February 2018 Funded Microgrants

February 2018 Funded Microgrants

23-1 ER Stress Response: Involvement in Marfan Syndrome Associated Aortic Aneurysm

Marfan syndrome (MFS) is a rare (1 in 5000) genetic disease of the connective tissue (the matrix between cells, which provides material and support for skeleton, muscles, and blood vessels), and it is caused by mutations in the fibrillin-1 gene leading to connective tissue abnormalities that impair skeletal, ocular, pulmonary and, most importantly, cardiovascular systems. MFS patients are always at risk of life-threatening problems such as damage to the valves in the heart or weakening of the large blood vessels leading from the heart (aorta). If the aorta weakens, it can balloon out (dilate) and break (rupture), leading to sudden death. So far, the best available treatment, other than major surgery, has been the use of drugs that lower heart rate and/or blood pressure. However, none of these approaches have been completely effective in treating the condition or blocking the growth of the aortic root in MFS patients, and there have been serious problems associated with the surgery. During the last few years, our laboratory has been searching for a more effective therapy that would affect targets within blood vessel wall in an attempt to block or reverse aortic dilation. The main objective of this study is to investigate whether a cellular stress response pathway (known as ER Stress) plays a role in initiating the destructive changes within the aortic wall in a well-established mouse model of MFS. We hope to better understand the mechanism underlying the aortic root dilation, and explore a more effective treatment that will not just slow arterial wall degradation, but also hopefully reverse the process.

23-2 Identification of Genetic Basis of Familial Non-Medullary Thyroid Cancer

Thyroid cancer (TC) affects less than 1 in 3000 individuals. Non-medullary TC (NMTC) arises from cells making thyroid hormone and accounts for most TCs. In up to 5% of NMTC there is a family history indicating a likely underlying genetic basis, causing familial NMTC (FNTMC). We have undertaken studies to look for gene alterations causing FNMTC in DNA from family members with FNMTC and identified a short-list of gene alterations which may be causing FNMTC. However, in all gene identification studies it is challenging to distinguish the likely causative gene alterations from those which are of no consequence; it is estimated that we all carry thousands of gene alterations which do not have medical implications. In order to narrow our short-list further, and so ton endeavour to pin-point the exact causative gene, we need to pursue additional investigations which we propose to do by a method known as immunohistochemistry (IH). Proteins cannot be visualized directly but can bind to a marker protein attached to a dye which can be seen through the light microscope. Whilst genes are the set of instructions within our cells that make proteins (the functional units within our cells), in any gene identification study it is important to understand the effect of the gene alteration on its protein. In IH studies, where there is no visible protein in the tumour this indicates loss of protein function and suggests an underlying damaging alteration in that protein’s particular gene. The identification of a novel FNTMC predisposition gene would be of significant benefit for these families. It would enable at-risk family members to be identified and surveillance with early interventions offered. The investigation of the cellular pathways involved may also assist the identification of new treatments for those individuals with advanced disease. Therefore funding for IH is requested to complete our studies.

23-3 Biomarkers of Allergic Bronchopulmonary Aspergillosis in Patients with Cystic Fibrosis

Cystic Fibrosis (CF) is the most common lethal genetic disease in Canada. Pulmonary exacerbations (PEx) are important to diagnose and treat early as they greatly reduce quality of life. Allergic bronchopulmonary aspergillosis (ABPA) is a hypersensitivity (allergic) reaction to the fungi, Aspergillus fumigatus, and can cause similar signs and symptoms to PEx caused by bacteria. It is estimated that 10% of CF patients develop ABPA and it is known that they experience worse outcomes during PEx. CF patients with overlapping ABPA can be difficult to recognize based on the current recommended CFF consensus guidelines due to shared clinical characteristics. These patients, however, could benefit from systemic corticosteroids alone (instead of antibiotics) or adjunctive to antibiotics to improve their response to PEx treatment and prevent irreversible lung damage. The basophil activation test (BAT) is a newer, non-routine blood test, that has been shown to be a reliable diagnostic and disease monitoring biomarker of ABPA in CF. This blood test entails stimulating and measuring the cells in blood, known as basophils, which are responsible for mediating allergic responses in our bodies. Therefore, by identifying CF patients with higher levels of basophils that are “stimulated” in the presence of Aspergillus, we can discriminate between CF individuals with and without ABPA. By using the current CFF consensus guidelines for ABPA as well as the BAT and blood protein levels of IL-5 and TARC, we will identify the subset of CF patients with ABPA. Moreover, by clinically distinguishing these patients, we will also be able to explore gene expression changes in the future between patients with and without ABPA and look for a genetic signature to suggest steroid responsiveness. To the best of our knowledge, this strategy is not currently being implemented to identify and monitor ABPA in CF patients.

23-4 Characterizing the functional impact of a new genetic cause of myopathy

Myopathies are a group of diseases characterized by progressive muscle weakness, which can be caused by hundreds of different genetic mutations. The vast majority of these mutations impair proteins involved in the stability of the muscle fibers, leading to muscle fiber fragility and muscle wasting. However, our team recently identified a patient having a new genetic cause of myopathy leading to a completely different phenotype. This patient is affected by a progressive decrease in muscle function leading to loss of ambulation (wheelchair bound by 6 year old) and respiratory dysfunctions. We identified a genetic mutation located in the gene PAX7, which plays a crucial role in the function of muscle stem cells, the cells responsible for muscle healing. Analysis of muscle biopsy revealed a complete absence of muscle stem cells, suggesting that impaired muscle regeneration could be the cause of the progressive loss of muscle function. To find a specific treatment for this patient, we need to further investigate this unknown disease. Therefore, to characterize this new myopathy, our team proposes 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. Thus, we will collect blood from the patient to generate iPSC and we will derive these cells into muscle cells. With this technique, we will be able to analyse how the formation of new muscle fiber is affected in this patient. Moreover, we will use this technique to test the therapeutic potential of compounds aiming to restore the expression of PAX7. Overall, this project will characterize the functional impact of a new genetic cause of myopathy; and it will allow testing new therapeutic avenues to treat this disease. This is the only project that has the potential to lead to clinical benefits for this patient affected by this uncharacterized rare disease.

