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Theses - Medical Genetics


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  • ItemEmbargo
    Genetic basis of inherited kidney and related tumours
    Andreou, Avgi
    Renal cell carcinoma (RCC) represents the 6th most commonly diagnosed cancer in males and the 10th most commonly diagnosed cancer in females, worldwide, with incidence rates rising. It has been estimated that 3-5% of RCC is inherited. The aim of this thesis was to improve the understanding of the genetic basis of predisposition to RCC by identifying the genetic architecture of inherited RCC, potential novel genetic causes and mechanisms of tumourigenesis. Additionally, evidence-based recommendations for the molecular investigation, germline variant interpretation and clinical management of patients with syndromic and non-syndromic inherited RCC are suggested. A variety of investigative approaches were utilised for this thesis. Firstly, a service evaluation was undertaken which showed a low diagnostic yield (2-3%) for a current clinical RCC panel consisting of 6 genes (*BAP1*, *FH*, *FLCN*, *MET*, *SDHB*, *VHL*). Following this I explored whether a larger panel consisting of 121 cancer susceptibility genes (CSGs) would increase the diagnostic yield and identify novel CSG and RCC phenotype associations in participants recruited with renal cancer (N=1,336) in the 100 000 Genomes Project (100KGP) which was unselected for inherited RCC risk factors. Whole genome sequencing (WGS) analysis identified 6.4% (95%CI [5.1, 7.8]) pathogenic/likely pathogenic variants (85 SNVs/indels and 3 CNVs) in a wider range of CSGs than previously recognised and mean RCC onset was 58.6 years. 24 ‘hot’ VUSs were also identified potentially increasing the diagnostic yield to 8%. Burden test analyses demonstrated excess of *CHEK2* variants in European RCC cases compared to controls (*P*=0.0019) and a trend of association with double strand homologous (DS HR) and nonhomologous repair (DS NHR) pathways. Candidate novel RCC genes were sought by familial studies on 13 trios recruited to the 100KGP and the Human Molecular Pathology of Genetic Disease (HumGenDis) study with WGS/WES data available. Trio analysis identified a novel *de* *novo* pathogenic variant in elongin C (*ELOC*), NM_005648.4:c.236A>G[p.Tyr79Cys] in a proband with a von Hippel Lindau phenotype. This variant occurs as a somatic hotspot mutation in RCC and disrupts pVHL-ELOC interaction mimicking the effects of pVHL inactivation on expression of hypoxia response elements. *ELOC* functions as a tumour suppressor gene and chromosome 8 deletion was demonstrated in the RCC tissue from the proband and other sporadic *ELOC*-mutated RCC tumours in the 100KGP and the literature. Five additional novel candidate genes (*CTTN*, *DMXL1*, *MUC17*, *GANAB* and *DET1*) were identified in other probands, all with missense *de* *novo* variants, which require validation in larger cohorts and/or additional functional studies to definitively determine their pathogenicity and clinical significance. To enhance clinical management of families with suspected inherited RCC, I explored the sensitivity, specificity and positive predictive value of immunohistochemical (IHC) staining for the fumarate hydratase protein expression and the antibody detection of S-(2-succino)-cysteine (2-SC) in determining FH variant pathogenicity. A clinical pathway of investigation was proposed for individuals presenting with leiomyomatosis. IHC for 2SC and FH used together had a 100% sensitivity, specificity and PPV in identifying P/LP FH variants. In addition, the frequency of candidate P/LP FH variants in the 100KGP cohort was explored and age-related RCC risks were calculated for participants with fumarase deficiency (FMRD) related variants (4%; 95%CI:0.88-1 by age 75 years). Overall frequency of candidate P/LP *FH* variants in the 100KGP cohort was 1 in 200; 1 in 5,000 for HLRCC-related variants and 1 in 250 for FMRD related *FH* variants suggesting that HLRCC is underdiagnosed and/or less penetrant than previously thought. In conclusion, about 6% of unselected RCC cases carry a P/LP variant showing that inherited RCC is more common than previously thought, thus I suggest expanding the existing clinical RCC panel and raise the age threshold for testing. *ELOC* is a novel candidate gene for VHL-disease related phenotypes and testing should be offered to VHL patients with no pathogenic VHL variant identified. I propose a management pathway for the investigation of suspected HLRCC in individuals with multiple leiomyomas and I showed that candidate P/LP FH variants are more common than previously thought with implications for RCC screening. Further work could involve validating findings in additional cohorts and with additional technologies like long read sequencing.
