Saudi Cultural Missions Theses & Dissertations
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Item Restricted Improving predictions of pathogenicity of CLCN variants using paralogue annotation(Saudi Digital Library, 2023-12-01) Alnakhlawi, Mona; Mannikko, RoopeWith the large expansion of genomic data, providing a genetic diagnosis has become a demanding task. Experimental approaches require funding, time, and expertise, establishing a bottleneck in classification of a variant and genetic diagnosis. This is particularly true in the genetic service environment, where family data is often not available. Current computational tools are too inaccurate in providing reliable predictions of pathogenicity, tackling biophysical changes, and providing precision-therapy solutions. In this project, I attempt to develop a framework for the CIC family based on paralogue annotation to improve and support pathogenicity predictions. Paralogue annotation consists of annotating paralogous genes, which are genes that have arisen from gene duplication. Paralogous genes are highly homologous, thought to have originated from gene duplication and have developed specialized functions in different tissues. Paralogue annotation is based on the assumption that excessively conserved residues are often vital to protein function. The CIC family of genes is subdivided into channels and transporters, and the validity of paralogue annotation across the family has not been established. By assessing sequence and structural alignment, I established core regions across the ClC-paralogues amenable for paralogue annotation, with 10% fully conserved and 26% partially conserved residues, and with full alignment for secondary structure elements in transmembrane and intracellular domains. Conservation was much higher among the channel and transporter subfamilies. Analysis of conserved residues indicated that they have a higher burden of pathogenic variants, as defined by their appearance in the human genetic mutation database (HGMD). The variants in HGMD were further manually annotated for pathogenicity. I further used the ratio of variants found in HGMD and of variants found in the general population (gnomAD) to establish regions of high and low impact within channel structures. High-impact regions include the subunit interface of the ClC dimer and sequences close to the chloride ion permeation/transport pathway. Low-impact regions are peripheral, distant from the subunit interface. These data establish the groundwork for paralogue annotation of the ClC family. I further contributed by functionally analysing variants identified in patients either at UCL or in ClinVar as variants of uncertain significance, and variants affecting residues with paralogue annotations, including the I238F, L497R, and V605A variants identified using paralogue annotation of ClinVar variants.15 0Item Restricted Investigating the function of RBMXL2 in spermatogenesis in humans and using mouse models(Saudi Digital Library, 2023-11-20) Aldalaqan, Saad; Elliott, DavidRBMX RBMXL2 and RBMY belong to a family of RNA-binding proteins. How important these proteins are in whole animal biology, what their global RNA targets and mechanisms of action are, and to what extent these proteins overlap in function has been poorly understood. Using histology I found that Rbmxl2 knockout phenotypes differed between mouse strains (C57BL/6 and Sv/129). Both strains, however, were infertile, with no sperm found, which indicates the importance of RBMXL2 in spermatogenesis. I performed an iCLIP analysis to investigate RBMXL2-RNA binding, which revealed that RBMXL2 directly binds to exons and introns of protein-coding genes. The strongest RBMXL2 binding was observed in genes associated with spermatogenesis and reproduction, specifically meiosis, such as Meioc and Esco1. Binding was also enriched within ultra-long exons to prevent the selection of cryptic splice sites. I attempted minigene experiments to try and model RBMXL2 function in transfected cells, but these experiments did not replicate RBMXL2 function in vivo. As an alternative I developed a stable cell line “rescue” approach to over-express RBMXL2 and test if this could replace endogenous RBMX. Using this approach, I demonstrated that RBMXL2 is likely to replace RBMX's function in meiosis when the X and Y are inactive. RBMXL2's ability to restore splicing control is dependent on its disordered domain not the RRM. RBMY, which is distantly related to RBMX, also replaces RBMX function in somatic cells.18 0Item Restricted Characterising Expression of Muscarinic Acetylcholine