Browsing by Author "Tarailo-Graovac, Maja"
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Item Open Access Driving mosaicism: somatic variants in reference population databases and effect on variant interpretation in rare genetic disease(2021-12-14) Avramović, Vladimir; Frederiksen, Simona D.; Brkić, Marjana; Tarailo-Graovac, MajaAbstract Background Genetic variation databases provide invaluable information on the presence and frequency of genetic variants in the ‘untargeted’ human population, aggregated with the primary goal to facilitate the interpretation of clinically important variants. The presence of somatic variants in such databases can affect variant assessment in undiagnosed rare disease (RD) patients. Previously, the impact of somatic mosaicism was only considered in relation to two Mendelian disease-associated genes. Here, we expand the analyses to identify additional mosaicism-prone genes in blood-derived reference population databases. Results To identify additional mosaicism-prone genes relevant to RDs, we focused on known/previously established ClinVar pathogenic and likely pathogenic single-nucleotide variants, residing in genes associated with early onset, severe autosomal dominant diseases. We asked whether any of these variants are present in a higher-than-expected frequency in the reference population databases and whether there is evidence of somatic origin (i.e., allelic imbalance) rather than germline heterozygosity (~ half of the reads supporting alternative allele). The mosaicism-prone genes identified were further categorized according to the processes they are involved in. Beyond the previously reported ASXL1 and DNMT3A, we identified 7 additional autosomal dominant RD-associated genes with known pathogenic single-nucleotide variants present in the reference population databases and good evidence of allelic imbalance: BRAF, CBL, FGFR3, IDH2, KRAS, PTPN11 and SETBP1. From this group of 9 genes, the majority (n = 7) was important for hematopoiesis. In addition, 4 of these genes were involved in cell proliferation. Further assessment of the known 156 hematopoietic genes led to identification of 48 genes (21 not yet associated with RDs) with at least some evidence of mosaicism detectable in reference population databases. Conclusions These results stress the importance of considering genes involved in hematopoiesis and cell proliferation when interpreting the presence and frequency of genetic variants in blood-derived reference population databases, both public and private. This is especially important when considering new variants of uncertain significance in known hematopoietic/cell proliferation RD genes and future novel gene–disease associations involving this class of genes.Item Open Access GPAD: a natural language processing-based application to extract the gene-disease association discovery information from OMIM(2024-02-27) Rahit, K. M. T. H.; Avramovic, Vladimir; Chong, Jessica X.; Tarailo-Graovac, MajaAbstract Background Thousands of genes have been associated with different Mendelian conditions. One of the valuable sources to track these gene-disease associations (GDAs) is the Online Mendelian Inheritance in Man (OMIM) database. However, most of the information in OMIM is textual, and heterogeneous (e.g. summarized by different experts), which complicates automated reading and understanding of the data. Here, we used Natural Language Processing (NLP) to make a tool (Gene-Phenotype Association Discovery (GPAD)) that could syntactically process OMIM text and extract the data of interest. Results GPAD applies a series of language-based techniques to the text obtained from OMIM API to extract GDA discovery-related information. GPAD can inform when a particular gene was associated with a specific phenotype, as well as the type of validation—whether through model organisms or cohort-based patient-matching approaches—for such an association. GPAD extracted data was validated with published reports and was compared with large language model. Utilizing GPAD's extracted data, we analysed trends in GDA discoveries, noting a significant increase in their rate after the introduction of exome sequencing, rising from an average of about 150–250 discoveries each year. Contrary to hopes of resolving most GDAs for Mendelian disorders by now, our data indicate a substantial decline in discovery rates over the past five years (2017–2022). This decline appears to be linked to the increasing necessity for larger cohorts to substantiate GDAs. The rising use of zebrafish and Drosophila as model organisms in providing evidential support for GDAs is also observed. Conclusions GPAD’s real-time analyzing capacity offers an up-to-date view of GDA discovery and could help in planning and managing the research strategies. In future, this solution can be extended or modified to capture other information in OMIM and scientific literature.Item Open Access GPAD: a natural language processing-based application to extract the gene-disease association discovery information from OMIM(2024-02-27) Rahit, K. M. T. H.; Avramovic, Vladimir; Chong, Jessica X.; Tarailo-Graovac, MajaAbstract Background Thousands of genes have been associated with different Mendelian conditions. One of the valuable sources to track these gene-disease associations (GDAs) is the Online Mendelian Inheritance in Man (OMIM) database. However, most of the information in OMIM is textual, and