22-1 Developing a Computer Simulation-Based Power Estimator for Design of Rare Disease Sequencing Studies
Many diseases including rare diseases (RD) are caused by unwanted genomic mutations that alter the functions of genes. Researchers have made efforts in deciphering such genetic basis of diseases for decades. A standard method is to sequence the DNA genomes of patients and their controls (e.g., siblings or parents) to identify mutations that show up more frequently in patients than controls. However, since any two individuals are different at many places throughout their genome, the study has to be carefully designed to ensure there is sufficient information to distinguish casual mutations signal from others. To address this question, a concept called “power”, the probability of identifying the genuine casual mutations in the study, has been utilized to assess the study design before carrying it out. In academia, estimating power of a study design is crucial not only for ethical/financial approval of the project, but also for justifying the output. Related to this, in genetic epidemiology (a field studying effects of genes on diseases), there is a saturation of statistical tools developed to estimate power of a study design. However, these tools are designed for common diseases and are ill-suited for RDs due to their small sample size. To mitigate the inherited constraint of RD studies, the sample rarity, researchers have to leverage biological knowledge to potential causal mutations. Likely due to the recognition of the importance of RD, many tools estimating pathological scores of putative causal mutations were published recently in Nature journals. However, with the involvement of these crucial tools, power estimation becomes unfeasible in the standard statistical framework. In this project we aim to develop a tool: an intuitive, simulation-based software that provides power estimations for prospective RD studies. Customizable parameters will allow researchers to realistically model the outcomes of study designs and provide guidelines to refine them.
22-2 The Natural History of Craniosynostosis: What Can We Learn from Unoperated Cases
Craniosynostosis is a condition in which the shape of the skull of newborns is abnormal due to premature closure of the gaps between skull bones. There is a strong relationship between the skull shape and the growth of brain. Therefore, it is believed that abnormal skull shape can drive or is a result of an abnormal brain growth. Therefore, traditionally this abnormality is corrected with an extensive surgical operation which. The goal of surgical operations for craniosynostosis is to normalize the shape of the skull and create room for brain growth where needed. The surgical operation is complex and has known risks such as bleeding and damage to brain structures. When there are no urgent medical indications for surgery, sometimes parents/caregivers decline surgical intervention. In this group of patients, they are usually monitored on regular basis by exam and 3D images, during childhood to ensure medical indications for surgical intervention do not arise. There is limited research conducted to assess what happens to children who do not get an operation for their abnormal skull shape. This study aims to examine if the severity of skull shape gets worse with time. This is a question parents and caregivers of craniosynostosis frequently ask. The study will review all the cases that did not have an operation and assess their 3-D photographs throughout their childhood. We anticipate this study will provide valuable information that can be used to scientifically answer parents and caregivers who wonder about the progression of this abnormality as their children get older. This study will also provide valuable information in our understanding about the disease and how does it progress. This is probably the first study of its kind and we anticipate a great local, national and international interest in the study findings.
22-3 Investigating the Genetic Mechanisms of Developmental Topographical Disorientation
Developmental Topographical Disorientation (DTD) refers to a newly discovered condition that affects the ability of individuals to orient and navigate in spatial surroundings. Individuals affected by DTD report getting lost in extremely familiar environments such as the neighbourhood (or workplace) where they have lived (or worked) for many years, and in some case their own homes; this condition is present from childhood despite well preserved general cognitive functions and in the absence of any apparent brain injury or neurological disorders.
Importantly, DTD has been recently shown to be inherited, indicating that there may be a genetic cause for the occurrence of this lifelong condition. Despite this familial evidence, however, to date, the genetic factors related to DTD remain unknown and fully unexplored. In this project we aim to conduct the very first study investigating how four specific genes that are known to be associated with spatial abilities are expressed in individuals with the DTD trait. Identifying genetic mutations related to the presence of DTD may be extremely helpful in the diagnosis of this condition, and, importantly, contribute to identifying the condition at a very early stage of child development. The benefits of early identification of DTD could be significant in light of a cognitive intervention aiming to the development of navigation and spatial orientation skills in children.
