24-1 How mutations in RanBP2 cause Acute Necrotizing Encephalopathy
Mutations in the RanBP2 gene are associated with Acute Necrotizing Encephalopathy 1 (ANE1), a rare condition where otherwise normal individuals overproduce cytokine proteins in response to influenza infections. Cytokines normally are required for individuals to mount an immune response, but cytokine overproduction can lead to problems. In ANE1 patients, the overabundance of cytokines can cause seizures, coma and a high rate of mortality. Four separate dominant point mutations in RanBP2 have been linked to this disease but are only 40% penetrant. Our work suggests that RanBP2 regulates the expression of cytokine genes. Gene expression begins in the nucleus, where a stretch of DNA (also known as a gene) is copied into mRNA. This mRNA is then packaged with proteins into a large complex and exported from the nucleus to the cytoplasm, where information in the mRNA is "translated" to make new proteins. To pass from the nucleus to the cytoplasm, the packaged mRNA must cross the nuclear pore. Previously we found that a nuclear pore protein, RanBP2, helps the translation of certain mRNAs into proteins. We believe that, as these particular mRNAs cross the nuclear pore, RanBP2 helps to remodel how they are packaged and this event changes how efficiently these mRNAs are used in the protein synthesis reaction. In preliminary data we have found that RanBP2 is required to decrease the translation of cytokine-producing mRNAs. This may explain why mutations in RanBP2 cause ANE1. We will collect nasal epithelial cells from ANE1-affected patients, relatives that are unaffected but either carry the mutation or not. We will determine the how viral infection of these cells affects cytokine release. We will use next generation sequencing technologies to determine how gene expression varies between the three groups. Our work will help to determine why mutations in RanBP2 cause ANE1 and help uncover other factors that determine susceptibility to this disease.
24-2 Unravelling the genetic causes of Woodhouse-Sakati syndrome, a very rare multi-organ syndrome
Woodhouse-Sakati syndrome is an extremely rare pediatric disorder that affects many organs, including the brain, inner ear, pancreas, nerves, and gonads. Symptoms can include intellectual disability, delayed or incomplete puberty, hearing impairment, poor hair growth, short stature, diabetes, and neurological problems. Genetic testing for mutations in the gene DCAF17 can confirm the diagnosis, but some children have negative genetic test results, leaving them without a confirmed diagnosis. A key reason for a negative genetic test result is that the disorder might be caused by more than one gene. A boy with intellectual disability, delayed puberty, sparse hair, delayed puberty, and hearing impairment, has been followed for many years by specialists, without ever receiving a diagnosis. He recently had full sequencing of his genes, which showed he had inherited both chromosome 8 copies from his mother. Normally, we inherit one chromosome copy from each parent. His chromosome 8 therefore had a double dose of any mutation in genes that are packaged in chromosome 8. We found a double dose for a rare, damaging variant in the gene DCAF13, which is not currently recognized as a cause for any syndrome. However, we realized that a family member of this gene, DCAF17, causes Woodhouse-Sakati syndrome, which closely matches his symptoms. Therefore, we suspect that DCAF13 may also be a cause of Woodhouse-Sakati. We seek to prove this hypothesis, which will allow us to finally provide this boy and his family with a diagnosis, and also help diagnose other affected individuals.
24-3 Restoring Extracellular Matrix Function in Marfan Syndrome by Augmenting Fibrillin-1 Deposition
Marfan syndrome (MFS) affects 2-3 in 10,000 newborns and is caused by genetic changes (mutations) in fibrillin-1 (FBN1). Patients with MFS have long bones, weak joints, eye problems, and are at high risk for the widening and fatal rupture of the biggest blood vessel in the body, the aorta. Some of the mutations in FBN1 result in weak connective tissue, because not enough FBN1 is made. In these cases, a strategy to improve tissue function is to use drugs that force cells to make more FBN1 by ignoring these mutations. The results of the proposed studies could lead to novel treatment strategy for a subset of patients with MFS.
