The study of human eye malformations: a zebrafish model
Chromosome microarray is a new technology recently implemented, allowing us to find very small chromosome abnormalities. Interpretation of these gains or loss of segments of the genome may be very difficult, since many of them are present in normal people. In a patient with iris coloboma (a slit in the iris which may pose vision problems), we found a loss of a segment of chromosome containing 2 genes. We do not know if this loss is responsible for this child's condition. Zebrafish is an excellent model to study eye development: these fish develop very quickly (4 days between fertilization and full development) and have very big and visible eyes. It is possible to simulate the loss of a gene in the zebrafish, using small chemicals named morpholinos, which specifically inactivates this gene during development. With such experiment, we hope to determine if the abnormality observed in this child is responsible for his eye condition, and, if so, to better understand how this lesion impacts the eye development. This will then serve as a model for future studies in other types of malformations.
Establishing high-throughput screening assays for chemical suppressors of nonsense and missense mutations
One of the severe impediments to treatment of rare diseases is their rarity. As rare conditions, these diseases individually are not a commercially viable market that can support pharmaceutical development. This calls for a paradigm shift for the treatment of rare diseases. By focusing on the classes of mutations causing these rare inherited diseases, therapies can potentially be developed that are applicable to thousands of disorders. This application requests seed funding to develop methods to screen for treatments for the two most common classes of mutations causing rare diseases.
This microgrant has enabled us to test over 30,000 chemicals for those capable of suppressing nonsense mutations in the yeast screening assay developed the previous year. This screen identified a single novel active compound, in addition to known aminoglycoside antibiotics. Unfortunately, this new compound showed no activity in cell lines bearing nonsense mutations that are derived from patients Schimke immuno-osseous dysplasia and X-linked chronic granulomatous disease. We are currently testing the hypothesis that drug combinations may be necessary to achieve efficient nonsense suppression in the yeast model system and in patient-derived cell lines.
Bringing new genomic technologies into direct patient care
Several severe disabling and fatal disorders have multiple genetic causes. This makes a genetic diagnosis difficult and expensive. Consequently, families often do not get the information they need for closure and to proceed with family and financial planning. The use of new genomic technologies has opened the door to new diagnostic possibilities for these families. We propose to show how using next generation sequencing we can provide families with a cost-effective diagnostic test. As an example of this, we present a family with a chronic and relentlessly debilitating condition that affects the muscles. Multiple costly and invasive clinically available tests did not provide a diagnosis. Using these new techniques we are now able to provide the family with an answer and improve the care for the affected children, and provide a cost-effective test for conditions that can be caused by many different genes.
Defining metabolite pattern in rare neurometabolic disorders by 1H-Magnetic Resonance Spectroscopy of the brain in children
Proton Magnetic Resonance Spectroscopy (1H-MRS) is one of the few techniques that can be used for the in vivo assessment of metabolic processes in the brain, by the measurement of specific brain metabolites such as creatine, choline, lactate, N-acetylaspartate and others. However, the clinical use of 1H-MRS in a pediatric setting, has mainly been limited to qualitative or semi-quantitative analyses. Our research aims to establish quantitative in vivo 1H-MRS of the brain in children as a standard of care at the Hospital for Sick Children. Furthermore, we aim to employ its use as a method of further defining and delineating diagnoses of rare known, as well as unknown, neurometabolic disorders. One of the main objectives that we would like to accomplish is to define and describe specific pattern of the metabolites measured on 1H-MRS, in known inborn errors of metabolism using reference data matched for age and localization. In addition, we also expect that we will be able to define new, previously undescribed neurometabolic disorders.
Complex I deficiency in children with microdeletion syndromes: Primary or secondary mitochondrial dysfunction?
Mitochondrial diseases collectively have an incidence of 1 in 5000 live births. Mitochondria are essential organelles, best known for their role in energy production via respiratory chain complexes in a process known as oxidative phosphorylation (OXPHOS).
There are a total of five large respiratory chain complexes (Complex I, II, III, IV and V) involved in the OXPHOS process, and each of these complexes is composed of multiple subunits. 13 of these approximately 80 OXPHOS subunits are encoded by maternally inherited mitochondrial DNA (mtDNA) and the rest are encoded by nuclear DNA.
