August 2017 Funded Microgrants

August 2017 Funded Microgrants

21-1 The Role of FAM 111B in the Pathogenesis of Hereditary Fibrosing Poikiloderma

We have discovered mutations in the FAM 111B gene as a cause of hereditary fibrosing poikiloderma (HFP), a multisystemic fibrosing (thickening/scarring) disease. HFP is a rare genetic disorder, characterised by tendon contracture, myopathy (muscle weakness) and fibrosing of the lungs, a resulting complication of the illness that has led to the death of affected family members. Previously we identified a total of three missense (nonsynonymous) mutations that span a total of five families of different ethnic origin. Recently, the number of cases have begun to grow, with more FAM 111B mutations identified and related clinical conditions (mostly fibrotic in nature) described. However, it not known how the identified mutations bring about these disease manifestations nor the biological function of FAM 111B in healthy individuals. Thus, we aim to study the gene product of FAM 111B (protein/enzymes) both in the normal and mutated state with the hope of designing a drug to treat or manage this condition.

21-2 Increasing patient empowerment in rare chromosome 6 disorders

Individuals with a chromosome 6 disorder experience a multitude of medical and developmental problems. As a consequence, they visit many professionals who often do not have a complete overview of the individual’s history at hand. Within our world-wide Chromosome 6 Project that is entirely driven by parents, we collect detailed information directly from parents. We want to adjust our online interactive system in such a way that parents can generate a detailed report on their child’s medical, biometric, developmental and behavioural data. This report can be used when visiting health care providers ensuring that they always have a complete overview of the individual’s condition.

The Chromosome 6 Project was started in 2013 as an initiative of a Facebook group. They teamed-up with the Center of Expertise for Rare Chromosome Disorders at the University Medical Center Groningen ( Via social media families were contacted to participate and an extensive online questionnaire was developed in seven languages. This resulted in a collection of detailed information on more individuals with chromosome 6 aberrations then ever described in medical literature. At present the collected data is analysed and publications revealing detailed information on the consequences of specific chromosome 6 aberrations will be published soon.

To know what can be expected if an individual has a rare chromosome disorder is extremely helpful information. However, as for all disorders, individual variability exists and knowing what can happen should not be seen as a rule for each individual. Having a personal report at hand will empower families to show what the condition does for their family member. The online questionnaire helps families to collect and up-date information and it would be wonderful if we could give them a detailed personal report in return to empower them and our research project.

21-3 Belimumab to treat interstitial lung disease in common variable immunodeficiency

Common variable immunodeficiency (CVID) is a rare disorder resulting from genetic impairment of the immune system. As a result of their immune system not working correctly, CVID patients have increased risk of infection. Additionally, as a result of this genetic disorder, the immune cells of CVID patients are predisposed to cause a severe form of inflammatory lung disease for which there is no adequate treatment. In the hope of identifying a much needed new therapeutic approach, we conducted the largest study of these patients ever in order to understand why lung disease occurs in CVID. The results from our study suggest that a protein called B cell activating factor (BAFF) is elevated in these patients and promotes lung disease. There is a medication called belimumab approved by the United States Food and Drug Administration and Health Canada that neutralizes BAFF and has been shown to treat diseases that are driven by BAFF, such as Lupus and Sjogren’s syndrome – a condition that causes a similar lung disease as in CVID. Belimumab has been studied for years and used in thousands of patients and has an excellent safety profile, but has never been tried in CVID. We propose to carry out a pilot study to investigate whether belimumab is a safe and effective option for CVID lung disease. The company that produces belimumab has committed to provide us the medication free of charge. With the support of this grant, we will conduct experiments in the laboratory using samples of patients’ blood both before and after belimumab administration. This will allow us to conduct vital biochemical and immunological analysis of the patients in this study to evaluate the effects of belimumab upon BAFF and its receptors in CVID. These experiments may help identify laboratory characteristics that distinguish patients that are likely to respond, or not respond, to the therapy as well as delineate the specific biological mechanisms that are altered by this novel therapy in CVID.