24-4 Improving the management of Marfan patients with improved pharmacotherapy: from mice to men
Marfan syndrome (MFS) is a genetic disorder that disrupts the body’s connective tissues. With an incidence of 1 in 5000 individuals, the most common life-threatening problems in those affected by MFS is aortic aneurysm, which can be described as a progressive rupture of the main blood vessel that carries blood out of the heart. While there is no cure for MFS, management guidelines support the use of blood pressure-lowering medications losartan or atenolol to decrease the physical stress exerted on the aorta. Unfortunately, both medications have a poor track record of preventing aortic complications, hence more research is needed. Recently, our group has shown that losartan does not attenuate aortic aneurysm in MFS mice by reducing blood pressure, but instead by activating a protective signaling pathway found endogenously, called endothelial function or Nitric Oxide release. This discovery is significant, since other losartan-like medications might be far superior at increasing endothelial function, something that has never been investigated. Finally, if this holds true in patients, a simple switch from losartan to endothelial function-optimized losartan-like medications could result in drastically improved management of the disease. Prior to studying losartan-like medications in Marfan patients, we will compare the effect of these existing but never studied medications to losartan in our Marfan mouse model to identify the one most likely to result in a protective effect in patients. For this, losartan vs other losartan analogues (from the same class of medications called Angiotensin II type 1 Receptor Blockers, ARBs) will be compared at blocking aortic disease in Marfan mice. This study will identify the top losartan-like medication for MFS patient management, which will be tested in patients within 1-2 years. This will lead to improved management of Marfan patients very soon, as these medications are already FDA-approve
24-5 Surgical Correction of Velopharyngeal Dysfunction in Children with 22q11.2 Deletion Syndrome
22q11.2 Deletion Syndrome (22q11.2DS) is a rare genetic syndrome (1:4000 live births) that affects nearly every organ in the body.1,2 In addition to their multiple care needs, many children with 22q11.2DS are unable to completely close the communication between their nasal cavity and mouth leading to functional problems around speaking, eating, and breathing. This is known as velopharyngeal dysfunction (VPD) and is treated through speech therapy and surgery. Although many surgical techniques for correcting VPD exist, children with 22q11.2DS experience lower success rates and poorer speech outcomes compared to those without a genetic syndrome. Additionally, there is a lack of evidence on what factors contribute to a successful surgical correction – an informed choice of surgical technique based on preoperative anatomy, or simply the technique itself. This study aims to compare the different surgical techniques for treating VPD in patients with 22q11.2DS over the last 20-years. We hypothesize that preoperative anatomy has no significant effect on outcomes, and some surgical techniques are more effective than others. The knowledge gained from this study may help inform how VPD is corrected in children with 22q11.2DS.
24-6 MMS19 Deficiency: Understanding the biogenesis of Fe-S proteins for better rare disease patient care
DPD (dihydropyrimidine dehydrogenase) deficiency is an autosomal recessive disorder classically due to inactivation of its encoding gene DPYD. In patients with DPD deficiency a considerable variability in clinical presentation had been noted, ranging from frequent [i.e. intellectual developmental disorder (IDD), epilepsy]1,2 to less frequent manifestations (i.e. growth retardation, microcephaly, dysmorphia, autism, hypotonia and ocular abnormalities). DPD deficiency has also been associated with an adverse reaction to the antineoplastic agent 5-fluorouracil, with potentially devastating sequelae.
Understanding the pathogenesis of a DPD deficiency is of utmost importance for appropriate care of patients with this rare disease, including the potential for development of novel therapeutic options. Here, we propose a study to unravel the role and contribution to disease of a novel gene, MMS19, identified using whole genome sequencing (WGS) in our cohort of DPD deficiency patients for whom the DPYD molecular analysis was negative.