Isolated defects in complex I activity are the most common type of mitochondrial disease. Typically in pediatric patients, following the identification of an isolated complex I deficiency a number of investigations ensue, including muscle biopsy, skin biopsy, sequencing of associated nuclear genes, and searching for mtDNA mutations that give rise to mitochondrial disease, as an attempt to elucidate the underlying etiology of the deficiency. Unfortunately, it is not uncommon for these invasive, expensive and time consuming investigations to be negative and not contribute to a more definitive diagnosis, beyond "Complex I deficiency".
In the Neurometabolic clinic at McMaster Children's Hospital, a significant proportion of patients that are assessed have probable mitochondrial disease, and a large proportion of which, have complex I deficiency (> 200 patients). One of our observations in our population, that has not been previously reported in the literature, is the presence of isolated complex I deficiency in patients with chromosomal microdeletions, detected by oligonucleotide microarray. Furthermore, the children we have made this observation in, often do not have the typical clinical findings that have been described to be associated with isolated complex I deficiency. These observations raise the question of whether the complex I deficiency is occurring in microdeletion syndromes as a secondary phenomenon?
In an attempt to further elucidate an explanation to this interesting phenomenon, we would like to perform a retrospective chart analysis on 200 patients who have had both mitochondrial complex I activity measurement and oligonucleotide microarray analysis completed as part of their diagnostic workup.
Development of a skeletal tool assessment and scoring system for MPS disorders
Mucopolysaccharidoses (MPS) are a group of rare conditions caused by the accumulation of complex sugars in different parts of the body (including joints and nerves) thereby affecting the overall body function. Patients with the disease have difficulty carrying out activities of daily living and demand continuous care with clinical follow up of the patient's functions. Currently available disease scoring systems are time-consuming and are not specific to MPS patients. At the Hospital for Sick Children (HSC) we have an MPS quality of life questionnaire which does not provide us with functional scores. We believe that we can make use of this questionnaire along with other available general score systems to create a short and specific score system that could be used for MPS patients. In addition to that, we will develop a standard video protocol for clinical assessment of MPS patients. This film is to include a 20 minute record of the child performing a number of activities done in each clinical visit and will be used to determine how MPS patients are responding to treatment.
Did not proceed with the project and did not use the grant money.
Permanent Junctional Reciprocating Tachycardia: A Multicentre Data Collection
Permanent junctional reciprocating tachycardia (PJRT) is a rare form of abnormal heart rhythm most commonly identified in infants and children. Medical therapy and catheter ablation can correct the abnormal rhythm and prevent complications, including heart failure and death. However, there is limited information for physicians regarding the natural history of this condition and the best options for management. This research seeks to define the course of PJRT in children and to determine optimal treatment approaches. Because PJRT is a rare condition and many centres have only a few patients per year, we intend to include cases from multiple pediatric centres. Our study is designed to build on multicentre relationships and data collection tools established for a previous study.
We are in the process of assembling the largest series of this condition ever. We hope to learn which patients need invasive treatment and which patients outgrow this condition.
Effect of NSD1 mutations on the DNA methylome in patients with Sotos Syndrome
Sotos syndrome is a genetic condition causing excessive growth in childhood, and a variety of neurodevelopmental and physical anomalies. The prevalence is unknown but several hundred patients have been identified. Sotos syndrome is caused by mutation in a gene called NSD1 which is known to change the 3-D structure of the DNA. Mutations in the NSD1 likely cause downstream effects on DNA modification that can be used as molecular targets for therapeutic intervention.
Evaluation of platelet serotonin transporter and other membrane proteins in two siblings with familial hemiplegic migraines and a neurodegenerative disease
We identified two sisters with severe migraines causing associated paralysis, who developed after the age of 15 years: difficulty walking, generalized muscle weakness, problems with balance, and speech, frequent urination, depression, and a sleep disorder. Magnetic resonance imaging (MRI) of the brain showed atrophy of the brain-stem and spinal cord. Biochemical investigations in spinal fluid and platelets revealed evidence of serotonin transporter dysfunction. Brain magnetic resonance spectroscopy (MRS) showed low levels of creatine (a chemical important in brain cell energy function), similar to what we see in creatine transporter disorders. Tilt table testing caused fainting with plasma stress hormone results suggestive of a norepinephrine transporter defect. The patients responded partly to treatment with 5-HTP (5-hydroxytryptophan), which is a serotonin precursor, by showing improved strength, and mood. Their episodes of migraines persisted and one of the sisters was in a prolonged coma for 5 weeks, suggesting a problem in the brain cell membrane calcium channel transport, similar to what has been described in other families with hemiplegic migraines. Overall these findings are suggestive of a complex membrane protein problem. So far we sequenced the genes that are encoding these individual proteins, and found no mutations. We will be collaborating with Dr. Blakely's Lab in Nashville, TN, who is a leading expert in serotonin an norepinephrine transporter research, and he agreed to evaluate the above mentioned protein expression in platelets.
