In vivo drug discovery using a zebrafish model of glycine encephalopathy
Glycine encephalopathy is a rare genetic disorder that leads to a generalized lethargy soon after birth, often followed by death. Surviving neonates depict strong intellectual disabilities, continuous tremor and have only poor interactions with their environment. The disease is linked with recessive mutations in glycine metabolising enzymes and no potent treatment is available. Using zebrafish, a forefront vertebrate genetic model of neural development, we generated animals that recapitulate the genetic mutation found in patients and aim at identifying potent small molecules that would attenuate or even rescue their condition. Ultimately, this project would allow the development of a treatment for glycine encephalopathy.
Aortic stiffness and coronary allograft vasculopathy in children with critical congenital heart disease requiring transplantation
Critical congenital heart disease requiring transplantation occurs in approximately 1 out of 10,000 infants and children. While patients are living longer than ever before, these children are at risk for post-transplant coronary artery disease called cardiac allograft vasculopathy (CAV). This progressive thickening of the coronary artery wall is very serious and can lead to death. Presently, CAV is found through a procedure (cardiac catheterization) using a special test call angiography (injecting dye directly in the coronary arteries). However this test only finds changes at a late stage of disease at which point there are not good treatment options. Our research group has found a more sensitive way to look at the coronary arteries, called optical coherence tomography (OCT), which can detect CAV earlier. Recently, the stiffness of the aorta (the large artery that transports blood from the heart to the body) has been shown to predict cardiovascular risk, and has been reported to be higher in children who have had heart transplants, and has been linked to the progression of CAV. We have developed a simple non-invasive ultrasound test to measure aortic wall stiffness that could be used to identify CAV earlier than we can now. We plan to assess aortic stiffness in children who have had a heart transplant, and compare these results with their OCT findings and determine how they are related.
Clinical Characteristics and Predictors of Survival Among Adults Newly Diagnosed with Cystic Fibrosis
Cystic Fibrosis (CF) is a fatal, genetic disorder mostly affecting children before adulthood. Due to this, most research about the characterization and prognosis of CF is focused on individuals <18 years of age. However, there is a unique subset of CF individuals (~10%) who are not diagnosed until adulthood. Little is known about the characterization, life expectancy and prognosis of CF in this population. We aim to fill this gap in the CF literature so clinicians can better understand adult onset (AO) CF patients and provide critical information to guide and improve current treatment strategies for this population. Additionally, our study will allow patients to better plan their lives and provide a sense of awareness about their condition based on evidence-based information.
Results - Cystic fibrosis (CF) is the most common genetic disease affecting children and young adults. More adults are being diagnosed with CF due to increased awareness among doctors and more sensitive diagnostic methods. Being diagnosed with CF as an adult is very rare; currently, they represent only 5-10% of all CF patients in Canada. These patients face unique challenges, issues, and needs compared to other CF patients diagnosed at younger ages. Information about life expectancy and prognosis is important, particularly for mature, older diagnosed patients. However, due to the rarity of this disease, doctors have little information to inform them. The main purpose of this study was to evaluate disease prognosis in this group of CF patients and we found that these patients have higher survival rates than previously reported. Also, being older at diagnosis and having low lung function at the time of diagnosis are important factors for poor survival. Having CF-related diabetes is also another potentially important factor. Doctors can now use this information to educate patients and guide treatment.
Link https://dx.doi.org/10.14288/1.0349192 (thesis link)
ICD use in Catecholaminergic Polymorphic Ventricular Tachycardia: Outcomes from a Prospective, Multicentre Registry
CPVT is a rare heart problem that causes abnormally fast heart rhythms. It is very dangerous but usually hidden, so young people can die despite appearing healthy. CPVT is familial, meaning that it can be passed down from parent to child. CPVT cannot be cured, and treatments include medications and restriction from sports. Our group has shown that CPVT patients still have life-threatening events despite medication, which often forces doctors to use extreme measures to prevent death. One heroic option is to surgically place an electronic device under the skin on the chest with wires that connect to the heart, known as an ICD. This device detects fast heart beats and can deliver a painful electrical shock to “re-set” the heart to a slower speed. Unfortunately, no one has properly studied ICDs in CPVT patients, yet half will have one put in. We found out in a past CPVT study that ICDs actually seemed to be ineffective and potentially harmful. One problem is called “electrical storm,” caused by painful, repeated and unnecessary ICD shocks to the heart, which can quickly lead to death.
An RDF grant will help us study ICDs in CPVT. Because the condition is rare, we started an international database whereby doctors from around the world can enter information on their CPVT patients. This registry will provide a large enough number of patients to answer this question. The results have the potential to significantly change our recommendations in CPVT, and improve and save lives.