21-4 Triggers for inheritable myotonia and periodic paralysis

Electrical signaling is a biological process vital for life; it controls nerve function, muscle contraction, and heartbeat. One of the main events in electrical signaling is the action potential. The action potential depends on a group of proteins called ion channels, which includes voltage-gated sodium channels (Nav). The genes that code for these proteins sometimes contain one or more mistakes. These mistakes, known as mutations, may get passed along, or inherited, to children from their parents. When mutations in ion channels cause disease, they are called “channelopathies”. Genetic mutations in the genes can result in channels and cells that are over- or under-excitable. Rare muscular channelopathies are classified as either non-dystrophic myotonias or periodic paralysis, with symptoms that occur within the first 20 years of life. Afflicted individuals suffer from life-long symptoms like muscle stiffness, weakness, or pain. Myotonic attacks involve sustained muscle contractions, whereas a paralytic attack results in an inability to move. These diseases have a prevalence estimated at 1:100,000. Interestingly, both these two seemingly contrasting conditions can be triggered by exercise. The goal of this project is to understand how a genetic mistake that causes incorrectly formed sodium channels results in muscular disorders, and how exercise can initiate either paralytic or myotonic attacks.

21-5 Whole genome sequencing to identify the genetic determinants of treatment-refractory ophthalmoplegia in myasthenia gravis

Myasthenia Gravis (MG) is a rare, treatable autoimmune disease characterized by fatigable muscle weakness resulting from acetylcholine receptor (AChR) auto-antibodies which target the muscle membrane and cause damage. While eye muscles are often affected early in MG, and respond to treatment much like other muscles, we have identified a unique subtype of MG characterized by severe treatmentresistant eye muscle weakness (ophthalmoplegia or OP-MG) presenting as squint and droopy eyelids. The visual impairment resulting from OP-MG causes substantial disability. Surgical elevation of droopy lids and surgical re-alignment for squint/double vision, is partially successful in a few cases. However, not all cases are eligible for this invasive approach. OP-MG most commonly affects subjects with African-genetic ancestry with otherwise characteristic AChR antibody-positive MG. It is more common in childhood onset MG compared to younger adults who develop MG. The underlying cause of OP-MG remains unknown. In this study, we will expand on our previous molecular genetic work in which we used DNA samples of the most severe juveniles with OP-MG (n=14) and compared their genetic make-up with juveniles who have MG, but who did not develop the subtype after several years of follow-up (n=11). This discovery group were matched for other variables such as age at onset and race, and differ only by their eye muscle treatment responsiveness. We have already performed whole genome sequencing (i.e. sequences of the genes and regulatory switches) on these individuals and started the data analysis to discover variants in genes that are found in OP-MG, but not in control MG, and which are predicted to alter the gene expression levels. We are postulating that the pathways related to eye muscle healing in response to the damage induced by the MG autoimmune attack, are perturbed in susceptible individuals. Understanding which pathways are involved will allow us to consider novel therapies.

Results - Myasthenia gravis (MG) is an immune-mediated disease in which antibodies target the nerve-muscle junctions. Although there are immune therapies to effectively control the disease in most patients, there is a subgroup of MG cases, (we’ll call them OP-MG) that develops treatment-resistant myasthenic paralysis of their eye muscles. This visually disabling condition frequently affects patients with childhood-onset MG. In our efforts to understand what causes OP-MG, we previously analysed the whole genomes (i.e. all the genetic material of individuals read by DNA sequencing) of a sample of our cases in which we chose the ‘extreme phenotypes’ i.e. the most severely resistant OP-MG cases compared to those who responded appropriately to therapy (control-MG). In this discovery sample we identified a number of gene variants which were predicted to impact on protein function and which occurred significantly more frequently among the OP-MG cases. To verify these associations with OP-MG, we specifcally sequenced these variants in a larger sample. The genes harbouring the OP-MG associated variants hold the instructions to make proteins which are important in a) maintaining healthy muscle membranes and b) securing proteins which require insertion into the muscle membranes for proper functioning. The next step is to study these proteins in eye muscle tissues derived from the OP-MG patients and controls because this is the specific tissue which is diseased in OP-MG.