22-4 Selective Nervous System Stimulation as a Treatment for Cardiac Defects with Popdc1 Gene Mutation
Regular beating of the heart, initiated by electrical signals that must travel through the heart in a consistent pattern, is crucial to effectively pump blood to the body. The beating rate of the heart is controlled in part by the nervous system. There is a portion of the nervous system located inside the heart itself, which is involved in helping to set the regularity of the beat, and its adaptation to stress. Arrhythmias can be caused by a variety of factors including loss of adequate nervous system control due to genetic mutations. We have recently identified malformation and dysfunction of nerves in the heart due to a mutation in the Popeye domain-containing (POPDC) genes, a family of genes important for formation and function of the heart. In human, POPDC mutation has previously been associated with cardiac electrical disturbances (electrical block and Long QT Syndrome) and structural abnormalities (specifically heart failure). Last year patients in a family with muscular dystrophy and cardiac arrhythmias were identified as having a specific defect in the POPDC1 gene. We have recently found this defect in the popdc1 gene results in a loss of normal nervous system control of the heart’s electrical activity that leads to arrhythmias during stress, which appear to be prevented by selective enhancement of the function of failing cardiac nerves, representing a novel therapeutic intervention. In this project I will use state-of-the-art genetic and imaging techniques to determine if enhancing the activity of specific elements of the nervous system in the heart can prevent Popdc mutation-related arrhythmias and possibly act as a preventative treaetment for structural defects. Overall, results of this project will reveal potential novel therapeutic targets and strategies for cardiac defects and arrhythmias that occur with Popdc1 mutation and arrhythmias caused by defects of nerves in the heart.
22-5 Epidermolysis Bullosa Simplex Gene Correction Using the Base Editing Approach
The Current genome-editing technology of CRISPR/Cas9 needs to introduce a double stranded DNA break (DSB) and a donor DNA template to repair mutations by induction of homologous recombination. This is inefficient to point mutation correction and typically induces an abundance of random insertions and deletions (indels). This is why the system was further improved and a base editing new approach was developed and allowing for the conversion of cytidine (C) to thymine (T), thereby effecting a C→T substitution without requiring double-stranded DNA breaks backbone cleavage or a donor DNA template. This genome editing method uses engineered fusions of Cas9 variants and cytidine deaminases. Epidermolysis bullosa simplex (EBS) is a rare autosomal dominant skin disease caused by different mutations of the KRT5 or the KRT14 gene resulting in non-scarring blisters and erosions caused by minor mechanical trauma. There is currently no efficient therapy for this hereditary disease. Our project aims to correct EBS mutations. In our EBS biobank, we have primary cells for seven patients for whom we previously characterized the mutational and transcriptomic profile. Three of these EBS patients carry as a mutation in their DNA a substitution of a T to C (T→C) as the cause of the disease. The correction of these three mutations will be achieved using the new editing approach that enables irreversible conversion of a specific DNA base into another. This will be done on patient-derived induced pluripotent stem cells (iPSCs) using a specific gRNA sequence and the fusion protein complex containing a catalytically defective Cas9, a cytidine deaminase, and an inhibitor of base excision repair. The repaired iPSCs will be differentiated into keratinocytes that will be used to generate autologous tissue-engineered skin in laboratory to treat affected skin areas of EBS patients. This will potentially avoid the patient’s immune system to reject skin tissue grafts.
22-6 The Use of Computerized Cognitive Batteries for Fast, Reliable and Sensitive to Change Assessment of Cognitive Abilities in Myotonic Dystrophy Type 1: A Study of Validity
Myotonic dystrophy type 1 (DM1) is a neuromuscular disorder with multisystemic involvement, including the muscular, cardiac and central nervous systems. It is the most common form of adult-onset muscular dystrophy. In the adult phenotypes of DM1, our team has recently demonstrated an early and accelerated decline in cognitive functions over 9 years. However, incoming treatments will aim to ameliorate cognitive abilities in short delays (such as 18-24 months). There is presently a lack of knowledge about short-time evolution of cognitive abilities in DM1. However, classic neuropsychological tests used so far in DM1 cannot support repeated evaluations in short period of time. Moreover, they are time and cost-consuming. For these reasons, we would like to introduce computerized cognitive tests in the assessment of DM1 cognitive abilities and their evolution, since they are known to be easy and fast to use, as well as highly sensitive and reliable in short-period repeated measures. Prior to their use, several metrological properties have to be tested and provided specifically in the DM1 population. In the present study, concurrent validity and test-retest reliability will thereby be tested in 30 adult DM1 participants. Beyond its utility in future CNS treatment trials (assess their efficiency), providing such properties will improve immediately the potential of cognitive testing in the patients’ clinical follow-up, since many neuropsychologists are hesitant to use computerized cognitive tests prior to such metrological data being available.