Results - The patient that is currently treated at the CHU Sainte-Justine in Montreal is the first case described of this disease. However, in the last year, we have found 4 other individuals across the world (Germany, Palestine, and Saudi Arabia) with pathogenic variants in the same gene. These findings from multiple patients allowed establishing causality. Moreover, it also helped to establish a more precise prognosis for these patients. These findings are now submitted in the peer-reviewed journal, Genetics in Medicine.  

The funding from the Rare Disease Foundation and the BCCHF also allowed generating induced-pluripotent stem cells from a blood sample of the patient. Thereafter, we differentiated these cells into myogenic cells to study how the formation of muscle is affected by the genetic variant. Our findings indicate that the absence of the protein PAX7 is partially compensated by an upregulation of a closely related isoform, PAX3, which allowed the production of myotubes (immature muscle fibers). These findings suggest that PAX3 could be a potential therapeutic target to treat this patient. We also investigated the therapeutic potential of the compound PTC124 (ataluren), which favours read-through of nonsense mutations, to restore the expression of PAX7. Unfortunately, our findings did not show any efficacy of this molecule. However, we are now testing the impact of CRISPR-Cas9 to rescue the genetic variants. Moreover, to better comprehend the impact of the lack of PAX7, we are now establishing a collaboration with a researcher from the University of Toronto, Dr Penney Gilbert, which engineered a 3D system to generate a muscle construct in which we can study muscle function and regeneration. Therefore, it is expected that, in addition to the manuscript that is already submitted to a top-tier journal in the field, this project will also lead to another publication characterizing the impact of the lack of PAX7 on muscle function. Our findings were also presented in local (1), national (3), and international (2) conferences. These presentations won different awards such as a poster prize at the international FASEB conference on satellite cells and skeletal muscle. More importantly, it won the Drew Lyall award of excellence granted to the top abstract (out of 250 abstracts) submitted to the Till and McCulloch meeting 2018 (annual meeting of the Stem Cell Network), which led to an oral presentation. 

Overall, the funding from the Rare Disease Foundation and the BCCHF led to a better understanding of that previously uncharacterized disease. It helped our patients to have a better diagnosis and established a more precise prognosis. It also opened new therapeutic avenues that are currently being explored. We are thankful to the Rare Disease Foundation and to the BCCHF for their support in this project. 

23-5 Comparison of 3D printed models with conventional imaging for pre-therapeutic evaluation of vein of Galen malformations

Vein of Galen malformations (VOGMs) is a rare disease of blood vessels in the brain that develops before birth and usually presents in infants and small children. These abnormalities are treated through blood vessels under X-Ray. Knowing the anatomy prior to treatment is crucial for accurate planning. 3D printed models have recently shown promise for surgical rehearsal. In this study, we propose to compare the information obtained from 3D printed models of VOGM against the current standard of imaging care for pre-treatment planning. This additional information is expected to help the treating physicians to better understand the structure of individual malformations and improve patient care.

23-6 Eye Exams for Mice to Optimize the First Gene Therapy for Aniridia

Aniridia is a rare eye disease affecting between 1:40,000 and 1: 96,000 people, which is in need of new treatments. People with a damaged copy of the paired box 6 (PAX6) gene are born with aniridia, which causes poor vision from birth. Vision gets worse during childhood and adolescence, often leading to blindness in young adulthood. A major cause of vision decline is clouding of the clear outer part of the eye (cornea). When the cornea becomes cloudy, light can no longer enter the eye. Approaches to treat corneal clouding typically involve trying to replace the cloudy tissue with a clear and healthy, or prosthetic, cornea. While improving vision in the short term, for many aniridia patients the effect is temporary, and the cornea becomes cloudy again within a few years. Furthermore, complications from such surgeries can lead to serious eye health problems. Consequently, new treatments are needed to provide a lifetime of vision to children born with aniridia. Breakthroughs in gene therapy (using genetic material to treat disease) are saving vision in people with rare vision diseases. Since aniridia is caused by a damaged gene, one way to treat the disease is to provide the eye with an undamaged copy of the gene using gene therapy. In 2017 the first gene therapy was approved by the FDA to treat an inherited form of blindness. Before this therapy was given to patients, studies were conducted in mice showing that the gene therapy was safe, and improved vision. Following this approach, we have developed the first gene therapy for aniridia using mice. This therapy treats the cornea by adding a new copy of PAX6. In this application, we are proposing experiments to optimize this new gene therapy. We will watch the mice that receive the therapy over time, giving them regular eye exams. These experiments are important next steps towards demonstrating our new therapy is a safe and effective treatment for aniridia.

23-7 Folic acid supplementation in children with sickle cell disease

Sickle cell disease (SCD) is a genetic blood disorder that affects ~2,500 Canadian children. Individuals with SCD cannot make healthy red blood cells, and have an increased risk of illness, infection, and death. Folate is a B-vitamin needed to make DNA and to help proteins function in our body. SCD causes children to have higher folate needs than healthy children. Currently, children with SCD take 1 mg/day of folic acid (the man-made form of folate), an amount six-times higher than that recommended for healthy children. There is growing concern that high-dose folic acid is harmful in the long term, as it may interfere with how folate works in the body, and it is associated with an increased risk of some cancers. Currently, the folate status of children with SCD in BC and the Yukon is unknown as the provincial government discontinued funding for folate testing over five years ago. Our goal is to determine if children with SCD have problems using the high doses of folic acid that are currently recommended. We will include 45 children with SCD (2-17 years) who are attending our hospital and consuming 1 mg/day of folic acid. We will measure the amounts of multiple forms of folate and folate metabolites in the blood. We will also measure other factors that influence folate, including vitamin B12 levels, some hereditary defects, and dietary intakes. This information will help inform the dose and form of folic acid supplementation that is best for Canadian children with SCD.