  • ItemEmbargo
    Deciphering the genetic and epigenetic basis of human imprinting and chromatin disorders
    Lee, Sunwoo
    Aberrant DNA methylation patterns, including disturbances in genomic imprinting, have been linked to congenital imprinting disorders as well as various disease conditions, including cancer, chromatin disorders, and neurodevelopmental disorders. Some imprinting disorders, however, result in alterations in multiple imprinted regions (MLID) for which their exact causes are unknown. The aetiology of rare disorders, including MLID, remains largely uncertain, requiring further investigations to elucidate their underlying molecular mechanisms and their potential association with sporadic congenital anomalies. Due to these reasons, the clinical diagnosis can be challenging, and genetic testing is vital in order to confirm the diagnosis. Unfortunately, current diagnostic methods have significant limitations. To address these issues, I used novel epigenetic assays (ImprintSeq and EPIC-NGS) and genomic testing to investigate the molecular basis of human imprinting and chromatin disorders. A major aim was to provide a more accurate diagnosis of imprinting and chromatin disorders in order to improve the care of children with epigenetic disorders. ImprintSeq has demonstrated high accuracy, sensitivity, and specificity in the detection of methylation alterations at multiple imprinted regions, and EPIC-NGS has demonstrated the capability to detect disease methylation episignatures (e.g., childhood-onset dystonia, Kabuki syndrome, Sotos syndrome, and Luscan-Lumish syndrome). In particular, methylation episignature analysis enabled the identification of epigenotype-phenotype associations and provided insights into pathogenetic mechanisms of copy number variants (duplicates or deletions) and variants of uncertain significance (VUSs). In addition, genome-wide methylation profiling showed the potential to identify candidate genes involved in epigenetic disorders. My PhD research has identified innovative diagnostic approaches for individuals afflicted by epigenetic disorders, highlighting their potential utility within clinical practice. Ultimately, my research aims to provide substantial benefits to individuals suffering from rare diseases by facilitating earlier and more precise diagnoses.
  • ItemRestricted
    Discovery of mechanisms regulating transcription factor EB (TFEB)
    Keshri, Swati; Keshri, Swati [0000-0003-3663-3574]
  • ItemEmbargo
    Characterising the roles of mammalian atlastins in the endoplasmic reticulum and beyond
    Zlamalova, Eliska
    Hereditary spastic paraplegia (HSP) is a group of currently incurable disorders caused by the degeneration of corticospinal upper motor neuron axons, which are among the longest in the body. Recent research suggests that a few unifying pathways could link many genetically distinct HSP subtypes. Investigating these pathways and the pathological mechanisms affecting them is crucial to advance the development of effective treatments for HSP and related neurodegenerative disorders, including Alzheimer’s and Parkinson’s disease, multiple sclerosis, peripheral neuropathies and various motor neuron diseases. This project focuses on endoplasmic reticulum (ER) proteins atlastins (ATLs), particularly ATL1, as *ATL1* gene mutations cause the most frequent childhood-onset form of HSP. Using CRISPRi, I engineered ATL1 knock-down (KD) into the i3 human induced pluripotent stem cells (iPSCs) and utilised iPSC-derived neurons to investigate the roles of ATL1 in the ER and other cellular compartments and processes commonly affected in HSP. Preliminary findings showed fewer ER junctions in ATL1 KD neurons, consistent with known atlastin’s role in ER tubule fusion. Additionally, ER proteins were highly enriched in a protein list with altered abundance in ATL1 KD neurons determined through proteomics. Moreover, abnormalities in the endo-lysosomal system were identified in ATL1-depleted neurons. They had slightly fewer and larger lysosomes with reduced degradative capacity. Additionally, neuronal endosomal tubules were elongated, pointing towards a potential tubule fission defect which could lead to missorting of diverse cargo, such as lysosomal hydrolases. Lipidomics and neutral lipid staining revealed higher triglyceride content in neurons lacking ATL1. Moreover, blocking lipolysis degradation led to lipid droplet (LD) accumulation, with significantly more LDs in ATL1 KD neurons. The cellular content of other lipid classes was also altered, indicating potential lipid imbalance resulting from ATL1 loss. Finally, developing ATL1-depleted neurons exhibited altered morphology, with a significantly longer dominant neurite. Despite the detected phenotypes, the lack of ATL1 had no effect on neuronal viability. These observations demonstrate that atlastin deficiency impacts cellular processes beyond its conventional role in ER shaping. In addition, the findings and tools developed in this study provide a foundation to investigate underlying mechanisms and pathways connecting the phenotypes resulting from ATL1 deficiency.