We believe that by finding the underlying defect we will improve treatment strategies in these two sisters and prevent further neurological deterioration and further episodes of coma (we can use not only 5-HTP, but also other medications like creatine, droxidopa, or verapamil, depending on the results of which of the proteins are affected in the membrane).
SERT molecular sequencing was negative, but abundant SERT protein was found in patient platelets. Through other microgrants, we identified the underlying cause and our patients' strength was improved significantly with serotonin replacement therapy.
Quantification of Bone Structure Integrity by Quantitative Ultrasound (QUS), Peripheral Quantitative Computed Tomography (pQCT) and Micro-Computed Tomography (Micro-CT) in a Rabbit Model of Inflammatory Arthritis / Bone Loss
Arthritis is a rare disease that causes significant joint disability in children. The disease itself and its treatment cause bone loss and thus, increased risk for development of fractures. Early diagnosis and treatment are essential to improve the quality of life of children with arthritis. We propose to evaluate a new non-invasive imaging technique called quantitative ultrasound to measure bone loss in knees of rabbits with arthritis that died previously and compared with a technique that is well-known to diagnose bone loss, micro-computed tomography. We hope to be able to use this non-invasive technique for early diagnosis of bone loss in children with arthritis in the future if this technique proves to be good for this purpose.
We are in process of finalizing the data and will submit our result to the Investigative Radiology journal soon.
Impact of the Complex Nutrition Team
Gastroschisis is a rare and serious birth defect in which the abdominal wall does not develop normally (rates in British Columbia have been increasing and are between 3.0-3.5 per 10,000 births). In the uterus, the intestines develop outside the abdomen, floating in the amniotic fluid. At birth, these infants will require surgery to return the intestines to the abdomen, and intensive care for nutritional support until the intestines are functioning well enough to support growth and development. These infants sometimes have difficulty feeding and growing because they may have shorter than normal intestines and/or some of their intestines have been damaged by the amniotic fluid, have been perforated, closed in areas, or have been damaged enough to die.
Following surgery, these infants will spend anywhere from 1 to 3 months, and in some instances more than a year, in intensive care to establish independent feeding. Feeding strategies are crucial to both short- and long- term outcomes. Problems can arise for these infants requiring increased length of hospital stay and prolonged use of intravenous nutrition which can result in liver toxicity and/or liver failure. Long-term growth, brain development and normal function of the intestines are all important concerns.
In May 2007, to more closely monitor the feeding strategies for these patients, we developed a specialized multi-disciplinary team (Team). The Team included: Dr. X (Neonatology), Dr. Skarsgard (Surgery), Dr. Jacobson (Gastroenterology) and Ms. Lalari (Dietitian) who rounded weekly to develop specific nutrition and feeding plans for patients with complex intestinal issues, such as those with Gastroschisis. We would like to assess whether this Team improves the care of these babies, helps their families, and helps the staff.
Establishing DNA testing criteria for children with possible Neurofibromatosis type 1
Neurofibromatosis (NF1) is a genetic condition causing serious medical problems such as tumors and high blood pressure. The signs of NF1 develop as a person ages. This makes it very challenging to diagnose young children without obvious signs and who are called `possible NF1'. Most, but not all people with a clinical diagnosis of NF1 will have a positive NF1 DNA test result. We want to see which children with "possible NF1" would most benefit from DNA testing by looking at the medical records of selected SickKids patients who have been classified as "possible NF1" and who have had DNA testing. This information will help develop guidelines for physicians to help decide which patients would benefit most from DNA testing.
