Mini-1 Intracellular Calcium Signaling in Patients with Severe Epilepsy
Epilepsy, characterized by periods of uncontrolled vigorous shaking, or seizures, affects approximately 1% of the population. Seizures can be very difficult to treat sometimes requiring several medications, while 30% of patients are unresponsive to treatment. Many cases of epilepsy are genetic in origin and a large number of causative genetic mutations have been identified. Recently there have been enormous advances in the development of new anti-seizure medications using the knowledge from genetics, and treating the condition of individual patients, thus developing and perfecting the notion of personalized medicine. For some novel genetic mutations associated with epilepsy, no disease mechanisms have yet been elucidated. These cases where we do not yet know how the genetic defect causes seizures are much more difficult to treat. One such mutation associated with epilepsy is in the RYR3 gene, which affects a specific calcium channel in the brain, but experimental evidence for how the mutation causes epilepsy is totally lacking. In other cases the genetic mutation puts patients at risk of dying unexpectedly, as in cases with mutations (SCN1A) in sodium channels. These mutated channels also alter calcium signaling in brain cells. We will perform experiments in cells derived from the patient’s blood that behave like brain cells and carry the RYR3 or SCN1A mutation to identify abnormalities in brain cell signaling caused by the mutated channels. Control experiments will be done on close relatives not carrying the mutations in question. The goal of these experiments would be to translate the knowledge obtained, into treating individual patients with epilepsy with medications that alter the calcium or sodium levels in the brain. This would not only mean a better quality of life without seizures, but ultimately saving the lives of those patients who are at risk for sudden death in epilepsy.
19-16 Establishing patient-derived cell lines for testing new therapies for Duchenne muscular dystrophy
Duchenne muscular dystrophy (DMD) is an inherited disorder caused by mutation of the dystrophin gene. This causes a deficiency of the muscle protein, dystrophin, which is essential for muscle integrity and function. This disorder is carried on the X chromosome and primarily affects boys, who experience progressive weakness leading to difficulty walking, running and jumping and all boys are in a wheelchair by the end of their teenage years. It also leads to progressive respiratory insufficiency requiring support for breathing as well as affecting the heart muscle. The mean age of death is about 28 years. About 10% of DMD patients have a type of mutation that creates a stop signal within the dystrophin gene so the dystrophin protein is cut short at the mutation and cannot function. Ataluren (PTC 124) is a drug designed to enable cells to ignore the premature stop signal and produce full-length dystrophin protein, a process called premature termination codon (PTC) read-through. In clinical trials, some patients receiving Ataluren showed slower disease progression while others did not. It is important to understand why patients respond differently. We will make immortalized cells from the blood of two DMD patients with different mutations. These cells will be differentiated into muscle cells and treated with various PTC read-through drug candidates to determine how much dystrophin they produce and if it is possible to predict which patients may be more sensitive to the drug. A positive outcome could lead to tests to predict which patients might benefit from the treatment.
19-21 GOT2 deficiency: studying a rare inborn error of metabolism to understand brain metabolism and targets for epilepsy treatment
Epileptic encephalopathies encompass a group of debilitating and potentially life-threatening neurologic conditions. Accurate diagnosis of the underlying cause, especially inborn errors of metabolism, is essential as this allows for targeted treatment, which can prevent brain damage and improve daily functioning and outcomes for affected children. Our team applied genomic technologies to study a 5-year old boy with severe seizures, developmental delay, and unexplained biochemical abnormalities in blood and spinal fluid. Excitingly, the team identified a novel rare neurometabolic disease characterized by mutations in the gene GOT2; this discovery allowed for treatment with nutritional supplements targeting the metabolic defects. The patient’s response is promising; seizures and head growth have improved although many serious challenges remain. Our goals are to understand the mechanisms underlying this rare neurologic condition which causes developmental delay and epilepsy, to enhance early identification and to develop better treatment strategies. We have already created cell lines missing GOT2 and now plan to study the chemical changes important to brain function. We aim to use the study results to develop more precise diagnostic and treatment methods for this rare epilepsy disorder. Central to our approach is an international collaboration between different disciplines, student training, family participation, as well as a translational focus from bed- to bench- to bedside.