24-7 Investigation of the role of phosphotidylinositol-3 kinase gamma (PIK3CG) in Common Variable Immune Deficiency
Common variable immune deficiency (CVID) is a rare disease of the immune system. Patients have low levels of antibodies and experience recurrent infections, autoimmunity, and digestive ailments. It has been reported that approximately 10% of cases of CVID have a genetic link. We identified a rare mutation in a gene encoding a phosphoinositide 3-kinase (PI3K) shared between a mother and son, both suffering from CVID with treatment-resistant inflammatory bowel disease. I hypothesize that the mutation causes PI3K to be overactive, leading to CVID in these patients. The goal of this project is to carry out functional studies in immune cells that contain normal or mutated forms of the gene. Findings from this study may provide the opportunity for personalized treatments through the use of the drug rapamycin, which can be used to reduce the activity of PI3K and alleviate CVID symptoms in these patients.
24-8 Acetylation of NPC2 and its potential as a new therapeutic target in Niemann-Pick Disease
Niemann-Pick Disease type C (NP-C) is a rare, inherited disease that is estimated to occur 1/120,000 live births. NP-C patients have an accumulation of lipids (cholesterol) in the cells of certain organs (liver and brain). This build-up of lipids causes cells to change shape, grow abnormally, or die. NP-C patients present with a wide spectrum of phenotypes including progressive neurological defects. Currently, there is no curative treatment. NP-C is divided into type 1 and type 2 depending on whether the genetic cause arises from mutations in the NPC1 gene (95% of cases) or the NPC2 (5% of cases) gene. These mutations can result in protein loss-of-function or a decrease of functional NPC1 or NPC2 protein, both of which are required for transport of cholesterol. Importantly, the interaction between the NPC1 and NPC2 proteins is essential for the transport of cholesterol. Recently our lab discovered that the NPC2 protein is modified in baker’s yeast. This protein modification, acetylation, adds an acetyl group to the NPC2 protein and is well known to increase a protein’s stability and can strengthen interactions with other proteins. We also observed an increase in NPC2 acetylation when we treated with a drug inhibitor of acetylation called nicotinamide. NPC2 protein sequence is conserved between yeast and human and it has been shown that yeast NPC2 can rescue the disease phenotype in NPC depleted patient cells.
Our novel hypothesis is that stabilizing the NPC2 protein through acetylation will strengthen its interaction with NPC1 and allow for improved function and cholesterol transport in NPC-2 patients that have reduced NPC2 protein levels. We aim to identify the players involved in NPC2 acetylation and investigate what effect acetylation has on NPC2 stability and its interaction with NPC1. Since, inhibitors of acetylation are already being pursued in clinical settings we believe our work will establish whether these drugs can be repurposed to treat NP-C.
24-9 Quantitative computed tomography in systemic sclerosis-associated interstitial lung disease
Systemic sclerosis (SSc) is a rare condition of the immune system with an unknown cause. It is characterized by thickening of the connective tissue throughout the body. Currently, SSc affects at least 20,000 Canadians. These patients often develop interstitial lung disease (ILD), a progressive scarring disease of the lung that causes shortness of breath and is a leading cause of death in patients with SSc. SSc-ILD has few effective and well-tolerated treatment options, including medications that are costly and have limited benefit. In addition, the current methods of measuring ILD severity are not specific, which decreases the chance that the disease is treated effectively. A new more direct method of measuring disease severity is through an imaging technique called computed tomography (CT), which provides detailed images of the entire lungs. Previously, CT scans have been visually assessed to evaluate disease extent. However, it is a subjective and time-consuming process, restricting its everyday use by doctors. More recently, computer algorithms have been used to automatically assess disease severity based on the lung density of CT scans. In the proposed study, we will develop a new computer-based approach to accurately identify areas of abnormal lung and track them over time. This new method will dramatically increase the ability to detect early or subtle changes in disease progression. Our work will provide a better understanding of how SSc-ILD develops, along with new strategies for disease monitoring. The findings of our study will have significant implications for both patient care and clinical trial design in SSc-ILD, with further implications for the management of other ILD subtypes.
