The day that I joined McMaster University and the Department of Health Research Methods, Evidence, and Impact as a Master student, I did not foresee the amazing journey I was embarking on or the impact of my academic achievements. I was continuously challenged to think critically and apply my ideas to real world problems. Seeing the opportunity to make a difference in clinical research and patient care inspired me to begin my PhD studies. Creating knowledge and applying it practically was a difficult task, however, the opportunities to share my research in a dynamic and complex field with the world fuelled my motivation. Completing a PhD has been an incredible privilege for which I will always be grateful. To my family – Mamma, Papa, Mark and Matthew – thank you for your undying and unconditional love, support, and encouragement. A pandemic forced us to come together, and having your support 24/7 (literally) made the journey more enjoyable. To my closest friends, thank you for being a part of my life and supporting my ambitions. We all recognized my ambitiousness would set me on a difficult path and completing a PhD was not my original plan. Thank you for inspiring me to take on the challenge and ensure my work was truly impactful. Thank you for encouraging mental strength, being understanding and willing to lend a hand, reminding me that I can achieve anything I set my mind to, and inspiring me to dare. To my supervisory committee members and independent study supervisor, thank you for your continued interest in my work and for always supporting my ambitions. I am grateful for your patience and belief that I would succeed in completing what I had sought to achieve. Thank you for the chance to work together, and for your constant support and mentorship throughout my PhD journey. To the faculty, staff and fellow students at the Department of Health Research Methods, Evidence, and Impact, and the McMaster Centre for Transfusion Research, thank you for providing the resources, friendship, and guidance I needed to achieve greatness. To the panel members of the ITP Emergency Management Guideline, thank you for believing in this project and making this work possible. I look forward to implementing the results of our efforts into clinical practice. To the ITP patient community, thank you for entrusting me with the task of improving patient care and supporting me along the way. You have enabled me to be a voice for change. To the rare disease community, I know the completion of my PhD journey will not be the end of our work together. To the funding agencies who allowed this project to be possible, the Canadian Institutes of Health Research and Platelet Disorder Support Association, thank you for supporting my PhD journey and the completion of this important work. To my doctoral supervisor, Dr. Donald Arnold, it has been an honour and privilege to have learned from you and received your guidance throughout my PhD journey. Since our initial meeting, when I hobbled into the McMaster Centre for Transfusion Research offices several minutes late, I felt that your kindness and honesty would be the perfect form of mentorship to push me towards success. Thank you for recognizing my ambition and reminding me to keep focused. I will continue to apply this lesson throughout my life and strive for ‘depth’. For many years, you have been my mentor who I reached out to for advice, support, honest feedback, and encouragement. Thank you for imparting your knowledge to me over my PhD journey and teaching me how to be an inspirational mentor who highlights the strengths of their students while simultaneously supporting their growth. Although I may have finally reached the destination in my PhD journey, I know that we will continue to collaborate for many years to come. / Developing clinical practice guidelines (CPGs) for rare diseases is methodologically challenging. As each disease has so few patients, published literature includes low-quality studies or studies that do not directly address the questions of interest. As a result, CPG panelists have limited evidence on which to base their recommendations. Historically, when no evidence was available, CPGs have relied on physician opinion. This does not align with the mandate of CPGs which transparently identifies, appraises, and relies on evidence. The challenges of developing CPGs for rare diseases are exemplified by immune thrombocytopenia (ITP), a rare autoimmune disease that affects approximately 1 in 8,000 people. It predominantly affects females and young adults, and is characterized by low blood platelets that increase the risk of bleeding. Bleeding emergencies in ITP patients are critical, life-threatening events that can cause life-long morbidity and associated health care costs. Treatment of ITP bleeding emergencies requires a rapid, coordinated approach that involves emergency department staff, hematologists, pharmacy, and the laboratory. However, there is no evidence-based CPG for the management of ITP bleeding emergencies. The objectives of my PhD thesis are (1) exploring the heterogeneity of ITP diagnosis using antiplatelet autoantibodies; (2) developing a standardized definition of ITP bleeding emergencies; (3) outlining the synthesis of existing evidence on the treatment of ITP bleeding emergencies through a systematic review; and (4) developing a novel methodology to address the lack of evidence in rare disease CPGs and applying it to develop a CPG for the management of ITP bleeding emergencies. / Thesis / Candidate in Philosophy / Guidelines for rare diseases can be hard to develop because of a lack of information. Doctors and researchers make decisions on rare disease management based on their experiences, which can be limited. Low blood platelets and emergency bleeding can be caused by a rare disease called immune thrombocytopenia (ITP). When emergency bleeds occur, patients need care from the Emergency Department immediately. The problem is that there is no standard way for doctors to treat these ITP bleeding emergencies. My PhD thesis project will fill an important gap for ITP emergency treatment. First, we will assess how ITP patients are diagnosed. Second, we will define an ITP bleeding emergency. Third, we will collect existing information about ITP bleeds. Fourth, we will overcome the challenge of not having enough information by collecting new data from patient records. The method we use to develop ITP guidelines can be used for other rare diseases.