  • ItemOpen Access
    Understanding the biological processes underpinning neurodegeneration in Xeroderma Pigmentosum
    Momen, Sophie
    Xeroderma pigmentosum (XP) is a rare genetic disorder of nucleotide excision repair (NER). XP is classified into eight complementation groups, XP-A to XP-G and V, depending on the protein component of the NER pathway that is defective. NER is split into two branches. Transcription coupled repair (TC-NER), which rapidly repairs DNA damage on the transcribed strand of actively transcribed regions of DNA. Global genome NER (GG-NER) is a slower process that repairs DNA damage on non-transcribed strands. Patients with defective NER are unable to repair Ultraviolet (UV) induced DNA damage. The somatic mutations not repaired cause the characteristic features of 10,000-fold increased risk of skin cancer, internal malignancies and neurodegeneration. Neurodegeneration occurs in patients with deficient TC-NER (complementation groups A, D, F and G). Since UV radiation does not reach the brain, the neurological disease in XP is thought to be the result of DNA damage that is normally repaired by NER, however the exact cause of neurodegeneration is not understood. In this thesis, I set out to understand the biological processes underlying neurodegeneration in XP. I studied multiple patients from a variety of complementation groups using a human induced pluripotent stem (hiPSC) model. Functional multi-omic characterisation at several stages of the directed differentiation of hiPSCs into neurons revealed an elevated endoplasmic reticulum stress response and dysfunction of the ubiquitin proteasome system (UPS) in XP neuronal models. I have also demonstrated an accumulation of multiple oxidised nucleosides, implicating oxidative stress as a contributing factor to the disease process. This study demonstrates that such hiPSC systems can be used to successfully model XP and collectively these findings offer new insights into the pathogenesis of neurodegeneration in XP. In the future, these results can be validated in patient-derived samples held in national brain biobanks and we can further explore these implicated pathways with the aim to determine how mutations in genes of the NER pathway result in the findings of this thesis. I have also explored the mutational signatures observed in XP and I present a case study of the clinical applications of WGS in an XP patient with metastatic angiosarcoma.
  • ItemOpen Access
    Variational Mixture Models for non-Gaussian observations: Applications to molecular data
    Gerontogianni, Stavroula
    Epigenetics is the field of biology that studies the changes in organisms due to alteration of gene expression rather than modification of the DNA sequence itself. DNA methylation is a well-studied type of epigenetic change, which results in gene silencing and can be dangerous when occurs at tumour suppressor gene loci. Many techniques have been developed to map the methylation pattern of individuals at several genetic loci, such as the HumanMethylation450 BeadChip, the EPIC BeadChip and the whole-genome bisulfite sequencing. Each of these DNA profiling platforms quantifies methylation occurrence in different ways, either continuously (rates of methylation intensity) or discretely (counts of methylated reads). Identifying subgroups of individuals with similar methylation patterns, as well as those genetic loci that discriminate the subgroups, is a crucial procedure that helps linking diseases to specific methylation patterns. Clustering analysis and posterior feature selection of the most important genetic loci that discriminate each subgroup of individuals are the two tools we suggest for achieving this venture. Clustering DNA methylation data though is not a trivial procedure since they are platform-specific and not normally distributed. In this thesis, we propose clustering DNA methylation data based on the data type (continuous or discrete) by fast model-based clustering methods, while we select the most important/discriminatory genetic loci by an a posteriori feature selection measure. Specifically, we apply variational non-Gaussian Dirichlet Process mixture models because they have infinite number of components that allow model-determination and are flexible to model any discrete or continuous data type. We also employ Variational Inference with the “annealing” extension that accounts for poor initialisation of the algorithm, due to its high speed in estimating the model parameters and its scalability to high-dimensional data. Our real applications on neonatal DNA methylation data measured in three different ways show that the discrete data types - number of aberrantly methylated genetic loci (counts) and whether a genetic locus is abnormally methylated or not (binary) - can be more informative than its continuous version (intensity of methylation per genetic locus) for revealing the association of artificial conception with the predisposition of developmental disorders.