Prepubertal Yolk Sac Tumors. Diagnostic and Surveillance Imaging: Is the radiation worth the price?
Extracranial germ cell tumors (GCTs) are rare in children, accounting for only 3% of childhood cancers and occurring at an overall incidence of 1 in 25,000 children. Yolk sac tumors (YSTs) are a particular type of malignant GCT that develops from yolk sac cells, important for a baby’s development in the womb. Pure YST almost exclusively affects young kids, those who have not reached puberty, and equally the GCTs of prepubertal children almost always contain some YST. YST produces a substance called alpha-feto protein (AFP), which is easily measured in blood. AFP levels can accurately tell if the tumor is responding to treatment, if there is a residual tumour, or if the tumour has come back. When a child is first diagnosed with YST, AFP levels are followed and CT scans are typically concurrently done to determine whether there is also cancer outside the primary site. After treatment for YST, AFP and CT scans are used to regularly monitor whether the cancer is back. Unfortunately, CT scans give high doses of radiation (the equivalent of 5 years of background radiation per CT), which can increase the future risk of second cancers and death.
Our objectives are to investigate whether CT scans can be safely omitted at diagnosis if AFP levels return to normal after surgery and remain normal during surveillance. The hypothesis is that rising AFP would detect all GCT recurrences on follow-up in prepubertal children, thus making CT scans redundant and hence avoidable. This strategy could ultimately cut health care costs, decrease potentially lethal radiation exposure, and minimize the long-term side effects of cancer treatment.
Proving Gain-of-Function STAT6 causes a novel Hyper-IgE syndrome that may be targeted with JAK-inhibition
We present the case of a young man who spent much of his childhood admitted to a children’s hospital as a result of chronic lung disease, gastrointestinal issues requiring a feeding tube for his nutrition, and a severe dermatological condition with itchy skin prone to repeated infection present from infancy. To this day his body is in “overdrive” with chronic inflammation of his skin, lungs and gut. His skin is red, dry and itchy. He must bath in soothing bath salts and soak in emolliating creams. He takes daily oral steroids that are making his bones fragile to the point where his bones have broken with minimal trauma. He rarely leaves the house during pollen season as it is intolerable for him. He eats a basic diet of meat and potatoes as if he strays from this, his bowels become inflamed. He has never known a day without feeling itchy, red, with chronic eye, sinus and chest discomfort.
Yet, we finally have hope on the horizon for this young man with a potential genetic answer and possible treatment in sight. We firmly believe that he has an “activating” mutation in a key gene (called STAT6) that has “inappropriately” turned up the signaling for the immune system in his body. With funds from the RDF, we plan to run experiments where we will put a “normal” STAT6 gene in some cells and a version of the “mutated” STAT6 in other cells and compare to see if the immune system is in relative “overdrive”. Then we will use medications that specifically “target” the STAT6 pathway to try to dampen down or turn off the response. The medicines we will be using (called ruxolitinib and tofacitinib) have already been used in adults for various other inflammatory conditions and have both been proven to be quite safe, with a low risk for side effects. Thus, if these work well here, they can be potentially used in future for the patient. The possibility that we may be able to “re-target” or “re-purpose” a pre-existing or approved medication for our patient with an incredibly rare genetic disorder is an extraordinary opportunity. We hope that you support our work towards proving a rare genetic cause for our patient’s disorder and early work supporting a promising potential therapeutic option for him.
Improving the collection of CSF neurotransmitters: a novel device
Producing a device to improve the quality of brain fluid collection for neurotransmitters will increase the diagnostic yield, as well as increase the likelihood of the test being performed due to the increased ease and simplicity of the technique. This may increase the success of diagnosing rare CSF neurotransmitter diseases, some of which are extremely successfully treated.
Results - Brain cells communicate using chemicals called neurotransmitters, which must be exquisitely regulated to maintain normal cognition, coordination and motor function. In some childhood diseases there is an imbalance of neurotransmitters, which can cause severe brain dysfunction. Diagnosing these diseases is challenging, and requires a lumbar puncture, or spinal tap, and collection of the spinal fluid in 5 small vials. Incorrect collection of the spinal fluid could lead to a missed diagnosis. Because the five tubes were very challenging to hold, the spinal fluid was collected correctly only about 10% of the time, according to a survey we conducted. To address this, microgrant funds were used to develop and manufacture a tube holder, which was then distributed throughout the hospital. Now the spinal fluid is collected correctly more than 90% of the time. The tube holder won an award at the British Columbia Quality Improvement awards, and has been patented. It is now standard of care in British Columbia.