23-8 Juvenile Breast Hypertrophy: Understanding the Molecular Pathogenesis to Target Treatment Approaches

Juvenile breast hypertrophy (JBH) is an exceedingly rare condition manifesting as rapid, extreme enlargement of one or both breasts at puberty with only 65 cases described. JBH causes debilitating breast and back pain, possible skin breakdown and infection and significant psychological and social challenges. Breast reduction surgery is preferred to try maintaining a naturally looking breast but increases risk of relapse when compared to mastectomy (removing the entire breast). Medications have been used, mainly after surgery, aiming to reduce the risk of recurrence but results are variable. The cause of JBH is unknown and nobody is researching this question. It is thought the breast tissue may be over-sensitive to estrogen (hormone that promotes breast growth). We think this may be the case, because these young women's breasts grow as though they are exposed to high estrogen levels despite having normal blood estrogen levels. Also, drugs that block estrogen help prevent recurrence and slow breast growth. We are caring for an adolescent girl with JBH who underwent breast reduction surgery. With consent, we preserved some breast tissue to attempt a better understanding of what causes this overgrowth. To investigate how estrogen sensitivity might be increased we plan to look at the genetic code and amount of certain proteins in this breast tissue, in particular investigating the estrogen receptor and proteins associated with its normal functioning. Currently, there is a risk of recurrence after surgery and we don't know the best drug to use, when, or how long to use it. An understanding of what really causes JBH could help direct the best treatment to slow breast growth, make surgery easier and prevent recurrence. Moreover, understanding basic mechanisms of breast tissue overgrowth may lend insights into understanding other, more common, breast diseases including fibroadenomas and breast cancer, potentially paving the way for new diagnostic and treatment options.

23-9 The Assessment of Liver Injury in Pediatric Fontan Patients

One percent of children are born with a structural heart abnormality. These children often require one or more surgical operations to repair their hearts. Children with the rarest and most serious heart conditions are born with only one single pumping chamber in their heart instead of two. To provide adequate circulation in these children, a series of three surgical procedures are required, concluding with the Fontan procedure. This surgical repair will allow the heart to pump a normal amount of oxygenated blood to the body. Unfortunately, this repair also places stress on the liver. Studies have shown significant structural and functional abnormalities in the liver following the Fontan procedure. The severity of liver injury with respect to time from the Fontan procedure is not well defined, and there is no consensus on the best tests for monitoring liver health in these patients. At our hospital we have a standard approach to evaluating liver health in these patients. We use blood tests, ultrasound imaging, a new non-invasive test of liver stiffness called “Fibroscan”, and liver biopsy. We will use this information to better understand how and when liver injury develops in these patients over time after their surgery. We hope to determine which non-invasive tests are best for these children by comparing simple test results to the liver biopsy results. By doing so we hope to improve the liver health of these patients and simplify their regular liver health monitoring tests.

23-10 Telling their story: Exploring future perceptions in young people with Complex Regional Pain Syndrome.

Complex Regional Pain Syndrome (CRPS), is a rare condition that affects 1 in 3800 people. CRPS is characterised by the presence of high levels of long-lasting pain, disability and emotional distress. Typically, the long-term prognosis for individuals with CRPS is uncertain and no clear cause for CRPS can be identified, making it a particularly challenging and difficult condition. Whilst CRPS is most frequently diagnosed in adults, it is also experienced by a sizeable number of children and adolescents. The experience of CRPS during adolescence can have a detrimental impact on individuals’ developmental progress. For instance, forming a stable sense of oneself, including future aspirations, is important for well-being yet, can be challenging for young people with CRPS. This may be due to the impact of CRPS symptoms (e.g. pain), on their education, work and social lives. Our recent work in adolescents with long-lasting pain highlighted the complexity of how pain can both stunt and accelerate young people’s development. Specifically, this work highlighted young people’s perceptions of being more emotionally mature than peers and their resistance to change their identity in the face of pain. Due to the distinct disruptive impact CRPS has on young people’s daily activities and the uncertain disease trajectory, young people with CRPS might be at a higher risk of experiencing difficulties with developing their sense of identity and self. To improve well-being in young people with CRPS, it is crucial to gain a better understanding of how they think about their future. To accomplish this, we will ask 50 young people with CRPS (14-25 years) to complete a story about their imagined future life and aspirations. In addition, a subset of participants will be interviewed to provide more detail on how their imagined future story is impacted by CRPS. The findings of the study will be shared with researchers and clinicians working with CRPS, both at a local and international level.

23-11 Study of the effects of Kat5-induced chromatin remodeling on neurological diseases

We identified three patients affected by a mutation on the Kat5 gene. All three individuals have short stature, cerebral malformations, seizures and global developmental delay or intellectual disability, with a significant speech disorder. Currently, we do not know how the mutations of this protein can be at the origin of the neurodevelopmental problems observed in the patients. However, one study measured the epigenetic effects induced by TIP60 and its effects on learning and memory. This work was done on Drosophila an showed, by TIP60 ChIP-seq, that there is a Tip60 enrichment for genes that have a function in cognitive processes. This study also showed that by mutating the HAT of TIP60, key genes responsible for learning and memory are poorly regulated in the brain. Our objective is to check if the neurodevelopmental problems caused by the mutations found in Kat5 are epigenetic. This could make it possible to identify a new protein crucial for the good development of the cortex, which can be a precursor to larger studies.