24-10 Implication of mutated DNMT3A in the Pathogenesis of Tatton-Brown-Rahman syndrome
Tatton-Brown-Rahman syndrome (TBRS) is a rare genetic disorder characterized by tall stature, intellectual disability, heart defects and dysmorphic facial features. This disorder is associated to a functional mutation in DNMT3A, an enzyme responsible for establishing DNA methylation modifications implicated in gene regulation, and vital for development. Currently, we do not know how functional mutations in the DNMT3A protein can be at the origin of the neurodevelopmental and other associated problems observed in patients with TBRS. Thus, there is an urgent need to develop human model systems to understand the molecular and cellular causes of TBRS, in order to find potential and specific treatments for patients. Therefore, we propose to use induced-pluripotent stem cells (iPSC), a technology that allows transforming the cells of a patient into stem cells, which can then be reprogrammed into any other cell types of the body. With this approach, we will be able to analyse how newly brain, muscle, bone and heart cells are affected. Overall, this project will uncover the functional impact of DNMT3A mutations and provide a functional model to test new therapeutic avenues to treat TBRS.
Results - In collaboration with clinical geneticists, we have identified 2 new heterozygous DNMT3A mutations in patients affected with Tatton-Brown-Rahman Syndrome (TBRS), a rare condition characterized by overgrowth, intellectual disability and facial dysmorphism. Using collected peripheral blood mononuclear cells (PBMC) from these patients, we generated iPS cell lines and produced the first cellular model of TBRS. We are now in the process of correcting the mutations in the patients iPS cell lines in order to get the isogenic controls. We are starting the differentiation process of these iPS cell lines into neural progenitors, which will then be terminally differentiated into neurons.
At each step of the lineage specification and differentiation process, various parameters (e.g., proliferative and differentiation potential) will be measured, and cellular identity and homogeneity will be assessed (e.g., lineage specific markers).
Our comprehensive approach to pathogenic DNMT3A mutations will help us to better understand the brain developmental defects observed in afflicted patients.
24-11 Development of allele-specific antisense oligonucleotides to treat Fibrodysplasia Ossificans Progressiva
Fibrodysplasia Ossificans Progressiva (FOP) is a rare and devastating genetic disease characterized by bone formation outside the skeleton, caused by mutations in a gene called ACVR1 or ALK2. The worldwide prevalence is approximately 1 case in 2 million individuals. There is no therapy available and the median lifespan of the patients is approximately 40 years of age. Although several drugs are currently under clinical trials or preclinical development, a major limitation is that most of these drugs cannot distinguish normal and mutated gene products (proteins), therefore, the normal gene function will be also affected. We propose to develop a new therapy using small DNA-like molecules (antisense oligonucleotides, AONs). These molecules are designed to target the mutated gene product only without affecting the normal gene function. In this project, we aim to employ AONs called 2’-O-methoxyethyl oligonucleotides or 2’-MOE. This type of AONs have a good safety profile in human body, as recently demonstrated by the first FDA-approved drug called Nusinersen (or Spinraza) for spinal muscular atrophy. Currently, two 2’-MOE AON drugs (Nusinersen and Mipomersen) are commercially available, and more than ten clinical trials of 2’-MOE-based drugs are ongoing. 2’-MOE is proven to be safe, and 2’-MOE AON drugs can be injected under the skin or into the vein directly. This is particularly beneficial for FOP patients because the virus vector-mediated gene therapy would activate immune responses and cause inflammation, which leads to more bone formation. We will examine the efficacy of newly designed 2’-MOE AONs in FOP patient cells. We expect that 2’-MOE is safe and prevents extra bone formation in FOP patients. This study will ultimately lead to the identification of a clinical trial drug candidate for FOP treatment. Therefore, this study will provide a significant positive impact on the lives of FOP patients.