BACKGROUND: The lysosomal storage disorder, Niemann Pick type C1 (NPC1), presents a variable phenotype including neurovisceral and neurological symptoms. 2-Hydroxypropyl-ss-cyclodextrin (HPssCD)-based therapies are presently the most promising route of intervention. While severe cerebellar dysfunction remains the main disabling feature of NPC1, sensory functions including auditory and olfactory ones are also affected. Morphological and functional anomalies of Npc1 (-/-) mouse retina have also been observed, although the functional integrity of the visual pathway from retina to visual cortex is still unsettled. We have addressed this issue by characterizing the visual evoked potential (VEP) response of Npc1 (-/-) mice and determining if/how HPssCD administration influences the VEPs of both Npc1 (-/-) and Npc1 (+/+) mice. METHODS: VEP elicited by a brief visual stimulus were recorded from the scalp overlying the visual cortex of adult (PN, postnatal days 60, 75, 85 and 100) Npc1 (+/+) and Npc1 (-/-) mice that had received repeated injections of either HPssCD or plain vehicle. The first injection was given at PN4 and was followed by a second one at PN7 and thereafter by weekly injections up to PN49. Cholesterol accumulation and myelin loss were finally assessed by filipin staining and myelin basic protein immunohistochemistry, respectively. RESULTS AND DISCUSSION: We have found that the transmission of visual signals from retina to visual cortex is negatively influenced by the loss of Npc1 function. In fact, the VEP response of Npc1 (-/-) mice displayed a highly significant increase in the latency compared to that of Npc1 (+/+) mice. HPssCD administration fully rescued this defect and counteracted the cholesterol accumulation in retinal ganglion cells and dorsal lateral geniculate nucleus neurons, as well as the myelin loss in optic nerve fibers and axons projecting to the visual cortex observed in of Npc1 (-/-) mice. By contrast, HPssCD administration had no effect on the VEP response of Npc1 (+/+) mice, further strengthening the treatment efficacy. CONCLUSIONS: This study pinpoints the analysis of VEP response as a potentially accurate and non-invasive approach to assess neural activity and visual information processing in NPC1 patients, as well as for monitoring the progression of the disease and assessing the efficacy of potential therapies.

近年來，許多專家學者廣泛探討偵測稀有疾病的發生率或稱為叢集上的空間或空間對時間的統計方法及模型。這些方法大部分都是處理個別資料或是只能偵測接近圓形的叢集。在這篇論文中，根據Choynowski在1959年所探討的方法，我們進一步提出針對整體資料去偵測非圓形叢集的方法，並且會將此方法與Nagarwalla’s Spatial Scan Statistic做比較。同時，我們會呈現模擬結果中的型一、型二誤差來衡量此方法的可行性。另外，我們也會將此方法實際應用到台灣的癌症死亡資料做探討。 / In recent years, many statistical methods have been proposed for detecting excesses of rare diseases, i.e., clusters, in space or in space-time. Most of these methods deal with case-event or individual-level data and can only detect clusters with shape close to circles. In this study, adapting Choynowski's (1959) idea, a simulation-based approach is proposed to detect non-circular clusters with aggregate or group-level data. The proposed cluster detection method will be used to compare with a frequently used method: Nagarwalla’s Spatial Scan Statistic. Computer simulation is used to illustrate the validity, with respect to Type-I and Type-II errors, of the proposed approach. In addition, the cancer mortality data in Taiwan area are also used as a demonstration of the proposed test.