21-6 Computer-Assisted Subcapital Correction Osteotomy in Slipped Capital Femoral Epiphysis Using Individualized Drill Templates: a Feasibility Study

Slipped capital femoral epiphysis (SCFE) is an adolescent hip condition that occurs when the head of the thighbone (femur) slips backward off the neck. In moderate-to-severe cases, patients need surgery to correct the position of the bone. However, the surgical technique with the best outcome is demanding, highly dependent on the surgeon’s experience, and requires considerable radiation exposure to the young patient. Using preoperative CT scans, we can develop a surgical plan and design corresponding drilling templates specific to the individual patient. These drilling templates will help the surgeons achieve higher accuracy in different steps of the surgical procedure and significantly reduce intraoperative x-ray exposure and surgery time.

21-7 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 (sometimes referred to as “dwarfism”). 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.

Results - Skeletal dysplasia is a rare genetic disease that affects bone development. This results in a range of ongoing health concerns, many of which are exacerbated by the increased prevalence of obesity in this population. Moreover, individuals with skeletal dysplasia are also at disproportionately elevated risk of obesity-associated negative health outcomes for any given level of body fatness. It is therefore remarkable that metabolic rate has never been measured in a large and representative cohort with skeletal dysplasia and therefore the inter-relationships linking nutritional requirements, obesity and cardio-metabolic health remain unknown. This funding from the Rare Disease Foundation enabled us to make the first such measurements to understand skeletal dysplasia better and inform future treatments that could involve diet, physical activity/exercise, and/or medication. Specifically, our results show that standard methods of predicting metabolic rate and therefore nutritional requirements (i.e. how much you need to eat) based on a person’s height, weight, sex and/or age may work well for average height individuals but generate inappropriate guidelines for people of disproportionate short stature. Our mathematical modelling of the relationship between metabolic rate and body fatness in the last year is already informing a more appropriate population-specific equation for individuals with skeletal dysplasia. In addition, we have gathered blood samples from this population and begun accumulating a database of systemic markers of metabolic and cardiovascular health. Our preliminary analysis reveals that the individuals tested may have slightly lower plasma concentrations of thyroid hormones (e.g. thyroxine; Free T4) that typically observed in the general population, which may be linked to our observations of the unique relationship between their body morphology and metabolic rate. However, notwithstanding the range participants tested in terms of total body fat content, other blood markers of health were on average not consistently outside the expected range. For example fasted insulin, glucose, C-reactive protein and glycated haemoglobin (HbA1c) were by no means elevated above the healthy range. In fact, total cholesterol was on average lower and high-density lipoprotein cholesterol was higher than in obese but otherwise healthy average height controls. Lastly, with regard to the link between obesity and appetite regulation, it was notable that the satiety hormone leptin was typical of healthy lean average height controls. These findings will be extended thanks to the follow-on funding provided by the Rare Disease Foundation for the coming year and will ultimately inform the first-ever guidelines for weight-management for this underserved population.

21-8 Metabolomics Study for Novel Disease Gene Discovery in Pyridoxine-Dependent Epileptic Encephalopathy

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 that cause biochemical defects in the brain. 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 these type of diseases, identified a consanguineous family who has multiple children affected which such rare form of epilepsy. Seizures started in most of these children early after birth and was controlled only by vitamin B6. Testing of genes that are known to cause this epilepsy was negative. We propose here to perform more comprehensive testing (known metabolomics) which will detect alterations in all small biochemical compounds in these children in one experiment. This will help to find out the defective biochemical pathway, identify causative disease gene and subsequently improve diagnosis and devise therapeutic interventions.