Receptors in Human Dental Pulp Stem Cells: An In-Vitro Study(Saudi Digital Library, 2023-08-12) Baradwan, Othman; Hamilton, Andrew; McLean, WilliamDental pulp stem cells (DPSCs) are a promising subgroup of mesenchymal stem cells (MSCs) that have the potential for regenerative applications. They have been reported to express a regenerative response for tooth structure by producing reparative dentine. One of the sources of DPSCs is the pulp of an extracted permanent tooth, which provides a convenient collection method. In addition, there are other groups of MSCs which share similar characteristics among them. Recent studies identified Acetylcholine (ACh) in some of the MSCs and the two major categories of Acetylcholine receptors (AChRs), muscarinic AChRs (m-AChRs) and nicotinic (n-AChRs). Although ACh is a neurotransmitter, there is evidence that it is produced by and influences non-neuronal cells. The involvement of ACh in the non-neuronal cells is called the non-neuronal cholinergic system (NNCS), which includes ACh-synthesizing enzymes, transporters, receptors, and degrading enzymes. Because DPSCs share similar characteristics with other MSCs, it is worth investigating the expression of m-AChRs in DPSCs. Also, to look into the role of m-AChRs in the regenerative function of DPSCs. There is potential for using DPSCs outside the tooth in regenerative applications, such as musculoskeletal regenerative medicine. The first part of the project started with m-AChRs by identifying messenger ribonucleic acid (mRNA) and protein expression. Secondly, to observe the influence of the most prominent m-AChRs in DPSCs, we attempted to generate a knock-out (KO) mutant in DPSCS using the Clustered Regularly Interspaced Palindromic Repeats (CRISPR)-associated protein 9 (Cas9) genome-editing system. The results showed that m-AChRs are identified on a genetic level through gene expression of mRNA in DPSCs and expressed the following genes CHRM2, CHRM3, and CHRM5. They are functional and can identify the following proteins: M2, M3, and M5. Then, we attempted to generate a CHRM2 KO mutant DPSCs and could only produce a low percentage of the mutant population of DPSCs, as we only managed to generate 13% of CHRM2 KO mutant DPSCs. Although the protocol proved its effectiveness on different types of cells, but it did not work with DPSCs in this experiment. Therefore, the protocol steps can be revisited for further investigation and optimisation to generate a sufficient population of CHRM2 KO mutant DPSCs to allow the exploration of the role of m-AChRs in DPSCs so it can be used in clinical applications in regenerative medicine.28 0Item Restricted Whole Exome Investigation Of Infertility In Men With Azoospermia(Saudi Digital Library, 2023-10-01) Sindi, Emad Rashad B; Jayasena, ChannaMale infertility is a disabling condition that affects 7% of men worldwide. The most severe form of male infertility is non-obstructive azoospermia (NOA) and approximately 20% of men with NOA have no identified cause. These patients are classified as idiopathic NOA. There is some data suggesting that the majority of men with idiopathic NOA have a genetic cause. The development of newer genetic sequencing technologies have allowed researchers to gain a better understanding of the genes contributing to spermatogenesis and those potentially causing NOA. In the present work, I have investigated clinical, biochemical, genetic and histological findings in men with NOA. I recruited 101 participants with azoospermia, and 48 of this cohort were diagnosed with idiopathic NOA. The cohort included 19 men with known causes of NOA and 24 men with obstructive azoospermia. Whole exome sequencing was performed on blood samples of men with idiopathic NOA for identification of candidate variants. I also recorded the available clinical factors, hormone profiles and testicular histopathology for all participants. The analysis of the sequenced data from the 48 men with idiopathic NOA identified 11 novel candidate variants. There were seven novel candidate Loss of Function (LoF) variants in men with NOA (MEIOB c.1140_1143del; FKBP6 c.469−2A>T; ANKRD36B c.2715T>G; FAM47C c.1536del; PKD2L2 c.1690C>T; FAM122C, c.427C>T; MCMDC2 c.108del) and four missense variants (YBX2 c.200G>A; MEIOB c.988A>C, PKD2L2 c.1391C>T; MCMDC2 c.119A>G). Two of the LoF (MEIOB and FKBP6) and two of the missense variants (MEIOB and YBX2) have functional evidence to support their association with azoospermia. The identified genes in NOA subjects showed a testicular phenotype matching the genes expression site during spermatogenesis. There