18-1 A Simpler Routine Procedure for the Diagnosis of Microvillus Inclusion Disease
Microvillus inclusion disease is a very rare and severe genetic bowel disorder that affects infants and young children. Patients suffer from unstoppable diarrhoea and do not tolerate normal food. The procedure to diagnose MVID involves highly specialized, expensive and time-consuming steps. An early and correct diagnosis, however, is critical to start appropriate treatment, without which patients die during infancy. We will use a novel technology to determine whether the most specialized and time-consuming step can be left out from the current diagnostic procedure, leading to a much easier and cheaper routine diagnostic procedure with equal reliability for this devastating disease.
18-2 Resolving Chronic Diarrhoea in Patients with Microvillus Inclusion Disease
Microvillus inclusion disease is a very rare and severe genetic bowel disorder that affects infants and young children. Patients suffer from severe unstoppable diarrhoea which complicates disease management and severely impairs quality of life. There is no treatment to resolve the diarrhea. Recently, a misbalance of three ion pumps in the cells that cover the bowel wall was found to contribute to the chronic diarrhoea in microvillus inclusion disease. In this study, we will use patients’ own cells to investigate the potential of FDA-approved drugs to modulate the activity of these ion pumps and resolve the chronic diarrhoea, and in this way improve patient care and quality of life.
18-8 Multiple Meningioma Predisposition Syndromes
Meningiomas are tumours that grow from the protective covering of the brain and spine. They account for approximately one third of all primary brain tumours. However, most meningiomas are isolated tumours that occur in older adults, while the occurrence of multiple meningiomas and meningiomas in children and young people is rare. The most common genetic cause of a predisposition to developing multiple meningiomas is called NF2. Three other genes, all belonging to the same cellular pathway, have been shown to predispose to small groups of non-NF2 associated meningiomas. These genes are called SMARCB1, SMARCE1 and SUFU. We plan to sequence the entire genome of people with familial and/or multiple meningiomas, who have been tested for mutations in the known predisposition genes and found to not have any of the known mutatons. We will use this data to identify new causes of meningioma predisposition. This information will be useful for providing patients with an accurate diagnosis and information on the associated risks of future tumour development.
Results - Meningiomas are the most common primary brain tumours in adults. However, most meningiomas occur in isolation, not associated with a genetic predisposition. In rare cases, meningiomas occur as part of a familial predisposition such as neurofibromatosis type 2, or familial clear cell meningiomas caused by mutation of the SMARCE1 gene. During our project, we have obtained the entire genome sequences of two related people with familial meningioma disease. Each of these people had undergone prior genetic testing for mutations in the known predisposition genes and was found to be mutation negative. We used this sequence data, along with the genome from another affected, but unrelated individual, to search for new causative genes for meningioma predisposition. We identified variants in two candidate genes, which are currently undergoing further analysis to determine whether they are causative of meningiomas in these people. In addition, we are currently applying for further funding to extend these studies and we plan to sequence the genomes of additional affected individuals to search for further causative genes.
18-12 Identifying the Genetic cause of Aicardi Syndrome through ‘Long Read’ Sequencing
Aicardi syndrome is a rare neurodevelopmental disorder with an estimated incidence between 1:105,000 and 1:167,000 in the United States. It is characterized by typical retinal lesions and brain malformations along with severe seizures in infancy. Many affected patients subsequently develop intellectual disability. This disorder has only been described in females or unusual males with two X chromosomes. Survival is variable, but most affected girls die before adulthood. No gene for this order has been identified, despite standard genetic testing of many patients throughout the world. Identification of the genetic cause of the disease is critical to understanding the nature of Aicardi syndrome and developing appropriate treatments. We aim to characterize the genome of a patient with Aicardi using state-of-the-art ‘long-read’ sequencing technology to look for the presence of a disease-causing mutation that cannot be detected by standard genome-wide testing.
18-13 Evaluation of Antisense-Mediated Knockdown to Treat Fibrodysplasia Ossificans Progressiva
Fibrodysplasia Ossificans Progressiva (FOP) is a rare and devastating genetic disease characterized by bone formation outside the skeleton. The worldwide prevalence is approximately 1 case in 2 million individuals. Currently, there is no therapy available and the median lifespan of the patients is approximately 40 years of age. We propose to develop a new therapy using small DNA-like molecules. These molecules can reduce the gene product that induces the bone formation outside the skeleton. We will measure the efficacy of these molecules on the signaling pathway that regulates bone formation. This study will identify new drug candidates that can treat FOP patients.