21-9 Development of Epidermal Progenitor Cell-Based Therapy for Hemophilia A

Hemophilia A is a genetic disorder with the deficiency of clotting factor VIII. Patients with hemophilia A suffer a series of symptoms including internal and external bleeding. Current treatment strategy relies on frequent venous infusion of factor VIII medication with complications including increased infection risk and neutralizing antibodies against factor VIII. Moreover, it causes a significant financial burden on patients and their families. Here I am proposing a new and cheaper experimental cutaneous cell therapy strategy on Hemophila A. My idea is to use artificial skin grafts to deliver factor VIII for Hemophilia A treatment. The artificial skin grafts will be generated using engineered epidermal progenitor cells of skin which stably express and secret factor VIII. Please refer to the below list for further details concerning the procedure of this therapy strategy.

1. Isolation & culture epidermal progenitor cells

2. Engineer epidermal progenitor cells to express and secret factor VIII

3. Evaluation of factor VIII secretion using standardized clinical methodology

4. Safety Evaluation of engineered epidermal progenitor cells

5. Culture of skin grafts using engineered epidermal progenitor cells

6. Transplantation of skin grafts on Hemophilia A mouse model

7. Evaluation of the efficacy, durability & safety

I have completed another project using the similar strategy to treat obesity on immunocompetent moue model. The results of that project have been accepted by the journal Cell Stem Cell for publication. The advantages of my strategy include lower cost, no frequent medical care such as venous infusion and safety. the skin graft can be easily removed through surgery. Since frequent infusion is avoided, the chance to develop neutralizing antibody against factor VIII would be quite low. I believe that my proposal brings a new idea into Hemophilia disease treatment.

21-10 Identification of a Hidden Cause of Autosomal Recessive Early-Onset Infantile Epileptic Encephalopathy (EIEE).

Early infantile epileptic encephalopathies (EIEEs) are a group of rare and severe brain disorders in infants and children which have a poor prognosis and high rates of morbidity and mortality. Many EIEEs are now recognized to have a de novo genetic cause.1 Identifying the genetic cause and making a genetic diagnosis can subsequently help direct genetic counselling within the family to avoid the reoccurrence of pregnancies with affected children, in addition to improving monitoring, and treatment options for their affected children. Furthermore, identifying genetic causes allows for insight into how and why the disease manifests, ultimately providing an evidence-base for interventional therapies. However, currently most families with children affected with EIEE, particularly those with autosomal recessive EIEEs (ar-EIEEs), are yet to have a genetic cause identified. This is due to the difficulty in studying such forms of genetic disease in outbred populations. Recent large-scale efforts to sequence the protein-coding part of the genome have not been successful in identifying causes in many patients, which raises the question of whether the genetic defect is present in the non-coding regions of the genome. In this study, we propose to use Whole Genome Sequencing (WGS) to reveal the elusive cause of the disease in one large consanguineous EIEE family with four affected children. We ultimately believe that any new findings from this study would lead to further diagnoses of many other undiagnosed patients with EIEEs.

21-11 Congenital Pseudarthrosis of Tibia - Exome Sequencing and Matching Model Organism

Congenital Pseudarthrosis of tibia (CPT) is a rare orthopaedic condition in children. It can occur as a fracture of the tibia which may be present at the time of the birth or during the first decade of life. CPT is often associated with neurofibromatosis-1, a disorder of nerve tissue. CPT is considered to be one of the most challenging conditions to treat in orthopaedics due to the difficulty in achieving bone healing. There is a high risk of post-treatment complications such as re-fracture, nerve compression, limb shortening and even limb amputation. Currently, there is no standard surgical treatment in orthopaedics that can successfully treat CPT. Gene therapy for stimulating bone healing shows promise in CPT. However, before moving on to gene therapy, we need to have information about the disease-causing gene. Therefore, we performed exome sequencing for two of our patients with CPT in order to obtain detailed information on the underlying gene. This information can help target treatment approaches for these children and a potential gene therapy in future. Now that we have discovered the genes in our two patients the next step is to apply to the Rare Disease Models and Mechanisms Network where this discovered gene will be matched to a model organism.