19-34 Chromium content of Pediatric Parenteral Nutrition solutions in Canada: Are Pediatric patients on Parenteral Nutrition exposed to toxic amounts?
Intestinal Failure (IF) occurs when a person’s gut cannot work well enough to absorb enough nutrients and fluids. In these cases, life-saving parenteral nutrition (PN) is given through their veins for months or years. Special bottles of certain nutrients are added together to make PN solutions. Unfortunately, these bottles have been proven to have too much chromium (Cr). Chromium is a trace element; the word “trace” meaning that we only need it in very small amounts. Chromium assists the hormone insulin, which helps the body use carbohydrate, fat and protein. While the body needs Cr to function properly, too much is unsafe. The nutrition solutions used in PN contain high amounts of Cr because of contamination. This level of contamination does not have to be measured or included on the label of the PN solutions. Patients on PN may therefore get enough or even too much Cr from the contamination of the PN fluids and may not need additional Cr in their PN. High levels of Cr in blood and tissue may have a negative impact on kidney function. We have observed that patients on PN have high amounts of Cr in their blood; up to 20 times higher than normal. Also, we have observed that patients on PN for long periods of time (over 6 weeks) have problems with their kidneys. It is possible that the excess Cr is linked to the kidney issues. The goal of this study therefore is to determine the amount of Cr in the PN solutions provided to patients with IF. We will collect 10 mL of sample from the discarded bags of PN from 45 IF patients in hospital and 45 patients on home PN. Then we will measure the amount of Cr in each sample and compare the measured amount to that which was ordered to determine the level of contamination. The information from this study will help us decide if we need to order Cr in the PN for our patients or whether the amount present from contamination is already enough or even in excess.
19-24 Treatment of Medically Refractory Epilepsy due to Focal Cortical Dysplasia with mTOR inhibitors
Focal cortical dysplasia (FCD) is a localized brain formation problem that often causes difficult to control seizures. Individuals with FCD can have a sudden worsening of seizures, requiring admission to ICU, and non-response to anti-seizure medications. This can cause a great burden for families and the healthcare system. Many require urgent epilepsy surgery, where the abnormal brain tissue is removed to improve seizures. However, additional treatments are needed in the interim. Research shows that the mTOR pathway, involved in cell growth and function, plays a role in FCD. Recently, mTOR inhibitors were proven to significantly reduce seizures with a good safety profile in patients with Tuberous sclerosis complex, who have multiple small FCDs. Therefore, mTOR inhibitors may be useful in treating seizures in patients with FCD and very difficult to control seizures, while they await epilepsy surgery. This is a pilot study of this treatment that will be conducted in one clinic.
19-7 Protein restoration therapy for children with inherited thrombophilia
Inherited thrombosis is a very rare genetic disease in which the liver does not make anticoagulant proteins, leading to a high risk of deep venous thrombosis and embolism (serious clots) in infants and young children. Patients require frequent and sometimes life-long treatment with blood thinners (anticoagulant drugs). These are difficult to dose correctly and carry the risk of inducing life-threatening bleeding. In this project we will determine the potential of new drugs that restore the body’s own production of anticoagulant proteins to better treat this disease without the risk of bleeding.
19-1 The gut microbiota as a target to resolve chronic diarrhea in children 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 diarrhea which complicates disease management and severely impairs quality of life. There is no treatment to resolve the diarrhea. Recent studies suggest a relationship between the diarrhea and the composition of the bacteria in the gut. Here, we will determine in detail the changes in the composition of the bacteria in the gut of these patients. We will identify targets for intervention in the composition of the bacteria in the gut to resolve the chronic diarrhea, and in this way improve patient care and quality of life.
19-33 Can phosphorylation of the E1alpha Subunit of Pyruvate Dehydrogenase Complex Help Predict the Prognosis and Direct Treatment of Pyruvate Dehydrogenase Complex Deficiency?