Results - Fibrodysplasia Ossificans Progressiva (FOP) is a rare and devastating genetic disease characterized by bone formation outside the skeleton. We propose to develop a new therapy using small DNA-like molecules. We established the system to measure the efficacy of these molecules on the signaling pathway that regulates bone formation, and found several good candidates that can reduce the amount of the gene products that cause FOP. These results pave the road towards clinical trials of the new drug candidates to treat FOP.
18-15 Proof-of-concept study: Rescue of a Missense/misfolding Defect in a Patient with Combined Immunodeficiency due to ICOSLG deficiency
Combined immunodeficiency syndromes (CIDs) are genetic disorders of lymphocytes, impairing the immune system and predisposing to recurrent infections. In its severe form (SCID), transplantation in early childhood leads to cure. In the less severe forms (CIDs), the best treatment is not known, but transplantation may actually be more dangerous. We have identified a young-adult patient with CID and recurrent infections due to a faulty ICOSLG gene. By characterizing at the molecular level the basis for why his ICOSLG gene is faulty, we have demonstrated that the mutation causes the protein to undergo degradation, instead of being delivered to the cell surface. We will test in our cellular model whether certain currently available drugs that are known to inhibit protein degradation can increase normal expression of the protein at the cell surface so as to restore cell function. If successful, this approach will be applicable to treat other genetic disorders with the same faulty mechanism.
18-18 Study of the Vascular Defects in Hutchinson-Gilford Progeria Syndrome Using Induced Pluripotent Stem Cells
Progeria is a rare genetic disease that affects ∼350 children around the world. Children born with Progeria age very rapidly and usually die in their teenage years due to cardiovascular disease. The only approved therapy is Lonafarnib, a drug that prolongs life expectancy by 1.6 years. This drug aims to avoid the accumulation of the mutant protein that causes the disease. To improve the quality of life of the patients and further prolong life expectancy, this drug could be combined with medications that target cardiovascular complications. Conventionally, these types of studies have been conducted using animal or cell-based models that do not fully reflect disease progression in humans. Instead, this study will use induced pluripotent stem cells generated from biopsies donated by Progeria patients. These cells will be differentiated into vascular endothelial cells and exposed to flow to mimic the conditions present in human arteries. The effect of drug combinations on these cells will be studied to identify better medications. This new way to study Progeria could avoid the use of animal models, better predict outcome in patients, and lead to new therapies to treat this rare genetic condition.
18-19 Proof-of-Concept Study: Rescue of Arginase-1 Deficiency in an Inducible Murine Model
Our research focuses on arginase-1 deficiency caused by mutation in the gene encoding the liver-type arginase-1 enzyme which involves in the detoxification of excess nitrogen. We have previously generated an arginase-1 deficient mouse model to study the pathological characteristics of this disorder. In this proposal, we are using this mouse model to facilitate the development of a gene correction therapy to treat this disease. We have successfully corrected the defective arginase-1 gene in laboratory settings using induced pluripotent stem cells derived from our arginase-1 deficient mice. The corrected cells were engineered to transform into liver cells for functional studies. To further validate our cellular model, the corrected liver cells will be transplanted into the arginase-1 deficient mice. We will determine if the transplantation can restore arginase-1 expression and facilitate rescue of function and phenotype of the disease model. Our work will be applicable to other scientists investigating their particular genetic disorder with the strategic gene-editing approaches being adaptable, especially to disorders amenable to liver targeting.