Results - Fibrodysplasia Ossificans Progressiva (FOP) is a rare and devastating genetic disease characterized by bone formation outside the skeleton, caused by mutations in a gene called ACVR1/ALK2. We propose to develop a new therapy using small DNA-like molecules (antisense oligonucleotides, AONs). These molecules are designed to target the mutated gene product only without affecting the normal gene function. In this project, we found that newly designed AONs (called 2’-O-methoxyethyl phosphorothioate oligonucleotides) reduced the amount of the gene products that cause FOP preferentially compared to the normal gene product. This study will ultimately lead to the identification of a clinical trial drug candidate for FOP treatment
24-12 Molecular Characterization of Olfactory Neuroblastoma
Olfactory neuroblastoma (ONB) is an uncommon and aggressive cancer that occurs at the back of the nose, or sinus. Current treatment strategies focus on removing the cancer with surgery, however, this can leave patients with major physical and sensational losses. Furthermore, surgery usually does not prevent the cancer from coming back and sadly, most patients die from their disease. To date, we know very little about the genetic material (so-called DNA), of ONB cancers; in other words, we do not understand the underlying features of what makes these cancers “tick”. This is partially due to the rarity of the disease and because granting agencies have less interest in funding research that will impact only a few patients a year. In spite of these obstacles, we intend to study ONB, precisely because it is an understudied cancer. To do so, we have we have assembled the largest cohort (n=40) on record of ONB cases, in collaboration with McGill University. Using material from two index ONB cases, the first part of our project will be to identify the differences in the DNA of healthy sinus tissue compared to ONB cancerous tissue. The first part of our project consists of reading out the DNA sequences of two ONB index cases using rapid and very accurate technology, so-called high-throughput sequencing. The findings from this initial study will subsequently be validated using material made by ONB cancer cells downstream of DNA, also known as RNA. With the knowledge we have gain from understanding the initial two ONB cases, we will expand our sequencing to include the entire cohort that we have assembled. Finally, we will be able to correlate our findings with ways of measuring ONB patient outcome. Together, our work will help unlock the mystery of what parts of altered genetic material lead to rapid ONB patient decline and crucially, will identify ONB patients who might need more aggressive treatment to ensure their lives are not cut short by this disease.
24-13 Role of Serca2a in pulmonary hypertension-associated Lung Fibrosis
Idiopathic pulmonary fibrosis (IPF) is a rare disease characterized by a progressive accumulation of scar tissue and a fibroproliferative process, leading to respiratory failure and ultimately to death within 3-5 years after the diagnosis. Among the PF complications, pulmonary hypertension (PH) occurs in 32 to 84% of patients with IPF and is associated with a right ventricular (RV) heart failure and an increase in pulmonary artery pressure. Medical therapy is ineffective in the treatment of PH-associated IPF; therefore, there is a critical need to identify new molecular therapeutic targets. Our recent studies have demonstrated that gene therapy targeting the calcium-handling protein SERCA2a in the lungs prevented the development of PH in multiple experimental animal models by preventing cardiac/arterial remodeling and improving heart function. Additionally, SERCA2a gene transfer has been extensively validated in the ventricular myocardium and shown promising results in clinical trial with patients with congestive heart failure. Here, we hypothesized that SERCA2a downregulation potentiates the development of PH in IPF and SERCA2a gene transfer inhibits PH-associated IPF. In this study, we discovered that SERCA2a was significantly decreased in a mouse model of PF induced by delivery of Bleomycin (Figure 2). In cells, we demonstrated that SERCA2a overexpression significantly decreased the proliferation of normal human lung fibroblasts and their differentiation as well as the expression of several fibrosis markers. Using an animal model, we will investigate whether intratracheal inhalation of the aerosolized human SERCA2a gene in a Bleomycin-induced PF model will reverse lung fibrosis and prevents the development of PH. Collectively, our study aims to investigate the role of SERCA2a and determine whether SERCA2a gene transfer may represent a new therapeutic strategy for treating patients with PH-associated IPF.