Pyruvate Dehydrogenase Complex Deficiency (PDCD) is a very serious condition that often results in death in infancy. It sometimes can present in a milder form with survival beyond infancy with development of learning difficulties or as dystonia, a disorder of movement where children are unable to control their limbs, torso or head. Sporadically it has been noticed that some children with PDCD are responsive to a vitamin called thiamine or a special diet. To date it is not well understood why the condition is more severe in one child over another or why only some children are responsive to treatment. We have attained samples from a male patient (6 years old) with a mild form of PDCD who is very responsive to thiamine. His debilitating symptoms of dystonia have all but resolved on thiamine supplementation and he can now walk home from school. We will use the samples from this patient to understand why thiamine was so successful in this child. This would help explain why mutations in PDC have such a variable effect, which will help doctors predict 1) the severity of the condition and 2) what therapy (thiamine versus special diet) should be used in treating the disorder.
19-37 Understanding the pathophysiology of vitamin B6-dependent epilepsy due to PROSC deficiency
Some forms of epilepsy in children are resistant to common antiepileptic medicines but can be stopped by treatment with vitamin B6. This disease is called GOT2 deficiency. These are designated as vitamin B6-reponsive epilepsies and attributed to inborn errors of metabolism. Recently a new form of these diseases was discovered that causes neurologic impairment in affected infants along with epilepsy. Little is known about the disease biology, and filling these knowledge gaps is important to improve diagnosis and treatment. We will create a yeast model for GOT2 deficiency to study the changes in biochemistry caused by the disease. Our goal is to gain more insights into the disease mechanism, and to define targets for intervention.
19-18 Role of lipid metabolic errors in frontotemporal dementia pathology
Frontotemporal dementia (FTD) is a fatal neurodegenerative disease that results in a selective degradation of neurons in the brain. Mutations in the granulin (GRN) gene, which result in low levels of its product progranulin (PGRN), have been identified as a major cause of FTD. Unfortunately, the mechanisms underlying this disease remain poorly understood. Our lab uses the nematode C. elegans as a powerful genetic system. We have created nematodes with mutations in GRN to study the effects of the loss of PGRN and to better understand howt his loss is connected to FTD. Our results so far suggest that a loss of PGRN may result in problems with lipid metabolism. These metabolic problems ultimately lead to cellular dysfunction and death. We will perform a mass-spectrometry based assay to characterize lipid metabolism in our nematodes that carry the GRN mutation. If this project is successful, we propose to validate our findings in patients with FTD by characterizing lipid metabolism in their tissues and provide clear, new insights into the pathology of this devastating disease.
19-29 Patient-Specific Modeling of Juvenile Idiopathic Arthritis-Associated Uveitis via Induced Pluripotent Stem Cells
Uveitis is a rare, chronic eye inflammatory disease of unknown cause that affects 7% of Canadian children with juvenile idiopathic arthritis (JIA), or 1:10,000 children. Uveitis is often painless, but leads to blindness, cataract and glaucoma when is diagnosed late. Why children develop uveitis and JIA is still poorly understood. Moreover, uveitis is often resistant to treatment. Therefore, new strategies are urgently needed to investigate the mechanisms of uveitis and find effective treatments for each patient. We will develop patient-specific induced pluripotent stem cells from children with uveitis and juvenile idiopathic arthritis, and then differentiate them into affected tissues. These are the uvea (the eye layer that become inflamed in uveitis), joint linings, cartilage, bone and endothelial cells. This will allow us to study the mechanisms of uveitis and arthritis directly on the affected sites, tests new medications and find individualized treatment for each child affected by this rare disease.