22-7 Developing a Handbook for Families of Children with Auto-inflammatory Diseases
Our team aims to improve care for children with auto-inflammatory diseases (AInD) and their families by developing and disseminating a Handbook for Families of Children with Auto-inflammatory diseases. Auto-inflammatory diseases (AInD) are rare disorders that cause repeated unprovoked attacks of inflammation starting early in childhood, with fever, rash, pain, and other symptoms. About 200 children in BC (1500 in Canada) suffer from AInD, with broad consequences to affected children and their families. For the family of a child with AInD, learning about their child’s condition is critically important yet extremely difficult, as parents identify lack of clear, understandable and available resources as a barrier. In addition, parents describe feeling ‘alone’, without connection to other families that have experienced a similar disease and problems. The Handbook for Families of Children with Auto-Inflammatory Disease will provide clear basic information about these rare conditions, advice regarding coping with these chronic relapsing conditions, and links to additional resources. We believe this resource will help families feel more confident in managing their child’s condition, and improve their understanding of these diseases and their treatment.
22-8 A Prospective Study to Assess Dynamic Deformation of The Femoral Head During Weight Bearing in Perthes Disease
Legg-Calve-Perthes (LCP) Disease is an important disorder that affects children’s hips and has lifelong consequences. However, little is known of its cause and which children would benefit from surgery. X-rays are used to diagnose and monitor the disease, providing information on its severity. Affected hips that are becoming deformed may benefit from surgery at appropriate time in an attempt to preserve normal structure. However, X-rays can only detect LCP disease and its severity after the collapse has started to occur. Thus, the prime opportunity for surgery may have been missed. MRI is a safe investigation with no radiation exposure. It offers earlier detection of LCP disease than X-rays and is well tolerated by children. Using a unique MRI system that allows children to have images taken while they are standing up may help doctors better understand the progression of the disease. We have completed a pilot study to test the feasibility of using this unique upright open MRI scans in children with LCP disease. We are requesting for funds to compile results from our pilot study and design a prospective study. The prospective study will compare MRI scans of hips affected by LCP disease with patient standing and lying down, to see if standing MRI scans show larger differences in how the disease progresses. This may help doctors to identify which patients would benefit from surgery.
22-9 Quantifying Single-cell CFTR Expression in Blood Monocytes and Nasal Epithelial Cells from Cystic Fibrosis Patients and Healthy Controls
Cystic fibrosis (CF) is a genetic disease that affects approximately 4,000 individuals across Canada. A protein known as the cystic fibrosis transmembrane conductance regulator (CFTR) found on the surface of cells that lines our airways and other organs is defective leading to abnormal thick and sticky mucus. The difficult to clear mucus in the lungs of CF patients causes blockage of the airways giving rise to repeated lung infections over a lifetime, and eventual respiratory failure leading to death at a young age. In nearly half of CF patients with a specific defect in their DNA or genetic code, there is only about 2% of normal levels of CFTR on the surface of cells. Fortunately, there are new but expensive therapies (costing $260,000/year) available capable of increasing CFTR (referred to as CFTR ‘modulators’). However, not all CF patients benefit from this treatment and therefore a test to quickly identify individual patients who are responding to this treatment are urgently required. In this project, we will develop a blood test to determine how much CFTR is present in the circulating blood cells of healthy volunteers and CF patients. If differences are found, we will use this blood test in a future study to determine if blood cells can be used to monitor changes in the levels of CFTR after initiating CFTR ‘modulator’ treatment.