23-12 Establishing an international registry for congenital pseudarthrosis of the tibia

Congenital Pseudarthrosis of tibia (CPT) is a rare orthopaedic condition in children. It can present 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 considered to be one of the most challenging conditions to treat in orthopaedics due to the difficulty in achieving bone healing. The fracture does not unite with standard methods of treatment adopted for fractures in children and the tibia can re-fracture even after satisfactory union of fracture 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 and management protocol in orthopaedics that can successfully treat CPT. The reasons for this include the rarity of the condition, the lack of high quality research studies and the paucity of studies with an adequate follow-up till skeletal maturity. In order to plan meaningful studies it is important that multicenter studies are designed to recruit adequate numbers of children for statistical analysis. Hence we propose the establishment of a multicentre registry for congenital pseudarthrosis of the tibia. We are requesting for funds to support the planning phase for the establishment of this multicentre study.

23-13 The prognostic significance of the 12-lead ECG in peripartum cardiomyopathy

Peripartum cardiomyopathy (PPCM) is a rare form of heart disease, in which women develop heart failure at the end of their pregnancy or in the first months post-delivery. Patients with PPCM usually complain of tiredness, shortness of breath and swollen legs. These symptoms develop when the heart muscle is not functioning well. Some PPCM patients gain complete recovery of their heart muscle function, whereas others have little long-term improvement. Good medical care would improve chances of recovery. The electrocardiogram (ECG) is a widely used test for patients with heart disease. In this project, we aim to study the features on the ECG that indicate poor heart muscle function and which may predict the outcome of PPCM patients. By identifying patients with PPCM who are at high risk of poor outcome through specific ECG patterns at baseline, we could help to improve their treatment regimen and improve follow-up care.

Results - In our study we evaluated the prognostic value of the 12-lead electrocardiogram (ECG) in women with peripartum cardiomyopathy.  We could show that ECG abnormalities were frequent at the time of diagnosis. For the first time, we could identify certain ECG features that predicted a poor outcome at the 6 months or 1 year follow-up visit. Having identified these predictive ECG features will help to designate better follow-up strategies and improve care for these patients at risk. Future studies are needed to understand the pathophysiology behind the observed changes on the ECG.

23-14 Improving the risk estimate of Wilms Tumor in children with Beckwith-Wiedemann Syndrome

Beckwith-Wiedemann Syndrome (BWS) is a rare disorder characterized by overgrowth and a predisposition to develop tumors – particularly kidney cancers known as Wilms tumors (WT). WT is treated with aggressive surgery, chemotherapy, and radiotherapy putting survivors at risk for renal failure and second malignancies. However, when caught early WT can be cured with reduced treatment intensity. Because of this benefit of early detection, children with BWS undergo regular tumor surveillance. There are several genetic mechanisms that can cause BWS each with a different cancer risk. The most common – an epigenetic change at chromosome 11 – is associated with a very low risk for developing WT. The low risk with the epigenetic change has led to suggestions that these children should no longer undergo tumor surveillance. On the other hand, inheriting two copies of chromosome 11 from the father rather than one from each parent carries a much higher tumor risk. Recently we reported cases where the common diagnostic test for BWS could not differentiate between the high-risk and low-risk mechanisms raising concerns that a significant number of children have a higher risk for tumor development than suspected by their physicians. Under the proposed reduction in tumor surveillance, these children would be diagnosed with a more advanced stage of WT and require more intensive therapy. It is therefore crucial to precisely measure the accuracy of the currently used diagnostic test (MS-MLPA). To do so we will review the children from our institution who were classified with the low-risk epigenetic change by MS-MLPA. We will use a test (SNP array) with high sensitivity for detecting inheritance of two copies of the paternal chromosome and determine how many cases had been misclassified. Based on the results from this pilot study we will expand our cohort to include multiple institutions. This work will directly inform diagnostic procedures and tumor surveillance protocols in BWS.

23-15 Evaluation of low density neutrophils as biomarkers of lung function and disease progression in cystic fibrosis

Cystic fibrosis (CF) is a rare, genetic disease that affects mainly the lungs and the digestive system. CF patients produce a thick mucus in their lungs that increases the risk of chronic respiratory infections. These infections cause pulmonary exacerbations that may lead to permanent loss of lung function and death. It is thus essential to identify patients that are more likely to suffer from exacerbations to tailor appropriate therapies to prevent these events. We have identified a potential predictor of pulmonary exacerbations and disease progression in the blood of CF patients. This biomarker is an immune cell known as the low density neutrophil (LDG). LDGs have been reported in other diseases such as cancer, and are not present in large numbers in healthy individuals. The presence of these cells in the blood is an indicator of disease severity. We identified two types of LDGs in CF patients that can be distinguished by the amount of Clec12A on their surface. LDGs with large amounts of the Clec12A protein on their surface were observed in patients with moderate to normal lung function. In contrast, patients with reduced lung function have LDGs with low levels of Clec12A. The level of expression of Clec12A on LDGs is thus a potential marker of lung function and predictor of pulmonary exacerbations. This finding is highly significant since we know the function of Clec12A, it inhibits immune cell activation. Immune cells with less Clec12A are thus more easily activated and more likely to affect lung function. The objective of this project is to confirm these findings by analyzing LDGs in a larger cohort of CF patients. We will also determine whether LDGs low in Clec12A predict a more aggressive disease evolution by analyzing clinical parameters. This research has the potential to identify a new marker of disease progression that will aid clinicians in offering a more targeted therapy to patients, and to provide insight into the possible role of LDGs in CF.

23-16 To enhance protective immunity in a rare neurological disorder

HSAN-I is a rare neurological disease. Patients having this disease do not feel pain. As a result, HSAN-I patients usually do not seek immediate medical treatment of small skin injuries. Because of neglecting these minor injuries, HSAN-I patients often have severe infections and ulcerations that may necessitate amputation afterward. Our preliminary study shows that a group of anti-infection immune cells, called “T cells”, have defects in activation and proliferation in HSAN-I patients. We will test a method to correct these defects by giving a chemical compound that is missing in HSAN-I T cells. We believe this novel method will enhance the anti-infection immunity of HSAN-I patients.