Results - Our research focuses on arginase-1 deficiency, a urea cycle disorder caused by mutation in the gene encoding the liver-type arginase-1 enzyme which involves in the detoxification of excess nitrogen. The loss of arginase-1 function leads to high amounts of the amino acid arginine in the blood and sporadic elevation of ammonia, accompanied by progressive mental retardation and failure to thrive. Since the disease is rare, it is difficult to study in humans. Our lab has previously generated an inducible arginase-1 deficient mouse model to study the pathological characteristics of this disorder. In the current work, we utilized this mouse model to facilitate the development of a gene correction strategy for proof-of-concept studies. We have published the first part of our work earlier this year, showing correct targeted gene repair using a powerful gene-editing tool called CRISPR/Cas9 system together with the induced pluripotent stem cells derived from our mice with disrupted arginase-1 function. The repaired cells were converted into liver cells for functional studies under laboratory setting. For the second part of our work, we transplanted the corrected liver cells into our mouse model for further validation of our cellular model. Our results showed that transplantation could restore arginase-1 expression and partially rescue the phenotype of the disease model. The survival of the arginase-1 deficient mice was extended by up to a week in some mice. Further experimental optimization is required to enhance the therapeutic efficacy. Our findings represent the first description of transplantation using cells derived from gene-edited stem cells for arginase-1 deficiency. The development of efficient targeted gene editing could open exciting new avenues for urea cycle disorders and other disorders amenable to liver targeting.
Publication and Dissemination of Research Findings
Sin YY, Price PR, Ballantyne LL, Funk CD. (2017) Proof-of-concept gene editing for the murine model of inducible arginase-1 deficiency. Sci Rep. 7, 2585.
Press release “A major step in treating genetic diseases” http://www.queensu.ca/gazette/stories/major-step-treating-genetic-diseases
Sin YY, Ballantyne LL, Richmond CR, Funk CD. Transplantation of gene-edited hepatocyte-like cells modestly improves survival of arginase-1 deficient mice. Submitted to Mol Ther Nucleic Acids (2017).
18-23 Metagenomic Analysis of Cerebrospinal Fluid to Detect Microbial Causes of Severe Spastic Cerebral Palsy with Microcephaly – Proof of Principle
Cerebral Palsy (CP) is a condition characterized by abnormal development or damage to parts of the brain that control movement, balance and coordination, and can take different forms depending on the severity, timing and location of the brain injury. CP is generally attributed to premature delivery, perinatal asphyxia (lack of oxygen), genetic causes, and maternal infections in pregnancy. Spastic CP is a form of the condition that is caused by damage to parts of the brain involved in controlling movement. A subset of children with severe spastic CP have spastic quadriplegia, a condition involving all four limbs, the torso, and face. This sometimes co-exists with microcephaly, a congenital malformation marked by a smaller brain size than typical newborns and resultant intellectual disability. Maternal infections may cross the placenta and result in this condition. The current evidence that Zika virus results in significant brain damage, including microcephaly, is a recent example of the potential impact of such maternal infections. Our objective is to try to identify causes of spastic quadriplegia with microcephaly by looking for genetic signatures of viruses and other microbial organisms in the cerebrospinal fluids of a few children with this condition. We anticipate that this will provide us preliminary data for a larger study. The immediate beneficiaries of this study will be children with CP and their families: Recognition of the underlying causes of CP will lead to improved clinical care, understanding of etiology and ultimately to means of prevention.
18-25 Effect of Eccentric-Training on Myogenic Capacity in Myotonic Dystrophy Type 1
Myotonic dystrophy type 1 (DM1), also known as Steinert’s Disease, is the most prevalent inherited neuromuscular disease in adults. This multisystemic disease is characterized by skeletal muscle impairment including muscle wasting. The development of rehabilitation interventions focusing on the improvement of quality of life of patients with DM1 is desirable. Slowing muscle wasting in this population using strength-training seems a promising strategy, but it remains unknown if it would trigger cellular and molecular responses, at skeletal muscle level, similar to the ones seen in healthy subject. This project aims to characterize the effect of a strength-training program on cellular and molecular skeletal muscle adaptations in patients with DM1.
18-26 Skeletal Dysplasia: Establishing Metabolic & Nutritional Requirements to Improve Healthcare
Skeletal dysplasia is a rare genetic condition that prevents bones from growing in the usual way. Most people notice that this results in short arms and legs but are unaware of the many other health problems that limit quality of life (e.g. breathing difficulties, back pain and spine surgeries, joint pain and difficulties moving around in a world designed for taller people). All these problems are made worse if a person is overweight or obese. This is important because people with skeletal dysplasia are more likely than those of average height to gain weight and so are also more at risk of life-threatening diseases like type 2 diabetes and heart disease. Nobody knows why being extremely short causes these problems. It is likely that metabolic rate might explain the links between body fat levels and health markers – but no scientific research has ever made these measurements to understand and help this unique group of people. Our project aims to address these important questions.