  • ItemOpen Access
    On the neurobiology of apathy and depression in cerebral small vessel disease
    Tay, Jonathan
    Cerebral small vessel disease (SVD) is a cerebrovascular pathology that affects the small vessels of the brain, resulting in heterogeneous brain tissue changes. These can lead to neuropsychiatric symptoms such as apathy, a loss of motivation, and depression, which is characterised by low mood and a loss of pleasure. Apathy and depression are both prevalent symptoms in SVD, but an understanding of the relationship between underlying disease processes and the expression of these neuropsychiatric symptoms remains poor. This thesis uses magnetic resonance imaging techniques to examine the neurobiological basis of apathy and depression in SVD. We show that apathy is related to focal grey matter damage and distributed white matter microstructural change. These microstructural changes underlie large-scale white matter network disruption, which is related to apathy, but not depression. We then show that depression, as a construct, can be dissociated into distinct symptoms which are associated with overlapping and distinct areas of cortical atrophy over time. This suggests that depression as a general syndrome may be characterised by atrophy in core structures, while different symptoms are associated with atrophy in more specialised areas. Consistent with these patterns of overarching tissue damage, we find that apathy, but not depression, predicts conversion to dementia in patients with SVD. Our findings suggest that different types of SVD-related pathology lead to apathy and depression. Diffuse white matter damage may lead to widespread network disruption, resulting in apathy and cognitive impairment. In contrast, depressive symptoms are associated with focal patterns of grey matter atrophy over time. This highlights the importance of differentiating neuropsychiatric symptoms, and paves the way for targeted treatment approaches.
  • ItemOpen Access
    Elucidating the constitutional genetic basis of multiple primary tumours
    (2019-10-26) Whitworth, James William
    Cancer predisposition syndromes are responsible for a significant minority of neoplasm occurrences and beget opportunities to mitigate the associated risks with clinical intervention. They are caused by constitutional genetic variation affecting tumour suppressor genes or proto-oncogenes and recent improvements in sequencing technology have led to greater capability to detect more affected individuals through diagnostic testing. Predisposition syndromes can be suggested by a variety of clinical indicators that can be used to prioritise cases for diagnostic assessment or research. These include the occurrence of multiple primary tumours (MPT) in the same individual, a phenomenon which forms the basis of this project and is observed more frequently in populations as cancer survivorship increases. A series of MPT cases was assembled through recruitment from clinical genetics centres. These had previously been assessed without a molecular diagnosis being identified. A heterogenous tumour profile was observed, apparently influenced by population frequency and pattern of referral to cancer genetics services. Comparison with a population based series emphasised these influences. Constitutional DNA samples from participants were subject to whole genome sequencing (WGS) and panel based sequencing to elicit causative variants in known cancer predisposition genes (CPGs) or at novel loci. Analysis of WGS data for variants affecting known CPGs showed that a pathogenic or likely pathogenic (P/LP) variant was detected in around 15% of previously undiagnosed cases. Just over 1% of these were structural variants, which would unlikely have been detected with nonWGS approaches. Over 40% of probands who harboured a P/LP variant had been diagnosed with a tumour that is not typical of P/LP variants in the relevant CPG, which may represent phenotypic expansion or coincidental tumours. Three individuals harboured two P/LP variants in two CPGs, a phenomenon which is reviewed in the context of cases previously reported in the literature. A clinical scoring system was devised and tested to predict which individuals had P/LP variants but this showed little potential for clinical utility. Panel data was interrogated for possible mosaic CPG variants but few were identified that were likely to be genuinely post-zygotic and causative. Various case control based analyses to identify novel loci associated with cancer predisposition were undertaken using single nucleotide variant calls and/or structural variant calls from WGS data. These focused on a number of putative coding and non-coding regions including genes recurrently mutated in somatic cancer studies, known or putative gain of function CPGs, telomere related genes, enhancers/promoters, ultra-conserved elements and expression quantitative trait loci observed in normal and cancer tissues. The potential efficacy of this approach was demonstrated by the over-representation of variants affecting known CPGs (CHEK2, PALB2, MAX, NF1) in some phenotypic subgroups. However, there was little evidence for novel loci relevant to cancer predisposition.