24-14 Dystonia Therapeutics Targeting Premature Stop Condons
Each gene translates its specific nucleotide sequence into a specific protein that is required for health maintenance. Genes bear a STOP trinucleotide that signals translation termination. Gene lesions (wrong nucleotides) can create a STOP signal early in their sequence, known as “Premature stop codon” (PTSC). These codons produce short, non-functional proteins and cause ~1800 diseases, such as muscular dystrophies, hemophilia, and dystonias. Numerous PTSC readthrough compounds have been identified and more are being developed by Canadian companies investing in PTSC therapeutics. These compounds prompt the translational machinery to “skip” PTSCs and continue until the normal STOP, restoring protein length/function. Their efficacy has been tested in cells, animal models, or clinical trials for various diseases, but not for dystonia. Dystonia is a group of devastating movement disorders associated with numerous mutations in various genes. Thirteen forms of childhood or adult onset dystonias are caused by PTSCs. We propose a Gaussia luciferase (Gluc) assay for measuring efficacy of PTSC compounds to readthrough dystonia PTSCs. Each of the examined PTSC will be linked upstream of Gluc. Thus, production of Gluc can only be achieved if a compound promotes readthrough of the preceding PTSC and can be simply assayed by adding Gluc substrate to the media of cultured cells, followed by luminescence reading. Monogenic dystonias are rare diseases and receive no attention for therapeutics. The present study has high probability of promoting therapeutics for 13 untreatable dystonia syndromes with minimal cost and expenditure of time, since it capitalizes on existing findings from other PTSC-diseases rather than exploring a series of untested compounds. Compounds that promote readthrough of dystonia-PTSCs in our reporter assay will attract future funding for testing them in patient derived cells and mouse models, and ultimately promote the most promising in clinical trials.
24-15 Differentiation of Frasier syndrome iPSC-derived podocytes for precision medicine
Frasier Syndrome is a rare congenital genetic condition that induces early onset kidney disease called idiopathic focal segmental glomerulosclerosis, or FSGS, where most patients develop kidney failure early in their adulthood. Mechanisms of FSGS are poorly understood; however, defects in glomerular cells, called podocytes, are thought to be primarily responsible for disease progression. While the genetic basis of the disease is known (mutation in the WT1 gene) no clear treatment options exist for the resultant chronic kidney disease and eventual kidney failure. In order to develop therapeutics, we need model systems to study disease mechanisms. Since Frasier Syndrome is exceptionally rare (with 50 reported case studies), it is difficult to develop model systems that can recapitulate the human disease processes. Here, we propose to use induced pluripotent stem cell (iPSC) technology to generate a patient-specific podocyte cell line for Frasier Syndrome and use this unique cell line to identify molecular, biochemical and biophysical changes in podocytes affected by this condition. We will re-program blood cells into iPSCs from a current Frasier syndrome patient enrolled in our clinic (31-year-old female with confirmed diagnosis). We will then use directed differentiation to generate a homogeneous population of human podocytes that can be compared to age-matched control human podocyte lines. As Frasier Syndrome is an exceptionally rare disease, a patient-specific podocyte line will not only help understand key disease processes, but will also serve as a drug screening platform for the patient that needs to be enrolled in clinical trials for FSGS.
24-16 The Youngest Patients with Catecholaminergic Polymorphic Ventricular Tachycardia: What About the Parents?
Each year, hundreds of young children in Canada die suddenly and unexpectedly. No cause of death is found in nearly half. Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT) is a rare heart disorder occurring in 1 in 10000 people and causes an abnormal heart rhythm during exercise or stress. CPVT causes 15% of unexplained sudden deaths in the young population. Without treatment, half of the people affected by CPVT die by the age of 35. Diagnosing patients with CPVT is very difficult because routine tests are normal. For this reason, there is a several year delay in diagnosing CPVT and many are left untreated. The most severe cases present before the age of 10, but some cases present in early adulthood. Studying the young, severe cases will help us understand the disease better. Genes are sets of instructions in the body, which determine a person’s characteristics and how their body functions. When there is a spelling mistake in a gene, it can affect a person’s health. Abnormal genes can cause heart rhythm problems. CPVT is caused by spelling mistakes, or “mutations” in the Ryanodine Receptor 2 (RyR2) gene. This gene is very important in controlling the normal heart beat and pumping of the heart. In order to treat CPVT more effectively, we need to understand the risk of abnormal heart rhythms. To learn more about why some family members are affected and others are not, and why some have mild disease and others have severe forms, we would like to study the parents of children with severe CPVT. By testing parents, we will learn if a parent has abnormalities in their heart and if they have signs of CPVT. We also want to determine whether children with severe CPVT inherited this condition from their parents. Our goal is to gain more knowledge about how CPVT affects different family members. This will help us prevent sudden unexpected death in the future.