Patient with severe autonomic dysfunction and CNKSR2 mutation
We are following a family with an affected child with developmental delay, epilepsy, and severe disturbances of his autonomic nervous system function. This has consequences on blood pressure and heart rate, varying between extreme highs and lows, bowel and bladder function, respiratory problems, sweating, tear production and salivation. This child has been in investigated and is in our care for over a decade, and has been admitted numerous times to hospital in hypertensive crisis, overwhelming infection from repeated bladder infections, because he was retaining his urine, and severe constipation. He now has a surgically opened bladder to avoid the bladder distension and has a bowel routine with an unconventional medication that has role in modifying chemical release in the nerves of the bowel. He also has signs of short-term memory loss, almost like an early onset Alzheimer’s disease, and has improved on an adult Alzheimer medication. His brain MRI’s were consistently normal. After many years of unsuccessfully looking for the cause of this condition, he was enrolled in a research study to look at his whole genetic make-up. The results are suggesting that a mutation in a gene on the X chromosome, CNKSR2 is causing his disease. This gene has been reported to be completely missing in some other children with developmental delay and seizures, but small changes in the gene have not been yet described. Validating this finding would mean not only that the family finally will have an answer after 16 years of search for a diagnosis, but could eventually lead to a novel treatment of the symptoms. If this gene is validated as the cause, there is good evidence that chemicals at nerve endings are missing and targeting this with medications could be beneficial.
Drug screening for PGRN-deficient FTLD
Frontotemporal lobar degeneration (FTLD) is a fatal neurodegenerative disease which results in a selective degradation of neurons in the brain. Mutations in the granulin (GRN) gene, which result in low levels of its product progranulin (PGRN), have been identified as a major cause of both familial and sporadic cases of FTLD. Our lab uses the nematode C. elegans as a powerful genetic system to study the effects of the loss of PGRN in a whole organism. Our results so far indicate that a loss of expression of PGRN results in age-dependent motility defects in C. elegans. We aim to perform a high-throughput drug screen to identify drugs which can restore the motility defects seen in our models. If our project is successful, we will further validate out compounds in other animal models which could open new treatment possibilities for FTLD-affected patients.
Seizure disorder caused by a rare SCN3A mutation
Childhood seizures are caused by a number of different factors. Some causes are acquired, like head injuries. Other causes include rare inherited defects, in which one or more genes contain mistakes, or mutations. One class of inherited defects is known as “channelopathies” because the affected genes code for proteins called channels. Channel proteins control the electrical excitability in nerves, muscles, and the heart. Mistakes in the genes can result in channels that are over- or under- excitable. Overly-excitable channels can cause seizure disorders and heart rhythm disturbances. Our goal is to understand how newly identified genetic mistakes, specifically in SCN3A that cause incorrectly formed NaV1.3 channels, led to severe seizures and neurodevelopmental impairment in 2 unrelated children. We intend to identify, at a molecular and biophysical level, why these children have seizures. Most importantly, this understanding will reveal targets for treatment and prevention, improving outcomes for patients with these rare diseases. This new knowledge can also be applied to other channelopathies, potentially helping many more patients with rare epilepsy around the world.
Role of empathetic T cells in a rare liver disease
T cells are a type of white blood cell that guides the response of the immune system to remove foreign material or to remove our own damaged cells. There are two main type of T cells: 1) αβ T cells, which are the best studied and function to recognize foreign pathogens invading the body, and 2) γδ T cells, which are unconventional in that they respond to dangers from both within and outside the body (such as viral infections and cancer) and are less understood. These γδ T cells have been referred to as being ‘empathetic’ due to their nuanced ability to differentiate friend from foe and the pathological from the benign to help us adapt swiftly and efficiently to life's many stresses. We have found abnormal γδ T cells in two children with a rare liver disease and propose to look at four more children with this very understudied condition to see if this is a common pathological feature. If so, we will use a preclinical approach to try to remedy the problem using a targeted immunotherapy strategy that has been used to fix a similar type of γδ T cell dysfunction in a different context. This work may provide insight into how this rare liver disease develops and could lead to a novel way to treat the cause of this disease which currently has no effective cure except transplantation.
Results - We came across a unique result - T cells displaying both αβ and γδ receptors on the same cell, which is very rarely seen and is not possible, according to the textbooks. These were present on 10% and 0.8% of cells in two patients with NOTCH1 pathogenic variants but on repeat testing, we only saw a very small proportion of such double positive cells. However, we found a higher proportion again on a follow up test in one of the patients and through questioning then recognized that the double positive cells seemed to appear only with illness - i.e. when there is expansion of the systemic T cell pool occurring. However, seeing as these dual TCR T cells do not persist, they may be removed from the system. Since the blood collection during illness was too complicated to obtain in another country, we were unable to pursue these studies further and test the therapy we had in mind.