18-28 CRISPR/Cas9-Mediated Gene Modulation on Merosin-Deficient Congenital Muscular Dystrophy Type 1A (MDC1A)
Merosin-deficient congenital muscular dystrophy type 1A (MDC1A) is a rare genetic disease that affects 1 in 150,000 children. It occurs when laminin-alpha-2 protein, which normally acts a shock absorber for muscles, is not produced sufficiently by the body due to mutations. Without laminin-alpha- 2, muscle fibers are progressively damaged and connective tissue begins to fill up the gaps left by dying fibers. Over time, the muscles of patients weaken and they lose the ability to walk, have trouble breathing, and die prematurely. Currently, there is no cure; however, treatments are available to manage symptoms. In this project, I will use CRISPR/Cas9 technology to increase laminin-alpha-1, which can substitute for laminin-alpha-2, and decrease the levels of osteopontin, which can prevent the accumulation of connective tissue in muscles of patients. I hypothesize that this strategy will ameliorate disease phenotypes. To fully assess therapeutic potential of this strategy, I will use adeno-associated viral vectors to package and deliver the CRISPR/Cas9 components to a mouse model of MDC1A. Successful completion of this project will provide critical pre-clinical data for a treatment strategy.
Results - Adeno-associated viruses (AAVs) have been readily used in human clinical trials, where they are often involved in gene transfer therapies. This microgrant funded the opportunity to optimize the use of AAVs in delivering CRISPR/Cas9 components for gene editing purposes to treat DMD caused by deletion mutations. The use of the AAV-DJ serotype was first used in vitro in HEK293T cells, and then adapted to patient myoblasts. Targeting efficiency and resulting gene editing was evaluated, validating the activity of CRISPR/Cas9 components in vitro after AAV
delivery. This research is key in validating AAV as a delivery vehicle for CRISPR/Cas9 components, where the vector does not interfere with gene editing efficiency. AAVs will be continued to be used for gene editing in vivo using DMD animal models in the development of a CRISPR/Cas9 gene therapy to treat deletion mutations causing DMD, which can eventually
be adapted to other disease caused by deletion mutations.
Merosin-deficient congenital muscular dystrophy type 1A is an autosomal recessive diseasecaused by mutations in the LAMA2 gene, which codes for the laminin alpha 2 chain of the laminin-211 cell adhesion molecule. Lama1 is a potential disease modifier gene for MDC1A that could be modulated to treat or prevent the disease phenotype. The Lama1 gene encodes the laminin alpha 1 chain protein, which is structurally similar to laminin alpha 2 chain. We have used the CRISPR/Cas9 system to upregulate expression of Lama1 and compensate for the deficiency of laminin alpha 2 chain in mice to re-establish the integrity of muscle fibers. We have evaluated the effectiveness of local intramuscular and systemic delivery of CRISPR/Cas9 components to modulate Lama1 expression in vivo to decrease disease severity in laminin alpha 2 deficient dy2j/dy2j mice. This was accomplished by packaging CRISPR/Cas9 gene activation constructs into adeno-associated viral (AAV) vectors, which were then delivered via injection into a muscle or the bloodstream. The treated cohort had a substantial increase in Laminin alpha 1 expression in skeletal muscle relative to the control cohort. Successful results from this project provide evidence to develop CRISPR/Cas9-mediated gene modulation as a therapeutic option for patients with MDC1A.
18-30 Phenotypic Delineation of Au-Kline Syndrome
Au-Kline syndrome (AKS) is a recently recognized genetic syndrome associated with intellectual disability and variable malformations affecting brain, skeleton, heart and kidney. Only a handful of patients are currently known, leading to uncertainty about the prognosis and the health problems affecting these patients. For example, some health issues that may be important for AKS patients include risk for aortic dilation and a very unclear risk for cancer. These issues, along with other health problems, need to be clarified. Our study aims to collect a larger group of AKS patients so that we can learn more about this syndrome. This information will help determine the best management and health surveillance for these patients and help maximize their developmental outcomes and quality of life. As part of this project, we will also encourage and aid the families in establishing a parent support network.