was a significant decrease in testicular volume (p= 0.0392) and a significant increase in serum FSH (p=0.0014) in men with NOA compared to men with obstructive azoospermia (OA). Additionally, data is showing that identified variants have testicular histology phenotypes matching their site of expression. FKBP6, MEIOB, PKD2L2, and MCMDC2 genes are normally expressed in spermatocytes. Patients identified with variants in these genes were found to have histology phenotypes of maturation arrest at spermatocyte level. This could potentially be used to predict the histology phenotypes without surgical intervention. I have identified novel genetic variants in men with idiopathic NOA but further studies are needed to confirm causality. Genetic analysis can potentially improve our understanding of fertility by identifying genes involved in spermatogenesis and novel variants in men with infertility.17 0Item Restricted Use of Genomic Enrichment and Long Read Sequencing to Investigate Variant Repeat Patterns of Myotonic Dystrophy Type 1 CTG Expansions(Saudi Digital Library, 2023-08-07) Asraf, Reem; Monckton, DarrenBackground: Myotonic dystrophy type 1 (DM1) is a multisystemic disorder caused by a CTG expansion of more than 50 repeats in the 3-prime untranslated region of the DMPK gene. The expanded repeat is unstable and expands in the soma and the germline. The length of the repeats and the presence of variant interruptions modifies disease onset and severity. No current available diagnostic methods are able to detect these interruptions with high accuracy at a low cost. In addition, complex variant sequences within the repeat often are undetectable using these methods. Demystifying these sequences will provide a better understanding of how their presence affects disease progression. Aim: To determine if Oxford Nanopore Technologies (ONT) long-read sequencing of multiple displacement amplification (MDA) enriched alleles can accurately detect repeat length and sequences in a subset of samples. the MDA enrichment is expected to generate sufficient coverage without the need for extra amplification. Method: The non-disease allele was removed using a Samplix Xdrop Sort and the expanded allele was enriched by MDA. Four samples were library prepped by debranching and then repairing before either being PCR amplified then sequenced, or directly sequenced by long-read nanopore sequencing. Through a bioinformatic pipeline, the resulting enrichment was calculated, and the length and sequence deciphered. Results: The resulting reads showed a high enrichment for the target region similar, if not better than, reported results. ONT was able to determine the length and sequence of the samples. However, non-CTG repeats were seen in the middle of the reads, possibly attributed to sequencing errors. Conclusion: Further research is needed to optimize the library preparation steps and explore the efficiency of other long-read sequencing methods with MDA enrichment.111 0Item Restricted Modeling the Heterogeneity and Targeting the Resistant Mechanism of Triple-Negative Breast Cancer(2023-06-23) Doha, Zinab; Rosalie, SearsTriple-negative breast cancer (TNBC) is a highly aggressive breast cancer subtype characterized by a remarkable molecular heterogeneity. Currently, there are no effective druggable targets and advanced preclinical models of human disease. This thesis investigates the role of MYC, a common TNBC deregulated oncogene, in TNBC microenvironment complexities, with a focus on oncogenic transformation of MYC driven therapeutic resistance as well as the functional consequences of targeting MYC in TNBC. Such understanding potentiates a critical need in the field to identify effective targeted therapy for TNBC and establishes a foundation for translating preclinical findings into effective human clinical trials. The studies are organized in the following outline; In Chapter 1, I delve into the background of MYC and its role in therapeutic resistance in TNBC. In Chapter 2 research, which has been accepted for publication in Nature Communications, we developed a novel heterogeneous TNBC murine model (Myc;Ptenfl) by mimicking two common TNBC mutations with high co-occurrence: amplification of the oncogene MYC and deletion of the tumor suppressor PTEN. In Chapter 3 research, which is in preparation for submission for publication, I use this TNBC preclinical model to address how oncogenic transformation of MYC can support therapeutic resistance in TNBC. Finally, in Chapter 4, I connect all the threads and provide thoughts on future directions of this research.15 0