Acute Atypical Psychosis Protocol
We aim to provide a protocol which can be used by psychiatrists to investigate children with features of atypical psychosis, with the goal of identifying a physical cause for their symptoms. Treatment of an organic cause may drastically improve the child’s prognosis.
Exome Sequencing in Congenital Pseudarthrosis of 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 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 propose exome sequencing for our two 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.
When genome-wide sequencing does not find the answer in rare diseases: unraveling somatic mosaicism
Next Generation Sequencing (NGS) has proven a powerful approach to the detection of single gene defects underlying rare diseases. Our TIDEX study applies this technology to patients with intellectual developmental disorder (IDD) and unexplained metabolic phenotypes; we recently reported a diagnostic yield of 38 diagnoses in 42 families studied, using whole exome sequencing (WES) and in 18 families the diagnosis impacted management beyond genetic counseling, including the discovery of 2 novel and 2 candidate novel neuro-metabolic diseases (NMDs) potentially amenable to causal therapy. To date, 3 families presenting with IDD plus either a lysosomal storage disease phenotype (coarse features, enlarged liver and spleen) or a ciliopathy phenotype (polycystic kidney failure, liver cirrhosis) remain undiagnosed, even after whole genome sequencing. We hypothesize that their multi-organ involvement disease is due to somatic mosaicism (a subset of the cells carry the DNA mutation, but not all), which can be detected by performing deeper coverage (≥200X) WES on affected tissues. We base this on similar reports in auto-immune and other rare multi-organ involvement conditions. To prove this and most importantly find a diagnosis for these families, we will extract DNA from stored liver and kidney biopsies stored for each patient and send out for ≥200X WES with interpretation via our Tidex pipeline. Using higher coverage will allow us to profile undiagnosed patients for presence of the causative variant even in a small number of cells of the affected tissues. Knowledge generated by this study will be disseminated via publication in peer-reviewed medical journals and presentations for the lay and professional public. Most importantly, if a diagnosis is established this will be discussed with the family along with proper genetic counseling and discussions with the managing physicians to optimize management and treatment.
Proof-of-concept study: Rescue of a Splicing Defect in a Patient with Combined Immunodeficiency due to Hypomorphic ZAP70 mutation
Combined immunodeficiency syndromes (CIDs) are genetic disorders of lymphocytes, impairing the immune system and predisposing to recurrent infections. In its severe form (SCID), transplantation in early childhood is urgently required for cure. In the less severe forms (CIDs), the best treatment is not known, but transplantation may actually be more dangerous. We have identified a young-adult patient with CID and recurrent infections due to a faulty ZAP70 gene. Because we have characterized the reason at the molecular level why his ZAP70 gene is faulty, we have developed a molecule intended to specifically negate the effect of his mutation. We will test in our cellular model whether this molecule increases production of normal protein and restores cell function. If successful, this approach will be applicable to treat other genetic disorders with the same faulty mechanism.
Results - We have identified the genetic mutation responsible for a 33-year-old patient’s serious health problems, for which the underlying cause had not yet been previously understood. We showed that the patient has a mutation in the gene ZAP70, which is critical for the proper function of the immune system by activating T cells. This specific mutation subtly affects the slicing of the gene but does not affect the amino acid sequence that synthesizes the ZAP70 protein. We developed a molecule called a Morpholino Antisense Oligonucleotide that exclusively blocks the mutation while allowing the protein to be synthesized, and demonstrated that treating the patient’s immune cells with the molecule corrected the patient’s mutation and re-allowed the cells to function properly.
Unraveling PIGG deficiency as novel cause of fetal akinesia
We present the tragic case of a young couple who successively lost two children to a devastating genetic neurodevelopmental condition with features of poor movement (fetal akinesia) in both. In addition, both children had unusual findings in the lung, making us highly suspicious that this family had a rare and unique genetic disorder. Despite extensive metabolic, genetic, and invasive investigations including muscle biopsies while their second child clung to life in the ICU, and full autopsies on their deceased children, a genetic answer could not originally be found. Now, with our efforts to fully sequence the whole exomes and genomes of the couple’s lost children, we are close to proving the genetic cause. Two variants in the PIGG gene have been found, present in both children. The parents each carry one mutation, consistent with an autosomal recessive condition (whereby parents are healthy carriers, and only when they both pass gene changes to the child, does the child become affected). Yet, the PIGG disorder is so new and so novel that no other children have been described with mutations in this gene. We do know, however, that the PIGG gene is an important part of the complex that synthesizes the “glycophosphatidyl inositol” or GPI-anchor, a complex molecular that is “stuck on” to the end of various other proteins (~ 150 different proteins). Thus, if PIGG isn’t doing its job to make the GPIanchor form, then many, many other proteins are affected. In order to prove that PIGG is the “right gene” gone horribly wrong, we have designed a set of experiments including “staining” the child’s affected tissue (like lungs and muscle) for PIGG to see if the protein looks different from “normal” tissues. We will also look at how much mRNA (the molecule that genes first make) and protein that is present in stored tissue, as well as the make-up of the complex sugars present in the tissue (“glycomics analysis”) from the affected child compared to a healthy sample. We hope with the RDF funds we will be able to provide the couple with a clear diagnosis, with the option for early prenatal diagnosis, and that we may shed further light onto the pathophysiology and causes for this unique disorder (PIGG-related) that sits within the broader spectrum of fetal akinesia disorders.