  • ItemOpen Access
    A study of succinate dehydrogenase deficient tumourigenesis: From functional assessment of variant pathogenicity to the discovery of new disease biomarkers
    (2019-07-20) Casey, Ruth
    A loss of function of the citric acid cycle enzyme complex succinate dehydrogenase (SDH) is associated with a predisposition to a spectrum of tumourigenesis including phaeochromocytoma, paraganglioma (1) (PPGL), gastrointestinal stromal tumours (GIST) (2), renal cell carcinoma (RCC) (3) and pituitary adenomas (4). Pathogenic variants in each of the four genes (SDHx) encoding the four sub-components of this complex (SDHA/B/C/D) have been associated with tumourigenesis. Germline pathogenic variants in SDHB account for up to 50% of patients with malignant PPGL and a 5 year survival of less than 50% in those with malignancy (5). Most SDHx variant carriers require life long surveillance for tumour development (6) but predicting malignant disease is challenging and histology is of limited assistance in this prediction. The advent of next generation sequencing (NGS) has been influential in this field of inherited neoplasia allowing more rapid and accurate identification of pathogenic variants in the SDHx genes. However the increased throughput achieved with NGS methodology has yielded more variants of uncertain significance in these genes which require additional assessment. New diagnostic adjuncts such as SDHB immunohistochemistry(7), have provided additional prognostic information and prediction of malignant risk but further biomarkers are needed. Furthermore there is a lack of effective treatments for malignant disease associated with SDHx variants (8) (9). This ‘multi-omics’ investigation has provided new insights into genotype-phenotype correlations in SDH deficient disease and has facilitated the translation of new techniques into clinical utility which will aid SDHx variant interpretation. This study has evaluated novel disease biomarkers and potential therapeutic targets in SDH deficient tumour
  • ItemOpen Access
    The use of whole exome sequencing data to identify candidate genes involved in cancer and benign tumour predisposition
    (2019-01-01) Fewings, Eleanor Rose; Fewings, Eleanor Rose [0000-0001-9012-3220]
    The development of whole exome sequencing has transformed the study of disease predisposition. The sequencing of both large disease sets and smaller rare disease families enables the identification of new predisposition variants and potentially provide clinical insight into disease management. There is no standard protocol for analysing exome sequencing data. Outside of extremely large sequencing studies including thousands of individuals, statistical approaches are often underpowered to detect rare disease associated variants. Aggregation of variants into functionally related regions, including genes, gene clusters, and pathways, allows for the detection of biological processes that, when interrupted, may impact disease risk. In silico functional studies can also be utilised to further understand how variants disrupt biological processes and identify genotype-phenotype relationships. This study describes the exploration of sequencing datasets from cancers and benign tumour diseases including: i) hereditary diffuse gastric cancer, ii) sweat duct proliferation tumours, iii) adrenocortical carcinoma, and iv) breast cancer. Each set underwent germline whole exome sequencing followed by additional tumour or targeted sequencing to identify associated predisposition genes. Variants within a cluster of risk genes that are involved in double strand break repair were identified as associated with hereditary diffuse gastric cancer risk via gene ontology enrichment analysis. This cluster included PALB2 within which, using externally collated data, loss of function variants were identified as significantly associated with hereditary diffuse gastric cancer risk. Germline protein-affecting variants in the myosin gene MYH9 were identified in all individuals with a rare sweat duct proliferative syndrome, suggesting a role for MYH9 in skin development, regulation and tumorigenesis. These MYH9 variants were analysed in silico to identify a genotype-phenotype relationship between the clinical presentation and variants in the ATP binding pocket of the protein. Tumour matched normal sequence data from adrenocortical carcinoma cases was used to elucidate the role of Lynch syndrome genes in disease pathogenesis. Within the breast cancer set, candidate genes were selected to undergo targeted sequencing in a larger set of cases to further explore their role in breast cancer risk. Risk associated genes identified within this study may ultimately aid in diagnosis and management of disease. This thesis has also generated multiple novel tools and sequencing analysis techniques that may be of use for further studies by aiding in the prioritisation of candidate variants. The described techniques will provide support to researchers working on rare, statistically underpowered datasets and to provide standard analysis pipelines for a range of dataset sizes and types, including familial data and unrelated individuals.