Characterization of FAM92A1 and RBM12B-like in a Danio rerio (zebrafish) model to identify their role in Nablus Mask-like Facial syndrome
Nablus Mask-like Facial syndrome (NMFS) is a rare disease with a distinct appearance as a result of thickened skin reported in less than a dozen individuals. Microarray analysis, a relatively new technology which identifies areas of gain or loss of genetic material, has allowed us to identify and refine a deleted region in individuals with NMFS. The critical region contains two novel genes, FAM92A1 and RBM12b, whose human function(s) is as yet unknown. We will examine developing zebrafish embryos to determine when and in which tissue these genes are expressed. We will attempt to mimic the human disease, NMFS, by producing a zebrafish deficient in the expression of these genes in order to understand more about their role. The zebrafish is an ideal animal model system for several reasons: short generation time, large numbers are produced at each mating, all stages of development can be investigated since fertilization is external, and embryo transparency allows visualization of internal structures. Identification of the causative gene will allow development of a diagnostic test to identify affected individuals and may provide clues about potential treatment.
First steps to the improvement of long-term outcome of GAMT deficiency by using an international database
Guanidinoacetate methyltransferase (GAMT) is a critical enzyme involved in production of creatine, which is a vital energy source in the brain. GAMT deficiency is a cause of intellectual disability, abnormal movements and uncontrollable seizures in children. The treatment of GAMT deficiency consists of oral creatine and ornithine supplementation and protein restricted diet. This therapy is effective to treat otherwise uncontrollable seizures and abnormal movements and to stop ongoing brain damage and should be started as combination therapy without a delay. GAMT deficiency is a very rare disease and there is no well established treatment protocol. A research database will help to collect data of the patients with this rare disease from the various numbers of countries. We will evaluate therapy and develop a universal treatment protocol for patients with GAMT deficiency. Our research institute (Child & Family Research Institute; CFRI) at the British Columbia's Children's Hospital has a database for this purpose.
Energy requirements in metabolic and mitochondrial disease
Metabolic diseases include inborn errors of intermediary metabolism that lead to an acute/progressive intoxication from the accumulation of toxic compounds proximal to the metabolic block, or deficiency in energy production. The wide range includes phenylketonuria, maple syrup urine disease, organic acidemia and mitochondrial respiratory chain disorders. Prevalence rates in children vary, for e.g. organic acidemias occur 1 in 50,000; mitochondrial disorders are 1 in 5,000. Treatment aims to restore homeostasis, including restriction of substrates (amino acids, fatty acids or glucose), removal of toxic metabolites, etc. Nutritional intervention is critical with provision of adequate energy for normal growth and development. The clinical standard for estimating energy requirement is by use of indirect calorimetry. Currently, for metabolic diseases the energy requirements are not measured but calculated using mathematical equations based on normal children. Studies have found large discrepancies between measured daily energy requirements and mathematical equations in several diseases. None of these studies have specifically targeted patients with metabolic disorders. The indirect calorimeter takes individualized measurements of respiratory gas exchange (Oxygen consumption, VO2 vs. carbon dioxide expiration, VCO2) to calculate each patient's specific energy need. From the VCO2 and VO2 values, the respiratory quotient (RQ) can be calculated. The RQ ranges in value from 0.7, 0.85 to 1.0, corresponding to fat, protein or glucose utilization, respectively. This specific knowledge can then be used to tailor total energy needs, as well as the type of energy substrate to feed the patient.
Chromosome translocation and angiomyolipoma
A mother affected by renal angiomyolipoma and her young daughter are both chromosome translocation carriers. The tumors can cause serious kidney damage. We do not know with certainty if the translocation is the cause and if the daughter requires life-long screening with ultrasound. We have good reason to suspect a particular gene is broken by the translocation, has partnered with a gene on the other chromosome, and is causing the tumors. We wish to pinpoint which genes are broken and fused, if any, and which biological pathway they could be acting through to cause the tumors. This is a serendipitous opportunity, in which little investment could have immense return.
We obtained custom-targeted contiguous sequencing of the cytogenetically estimated breakpoints of a balanced translocation in a woman with renal angiomyolipomas. One of the chromosomal regions is recurrently implicated in mesenchymal tumors. We were looking to see if a fusion gene was created with the translocation or if a tumor suppressor was disrupted. We did not find the breakpoint in the predicted location---even after a de novo assembly using Abyss. Once the cost of whole genome sequencing dropped considerably, we re-applied to Rare Disease Foundation in 2011 to try again with a second approach.