21-12 Understanding Patterns of Disease Progression and Survival after Huntington's Disease Diagnosis

Huntington's disease (HD) is a progressive and fatal condition that causes a breakdown of the nerve cells in the brain. Although there is no known cure, treatments that focus on reducing symptom burden have been shown to enhance quality-of-life for HD patients and their families, as well as slow the disease progression process. However, identifying appropriate treatments for patients with HD is limited because we know very little about the disease clinically. Because the condition is so rare (approximately 5.7 cases per 100,000 individuals), few opportunities are available to study large groups of patients to better understand common clinical features, such as how many patients experience certain known symptoms (e.g. seizures, abnormal muscle contractions, sleep disturbances); and discover whether there may be important, but unrecognized symptoms. Finally, we know very little about how the disease progresses over time. Current estimates suggest that HD is fatal between 10 - 30 years after diagnosis, but survival is variable and the specific factors that influence disease progression are not well understood. To address these unanswered questions, we propose to leverage our access to a massive database of approximately 67 million de-identified patient records to examine the clinical features, and predictors of enhanced survival, in a large sample of patients diagnosed with Huntington’s Disease (n=3700). Results from the proposed study will provide fundamental knowledge regarding the nature and timing of HD symptomatology and the factors that affect disease progression over time. This information has the potential to provide essential information to doctors and patients with HD, and can help us identify new targets for potential treatments. As there is no known cure for HD at present, developing tools to that can minimize the devastating consequences of this disease is a major priority that can positively impact HD care for patients and their families.

Results - Because Huntington’s Disease (HD) is exceedingly rare, many basic epidemiologic characteristics of the disorder remain unclear. We sought to describe the diagnostic frequency, incidence and sociodemographic features of HD in a large U.S.–based cohort and to examine whether previously reported racial/ethnic differences in between-country estimates hold in a diverse population within a single country. Using an administrative cohort of approximately 67 million, privately insured individuals, we retrospectively identified 3,707 individuals diagnosed with HD between 2003-2016 using International Classification of Diseases (ICD) codes. We calculated annual incidence, diagnostic frequency, and differences by sociodemographic characteristics. The diagnostic frequency rate of 5.49/100,000 persons (95% CI: 5.31, 5.66) and the incidence rate was 1.59/100,000 persons (95% confidence interval (CI): 1.53, 1.65); several important differences in disease rates were noted by self-reported gender, race/ethnicity, and income. It is widely accepted that Huntington’s Disease rates vary by race and ethnicity, however, disease trends also appear to vary by several other characteristics. Huntington’s disease patterns may be more driven by social or environmental factors than has been previously discussed. Results further indicate the potential utility of large insurance datasets in understanding the epidemiology of rare diseases when other means are unavailable.

21-13 Co-developing rehabilitation guidelines for children with arthrogryposis multiplex congenita

Arthrogryposis multiplex congenita (AMC) is a term used to describe joint contractures present in two or more body areas. This rare condition is present at birth and affects 1 in 3000 live births. AMC can affect the upper and lower limbs as well as the jaw and spine. The contractures are typically non-progressive but are severe enough to limit independence in mobility and daily life activities, especially in the areas of upper extremity function, transfers, mobility, sports and physical activity. Despite early intervention, the deformities often come back, particularly in young children. Early intensive rehabilitation is necessary for children with AMC, but there are currently no rehabilitation guidelines to inform best practice for this population. The aim of this project is to develop practice recommendations for rehabilitation together with youth with AMC, family representatives, and clinicians. This process will ensure the practice recommendations are family-centered. The practice recommendations will focus on the five following priorities: i) promoting independence in daily activities, ii) access to rehabilitations services, iii) addressing psychosocial needs (e.g., self-esteem, motivation), iv) managing pain, v) addressing physical needs (e.g., getting around, endurance). Three sets of focus groups will be conducted with youth with AMC, caregivers and clinicians to co-develop the content and the format of the practice recommendations. The findings will be shared with clinicians from rehabilitation and hospital centers, as well as with youth with AMC and families. It is expected that this project will improve the care provided to children with this rare condition by promoting evidence-based, coordinated services, and communication among clinicians and families.

