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest theses.
The pursuit of precision medicine can be achieved via pharmacogenetic testing prior to treatment onset thanks to the genomic revolution. It has allowed for large-scale genetic testing to be done relatively inexpensively resulting in specific genetic variants to be linked with a phenotype or disease of interest. While beneficial, there remains a burden of proof before translation of knowledge from bench to bedside in order to provide the best possible healthcare. In this study, novel in vitro methods were used with the goal of functionally exploring the role of UGT1A6 in relation to a patient’s risk of the adverse drug reaction (ADR) known as anthracycline-induced cardiotoxicity. More specifically, the variant in UGT1A6 known as rs17863783 has been previously shown, along with other genetic and clinical risk factors, to put a patient at increased risk for cardiotoxicity following anthracycline treatment onset. HEK293 cells were used to create stably transfected expression systems of various haplotypes of UGT1A6. These cells were validated to be expressing the correct genotype via PCR and Sanger Sequencing and subsequently treated with the anthracycline known as doxorubicin (DOX) for viability investigation. Transiently transfected HEK293 cells were also used to express UGT1A6 haplotypes of interest for an investigation of cell viability in addition to UGT1A6 enzyme activity via a 4MU general glucuronidation assay. Despite inherent limitations of in vitro analysis, we conclude that UGT1A6 1) plays a role in anthracycline-induced cardiotoxicity 2) the various haplotypes, such as rs17863783, alter functionality providing additional evidence to support prospective pharmacogenomic testing of patients along with serving as a knowledge base for future research and 3) not all model systems effectively model in vivo phenomena. This thesis will detail the methods and results obtained over the course of investigation into the role of UGT1A6 and anthracycline-induced cardiotoxicity.
Gene editing is a potential treatment for genetic diseases, but current gene editingtechnologies are limited in their use and safety. CRISPR-Cas9 installs double stranded breaks inthe genome which can be hard to fix, as the homologous-driven repair mechanism it uses has lowefficiency. Newer gene editors have more specificity, such as CRISPR/Cas9-derived baseeditors, which enzymatically convert a target base to another. However, base editors are alsocapable of creating unwanted mutations, known as off-target edits, which can potentially lead totumourgenesis. The difficulty of detecting these base changes has resulted in a poorunderstanding of factors involved in off-target edits.For my study, I successfully developed a human cell fluorescent model system using amutated Green Fluorescent Protein (GFP) reporter gene with a premature stop codon. Aftersuccessful base editing to correct the mutation, the cells are analyzed using flow cytometry toquantify the editing efficiency. To simulate guide RNA (gRNA)-dependent off-target editing, Idesigned multiple mismatched sgRNAs with intentional mismatches to the target GFP sequence.These mismatched sgRNAs mimic the true GFP sgRNA matching incorrectly to sequencessimilar to GFP in the genome.I tested the model with three adenosine base editors with different deaminases: the newbase editor ABE8e, which is known for its promiscuous editing activity, an ABE8e containingthe safety mutation V106W to reduce off-target edits, and the standard ABE7.10. I hypothesizedthat using ABE8e V106W with off-target mismatched sgRNAs would have reduced genomeediting compared to ABE8e.ivUsing this model system, ABE8e was shown to correct GFP at 25.5%, ABE8e V106W at28%, and ABE7.10 at 34%. ABE8e and ABE8e V106W showed no editing difference betweenthe mismatched and matched sgRNAs, but ABE7.10 could not tolerate the mismatched sgRNAs.Sanger sequencing showed two bystander edits with ABE8e and ABE8e V106W, potentiallyreducing fluorescence of GFP. These results show the model is capable of modelling sgRNA-dependent off-target edits and was able to detect differences between versions of base editors.This mismatched model could be used to investigate off-target edits by base editors tohelp develop safe, effective gene therapies for treating genetic diseases.
Lipoprotein Lipase (LPL) is responsible for the clearance of triglyceride-rich lipoproteins from the blood. LPL Deficiency is an autosomal recessive genetic disease caused by mutations in the LPL gene that disrupt normal LPL enzyme function resulting in severe hypertriglyceridemia and pancreatitis. Previously, our lab developed an AAV-based gene therapy to treat LPL deficiency by delivering the LPL gene into patients demonstrating proof of concept for viral-based gene therapy. This treatment became the first gene-augmentation therapy to receive regulatory approval (Glybera®). One limitation of this approach, however, is the dysregulated expression of the delivered therapeutic transgene. Gene editing may overcome some limitations of gene augmentation gene therapies, such as dysregulated transgene expression. We hypothesize that CRISPR/Cas9 base editing delivered via lipid nanoparticles can repair the mutant LPL gene and demonstrate a proof of concept for this novel therapeutic approach. We investigated the use of CRISPR/Cas9 based editing composed of a partially deactivated Cas9 (nCas9) protein with an adenine deaminase to directly repair the common P207L mutation in the LPL gene. We generated Flp-In T-RExTM 293 cell lines stably expressing either LPLP207L and wildtype LPL as model systems to explore the gene editing repair of LPLP207L. The effectiveness of base editing was measured both by Sanger DNA sequencing and measuring the restoration of LPL enzyme activity. After optimization of this process, we observed an approximately 50% correction of the LPLP207L mutation by Sanger DNA sequencing, which corresponded with a 52% restoration of LPL enzyme activity compared to wildtype LPL, using an NG-ABE8e base editor. These results demonstrate a proof-of-concept for DNA base editing as a novel treatment strategy to directly repair the LPLP207L mutation that causes LPL deficiency.
Cisplatin is an effective chemotherapeutic agent used for a variety of solid organ malignancies in children and adults. However, its clinical use is limited by the high incidence of cisplatin-induced ototoxicity (CIO), which can affect up to 40-60% of children treated. To date, the genetic basis for CIO has been studied with only focused candidate-gene approaches. Here we report the findings of the first genome-wide association study (GWAS) of cisplatin-induced ototoxicity in children. We examined 738,432 genetics markers in a discovery cohort of 282 Canadian paediatric patients treated with cisplatin, followed by a replication study in an independent Canadian cohort of 82 children. In addition, clinical, therapeutic, and demographic characteristics of cases and controls were analysed to identify clinical factors that may also contribute to the susceptibility to CIO. The genome-wide analyses identified a significant association within the toll-like receptor 4 (TLR4) gene on chromosome 9. The most highly associated single nucleotide polymorphism (SNP) rs960312 conferred a highly protective effect against cisplatin-induced hearing loss (P = 1.19x10-⁸ , odds ratio = 0.22). This variant was subsequently replicated in an independent paediatric cohort (P = 0.018, odds ratio = 0.25). This variant is a tag SNP for a TLR4 promoter haplotype reported to have significantly altered transcriptional efficiency of TLR4. In both cohorts, CIO is significantly associated with younger age (P = 3.41x10-⁶), concomitant vincristine use (P = 2.03x10-¹²), and germ-cell tumour type (P = 4.50x10-⁶). After correcting for these clinical factors, TLR4 rs960312 remains highly associated (Uncorrected P = 1.16x10-⁹ ; Corrected P = 1.01x10-⁹). Several lines of evidence from in vitro and in vivo studies have implicated TLR4 in cisplatin-induced cochlear toxicity and hearing loss. Here we provide the first evidence linking TLR4 and CIO in human patients treated for cancer, leading to new insights into the mechanism underlying this pervasive and clinically limiting adverse drug reaction. The identification of additional markers that contribute to the susceptibility of CIO can be used to develop individualized patient treatments, which can potentially improve safety and treatment outcome of cisplatin.