heterogeneous (e.g. summarized by different experts), which complicates automated reading and understanding of the data. Here, we used Natural Language Processing (NLP) to make a tool (Gene-Phenotype Association Discovery (GPAD)) that could syntactically process OMIM text and extract the data of interest. Results GPAD applies a series of language-based techniques to the text obtained from OMIM API to extract GDA discovery-related information. GPAD can inform when a particular gene was associated with a specific phenotype, as well as the type of validation—whether through model organisms or cohort-based patient-matching approaches—for such an association. GPAD extracted data was validated with published reports and was compared with large language model. Utilizing GPAD's extracted data, we analysed trends in GDA discoveries, noting a significant increase in their rate after the introduction of exome sequencing, rising from an average of about 150–250 discoveries each year. Contrary to hopes of resolving most GDAs for Mendelian disorders by now, our data indicate a substantial decline in discovery rates over the past five years (2017–2022). This decline appears to be linked to the increasing necessity for larger cohorts to substantiate GDAs. The rising use of zebrafish and Drosophila as model organisms in providing evidential support for GDAs is also observed. Conclusions GPAD’s real-time analyzing capacity offers an up-to-date view of GDA discovery and could help in planning and managing the research strategies. In future, this solution can be extended or modified to capture other information in OMIM and scientific literature.Item Open Access Identifying the Genetic Factors in Natural Genome Backgrounds That May Modulate Phenotypic Outcomes in C. elegans(2023-07) Chida, Afiya Razia; Tarailo-Graovac, Maja; Hansen, David Donald; Long, Quan; Sameul, MarcusLoss-of-function mutations in essential genes can lead to a severe phenotype characterized by reproductive stress or hindrance in development and survival. Whilst in some individuals these lethal mutations cause severe phenotypes, in others, phenotypic variability is observed. One of the factors causing this phenotypic heterogeneity is the genetic background that affects the primary mutation. This background is characterized by genetic modifiers - genes that interact and influence the role of causative gene by ameliorating or exacerbating a trait or disease. This thesis aims to explore the genetic modifiers of two essential genes– mat-1 (metaphase-to-anaphase defect) and cgh-1 (conserved germline helicase) in Caenorhabditis elegans, using natural isolate backgrounds. Both, cgh-1 and mat-1, contain hypomorphic temperature-sensitive alleles that allow propagation at a permissive temperature (15°C) while causing embryonic arrest at a restrictive temperature (25°C) in the reference N2 background. Previous studies revealed that phenotypic outcomes in case of CGH-1 and MAT-1 deficiencies are variable in two different genetic backgrounds (CB4856 and N2), suggesting the presence of genetic modifiers. We observed phenotypic variability of temperature sensitive variants in these genes across six natural isolates of C. elegans that are obtained from diverse geographical locations such as CB4856 (Honolulu, Hawaii), JUI400 (Seville, Spain), AB1 (Adelaide, Australia), GXW1 (Wuhan, China) and KR314 (Vancouver, Canada), along with N2 (Bristol, England). Here, using advanced bioinformatics approaches, I identified and validated the spectrum of variants present in these six C. elegans genomes, including Single Nucleotide Variants (SNVs) and complex Structural Variants (SVs) such as deletions, duplications, inversions, as well as complex genomic rearrangements that may act as potential genetic modifiers. This study is the first to explore the SV category in natural isolates of C. elegans. We prioritized the candidate modifiers (both in SNV and SV categories) from an extensive list of variants by analyzing the genetic interaction networks. With the help of wet-lab personnel using molecular biology techniques, these modifiers will be tested for their either supressing or enhancing effect on the primary mutation. Understanding the effect of genetic background on phenotypic outcomes is important, but could be a difficult task, especially in organisms like human. In this thesis, using model organism C. elegans, I contributed to the field by uncovering a spectrum of genetic variants (SNVs and SVs) in wild isolate genomes, and utilizing different approaches to find candidate genetic modifiers across the mutation spectra of natural isolates. Importantly, methods developed in this project could be helpful in translational research for devising better SV and modifier prediction strategies in humans.Item Open Access Investigating Multiple Modifier Network Candidates as Suppressors of zyg-1/PLK4 in C. elegans(2022-09) Stuart, Amanda Rhae; Tarailo-Graovac, Maja; Shutt, Timothy; Hansen, David DonaldGenetic modifiers are second-site alleles that affect the function or regulation of a primary allele to either suppress or enhance the associated phenotype. The traditional methods of identifying and studying these modifying interactions have been useful for the consistent elucidation of single modifier activities, which has expanded the known single gene interactions significantly. However, these methods have been limited in their capability to identify true cases