Results - Rationale: Arthrogryposis is a term used to define congenital joint contractures in two or more different areas of the body and occurs in 1 in 3000 live births. Contractures can lead to decreased movement and strength, and may affect getting around and doing activities independently. Early intensive occupational and physical therapy (OT/PT) is recommended to improve independence in mobility and daily life activities, and to improve function. Since arthrogryposis is a rare condition, OT/PT in community settings may feel unprepared and uninformed when providing care. 

There are currently no practice guidelines for rehabilitation for this population. Methods: The development of rehabilitation guidelines involves six phases: Identification of rehabilitation needs; Literature review; Expert opinion; Integration of evidence and expert consensus; Knowledge Translation; and Implementation and evaluation. 

Rehabilitation priorities for the guidelines were identified through individual interviews, focus groups and a semi-structured questionnaire. These priorities are: 1. Daily activities, 2. Muscle and joint function, 3. Mobility, 4. Pain management, 5. Participation. The rehabilitation guidelines will be shared using various tools (e.g., infographic, flowchart, manual) which will be comprehensive, clear, and easy for OT/PT to use. The development of the rehabilitation guidelines for arthrogryposis was conducted with input from youth, parents and OT/PT. Practice Implications: Family-centered rehabilitation guidelines will promote evidence-based, coordinated services, and improve the rehabilitation care provided to children with this rare condition. These guidelines will inform OT/PT, and prepare youth and families for their rehabilitation journey.

21-14 A National STXBP1 Registry & Family Resource

We wish to establish a secure Canadian registry & family resource for patients who are affected by STXBP1 gene mutations. These mutations cause a rare neurodevelopmental disorder that starts in early childhood with drug-resistant epilepsy, a movement disorder, intellectual disability & autism spectrum disorder. With very few patients known to us across the country, we do not have a complete characterization of this disorder, nor are there any personalized treatments or any family support or patient advocacy groups in Canada. The registry will facilitate a better understanding of how we can make the greatest impact on the patients’ quality of life by understanding the experiences across the lifespan from the patients, their families & clinicians. We hope to create a safe, secure meeting point for patients & their caregivers to reach out to each other for support, advocacy & the sharing of ideas.

We want to be able to update clinicians & families on the latest advances in the treatment of STXBP1 disorders. As the field of personalized medicine in genetic disorders quickly progresses, we wish to have patients organized as a group such that they may attract the attention of those designing potentially life-changing treatments for this severe diagnosis. Our Canadian clinical & research team have experience in diagnosing & managing patients affected by STXBP1 mutations. Our combined cohort consists of 15 of these patients across Canada, spanning both pediatric & adult populations. We hope that setting up the registry will identify an equal number again at other centres across the country.