23-17 Investigating the therapeutic potential of CRISPR/Cas9 gene-editing for Mucopolysaccharidosis IIIB

Mucopolysaccharidosis (MPS) IIIB is a rare genetic disease that results from a single mutation that causes an essential enzyme to become non-functional. Patients with MPS IIIB experience severe neurodegeneration, in addition to cardiac issues, sleeping problems, and joint stiffness. Those afflicted with MPS IIIB usually succumb to the disease in the first few decades of life. Currently, treatment of MPS IIIB symptoms is limited due to a protective barrier between the brain and the body that blocks access for enzyme replacement therapies, a common treatment of other related diseases. We propose the use of a gene-editing technology, which works like ‘molecular scissors’ to cut DNA, to target and remove the disease-causing mutation. If successful, correction of the mutation in stem cells derived from patients with MPS IIIB would allow for the production of functional enzyme. Through transplantation of gene-edited cells back into the patient, a long-term supply of functional enzyme would be produced within the brain. This would result in a reduction of disease symptoms and an increased quality of life for patients with 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. 

23-18 Evaluation of novel therapeutics against Ewings sarcoma

Ewing's sarcoma (ES) is a rare, highly metastatic bone-associated cancer mostly affecting children and young adolescents (incidence: ca. 1.6x10^6). Currently, established therapies have limited success despite high toxicity. Hence, selective and less toxic drugs are needed to reduce the toxic burden of cure. We propose to continue the development of a novel engineered antibody called S4 against a highly expressed protein (STEAP1) on the cell membrane of ES cells. We showed that this antibody, linked to a drug used to treat cancer, was directly delivered into and killed ES cells. The success of our approach will lead to development of novel treatment for ES with improved efficiency and reduced toxicity.

23-19 PSMB11 deficiency: A novel inborn error of immunity due to thymoproteasome defect

Primary Immunodeficiencies (PIDs) are a group of disorders in which the immune system fails due to a faulty gene. Consequently, affected patients can get various manifestations, including increased risk of certain infections and autoimmune conditions. We have identified a young-adult patient with diffuse, disfiguring warts and inflammatory bowel disease (IBD) due to a mutation in the gene, PSMB11. By characterizing at the molecular level the basis for why his PSMB11 gene is faulty, we have demonstrated that the mutation affects one copy of the gene, resulting in an abnormally long protein. The other copy of the gene is intact, producing a regular-sized protein. We will test in our cellular model whether the abnormally long protein directly interferes with the function of the normal one. We will also develop a molecule that will specifically negate the effect of the abnormally long version and assess its capacity to increase production of the normal protein and restore cell function. If successful, this approach will be applicable to treat other genetic disorders with the same faulty mechanism.

23-20 Therapeutic potential of PARP inhibitors in Amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS), is a progressive neurodegenerative disease characterized by the loss of the motoneurons. ALS is an incurable disease which causes muscle weakness, paralysis of the voluntary muscles and eventually death, 3-5 years after the first symptoms. The only approved drugs for ALS are Riluzole and Radicava, having a modest influence on survival (6 months) or symptoms, which shows the importance for new drug discoveries. In our lab, we use a translational approach to drug discovery by using the advantages of simple and higher ALS models such as C.elegans and mice. This approach has shown to be effective in identifying new disease modulating drugs. In the current project, we identified the gene parp-1, coding for Poly (ADP-ribose) polymerase 1 (PARP1), to enhance ALS pathology. Our preliminary data in our C.elegans model show that PARP inhibitors reduce ALS phenotypes, suggesting therapeutic potential of PARP inhibitors. a wide array of well-studied PARP inhibitors exist, as they are exhaustively studied in cancer research. The discovery of a therapeutic effect of PARP inhibitors in ALS is consistent with other pioneer experiences in other models of neurodegenerative diseases such as Huntington’s, Parkinson’s and Alzheimer's disease. Currently, we are the first to show that PARP inhibition by the PARP inhibitors Olaparib and 3-aminobenzamide (3-AB) is protective in ALS. To further confirm our hypothesis, we seek translational validation in an ALS model closer to the human. Therefore, our next step is to confirm that PARP inhibitors slow down ALS pathology in a mouse model of ALS.

Results - In the current project, we identified the gene parp-1, coding for Poly (ADP-ribose) polymerase 1 (PARP1), to enhance ALS pathology.  Our preliminary data in our C.elegans model show that PARP inhibitors reduce ALS phenotypes, suggesting therapeutic potential of PARP inhibitors. a wide array of well-studied PARP inhibitors exist, as they are exhaustively studied in cancer research. The discovery of a therapeutic effect of PARP inhibitors in ALS is consistent with other pioneer experiences in other models of neurodegenerative diseases such as Huntington's, Parkinson's and Alzheimer's disease. Currently, we are the first to show that PARP inhibition by the PARP inhibitors Olaparib and 3-aminobenzamide (3-AB) is protective in ALS. To further confirm our hypothesis, we seek translational validation in an ALS model closer to the human. Therefore, our next step is to confirm that the PARP inhibitor 3-AB slows down ALS pathology in a mouse model of ALS.

The Rare Disease Foundation microgrant, allowed us to take this step by using mouse models of ALS to evaluate the therapeutic potential of the PARP inhibitor 3-AB. These mouse models have been treated with 3-AB and evaluated for disease progression such as lifespan, weight loss, disease onset. Furthermore, this microgrant allowed us to gather brain and spinal cord tissue of treated and untreated mice. These tissues will be used to analyze ALS related pathologies such as inflammation or neuronal death. These experiments will allow to determine the therapeutic potential of 3-AB for ALS patients. The grant helped to evaluate another treatment that could eventually lead to a better understanding of the disease and potentially a new and easily accessible drug.