of modifier networks within a genome because of the use of backcrossing to minimize the regions of the genome to be examined for modifiers. By limiting our knowledge of multiple modifier networks, we lack knowledge on the intrinsic complexities of the genome, and how the larger genomic context may be influencing phenotypic outcomes. This thesis explores the use of high-throughput mutagenesis-based suppressor screen methods of a temperature-sensitive, lethal C. elegans allele, zyg-1(it25); these methods include whole genome sequencing and bioinformatic analyses without backcrossing steps, followed by candidate modifier validation using CRISPR/Cas9 methods to study potential multiple modifier networks. Our results show that ~20% of all mutagenized strains have more than one candidate modifier identified after bioinformatic analysis, and CRISPR/Cas9 validation of the candidate networks derived from four mutagenized strains showed that 50% of tested strains carried true multiple modifier networks. Six individual modifier alleles of zyg-1(it25) were validated while testing the candidate networks, including a novel suppressor. Furthermore, study of the candidate alleles in a wild-type background showed that all modifying variants induced their own phenotype, reducing the population viability of these strains. Finally, this study explores the mechanism for suppression of zyg-1(it25) from the validated modifiers, most of which are components of the Anaphase Promoting Complex; all modifying variants show delays of the cell cycle, particularly of anaphase-onset, indicating that increased mitotic time is allowing embryos to overcome deficiencies of ZYG-1. With the ZYG-1 human ortholog, PLK4, being implicated in symptomatic microcephaly, elucidation of the modifier networks and phenotype-altering interaction may lead to further understanding of the mechanisms that cause developmental microcephaly and can have expanded applications to other genetic diseases.Item Open Access Phenotypic variability in C. elegans natural isolates reveals plasticity of gene essentiality for mat-1 and cgh-1(2024-03-01) Rodrigues Alves Barbosa, Victoria; Tarailo-Graovac, Maja; Grewal, Savraj; Hansen, DavidEssential genes are deemed crucial for survival and/or reproductive success, hence, their loss of function leads to lethality. However, gene essentiality is not static, but dependent on multiple factors, including the genetic background. The genetic background affects gene essentiality through genetic modifiers, second-site variants capable of interacting with the primary variant and altering phenotype. Genetic modifiers can act as suppressors, alleviating the phenotype, or enhancers, exacerbating the phenotype. The plasticity (modification) of gene essentiality caused by genetic modifiers has been shown in multiple species. In Caenorhabditis elegans, recent evidence shows phenotypic variability for knock-down of essential genes in two natural isolates: N2 and CB4856. N2 represents the laboratory-cultivated strain, while CB4856 is one of the strains with most diverse genome in comparison to N2. Much has been explored for these two genetic backgrounds, but little is known about the plasticity of gene essentiality in other C. elegans wild isolates. Thus, I here explore the effect of the genetic background on the plasticity of two genes, known as essential in the N2 background, but potentially dispensable in CB4856: the Metaphase-to-Anaphase Transition Defect gene (mat-1), and the Conserved Germline Helicase (cgh-1). These are involved in cell division and posttranscriptional regulation, respectively. Here, I further investigate the plasticity of mat-1 and cgh-1 in N2 and CB4856 backgrounds, and also include four other wild isolate backgrounds from diverse geographical locations (GXW1, KR314, JU1400, and AB1). Using hatch rate and propagation assays, I explored the phenotype of mat-1 and cgh-1 knockdowns across the six natural isolates using temperature-sensitive alleles and uncovered phenotypic variability for the embryonic lethal phenotype, suggestively due to influence of each genetic background. Next, bioinformatics and genomics tools were utilized for identification of candidate genetic modifiers for mat-1 and cgh-1 and led to prioritization of eight extragenic variants. Out of these, none showed notable modifying activity when studied in isolation. Importantly, this work shows the challenges associated with identifying true genetic modifiers in genomes with substantial variation. Undeniably, understanding more about a gene and its phenotype under different conditions and genetic backgrounds may be fundamental for elucidating fixed and plastic genetic interactions.Item Open Access Rare disorders have many faces: in silico characterization of rare disorder spectrum(2022-02-22) Frederiksen, Simona D.; Avramović, Vladimir; Maroilley, Tatiana; Lehman, Anna; Arbour, Laura; Tarailo-Graovac, MajaAbstract Background The diagnostic journey for many rare disease patients remains challenging despite use of latest genetic technological advancements. We hypothesize that some patients remain undiagnosed due to more complex diagnostic scenarios that are currently not considered in genome analysis pipelines. To better understand this, we characterized the rare disorder (RD) spectrum using various bioinformatics resources (e.g., Orphanet/Orphadata, Human Phenotype Ontology, Reactome pathways) combined with custom-made