18-31 Porphyria Registry
Porphyrias are a group of rare inherited disorders caused by deficiencies of enzymes needed in the synthesis of the heme molecule. The deficiencies lead to the accumulation of porphyrins or their precursors in the body. Symptoms vary between different porphyria types, but most commonly, the skin and nervous system are affected. Patients may experience light-sensitive, fragile skin that burns, blisters and scars easily, as well as episodes of severe abdominal pain, nausea, vomiting, hallucinations, seizures or other neurological manifestations. Diagnosis is often difficult and slow. A previous grant from the RDF funded the development of Canadian Porphyria Registry (CPR) questionnaire. This questionnaire will now be used to assess confirmed and suspected cases of porphyria if the patients consent to be enrolled in the registry. The ultimate goal is to directly help porphyria patients by, at the very least, providing them access to viable treatment options. There are treatments currently available but inaccessible to Canadian porphyria patients: as there is no clearly defined health care network in place for porhyria patients, companies developing treatments are wary of entering the Canadian market. The registry will link specialists, patients and treatment developers to improve access and expedite the process of finding viable treatment protocols, along with providing an important resource for future researchers. By reaching out to physicians and specialists, we will also be able to clarify issues that are frequently encountered during biochemical testing of porphyria specimens, thereby helping to improve diagnosis and reduce the number of misdiagnosis of porphyria patients. Once the registry is under way, the prevalence of the different types of porphyria within Canada will finally be known. As a whole, the registry is not only a means of gathering pertinent statistics, it harbours much potential to become an essential tool for aiding Canadian porphyria patients in a direct and tangible way.
18-32 A prospective study of the efficacy and tolerability ketogenic diet in patients with West Syndrome who are refractory to the first line treatments
West syndrome (WS) is a rare age-specific epilepsy of early infancy characterized by spasms, abnormal brain wave patterns called hypsarrhythmia and intellectual disability. The prognosis of WS is usually unfavorable with respect to treatment and developmental outcomes. The neurodevelopmental outcome of WS patient are poor even after following current treatment guidelines, and about 30% of patients do not respond to treatment. The ketogenic diet (KD) is a high-fat, low-carbohydrate, and adequate-protein diet well-established as treatment option for drug resistant early childhood epilepsies. There are few studies of the efficacy and tolerability, and no study of the safety of the ketogenic diet in treatment of infantile spams. The KD is resource intensive treatment, requiring a multidisciplinary team, including dietician, nurse, physician and parents. The aim of this study is to evaluate the efficacy, tolerability and safety of the early use of the KD in WS patients who do not respond to conventional treatment. Information obtained from this study is necessary to optimise choices in WS treatment, aiming to improve outcomes and thus determine whether and when the KD should be used.
Funding for the above project was made possible by a grant from the Canucks for Kids Fund.
18-33 SPROUTY 1 Deficiency:A New Disorder with Over-Activation of RAS and FGF Signaling?
Undiagnosed rare disease is a major problem that strands patients in the medical system without a care home or care plan. A genome-wide sequencing program at our clinic is providing a diagnosis to nearly half of children enrolled. However, this means that half of children still remain undiagnosed. A large portion of these undiagnosed children have a striking abnormality in one of their genes that is probably the cause of their disorder. Through new global networks of geneticists (eg., GeneMatcher and DECIPHER), it is now possible to put together patients with very rare syndromes and changes in the same gene. We have gotten this far for a boy with problems affecting his hearing, teeth, growth, and development. He has lost one of his two copies of a gene called SPROUTY1 (SPRY1), and the same thing was found in two other children with similar problems living in the UK and in the US. Because the SPRY1 gene is involved in two major cell signaling pathways, the RAS pathway and the FGF pathway, our project aims at demonstrating that the problem in patients with a SPRY1 gene mutation is over-activation of these pathways, and then show that certain existing medications (eg., salirasib, deltarasin, or tipifarnib) suppress the over-activation.
Funding for the above project was made possible by a grant from the Canucks for Kids Fund.
18-41 Is Endogenous Retrovirus-K a Therapeutic Target for Spinal and Bulbar Muscular Atrophy?