Protein phosphorylation is an essential regulatory mechanism employed by many key pathways in the eukaryotic cell. This thesis explored two examples of protein phosphorylation, one in translation and one in cell cycle regulation. TRNT1 is the RNA polymerase that adds the 3’ CCA nucleotides to all tRNA and is required for aminoacylation, tRNA quality control, and protection of the translation machinery (Aebi et al., 1990; Vörtler & Mörl, 2010; Wellner et al., 2018). Mutations in TRNT1 cause SIFD, Sideroblastic Anemia With B-Cell Immunodeficiency, Periodic Fevers, And Developmental Delay, a rare mitochondrial disease (Chakraborty et al., 2014). In addition to oxidative phosphorylation defects previously reported (Liwak-Muir et al., 2016; Sasarman et al., 2012; Wedatilake et al., 2016), I show that defects in TRNT1/Cca1 induce the eIF2α-P translation stress response to slow cytoplasmic translation but is only beneficial when mitochondrial translation is required. This data suggests that impairment of TRNT1/Cca1 function results in an imbalance of translation homeostasis and that altering the balance between mitochondrial and cytoplasmic translation may be an effective therapy for SIFD. In another example of protein phosphorylation regulation, Cdc25-dependent dephosphorylation of Cyclin dependent kinase (Cdk1) is a universally conserved mechanism regulating mitotic entry. In budding yeast, Cdk1 is dephosphorylated when the cdc25 homologue, MIH1, is deleted, indicating an additional phosphatase functions redundantly with Mih1. I identified two phosphatases, Ptp1 and PP2ARts1, that function in conjunction with Mih1. This redundancy suggests novel mechanisms for how cells may activate pools of Cdk1 at different times and in different cellular locations.

[ES] Durante las últimas décadas, se han realizado importantes avances en el estudio de las causas genéticas de enfermedades raras y comunes, donde un gran número de variantes han sido identificadas y asociadas a múltiples enfermedades. Con las tecnologías de secuenciación de nueva generación, hoy en día somos capaces de investigar, con un alto rendimiento, la contribución de variantes de alta y baja frecuencia a distintos tipos de enfermedades, permitiéndonos así estudiar su importancia en el desarrollo de las mismas. En ésta tesis se ha utilizado la secuenciación del exoma como tecnología para el estudio de variantes raras en una enfermedad compleja, la alergia gastrointestinal inducida por múltiples alimentos. Para ello, se realizó la secuenciación del exoma completo de una cohorte de 31 individuos (ocho afectados y 23 no afectados) provenientes de siete familias diferentes. Se desarrolló un flujo de trabajo para procesar los datos generados a partir de diferentes librerías e instrumentos de secuenciación, así como un control de calidad exhaustivo con el fin de maximizar el número de variantes de alta calidad. Diferentes tipos de mutaciones fueron investigadas, incluyendo polimorfismos de nucleótido único, inserciones/deleciones, variantes del número de copia y haplotipos HLA, y se realizaron diferentes métodos de filtrado para su interpretación. Finalmente, se encontraron una serie de mutaciones que podrían estar asociadas con la enfermedad y se describe su posible papel en la patogénesis de las alergias gastrointestinales. Los resultados de esta tesis suponen importantes avances en el estudio de la compleja arquitectura genética de las alergias gastrointestinales y abren las puertas a futuras líneas de investigación, que serán necesarias para entender completamente las bases genéticas de esta enfermedad. / [CAT] Durant les últimes dècades, s'han realitzat importants avanços en l'estudi de les causes genètiques de malalties rares i comunes, on un gran nombre de variants han sigut identificades i associades a múltiples malalties. Amb les tecnologies de seqüenciació de nova generació, avui en dia som capaços d'investigar, amb un alt rendiment, la contribució de variants d'alta i baixa freqüència a diferents tipus de malalties, permetent-nos així estudiar la seva importància en el desenvolupament de les mateixes. En aquesta tesis s'ha utilitzat la seqüenciació del exoma com a tecnologia per a l'estudi de variants rares en una malaltia complexa, l'al·lèrgia gastrointestinal induïda per múltiples aliments. Per això, es va realitzar la seqüenciació del exoma complet d'una cohort de 31 individus (vuit afectats i 23 no afectats) provinents de set famílies diferents. Es va desenvolupar un flux de treball per a processar les dades generades a partir de diferents llibreries e instruments de seqüenciació, així com un control de qualitat exhaustiu amb la fi de maximitzar el nombre de variants d'alta qualitat. Diferents tipus de mutacions foren investigades, incloïent polimorfismes de nucleòtid únic, insercions/delecions, variants del nombre de còpia i haplotips HLA, i es realitzaren diferent mètodes de filtrat per a la seva interpretació. Finalment, es trobaren una sèrie de mutacions que podrien estar