23-21 Quality of life in patients diagnosed with Moyamoya disease: cross-sectional study

Moyamoya disease is a rare and chronic disease characterized by the progressive narrowing and blockage of proximal vessels in the brain. Medical therapy has not been proven effective for the disease. In selected patients, revascularization with indirect or direct surgical techniques, have proven to reduce ischemic and hemorrhagic events. Direct revascularization appears to be more effective by providing robust re-perfusion but there must be balanced against a higher perioperative risk than indirect revascularization. The subjective experience of patients with Moyamoya disease is lacking from literature. Only one study has reported patient-reported quality of life assessment in the pediatric population in patients treated with only direct bypass. A recent systematic review revealed that an extensive look at QOL in adult patient has not yet been performed. Chronic illness that cannot be cured such as Moyamoya disease can cause a burden on a patient’s life. The threat of further deterioration due to stroke or hemorrhage can be stressful to a patient. The goal of this project is to determine the effect the diagnosis of Moyamoya disease has on patient’s quality of life and functional outcome. Also, the goal is to determine how the mode of presentation (incidental, ischemia or hemorrhage) and treatments undertaken (medical management and revascularization) impact the quality of life and functional outcome of these patients over time. This data will help patients and treating physicians understand the impact of the disease and help inform treatment decisions. The main goal of the present study is to identify how does the diagnosis of Moyamoya disease, its different clinical presentations and its subsequent treatment impact quality of life as measured by quality of life surveys (SF-36, EQ5D, 12-item Stroke-Specific Quality of Life Scale (SSQOL)) and functional outcomes as measured by the Modified Rankin Scale in patients.

23-22 Whole-Genome Sequencing Pipeline for Genetic Testing of Patients with Short-Tandem Repeat Expansion Disorders

Mutations in the DNA of an individual can result in disease, and identifying the underlying genetic cause is necessary for optimal clinical management. The advent of whole-genome sequencing (WGS) has led to the identification of a variety of mutations in the genomes of patients with diseases, yet nearly half of patients with suspected genetic disease who undergo WGS do not receive a diagnosis. This is perhaps because current clinical WGS pipelines are limited to analyzing certain classes of genetic mutations. One class of mutations that clinical WGS pipelines do not reliably detect is the short-tandem repeat (STR) expansion, despite the fact that STR expansion mutations have been implicated in ~40 neurological disorders, including fragile X syndrome and Huntington disease. Current tools for STR analysis do not provide reliable repeat size estimates of STR loci. We aim to develop a bioinformatics pipeline that would facilitate accurate genotyping of STRs in WGS data, and validate the performance of our pipeline on experimental data from reference samples with known STR expansion disorders. This research would facilitate the development of a robust WGS pipeline for diagnosing patients with disease-causing STR expansion mutations.

23-23 SRRM2 gene conditional knock-out mouse model

We can gain insight into most rare human diseases by analyzing the  equivalent genes in experimental organisms. Mouse models help to better understand the basic molecular function of genes and specific gene mutations. Several families in the world, including one family from BC, presenting with complex neurological features have been found to have variants in a gene, called SRRM2, but no centre has confirmed its association with a disease. This gene encodes a protein with a very important genetic mechanism; it produces the code for making proteins from many other genes. Mutations in this gene could explain the multitude of symptoms in the patients. Since this genetic disorder has never been characterized before, we propose to produce a mouse model, the first in the world for this gene. This will allow us not only to validate a new disorder in the family followed in BC, and in the other families in other centres, but also would allow us to continue the research in order to gain insight into the process of the neurological deterioration that is also associated with this condition. Producing a mouse model is the expertise of the Co-PI and full funding for the project will be secured through other grants. The RDF microgrant would be used to cover the amount required to purchase the cell lines that are necessary to start the experiments.

23-24 Characterizing a novel patient mutation underlying Niemann-Pick disease type C

Niemann-Pick disease type C, (NPC), is an autosomal recessive disorder characterized by progressive and fatal neurodegeneration that affects approximately 1 in 150000 live births. In 95% of cases, patients carry mutations in the NPC1 gene, ultimately resulting in a pathological accumulation of intracellular cholesterol. Presently, there is no cure for Niemann-Pick disease type C, with treatment options limited to symptom management. Two sibling NPC patients sharing two unique mutations, one in each copy of their NPC1 gene, have been identified. One mutation results in the pathological insertion of a premature stop signal, resulting in a significantly shortened and non-functional NPC1 protein. The other mutation results in an amino acid substitution in the NPC1 protein. The latter mutation has not yet been characterized and has been classified as a variant of unknown significance. Using patient-derived fibroblasts, I aim to characterize and determine the pathogenicity of the variant of unknown significance mutation. Using a variety of molecular and biochemical techniques, I will determine exactly how this mutation causes or contributes to disease in these patients. Understanding the precise impact of this mutation on the NPC1 protein will reveal specific points of intervention pertinent to restoration of proper NPC1 function in these two patients.

23-25 Synaptopathy of Nf1-deficient neurons generated from human pluripotent stem cell

Neurofibromatosis type 1 (NF1) is an autosomal dominant neurological condition that affects ~ 1/3000 people worldwide. Caused by loss-of-function mutations in neurofibromin 1 (NF1) gene, NF1 is recognized as a tumor predisposition syndrome characterized by a high incidence of cognitive symptoms, including learning disabilities, attention defects, executive function deficits, and motor coordination problems. Moreover, 30–40% of children with NF1 meet diagnostic criteria for autism spectrum disorder and exhibit poor social and behavioral adaptation. NF1 has been a subject of nearly a century of intense investigation, however, studies examining NF1 function in human neurons are lacking due to limited availability of human neuronal cells. NF1 mouse models show behavioral phenotypes related to learning disabilities and are instructive in understanding disease pathogenesis. However, some deficits displayed by NF1 mouse models have been difficult to reconcile the clinical symptoms of NF1 patients. For instance, lovastatin that reversed learning and attention impairments in mouse NF1 models failed to produce detectable improvements in attention in patients. These observations underscore the urgent need to develop human model systems to study human neurodevelopment and function. The recent advances in induced pluripotent stem (iPS) cell technology and the differentiation of iPS cells have inspired us to model neurodevelopment. Especially, the induced neuronal (iN) cell approach recently developed by our group can rapidly generate pure neuron populations from human iPS cells that are fully functional and enable the characterization of defects in human neuron transmission. We will create human cell models that carry mutations found in NF1 patients and investigate how NF1 alterations impact neuron transmission. Such cellular models can be used to explore the mechanisms of synaptopathy and perform large-scale screens for novel therapeutics of neuronal deficits in NF1 patients.