  • ItemOpen Access
    Vinexin regulates autophagy through YAP/TAZ: implications for health and disease
    (2018-04-28) Frake, Rebecca Astrid
    Macroautophagy (hereafter referred to as autophagy) is a highly conserved cellular process that promotes cytoplasmic homeostasis via lysosomal degradation of proteins and organelles. Dysfunctional autophagy occurs in numerous human pathologies, including neurodegeneration and cancer. Vinexin (encoded by SORBS3) is a physiologically important adaptor protein for two main reasons: 1. SORSB3 mRNA expression increases in normal human brain ageing, 2. SORBS3 is a candidate tumour suppressor in hepatocellular carcinoma (HCC). This dissertation builds on published data from an siRNA screen for autophagy regulations under basal conditions, which indicates vinexin knockdown upregulates autophagy. I replicate this finding in multiple cell lines, before characterising the impact of siSORBS3 treatment on autophagy; autophagosome biogenesis is increased, while flux through the autophagy pathway remains intact. Having excluded several possible mechanisms suggested by the literature, I focus on the transcriptional coactivators YAP and TAZ. The rationale here is: 1. YAP/TAZ activity is implicated in autophagy, 2. YAP/TAZ and vinexin are both linked to HCC. My data show that YAP/TAZ transcriptional activity is upregulated upon vinexin depletion. Moreover, increased autophagy following siSORBS3 treatment requires YAP and TAZ. A key focus of this dissertation is the mechanism by which vinexin knockdown upregulates YAP/TAZ and hence, autophagy. This centres on altered actin cytoskeleton dynamics; an increase in F-actin structures appears to compete with YAP/TAZ for binding to angiomotins, established sequesterers of YAP/TAZ in the cytosol. In this way, siSORBS3 treatment facilitates YAP/TAZ nuclear localisation and consequent transcriptional activity. Angiomotin overexpression therefore ameliorates the increase in autophagy caused by vinexin depletion. Published RNA sequencing data is used to confirm that SORBS3 mRNA expression increases in normal brain ageing, not only in the frontal cortex (as previously published), but also in the hippocampus. This sits alongside decreased expression of core autophagy genes in both tissues. Accordingly, vinexin could contribute to the decline in autophagic potential thought to occur in normal brain ageing. With regards to SORBS3 as a candidate tumour suppressor in HCC, I show that stably re-expressing vinexin in a HCC cell line downregulates YAP/TAZ and hence, autophagy. These cells also show reduced clonogenicity. My data therefore support the hypothesis that SORBS3 is a tumour suppressor in HCC; YAP and TAZ are well-known to increase proliferation and resistance to apoptosis, while autophagy can enable tumour cells to survive stressors such as nutrient starvation. The conclusions of this dissertation are that vinexin depletion upregulates autophagy in a YAP/TAZ-dependent manner and that this has physiologically important implications, especially with regards to HCC.
  • ItemOpen Access
    Understanding transcriptional regulation through computational analysis of single-cell transcriptomics
    (2017-10-05) Lim, Chee Yee; Lim, Chee Yee [0000-0003-3553-3950]
    Gene expression is tightly regulated by complex transcriptional regulatory mechanisms to achieve specific expression patterns, which are essential to facilitate important biological processes such as embryonic development. Dysregulation of gene expression can lead to diseases such as cancers. A better understanding of the transcriptional regulation will therefore not only advance the understanding of fundamental biological processes, but also provide mechanistic insights into diseases. The earlier versions of high-throughput expression profiling techniques were limited to measuring average gene expression across large pools of cells. In contrast, recent technological improvements have made it possible to perform expression profiling in single cells. Single-cell expression profiling is able to capture heterogeneity among single cells, which is not possible in conventional bulk expression profiling. In my PhD, I focus on developing new algorithms, as well as benchmarking and utilising existing algorithms to study the transcriptomes of various biological systems using single-cell expression data. I have developed two different single-cell specific network inference algorithms, BTR and SPVAR, which are based on two different formalisms, Boolean and autoregression frameworks respectively. BTR was shown to be useful for improving existing Boolean models with single-cell expression data, while SPVAR was shown to be a conservative predictor of gene interactions using pseudotime-ordered single-cell expression data. In addition, I have obtained novel biological insights by analysing single-cell RNAseq data from the epiblast stem cells reprogramming and the leukaemia systems. Three different driver genes, namely Esrrb, Klf2 and GY118F, were shown to drive reprogramming of epiblast stem cells via different reprogramming routes. As for the leukaemia system, FLT3-ITD and IDH1-R132H mutations were shown to interact with each other and potentially predispose some cells for developing acute myeloid leukaemia.