19-31 Characterization of Pediatric Idiopathic Recurrent Pericarditis in Canada
Childhood idiopathic recurrent pericarditis (IRP) is a rare and often unrecognized chronic inflammatory disease of the lining of the heart. IRP has not been yet investigated systematically in Canada. We will ask Canadian pediatric specialists how many cases of IRP they diagnose in a year, and analyze the patients’ characteristics, risk factors and treatment regimens. This study will increase our understanding of this rare pediatric disease and provide the much needed clinical care guidelines that will improve the diagnosis and outcome in children diagnosed with IRP in Canada.
19-9 Mucopolysaccharidosis IIIB: Improving enzyme replacement therapy utilizing protein transduction domains.
Mucopolysaccharidosis IIIB is a genetic disease characterized by the accumulation of un-degraded cellular products in the brain causing neural degeneration and a significantly shortened lifespan. This disease results from mutations in the α-N-acetylglucosaminidase (NAGLU) gene. Because of these mutations, the NAGLU enzyme is missing in patients. Treatment by injection of the missing enzyme is ineffective as the administered enzyme fails to reach the brain because it is unable to cross the blood-brain barrier. Protein transduction domains are relatively small positively charged peptides that can be fused to biological enzymes. These peptides are able to facilitate the delivery of enzymes across biological membranes, including the blood-brain barrier. The fusion of the missing enzyme (NAGLU) to the protein transduction domain, PTD4, has the potential to improve delivery of active NAGLU across of the blood-brain barrier and therefore improve enzyme replacement therapy for the treatment of MPS IIIB.
Results - We created the Naglu-PTD4 DNA construct and introduced it into the genome of Sf9 insect cells. These Sf9 insect cells acted like a ‘biological factories’ by producing quantities of active Naglu-PTD4 enzyme that could be used for further testing. Naglu-PTD4 was purified from the Sf9 insect cells using fast liquid column chromatography with a yield of 11%. Preliminary uptake studies with Naglu-PTD4 and MPS IIIB fibroblasts showed a detectable increase in active Naglu within the cell (activity of Naglu in control fibroblasts was ≤ 0.3 U/mg and activity of Naglu in fibroblasts treated with Naglu-PTD4 was ≈ 5.3 U/mg). This preliminary data suggests that active Naglu-PTD4 was able to successfully cross fibroblast cell membranes. Successful uptake studies with Naglu-PTD4 on MPS IIIB fibroblasts are the first step in testing the delivery capability of PTD4. In future, we plan to conduct blood-brain barrier penetration studies on mice to test the ability of PTD4 to deliver the missing enzyme Naglu across the blood-brain barrier.
19-13 Locus heterogeneity in Niemann-Pick disease type C
Niemann-Pick disease type C (NPC) is a genetic condition that affects the body’s ability to transport and handle cholesterol. In patients with NPC, the cholesterol accumulates in a variety of tissues and causes signs and symptoms that can develop at any age and generally worsen over time. It can cause neurological problems, including difficulty coordinating movements (including of the eyes), speech problems, swallowing problems, stiffness, and dementia. Other problems can include severe liver and lung disease. Two genes are known to cause the disease, NPC1 and NPC2. It is important to diagnose NPC early since there is a treatment available to slow the progress of the disease. Our clinic follows a patient who has a very large spleen and laboratory findings that are typical for NPC, but who does not have any of the neurologic problems. His genetic testing for changes in NPC1 or NPC2 was negative, suggesting he may have a novel condition that mimics NPC. In order to provide our patient and his family with a genetic diagnosis, we will aim to identify the causative gene and gain some understanding of its function. Finding this gene may help the 10% of patients and families with NPC who do not have a mutation in NPC1 or NPC2. It may also provide insights into the cause of the neurologic problems we see in this condition.
19-38 Genetic basis of pyridoxine-dependent neonatal epilepsy in a consanguineous family
Some forms of epilepsy in children are resistant to common antiepileptic medicines but can be stopped by treatment with vitamin B6. They are designated as vitamin B6-dependent epilepsies and are attributed to inborn errors of metabolism. This study of families afflicted with these forms of treatable neonatal epilepsy will help to identify their genetic causes and improve diagnosis and treatment. Our team, specialized in research of this type of diseases, identified a consanguineous family who has a child affected which such a rare form of epilepsy. Seizures started in this child in day 3 of life and were controlled only by vitamin B6. Testing of a gene that is known to cause one form of this epilepsy was negative. We will perform more comprehensive testing (known as whole-exome sequencing) which will test all genes of this child in one experiment. This will help to identify the causative disease gene and subsequently improve diagnosis and help to devise therapeutic interventions.