associades amb la malaltia i es descriu el seu possible paper en la patogènesis de les al·lèrgies gastrointestinals. Els resultats d'aquesta tesis suposen importants avanços en l'estudi de la complexa arquitectura genètica de les al·lèrgies gastrointestinals i obrin les portes a futures línies d'investigació, que seran necessàries per entendre completament les bases genètiques d'aquesta malaltia. / [EN] The study of genetics has been making significant progress towards understanding the causes of rare and common disease during the past decades. Across a wide range of disorders, there have been hundreds of associated loci identified and associated with multiple disorders. Now, with the advent of next-generation sequencing technologies, we are able to interrogate the contribution of high and low frequency variation to disease in a high throughput manner. This provides an opportunity to investigate the role of rare variation in complex disease risk, potentially offering insights into disease pathogenesis and biological mechanisms. In this thesis, it has been assessed the use of whole-exome sequencing technology to investigate the role of rare variation in a complex disease, gastrointestinal food allergy induced by multiple food proteins. For that, a cohort of 31 individuals (eight affected and 23 non-affected) from seven different families was whole exome sequenced. Data obtained from multiple sequencing systems and libraries were analysed, and a workflow was developed, focusing on a comprehensive quality control to maximise the number of real positive calls. Different types of genome variations were investigated, including single nucleotide variants, insertions/deletions, copy number variants and HLA haplotypes. By approaching different methods of variant filtering, a set of rare variants that could be associated with the disease was identified. The possible role of these candidate variants in the pathogenesis of gastrointestinal food allergies was also discussed. These results reveal important insights into the genetic architecture of gastrointestinal food allergies and lead to additional lines of investigation that will be required in order to fully understand the genetic basis of this disease. / Sanchis Juan, A. (2019). Exome Sequencing in Gastrointestinal Food Allergy Induced by Multiple Food Protein [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/134361 / TESIS

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21631号 / 医博第4437号 / 新制||医||1034(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 川上 浩司, 教授 松田 文彦, 教授 黒田 知宏 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Rare diseases currently impact over 250 million people worldwide, accounting for over 3.5% of the global population. Clinicians caring for individuals living with rare diseases face difficulties providing accurate diagnosis and effective treatments. The low prevalence of individual rare diseases, and limited data and constrained resources available for research, makes it challenging to develop useful clinical guidelines. The objective of this thesis is to share our experience in conducting evidence synthesis for the guideline development of a rare disease—chronic hypoparathyroidism, and show how we addressed the challenges encountered during the review process. The thesis begins by describing the challenges of evidence synthesis for guideline development in the context of rare diseases. I then present our strategies to overcome these challenges in three systematic reviews prepared for a chronic hypoparathyroidism guideline. The thesis ends by summarizing the challenges and solutions, highlighting strengths and limitations, and describing opportunities and challenges for future research in evidence synthesis for rare diseases. / Thesis / Candidate in Philosophy

Objectives: We investigated pediatric rare disease researchers’ experiences and perspectives with research collaborations involving industry partners. Methods: This mixed methods study included a cross-sectional survey of academic/hospital-based Canadian pediatric rare disease researchers which informed semi-structured interviews with a subsample of survey participants. We analyzed survey data descriptively and interview data thematically, integrating findings narratively. Results: Of 126 survey respondents, 59 (47%) reported research collaborations with industry; we interviewed 10 of these researchers. Important benefits to collaborations with industry reported by survey participants and interviewees included access to funding and resources, while disadvantages stemmed from perceptions that partners had different motivations. Interviewees provided advice for future researchers including careful selection of an industry partner, relationship building, clear expectations, and utilizing supportive institutional structures. Conclusion: Our findings provide insights into the experiences of pediatric rare disease researchers and offer suggestions on how to conduct successful collaborative research with industry.