Results - In the proposal, we hypothesized that heterozygous null mutations of NF1 in human neurons distinctively impact excitatory and inhibitory synapse structure and function. During the past year, to look into the function of Neurofibriomin 1 in neurons, we generated control and mutant (carrying heterozygous loss-of-function mutation) excitatory and inhibitory neurons directly from human induced pluripotent stem cells. The neurons were then exposed to an elevated level of potassium chloride (KCl), which led to membrane depolarization and an influx of calcium through L-type voltage-sensitive calcium channels. The resulting increase in intracellular calcium level then triggered several calcium-dependent signaling pathways that ultimately lead to changes in gene expression. This in vitro neuronal culture system has been extensively used to study activityregulated gene expression and chromatin regulation in primary neurons, therefore, we adapted this method for human neuron cell culture. Based on previous studies of gene expression in neurons and other cell types, it is clear that depolarizing stimuli can induce the MAPK/ERK intracellular kinase cascade, which then activates TFs to initiate transcription of canonical early-response genes. Indeed, we observed robust activation of MAPK/ERK pathway and the expression of activity response genes including FOS, FOSB and NPAS4. More intriguingly, we found elevated activation level of MAPK/ERK pathway and elongated expression of FOS protein in the mutant excitatory neurons. We are now currently working on whether the same phenotype is shown in inhibitory neurons and whether the impaired early response-gene expression leads to long lasting impact on late response gene expression and the plasticity of synapses.  Neurofibromatosis type 1 (NF1), caused by a germline mutation in a single gene shows extreme clinical heterogeneity in a disease. In this regard, the overall goal of this project to identify a robust phenotype shared by in vitro derived neurons from induced pluripotent stem cells of patients with cognitive deficits.  Once accomplished this work will provide a more systematic understanding of the cellular and molecular factors that underlie disease heterogeneity in NF1, specifically those affecting the nervous system, and will potentially be used to predict whether patients with certain mutations have higher-risk to development of deficits in cognitive function. 

23-26 Cartilage, Bone and joint integrity genes as aetiological indicators in the rare Mseleni Joint Disease

In 1970, a rare hip disease was reported in Maputaland, KwaZulu-Natal, South Africa. This disease appeared to be prevalent in a geographically isolated community located near the Mseleni Mission Hospital and was therefore named, Mseleni Joint Disease (MJD). MJD is a rare disease with a country-wide prevalence of 1 in 84 000 people and a 28% prevalence in the Mseleni community. This disease manifests as a bilateral, uniform joint osteoarthritis, most severely localised to the hip region, although joint pathology has been observed at the knees, ankles, wrists, shoulders and elbows. MJD manifests in children as young as six years old and is progressively degenerative, resulting in a physical handicap by adulthood. Many Mseleni community members depend on subsistence farming and due to the debilitating effects of MJD, sufferers depend on their family for survival. Children, especially young females (due to cultural partitioning of labour), forgo school attendance to assist with agricultural subsistence, further perpetuating poverty and the lack of education in this community. Currently, pain management and surgical hip replacement are the only available treatments for MJD. Understanding the aetiology of MJD could inform treatment regimens and preventative therapies, thereby providing a better quality of life for patients, in addition to reducing the socio-economic burden of this disease. This study will assess the aetiology of the disease, using whole genomic (DNA) and transcriptomic (RNA) analyses, with the purpose of evaluating epigenetic changes in genes associated with cartilage, bone and joint integrity. Results may be relevant to other forms of osteoarthritis and could yield gene targets for future drug development studies, helping individuals in several communities.

23-27 Genomic analysis of brain metastases from the head and neck

Squamous cell carcinoma of the head and neck (HNSCC) is a type of cancer that affects nearly 500,000 individuals globally each year; however, this type of cancer spreads to the brain in less than one percent of all cases. Some types of HNSCC are related to previous exposure to a virus and have an increased likelihood of spreading throughout the body, and the brain. Patients with HNSCC brain cancer are routinely excluded from participation in clinical trials since they typically don't have long to live. Currently, there aren't sufficient options to treat this type of cancer. Our goal is to find better treatment for these patients. We will look at the DNA of these patients to see if we can find a pattern as to why and how this type of cancer spreads to the brain and create a way to better treat this deadly disease.

Results - The supplies ordered supported successful organization of samples, extraction of DNA, DNA quantification, and sample submission of brain metastases and matched primary-normal from the head and neck. We performed whole exome sequencing of 46 samples from 11 patients with brain metastases from the head and neck. Phylogenic analyses revealed divergent evolution and potential therapeutic targets in the brain metastases. Additional analyses and validation of our findings are currently underway.

23-28 CRIPAK Deficiency: A novel Genetic Immunodeficiency with Susceptibility to Influenza

Primary Immunodeficiencies (PIDs) are a group of disorders in which the immune system fails due to a faulty gene. Consequently, affected patients can get various manifestations, including increased risk of severe infections. These rare individuals provide lessons about the human immune system, allowing for the development of new therapies to treat such infections in the greater population. We have identified a young boy who has had two life-threatening infections with influenza A (flu) virus, from whom we identified a mutation in the gene, CRIPAK. By characterizing at the molecular level the basis for why his CRIPAK gene is faulty, we have demonstrated that the mutation affects both copies of the gene, resulting in no protein. CRIPAK controls activation of another protein, PAK1. It has been previously shown that PAK1 activation permits the flu virus to infect and multiply. We will test the impact of the mutant CRIPAK gene on patient's cell response to flu infection and its ability to control viral replication. We will also obtain and test a drug in development that regulates PAK1, to determine if it can specifically negate the effect of the absent CRIPAK protein and restore ability to his cells to combat the flu virus. This project will help define a new human disorder of the immune system, making it easier for other patients with this condition to be accurately diagnosed. Moreover, our work will provide this family (as well as future identified patients) a promising avenue to treat the infection specifically associated to this genetic immunodeficiency.