A pilot study to assess prevalence of GAMT deficiency in British Columbia
Guanidinoacetate methyltransferase (GAMT) is a critical enzyme involved in production of creatine. Creatine is a vital energy source in the brain. Oral creatine and ornithine supplementation and protein restricted diet are the treatment of choice for the GAMT deficiency. This therapy is effective to treat otherwise uncontrollable seizures and abnormal movements and to stop ongoing brain damage. However it is not effective to prevent intellectual disability that occurred before diagnosis. Early identification and therapy in the neonatal period has been shown to prevent intellectual disability. With this pilot study we will identify prevalence of GAMT deficiency in British Columbia. This will allow us to apply for future research projects for the development of methods and setting up newborn screening for GAMT deficiency in British Columbia.
To find an answer to this question, we performed a pilot study to assess the carrier frequency of GAMT deficiency in the newborn screening population in British Columbia. We randomly chose 3000 anonymized newborn blood dot spot cards to look for the two most common mutations (c.59G>C and c.327G>A) in the GAMT gene. Neither mutation was detected in any newborn. However, we found two abnormally sized amplicons of two newborns showing two novel heterozygous mutations; c.283_285dupGTC (p.Val95dup) and c.278_283delins CTCGATGCAC (p.Asp93AlafsX35). Sequencing of the complete open reading frame identified no additional mutations in both newborns. Carrier frequency of GAMT deficiency was therefore calculated as 1/1475 in randomly selected newborns. We presented our results at an international metabolic genetics conferences as a poster presentation. Our study is the first study to report carrier frequency of GAMT deficiency in the newborn population. We established an evidence base for the first steps towards newborn screening for this rare treatable neurometabolic disorder.
Can We Predict Sinonasal Disease in Cystic Fibrosis Patients?
Individuals with cystic fibrosis (CF) have a defect in a protein known as cystic fibrosis transmembrane regulator (CFTR) which is essential for hydrating the mucous in the sinus cavity. Unfortunately, the thick mucous environment that results can encourage chronic infection and inflammation of the sinus cavities known as sinusitis. Patients with sinusitis often have nasal discharge, congestion, facial pain, headache, and decreased sense of smell. We do not currently have a good understanding of how many children with CF suffer from these symptoms, and how to predict which children would be most affected. If we could predict which children are more likely to develop severe sinus disease and nose-related symptoms, then treatment for these children can be obtained earlier. This may reduce suffering in these by children through earlier intervention, closer follow-up, and may reduce the risk of severe complications of sinus disease such as spreading of the disease into the eye, brain, bone, and vascular system. Our study aims to gain a better understanding of the severity of sinus-related symptoms in children with CF and to explore potential predictors.
New insights into Failure to Thrive
Up to 10% of all babies suffer from growth failure. A subset of these babies continues to have growth delay throughout life, associated with developmental delay. Both genetic and non-genetic cases have been associated with this "failure to thrive", however in many children we are unable to understand why they do not grow despite adequate food intake, and why they have intellectual disability. We have identified a gene that is not properly expressed in a child with failure to thrive and developmental delay. This gene, SLC2A13, is poorly understood, although we suspect that it may regulate modification of proteins needed for growth. We propose to study the role of this gene and the protein it encodes with the ultimate goal of providing new understanding of failure to thrive and potential targets for treatment.
Markers of Parenteral Nutrition (PN) Associated Metabolic Bone Disease in Children on Long-Term PN for Intestinal Failure
Parenteral nutrition (PN) is the provision of nutrients via the intravenous route. Parenteral nutrition associated metabolic bone disease (MBD) was first described in children in the 1980s. Since then, there has been little to no research into the underlying relationship and as a result, little evidence on which to base clinical care. In adults, MBD is associated with increased fractures. At the Hospital for Sick Children in Toronto, an intestinal failure program has been set up since 2003. This is the only intestinal failure program in Canada and receives country-wide referrals. Most of the patients have short bowel syndrome (SBS) and require PN for prolonged periods, or for life. About 90% of these patients have MBD, and some have had bone fractures. An understanding of the etiology of MBD would provide information to guide care, and prevent this condition. Funding for this area of research however is challenging because intestinal failure requiring long term PN is a rare condition, accounting for approximately 200 - 300 children in all of Canada. The goal of this study therefore is to gather pilot date on markers of MBD in children on long term PN, and to compare these markers to age and gender matched control patients who are fed by mouth or feeding tube. The information gathered from this study will help us begin to understand what is actually happening in the bones of children on long term PN and will form the basis for future studies and improved clinical care.