R scripts. Results Our in silico characterization led to identification of 145 borderline-common, 412 rare and 2967 ultra-rare disorders. Based on these findings and point prevalence, we would expect that approximately 6.53%, 0.34%, and 0.30% of individuals in a randomly selected population have a borderline-common, rare, and ultra-rare disorder, respectively (equaling to 1 RD patient in 14 people). Importantly, our analyses revealed that (1) a higher proportion of borderline-common disorders were caused by multiple gene defects and/or other factors compared with the rare and ultra-rare disorders, (2) the phenotypic expressivity was more variable for the borderline-common disorders than for the rarer disorders, and (3) unique clinical characteristics were observed across the disorder categories forming the spectrum. Conclusions Recognizing that RD patients who remain unsolved even after genome sequencing might belong to the more common end of the RD spectrum support the usage of computational pipelines that account for more complex genetic and phenotypic scenarios.Item Open Access The Variation of STRIPAK Components in Regulating Katanin Microtubule-severing in the Caenorhabditis elegans Embryo(2021-06-29) Lu, Tammy; Mains, Paul; Brook, William; Tarailo-Graovac, MajaMicrotubule-severing plays important roles in organism development, including for cell structure and cell division. Using C. elegans, I studied the regulation of katanin, a conserved microtubule-severing complex. Katanin is required for oocyte meiotic spindle formation; however, it must be inactivated for mitosis to proceed as continued katanin expression is lethal. In addition to two ubiquitin-based protein degradation pathways, another ubiquitin ligase, HECTD1 ubiquitin ligase homologue (HECD-1), regulates katanin activity without affecting katanin levels. Most regulators of katanin act as either a katanin activator or inhibitor; however, hecd-1 switches from being an activator in meiosis to an inhibitor in mitosis. My project set out to characterize the mechanism of hecd-1 mediated regulation of katanin. I used a CRISPR FLAG-tagged HECD-1 to compare the levels and localization of HECD-1 in meiosis and mitosis. HECD-1 showed ubiquitous expression in the cytoplasm throughout meiosis and early development and its levels remained unchanged. In other organisms, HECD-1 is a component of the striatin-interacting kinase phosphatase (STRIPAK) complex. This complex has been known to affect blood vessel formation, apoptosis, cell proliferation and a variety of signalling pathways. Using double mutants and genetic knockdowns, I found the STRIPAK complex components affected katanin activity differently. STRIPAK core components (farl-11, cash-1, let-92, gck-1), except for ccm-3, were katanin inhibitors in mitosis and activators in meiosis, much like hecd-1. In contrast, variable components (M4.1, otub-2) functioned as activators of katanin activity in meiosis and mitosis, while other components (mob-4, C49H3.6) were not involved in katanin microtubule-severing. These experiments outline separate functions for the STRIPAK complex and suggest a paradigm in which the addition of variable components alters the function of the complex. Additional experiments indicate that katanin may be involved with the central spindlin or a tubulin chaperone pathway. My research elucidated the interactions of nearly all the STRIPAK complex components in a single in vivo system and revealed their roles in katanin microtubule severing and cell division.Item Open Access Whole genome sequencing facilitates intragenic variant interpretation following modifier screening in C. elegans(2021-11-13) Jean, Francesca; Stasiuk, Susan; Maroilley, Tatiana; Diao, Catherine; Galbraith, Andrew; Tarailo-Graovac, MajaAbstract Background Intragenic modifiers (in-phase, second-site variants) are known to have dramatic effects on clinical outcomes, affecting disease attributes such as severity or age of onset. However, despite their clinical importance, the focus of many genetic screens in model systems is on the discovery of extragenic variants, with many labs still relying upon more traditional methods to identify modifiers. However, traditional methods such as PCR and Sanger sequencing can be time-intensive and do not permit a thorough understanding of the intragenic modifier effects in the context of non-isogenic genomic backgrounds. Results Here, we apply high throughput approaches to identify and understand intragenic modifiers using Caenorhabditis elegans. Specifically, we applied whole genome sequencing (WGS) to a mutagen-induced forward genetic screen to identify intragenic suppressors of a temperature-sensitive zyg-1(it25) allele in C. elegans. ZYG-1 is a polo kinase that is important for centriole function and cell divisions, and mutations that truncate its human orthologue, PLK4, have been associated with microcephaly. Combining WGS and CRISPR/Cas9, we rapidly identify intragenic modifiers, show that these variants are distributed non-randomly throughout zyg-1 and that genomic context plays an important role on phenotypic outcomes. Conclusions Ultimately, our work shows that WGS facilitates high-throughput identification of intragenic modifiers in clinically relevant genes by reducing hands-on research time and overall costs and by allowing thorough understanding of the intragenic phenotypic effects in the context of different genetic backgrounds.