23-29 Screening the RPGR gene mutation hotspot in indigenous African patients with inherited retinal diseases

Inherited blindness is a debilitating disorder and can be caused by mutations in many different genes. The knowledge of the specific genetic reason for blindness in a family not only allows for diagnostic closure but also enables calculation of the risk of developing the disease, facilitating decision-making and life choices. Importantly, a molecular diagnosis may also determine eligibility for a potentially available gene-based therapy and/or other treatments and interventions to preserve vision. Through a research project into the genetic causes of RP in indigenous black African patients, we have found that a mutation hotspot, which is challenging to analyse, may be a significant contributor to the local burden of disease. We have identified a collaborator to assist us in screening our larger indigenous African patient cohort, with the ultimate aim of setting this up locally. Patients from this population group have been under-represented in genomic medicine research and are a valuable resource. We anticipate that a significant number of the 96 samples selected for screening, will have a mutation identified in this hotspot, which will not only greatly benefit our research but also impact on the care and counselling for these patients and their families.

Results - Inherited blindness may be caused by mutations in many different genes. The knowledge of the specific genetic cause of blindness in a family allows for diagnostic closure and enables risk calculation, in turn facilitating decision-making and life choices. Importantly, a molecular diagnosis may also determine eligibility for a potentially available gene-based therapy and/or other treatments and interventions to preserve vision. Our previous research in indigenous black African patients hinted that a mutation hotspot in the RPGR gene, which is technically challenging to analyse, might be a significant contributor to the local burden of disease. 

Closer scrutiny of the RPGR mutation hotspot formed the focus of this study, done in collaboration with Prof Wolfgang Berger at the University of Zurich. Sequencing the mutation hotspot in 82 affected, unrelated African patients revealed the likely causal mutations in 11 individuals (~13.4%). The genetic results will therefore impact the care and counselling for these 11 patients and their extended families. Interestingly, three recurrent mutations have been identified (occurring in two, three and four families respectively), providing a possible target for an initial, cost-effective diagnostic screen. Two of the mutations are novel, having never been reported elsewhere, which highlights that African patients, who have historically been under-represented in genomic medicine research, are a valuable resource and contribute towards a more comprehensive global catalogue of disease-causing mutations. 

The individuals lacking a hotspot mutation are now eligible for inclusion in further research projects to screen the many candidate genes remaining, in the hopes of finding the genetic causes of blindness. The sophisticated technology used for simultaneous screening of multiple genes is expensive and does not capture this specific mutation hotspot.  Therefore, using this initial test to pre-select the cohort i.e. exclude patients with hotspot variants from further testing, will save costs and greatly benefit our research. Our next aim is to establish a test locally to screen the RPGR mutation hotspot for diagnostic purposes. This will also permit validation of the results of this pilot project, as we will test family members of the 11 individuals identified with mutations, in order to draft a manuscript for publication. We will submit a reference for any publication arising from this work.

23-30 Evaluation of long-term surgical outcomes and quality of life after facial reanimation in pediatric facial paralysis

Facial paralysis is a rare and devastating condition that impairs a child's ability to chew, speak, and express emotion by smiling. This has major effects on a growing child’s self-image, emotional development, social integration, and quality of life. When spontaneous recovery is not expected, surgical reconstruction may be undertaken. Facial reanimation involves transplanting a muscle from the thigh into the cheek and connecting small nerves to improve facial symmetry and to enable a child to smile. While outcomes of facial reanimation are generally good, it is not known whether a child’s ability to smile improves, worsens, or remains stable over time, as very few studies have looked at long-term follow up. Furthermore, there are few scientific studies that have examined outcomes reported by patients—are they satisfied with having undergone the procedure? Do they feel the procedure has improved their life? Are they able to smile in response to a funny joke without thinking or do they have to consciously think about smiling? The objective of this study is to evaluate the long-term outcomes (at least 10 years after surgery) of smiling and facial symmetry following smile reconstruction facial reanimation. This technique of facial reanimation was pioneered at our hospital, and we have among the largest experience in the world with this procedure, with many of our patients operated in the 1980s and 1990s now healthy working adults. Our hospital is thus in a unique position internationally for this rare condition. With the support of the Rare Disease Foundation grant, we hope to invite these former patients back for a long term follow up assessment to quantify and qualify their experience with this surgery. This is the first study of its kind and the results will be of great interest and benefit to surgeons, pediatricians, psychiatrists, and patients around the world.

23-31 Improving the research consent process to build partnership relationships with undiagnosed patients and caregivers

Patient partnership in research has begun to take a more prominent place in discussions about medical research broadly and rare disease research specifically. Engagement and research partnerships are seen as a way to both improve patient care and improve the quality and directions of research. It is unsurprising that the research consent process takes also a central place in these conversations. Consent procedures can influence the quality of consent and impact participation in research studies, supporting or hampering patient engagement efforts. Consent is about more than just the written consent forms, however. It is an ongoing process and an essential part of building an engaged partnership relationship with patients and families. This is especially true for undiagnosed patients and families where participation in research is often deeply entwined with seeking diagnosis, treatment or management options. This creates a complicated tension between informed consent and the role of patients and families as engaged partners both in research and their own health care management. This study will use interviews (n=20-30) to examine the research consent experiences of undiagnosed patients and caregivers, especially in relation to research that also seeks diagnostic or treatment and management information. Those interviews will be used to create recommendations for how the ongoing consent process between researchers, physicians, patients and families can contribute  to creating a positive and engaged partnership relationship.

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