19-6 Surgical Outcomes Of Rare Types of Strabismus: Development of a Canadian Registry
Strabismus is an eye condition where the eyes are not straight. Strabismus can lead to loss of vision in children or severe functional difficulties in adults by causing double vision. Rare causes include abnormal nerve supply to the eye muscles from genetic or acquired diseases. The resulting paralyzed or stiff muscle cannot move in sync with other muscles. Surgery to align the eye muscles are often not successful in such patients, and many different techniques are practiced. Because of the rare nature of these types of strabismus, each hospital may only see a handful of cases per year. Therefore, we do not know which procedures work best. Our goal is to set up a national database to collect information from multiple hospitals and eye care centres on these rare forms of strabismus. A collaborative effort across health care institutions will allow us to pool information and examine how effective different procedures are. An online database can be used to provide long overdue evaluation of surgical techniques used in rare strabismus. We will be able to statistically analyse the best surgical procedure by having enough numbers of these difficult cases which should ultimately result in stronger patient-centred care.
19-2 Cardiovascular Outcomes of Pregnancy in Turner Syndrome: A Multi-Center Retrospective Case-Control Study
Turner Syndrome is a genetic condition affecting 1/2000 live born girls where there is partial or complete loss of one of the X sex chromosomes . As a result, these women are mostly infertile, however, with newer technology they are able to achieve pregnancy through egg donation. The risk of pregnancy is thought to be high with 2% death rate quoted from research in small numbers of patients. Up to 50% of women with Turner Syndrome have heart abnormalities. The presence of these abnormalities has been shown to increase the risk of pregnancy. More research is needed to better understand the risk of pregnancy in this group. We have organized a network of researchers who look after these women to collect pregnancy outcomes data so that a clearer picture of risk can be presented to patients. We have identified approximately 155 women who have had a pregnancy at one of these sites, and this will be the largest group studied to date.
Results - Turner Syndrome affects 0.5% of women, and although many women with Turner Syndrome are keen to become pregnant, difficulties and risk surrounding pregnancy exist. With advances in reproductive therapies, more women with Turner Syndrome are able to become pregnant. This study aims to better understand the cardiovascular outcomes of pregnancy in Turner Syndrome. To date, 32 pregnancies have been included in the study. We plan to include up to 100 pregnancies and compare them to non-pregnant women with Turner Syndrome. The results of this study will improve our understanding of the cardiovascular changes that may occur during pregnancy, and the researchers are grateful to the Rare Disease Foundation for supporting this study.
19-14 Characterization of a novel mitochondrial disorder resulting in metabolic crisis
We have been caring for two young cousins who have both presented throughout childhood with episodes of devastating illness requiring repeated admission to the paediatric intensive care unit for resuscitation and emergency management. Each episode has been precipitated by a mild infection or slight dehydration. In between episodes the children are completely well and attend full time education with no difficulties. Recent studies by our team have identified the specific genetic change responsible for this illness in the boys. The change (one copy inherited from each of the children’s parents) alters a gene that makes a protein that we know is important in the energy producing bundles in the cells called the mitochondria. We plan to 1. Identify other children that are similarly affected due to changes in this gene; 2. Undertake analysis to look at the different types of tissue (e.g. muscle, brain,) where this gene works and 3. Use yeast models to study the function of this gene in more detail. These complimentary studies will help us to understand why this gene change leads to this devastating, and likely under-recognized, condition and to develop strategies to avoid future episodes of crisis in the boys and other affected families. The work will also allow appropriate counseling of family members who are considering having children about the risks of having an affected child and establish the first steps in developing an effective treatment.