Isolating and Storing Circulating Cell Free DNA in Pediatric Cancer for Biobanking Purposes
Circulating cell-free DNA (cfDNA) found freely circulating in plasma and serum is a potential biomarker for the presence of tumor cells. Though most of the research regarding cfDNA has been performed on adults with cancers, detection of cfDNA in pediatric cancer is also possible. Since a non-invasive method to diagnose and monitor pediatric cancers would be very helpful, it is crucial to efficiently isolate and store this valuable biomarker. This study will investigate the best sample type and storage conditions for cfDNA from blood of pediatric cancer patients as well as whether cfDNA levels correlates with tumor progression in a mouse model.
Adding Strength to Variant Interpretation: Enabling Cascade Screening to Identify those at Risk for Sudden Death
Long QT syndrome (LQTS) is a condition that affects the electrical system of the heart, causing fainting and sometimes cardiac arrest in young healthy individuals. Knowing that you have LQTS is important because there is a simple treatment available that lowers the chance of having a cardiac arrest. LQTS runs in families and is often passed on from parent to child. A spelling mistake (called a variant) in an instruction (gene) that controls the heart’s electrical system can cause LQTS. These variants can be found through a blood test (genetic test). If a variant that is known to cause LQTS is found in an affected person, each of their family members can be tested to see whether they have the same variant. Those who have it are at risk to have LQTS, those who don’t are not. This type of testing can only be done when doctors are sure that the variant found causes LQTS. There are 16 genes that can cause LQTS but doctors and researchers know a lot about only 3 of these. If someone has genetic testing for LQTS is found to have a variant in a less common gene, there is a good chance that doctors will not definitely know what it means and so, there isn’t a blood test available for family members. We can learn more about the rare LQTS genes and variants by asking scientists to do tests in a laboratory. These tests recreate the heart’s electrical system and can show if these variants actually cause LQTS. Our team would like to do this for a rare LQTS gene called CACNA1C. A variant in this gene has been found in two families so far but there could be more. The results of the tests can make doctors more confident about the variant interpretation and offering testing to family members.
Establishing the Definition of Complexity for Program Development at BCCH
This study will address the challenge of providing care to children with complex health conditions. These children require care from multiple subspecialty teams. They need a coordinated care team, an integrated care plan and assistance with navigating the health system. However, since they often have rare diseases, or are undiagnosed, they do not fit well within the traditionally available clinics. To address this gap, we aim to develop a definition of “complexity” that can be used at BC Children’s Hospital to identify and target these children. We conducted a chart review of children with complex conditions to identify indicators and develop a conceptual model to describe their complexity. This was followed by interviews with expert clinicians who helped to refine and validate the model. The next step, in order to make this work applicable in a practical setting, is to generate a statistical model that can be applied to provincial databases. The information obtained can then be scaled to determine how many patients can be served with the amount of resources available. This will be the foundation of growing complex care services in British Columbia.
Role of [18F] FDG PET/CT in the Staging of Pediatric Malignant Germ Cell Tumours
Pediatric malignant germ cell tumours are rare form of childhood cancer that can be devastating for patients and their families. Since this is such a rare tumour, there are relatively few studies evaluating how to stage this type of cancer. Staging cancer is very important because cancers that have spread (i.e. metastasized) are treated differently than those that have not. One way to stage pediatric cancers is through PET/CT scans, which are a type of radiology exam that can distinguish cancer cells from normal cells. The goal of this study is to determine the role that PET/CT scans play in staging pediatric malignant germ cell tumours. A RDF grant will help us to better understand how to more accurately stage patients with this rare and devastating type of childhood cancer. This information will help oncologists to better plan treatment for these patients and hopefully be able to improve survival outcomes.