21-15 Advancing Surgical Care of the Neonate with Sacrococcygeal Teratoma: Evidence, Action, and Advocacy

Sacrococcygeal teratoma (SCT) is a rare congenital disease affecting 1:35,000 – 1:40,000 newborns. Babies who are born with a SCT diagnosis have a strong chance of survival. Survival is dependent upon timely diagnosis, referral to an interdisciplinary pediatric surgical team within a tertiary setting, and treatment which involves complete resection of the primary tumor and coccygectomy. Perioperative mortality is not uncommon with late presentation of a SCT diagnosis. Benign SCTs have a high recurrence rate, which require follow-up for more than three years post-operatively. Risk of mortality and recurrence are key concerns among pediatric surgeons for babies born with this rare surgical diagnosis. Uganda has the third highest birth rate in the world with 43.4 live births/1,000 and the fifth highest fertility rate with 5.7 births per mother. As such, our international team have observed that SCT is a rare diagnosis more commonly seen and surgically treated in Uganda than in Canada. Within the Pediatric Surgical Camp setting, most infants respond well to surgical treatment, however late presentation within a low-income setting leads to unnecessary mortality. Our study team sees an important opportunity to improve survival of infants presenting with a SCT diagnosis through evidence, timely referral to surgical care, and advocacy. Timely referral and access to surgical care are in keeping with the goals of the Lancet Commission on Global Surgery.

21-16 A New Target for Developing Treatments for Duchenne Musculat Dystrophy

Duchenne muscular dystrophy (DMD) is a lethal degeneration of muscles with replacement with fibrotic (scar) tissue and adipose tissue. It afflicts about 1 in 3500 boys and leads to death, usually in their twenties. Mutations in the DMD gene (which codes for the protein called dystrophin) on the X chromosome are responsible, and there is no cure (ref 1). Part of the normal cycle of wear and tear which occurs as muscles are used involves the laying down of a tissue matrix which, once it has served its purpose is removed by macrophages, specialized cells whose function (among others) is to remove debris. If the matrix is not removed, it accumulates and develops into the fibrotic tissue which contributes significantly to the pathology. We have recently made the remarkable finding that the DMD gene product is necessary for the activity of the macrophages; this discovery coupled with the existence of a very similar gene in our genomes (called utrophin, or UTRN) immediately opens the door to a new target for development of therapies, namely ‘turning on’ the UTRN gene in patients’ macrophages.

Proposed patient intervention (This is contemplated once the experiments proposed here are completed, in the event we obtain a positive outcome): Since UTRN is not expressed in macrophages, we hypothesize that removing macrophages from the blood of patients by the standard procedure of apheresis, treating them with well-known techniques to enhance UTRN expression ex vivo (outside the body) and reintroducing them into the boys’ bloodstream may have a beneficial effect on the state of the patients’ muscles by reducing fibrotic tissue accumulation. The proposed experiments involve preclinical work to test the effect of normal macrophages (which express functional dystrophin) on fibrosis in mice carrying mutations which mimic the condition in DMD patients.

21-17 The Effect of Positioning Error on the Hilgenreiner Epiphyseal Angle as Compared to the Femoral Neck-Shaft Angle in Cerebral Palsy: A Geometrical Modeling Study

Cerebral palsy (CP) is a brain disorder that affects muscles, often causing them to stay tight most of the time. This can cause the upper end of the thighbone (femur) to grow with less bend than normal, which can cause problems with the hip displacing (slipping/staying out of place). Sometimes surgery is needed to add more bend to the bone. Surgeons use a particular measurement from X-rays to determine how much bend to add and how the bend changes with surgery and as children grown further. However, many patients’ hips cannot be placed in the ideal position for X-rays due to their tight muscles. It is not clear how much measurement error these non-ideal X-rays cause. With computer models, we can find out how much error there is in non-ideal positions and compare it to the error in an alternative measurement. This will tell us if the current measurement works well, or if the alternative measurement is more reliable in the surgical planning and post-surgical treatment for children with CP.