The diagnosis of genetic illnesses has undergone a revolution with advancements in sequencing technology. Next-generation sequencing (NGS) has become a standard practice in genetic diagnostics, enabling the identification of various genetic variations. However, distinguishing causative variants from a vast number of benign background variants presents a significant challenge. This study focuses on improving the rank score model used in genetic rare-disease diagnostics at a clinical genomics facility in Stockholm. The objective is to develop a more effective and optimized model through the utilization of exploratory data analysis techniques and machine learning methods, investigating the strengths and weaknesses of various existing annotation scores to identify suitable features and enhance the model's classification performance. The research methodology involved analyzing publicly available ClinVar data, utilizing statistical methods such as principal component analysis (PCA), heatmap, Welch's t-test, and Chi-Square test to evaluate the correlation, patterns, and classification abilities of different variant types. In addition, the study employed a machine learning approach that combines allele frequency filtering and logistic regression trained on both public and in-house datasets to prioritize single nucleotide variants (SNVs) and insertions/deletions (InDels). The resulting model assigns binary class labels (benign or pathogenic) and provides scores for variant classification. Promising performance was observed in both the ClinVar dataset and the unique patient datasets, demonstrating the model's potential for clinical application. The findings of this study hold the potential to enhance genetic rare-disease diagnostics and contribute to advancements in rare disease research.

La perte auditive est le trouble sensoriel le plus commun avec 40 % des personnes de plus de 65 ans affectées, entraînant, chez ces patients une dégradation de leur qualité de vie et un isolement sociale. Les principales causes sont le vieillissement ou l'exposition au bruit, mais les mutations génétiques sont aussi à l'origine de déficits auditifs. Parmi ces surdités, le Syndrome de Chudley McCullough (CMCS) est une maladie rare caractérisée par une surdité sévère et précoce associée à des anomalies cérébrales (Chudley et al., 1997). Récemment, des mutations du gène GPSM2 (G protein signaling modulator 2) ont été décrites comme étant responsables de cette pathologie sans que l'on en connaisse les mécanismes (Walsh et al., 2010). A l'aide d'un modèle d'étude murin de cette pathologie, nous avons identifié les bases moléculaires de la pathologie ainsi qu’une nouvelle fonction moléculaire pour Gpsm2 sur la modulation du cytosquelette d’actine. La perturbation de cette fonction affect à la fois la maturation des cellules auditives et la croissance des jeunes neurones, pouvant expliquer les surdités et l’hypoplasie du corps calleux décrits chez ces patients (Mauriac et al., 2017). De plus, nous avons identifié les partenaires de Gpsm2, les protéines Gαi, comme indispensables à la fonction auditive (Beer-Hammer et al., 2018). Au niveau moléculaire, nous avons découvert une nouvelle interaction de Gpsm2 avec une protéine essentielle à la maturation des cellules auditives et impliquées dans les surdités de type Usher, la Whirlin.Par conséquent, notre étude a permis de clarifier l’étiologie du CMCS et de montrer que sa complexité et son aspect multisyndromique sont dus au rôle multifonctionnel du complexe Gpsm2/G⍺i non seulement sur la dynamique de la tubuline dans des cellules en prolifération et en post-mitotiques (Ezan et al., 2013), mais aussi sur la dynamique d’actine (Mauriac et al., 2017). / Hearing loss is the most common sensory disorder, affecting 40% of people over 65 years old, leading for these patients, to the deterioration of their quality of life and to their social isolation. The main causes are aging or exposure to noise. However, many genes can also cause deafness. Among these deafnesses, the Chudley McCullough Syndrome (CMCS) is a rare disease characterized by severe and early deafness associated with brain abnormalities (Chudley et al., 1997). Recently, mutations in the GPSM2 (G protein signaling modulator 2) gene were found to be causative of this pathology, but the molecular basis were unknown (Walsh et al., 2010). Using a murine model of this pathology, we identified the molecular basis of this pathology as well as a new molecular function for Gpsm2 on the modulation of actin cytoskeleton. The disruption of this function leads to defect of the maturation of auditory hair cells and the reduction of the outgrowth of young neurons which may explain the deafness and the hypoplasia of the corpus callosum described in these patients (Mauriac et al., 2017). In addition, we identified partners of Gpsm2, Gαi proteins, as essential for auditory function (Beer-Hammer et al., 2018). At the molecular level, we have discovered a new interaction of Gpsm2 with a protein essential for the maturation of auditory cells and involved in Usher type deafness, Whirlin.Therefore, our study clarified the etiology of CMCS and show that the complexity and multisyndromic aspect of this pathology is due to the multifunctional role of the complex Gpsm2/G⍺i not only on tubulin dynamics in proliferating cells and post-mitotic cells (Ezan et al., 2013), but also on actin dynamics (Mauriac et al., 2017).