22-10 Exploring the Potential of CRISPR/Cas9 for the Treatment of Pelizaeus-Merzbacher Disorder
Pelizaeus Merzbacher Disorder (PMD) is an X-linked paediatric disorder, that affects approximately 1000 children in the United States. The disease is associated with severe motor and cognitive impairment and limited life expectancy. Currently, there is no cure and treatment options are limited to symptoms management. PMD is caused by mutations in the PLP1 gene, encoding proteolipid protein 1, one of the main components of myelin. In healthy individuals, myelin forms an insulating layer around the nerve fibers to allow fast and efficient signal conduction in the central and peripheral nervous system. When PLP1 is mutated, myelin degenerates causing PMD symptoms. 70% of PMD cases are caused by duplications of the of the X chromosome region containing the PLP1 gene. Here utilizing the novel genome editing technology, called clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9), I propose to develop a clinically relevant genome editing strategy to correct the duplication mutation and to ameliorate the disease’s manifestations in a PMD mouse model. The CRISPR/Cas9 system acts as “molecular scissors” that cleaves DNA at a specific location with the potential to target and edit virtually any site in the genome. In preliminary work, we successfully removed the duplication in cells derived from the PMD mouse model. Here, we will test this approach directly in mice. The CRISPR/Cas9 components will be administered using adeno-associated viral vectors into PMD mice and the successful duplication removal will be evaluated at behavioural, histological and molecular levels. Successful completion of this project will provide pre-clinical data to support the development of a new genome editing strategy as a potential one-time treatment for individuals affected by PMD caused by duplication mutations and, generally, to prove the efficacy of CRISPR/Cas9 system for the cure of different inherited diseases caused by genomic duplications.
22-11 Development of Novel CRISPR/Cas9-Mediated Exon Skipping Strategy for the Treatment of Duchenne Muscular Dystrophy
Duchenne muscular dystrophy (DMD) is an X-linked recessive neuromuscular disease, affecting approximately 1 in 5000 boys. Due to mutations in the DMD gene, DMD patients lack expression of the dystrophin protein, leading to disease manifestation such as progressive muscle wasting, loss of ambulation, and cardiorespiratory failure, which results in a limited life expectancy. While symptomatic management is available, there is no cure for DMD. Deletion mutations account for 68% of the DMD mutation spectrum, which disrupts the open reading frame (ORF) and leads to the lack of dystrophin expression. One approach to treat these deletion mutations is to restore the ORF by removing an additional exon, producing a shorter but functional dystrophin protein representative of the milder Becker muscular dystrophy (BMD). Here, I propose that deletion mutations causing DMD can be corrected using a genome engineering technology called clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) to produce a shorter but functional dystrophin protein. I have restored the ORF in muscle cells derived from the novel DMD mouse model containing the deletion mutation of Dmd exons 52-54 (Dmd del52-54). This strategy will be adapted to the Dmd del52-54 mouse model in vivo through adeno-associated virus (AAV) delivery. Successful restoration of a truncated dystrophin and functional improvement in the Dmd del52-54 model will establish the tremendous therapeutic potential of this strategy in treating DMD patients carrying deletion mutations.
22-12 Opioid Addiction, Overdose and Associated Deaths in Patients with Sickle Cell Disease Treated with Opioids in Childhood: A Population-based Cohort Study
Sickle cell disease (SCD) is a rare genetic disease that primarily affects people of African descent. It causes red blood cells to have an abnormal shape and get stuck in small blood vessels, especially in the bones. When this happens, patients experience pain that can be so excruciating that they require admission to the hospital, where they often receive treatment with strong painkillers called opioids (such as morphine, hydromorphone and others). Unfortunately, we now know that such medications can lead to addiction when taken regularly. Addiction to opioids has become a true health crisis in Canada and was responsible for almost 3000 Canadian lives lost in 2016. Because patients with sickle cell disease receive opioids frequently from early childhood, we are concerned that our treatments may predispose to drug addiction, overdose and death from opioids later in life. Surprisingly, no one has investigated this problem yet. We will study the extent to which children with SCD are at increased risk of addiction to and harm from opioids later in life, compared to the general population.
22-13 Natural History of Morquio B Disease –Protocol Development for Orthopaedic Outcomes
Morquio B Disease (MBD) is a rare genetic disorder with less than 50 patients known worldwide. The patients are usually diagnosed during their first decade of life due to severe orthopaedic problems and other growth related issues. Some of the symptoms may include: improper formation of the bones, the loosening of the ligaments and tendons and shortened stature. Many of these patients end up undergoing hip replacement, ankle fusion and spinal disc fusion in the neck region. The quality of life of these individuals deteriorate over time due to limited mobility, reduced body height, corneal clouding affecting vision and cardiac complications. The knowledge on MBD’s natural history and correlation between genetics and physical symptoms is limited. Without this knowledge it is not possible to identify endpoints for studies or compare data needed for clinical trials, which are required when potential treatments are identified. Hence, the applicant is planning a natural history study to determine measurable outcomes for the anticipated clinical trial of a drug which could potentially treat patients with MBD. An orthopaedic surgeon and co-investigator for this study will be designing the protocol involving the measurement of orthopaedic outcomes including function and quality of life of patients with MBD.