21-18 Investigation of Novel Targets for CRISPR/Cas9-based Genome-editing to Treat Dysferlinopathy

Dysferlinopathies are rare muscular dystrophies caused by mutations in the dysferlin gene, and include limb-girdle muscular dystrophy IIB (LGMDIIB), Miyoshi myopathy, and distal myopathy with anterior tibial onset (DMAT). The prevalence is approximately 1/100,000-200,000, depending on the populations. Currently, no specific treatment is available for dysferlinopathy. Dysferlin is required for efficient cell membrane repair. We aim to develop a new therapy for dysferlinopathy using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system-based genome editing. Dysferlinopathies have not yet been tackled by therapeutic CRISPR/Cas9 genome editing. Because shorter forms of Dysferlin are associated with milder symptoms and can ameliorate the symptoms of the model mice, we will rescue dysferlinopathy by removing the mutated part and creating shorter but functional forms of Dysferlin using CRISPR/Cas9 system. To identify the best target sites of the CRISPR/Cas9 genome editing, we will make a series of shorter forms of Dysferlin (micro-Dysferlins) and test the function in dysferlinopathy patient cells. To evaluate the membrane repair ability, the cells introduced with the micro-Dysferlins will be damaged by a laser, and the repair process will be monitored by microscopy in real-time. The micro- dysferlins which effectively restore the ability of membrane repair in the patient cells will be chosen to design CRISPR/Cas9 genome editing. Successful completion of this study will identify the parts required for the membrane repair function of Dysferlin, and provide a proof of principle for applying CRISPR/Cas9 genome-editing therapy for dysferlinopathy.

Results - We aimed to rescue dysferlinopathy by removing the mutated part and creating shorter but functional forms of Dysferlin using CRISPR/Cas9 system. In this project, we found that removal of Dysferlin exons 26-27 and 28-29 restores membrane resealing ability in dysferlinopathy patient cells. These results will be published online (Mol Ther Nucleic Acids, in Press)

21-19 CIAO1 Deficiency: Investigating a Rare Neurometabolic Disease to Enhance Care for the Rare

Here we propose to unravel new mechanisms underlying a debilitating disorder presenting with intractable seizures, developmental delay and poor growth in 2 unrelated patients showing a functional DPD deficiency but without mutations in the gene encoding the pyrimidine degradation defect. Whole genome sequencing revealed potentially pathogenic mutations in both patients in a gene called CIAO1, which is required for maturation of DPD as shown by previous experiments. In patients with a complete DPD deficiency, a considerable variation in the clinical presentation has been observed. Psychomotor retardation and convulsive disorders are relatively frequent manifestations whereas growth retardation, microcephaly, dysmorphia, autism, hypotonia and ocular abnormalities are less frequently observed. They are vulnerable to severe, potentially life-threatening toxic reactions to certain drugs called fluoropyrimidines that are used to treat cancer. The pathogenesis of a DPD deficiency is poorly understood and insight herein is of crucial importance for the development of potential therapeutic options. Various organs and tissues have been implicated to be affected by a DPD deficiency but a consistent phenotype has not yet emerged. Epileptic encephalopathy, resistant to multiple anti-seizure medications, is a severe clinical condition as it always has a negative impact on psychomotor development, and often is accompanied by multi-organ involvement. Etiology is heterogeneous and thus the differential diagnosis vast. Reduction of unnecessary diagnostic delay along with more insights into pathophysiology of this specific condition with key features such as failure to thrive and the pattern of DPYD deficiency, via model organism studies will reveal treatment targets and ultimately result in better management and improved patient outcomes. We shall further unravel this metabolic pathway, find a diagnosis for these 2 patients and develop treatments.

21-20 Next Generation RNA Sequencing for Novel Disease Gene Discovery in Pyridoxine-Dependent Epileptic Encephalopathy

 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 that cause biochemical defects in the brain. 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 these type of diseases, identified a consanguineous family who has multiple children affected which such rare form of epilepsy. Seizures started in most of these children early after birth and was controlled only by vitamin B6. Testing of genes that are known to cause this epilepsy was negative. We propose here to perform a new comprehensive testing (known as Next Generation RNA Sequencing) which will detect alterations in expression of all genes in these children in one experiment. This will help to identify the causative disease gene and subsequently improve diagnosis and devise therapeutic interventions.

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