Amazingly, over 8% of human DNA is of retroviral origin – scattered inside our genome are thousands of retrovirus-like sequences called endogenous retroviruses. We have shown that there is active endogenous retrovirus-K (ERVK) in patients with the neurodegenerative disease Amyotrophic Lateral Sclerosis (ALS). Another motor neuron disease, Spinal Bulbar Muscular Atrophy (SBMA) is caused by a mutation in the androgen receptor (AR). As the ERVK promoters contain binding sequences for the AR, we hypothesize that ERVK expression is also increased in SBMA and contributes to this disease. The identification of ERVK-mediated pathogenesis in SBMA would provide novel therapeutic options, utilizing anti-inflammatory and anti-viral drugs which are currently in clinical use.
Results - Our findings were presented at the ALS Canada Research Forum (Toronto), Manitoba Neuroscience Network meeting, Undergraduate Neuroscience Conference (Edmonton), and UW Randy Kobes Undergraduate Symposium in 2018. We show that patients with SBMA exhibit increased expression of ERVK in their PBMC than age & sex matched controls. This work is in the process of being submitted for an invited paper for Frontiers in Neurology.
18-42 Effect of Conotoxin-like Protein on Cerebral Organoids: A Novel Model of ALS
Endogenous retrovirus (ERV) sequences are found throughout our genome as remnants of ancient retroviral infections. Our lab has shown that ERVK conotoxin-like protein (CTXLP) is toxic to cells, and that it is expressed in Amyotrophic Lateral Sclerosis (ALS). We propose to use a cerebral organoid (mini-brain) model to investigate how CTXLP impacts brain cell health and organization. Our research will provide insight into the mechanism of CTXLP-mediated neurotoxicity and lead to new therapeutic targets for the treatment of ALS.
Results - Select findings are found as part of a submitted paper. The Rare Disease Foundation has been acknowledged as a support of this work.
18-45 Identification of a Gene Causing Inherited Juvenile Cataract
We have identified a family with a rare inherited disease that causes juvenile cataracts. Preliminary experiments are consistent with the hypothesis that a novel gene is involved. Its identification will allow the family to better understand their condition, and may enable better treatments not only for cataracts affecting children, but also for the much more common form affecting the elderly.
18-53 Cystic Fibrosis in BC: Transitioning from Pediatric to Adult Care
Cystic fibrosis (CF) is a fatal genetic disease that most commonly affects children and young adults. CF is caused by a genetic mutation that results in production of a defective protein called the CF transmembrane conductance regulator (CFTR). CFTR is found in many tissues of the body, and plays an important role in the airways and sweat glands. CF patients in British Columbia (BC) are required to transition to adult care facilities after reaching adulthood. During this transitioning period, many patients can be temporarily or permanently lost to follow up, which can have detrimental effects on their health. Therefore, our study will examine the characteristics of patients who are at high risk for loss to follow up and evaluate how gaps in care during transition can adversely impact CF health outcomes.
18-56 Understanding a Founder Population of Individuals with Phospholamban (PLN) Cardiomyopathy
Phospholamban (PLN) cardiomyopathy is a rare heart condition (1:2000 – 1:5000) affecting the heart muscle that can lead to loss of consciousness, cardiac arrest, and sudden death in children and adults. It can be difficult to diagnose, and one can be asymptomatic despite significant disease. Unfortunately, sudden death of an otherwise healthy young individual can often be the first sign that something is wrong. After a sudden death in the family, relatives need to undergo screening and assessment by a heart rhythm specialist and genetic counselor to determine if others are also affected. Affected family members who carry the genetic mutation may also suffer from sudden death and progressive heart weakening. There are many questions that remain unanswered in Phospholamban cardiomyopathy. A better understanding of disease can allow for individuals at-risk to be identified earlier and treated safely. In our project, we will identify individuals with Phospholamban cardiomyopathy within a network of Inherited Arrhythmia Clinics across Canada. We will review all of the cardiac testing performed, to see if there are earlier clues for diagnosis. Earlier clues for diagnosis can prompt earlier genetic testing for the mutated gene. This process will ultimately allow for earlier identification and prevention of sudden death in individuals at-risk.