22-14 Defining the Role of a Novel BCL11b Variant in Atopy and Immune Dysregulation
Our team has recently identified a young girl with severe food allergies, asthma, eczema, autoimmune hair-loss (alopecia totalis), and brittle nails. Examining her genetic make-up using sophisticated next-generation DNA sequencing uncovered that she is only the second known person worldwide to have alterations in a gene called BCL11b, a regulator of immune cell function. Both allergies and alopecia are caused by a ‘confused’ immune system. In the case of allergies, these cells respond to substances (such as those found in common foods) that are harmless to most people, while in alopecia, the confused immune system attacks the patient’s hair follicles. These reactions involve immune cells that are directly regulated by BCL11b. Thus, we believe that this genetic change results in either increased numbers or activity of these cells, leaving her excessively sensitive to certain stimuli. In the proposed research, using state-of-the-art technologies capable of analyzing rare cell populations that might be missed using less sensitive approaches, we will work to carefully understand how this genetic alteration in BCL11b has confused her immune system by measuring the numbers and function of key immune cells. As this girl is only the second ever known case in the world, this study will not only allow us to identify novel treatment options for her, it will also help inform the therapy and care of future patients identified with changes in the same gene or patients afflicted with related allergic disorders.
22-15 Identification of genetic risk factors for SDS-associated leukemia in pediatric populations.
Shwachman-Diamond syndrome (SDS) is a rare disease that is characterized by pancreatic insufficiency and decreased production of white blood cells. Individuals are also at an increased risk of developing leukemia, typically after the age of 20. We are currently following a family with a young patient with SDS, who developed acute myeloid leukemia at 5 years of age. His brother also has the same causative mutation for SDS but has not developed leukemia or other blood complications. This leads us to believe that the patient may have acquired other mutations that, together with the SDS mutation, caused the leukemia. We want to use new DNA sequencing technology to identify any additional mutations that may be present in the leukemic brother's DNA, that may have contributed to his leukemia at such at a young age. In order to do so, we will be sequencing the DNA of his bone marrow and comparing it to his sibling’s non-leukemic bone marrow. We will also be sequencing the parents’ DNA to help identify inherited mutations. This information will help us to understand the genetic reasons why some individuals with SDS may be at higher risk of developing leukemia.
22-16 Investigation of Vascular Disease Progression in Neurofibromatosis Type 1 Patients Using Serum Biomarkers and Assessment of Vascular Function
Neurofibromatosis 1 (NF1) is a rare genetic disorder characterized by multiple non-cancerous tumors, bone abnormalities, learning difficulties, and vascular disease. NF1 vascular disease may present as high blood pressure, stroke, heart attack, or arterial weakness or rupture. Vascular disease is a significant cause of death in patients with NF1. Many patients with NF1 vascular disease are asymptomatic, with the first indication of disease being a life-threatening event such as a stroke. Therefore, diagnosing vascular disease earlier, prior to the occurrence a life-threatening event, is vital. In people without NF1, vascular disease can be assessed by examining markers of cardiovascular risk in blood and by special tests called flow-mediated dilation (FMD) and a measurement of the artery intima-media thickness (IMT), which examines the vessel’s ability to dilate and contract and its thickness respectively. The value of these tests in assessing the risk of vascular disease in people with NF1 is unknown. In 2011, we examined 20 young adults with NF1 utilizing these tests and found that young patients with NF1 and no symptoms of cardiovascular disease did indeed have both blood markers of increased cardiovascular risk and evidence of diminished vascular function on the flow mediated dilation test. However, it is not clear whether these risk factors actually predict cardiovascular disease in NF1 patients, as they do in the general population. To address this issue, we will bring the same patients back for another assessment using these same tests and also a cardiovascular assessment using standard clinical means. This study will determine whether screening for vascular disease in young asymptomatic NF1 patients might identify those who would benefit from interventions to prevent subsequent development of serious vascular complications.