November 2012

November 2012 Funded Microgrants

Discovery of an unknown serum factor that suppresses B cell development

We are involved in the care of a 6-year old girl who has chronic lung disease and does not make any antibodies. This makes her very susceptible to infections. Antibodies are produced by a kind of white blood cell called a B cell. When we examined her B cells we found that there was a block in their development. She was unable to make mature B cells capable of making antibodies. Further testing suggested that there was a specific factor in her blood that causes this block. When her blood was incubated with normal B cells the same effect was observed. This has never been described before. We also believe that this factor is present in several rare diseases involving problems with B cells and antibody production. We would like to figure out what this factor in the blood is. Identifying this factor could provide a new target in the treatment of this patient and others with B cell defects.

Results - 

We investigated a child in whom there was a lack of mature B cells resulting in an absence of antibody production. Our work resulted in two important discoveries. Firstly, using whole exome sequencing, we were able to discover a novel mutation in a gene which encodes a protein called phospholipase C-gamma 2 (PLCg2). This protein triggers the movement of calcium into B cells, which is an essential cofactor in the function of many other proteins. The mutation we identified caused a gain of function in the protein leading to the premature death of developing B cells and provide novel insights into early B cell development. Secondly, we discovered that elevated levels of a modified version of B cell activating factor (BAFF), normally an essential growth factor for B cells, also negatively affects the development of early B cells. It appears that this phenomenon is not unique to this one patient and may be present in a large number of inflammatory and autoimmune conditions making it a promising therapeutic target.

Carbonic Anhydrase VA deficiency: Improving diagnostic and therapeutic options of a novel inborn error of metabolism

Previous Rare Disease Foundation funding allowed our TIDE-BC group ( to confirm that the mutations detected in the CA5Agene through whole exome sequencing were indeed disease causing. As a result, we have been able to resubmit our manuscript on the exciting discovery on this novel inborn error of metabolism to highly esteemed journal meeting the editor's demands.  The next step is to identify other patients around the world presenting similarly with this rare condition causing life-threatening symptoms of episodic lethargy, low blood sugar and high levels of blood lactic acid and ammonia. We have set up the gene analysis to do so in BCCH/CMMT; for 3 patients samples have been sent to us by international colleagues. In parallel we shall pursue laboratory experiments in collaboration with experts in St Louis to better characterize this condition at a cell and enzyme level, allowing us to optimize available treatment strategies (zinc, carglumic acid) and search for specific biomarkers which might allow high-throughput screening for patients as part of our TIDE diagnostic protocol for treatable intellectual disabilities.

Results - 

Carbonic anhydrase VA deficiency was discovered and published in 2014 by Dr van Karnebeek and the Omics2TreatID team as a novel cause of life-threatening hyperammonemia in the newborn (ref: This microgrant has allowed us to identify another 10 families around the world affected by this rare inborn error of metabolism and collect their clinical, biochemical and genetic data in a structured way. Most importantly this research has allowed us to establish ammonia scavengers and emergency medical diet as the treatment of choice to allow for healthy development and avoid brain damage. Furthermore it has fueled a global collaboration to identify biomarkers for newborn screening; addition of this rare disease to the current panel will allow for early detection, treatment and prevention of the complications seen in children who remain undiagnosed.

Rare Syndromes Informing Common Disease: Familial Systemic Arthritis and Type 1 Diabetes

Lupus is a serious disease where a person's own immune system attacks itself. Lupus is rare but important disease to study because it has no cure, it often strikes women in their childbearing years and has serious long-term health consequences. We follow a large family with many members that all have lupus, and several of them got lupus as children. When a disease shows up so early and in so many family members, a damaged gene is almost always responsible. We have already collected samples from healthy family members and from those with lupus, and started basic "gene-mapping" studies to narrow down the location of the broken gene. To identify the exact gene, we will use a technique called "exome sequencing," which finds all of the major mutations that are part of someone's genes. We hope that finding this gene will let us use existing drugs in better ways to help this family and lead to new treatments that would be useful for everyone with lupus.

In vitro study of substrate reduction and enzyme replacement

Action myoclonus renal failure syndrome (AMRF) is a very rare devastating disease characterized by progressive myoclonus epilepsy and renal failure. Most patients do not survive 30 years of age and die from renal failure or from the progression of myoclonus and epilepsy. The clinical features are caused by deficiency of the SCARB2 protein which is the transporter of -glucocerebrosidase (-GC), the enzyme that is deficient in Gaucher disease GD). Since there is no treatment for this disorder, we aim to undertake an evaluation of the ability of an enzyme replacement treatment of the -GC in cells of AMRF patients.

Results - Funds went unclaimed by the applicant.

Gene-negative Suspected Hyper IgM Syndrome: Moving Care Forward With Research

A 19-year-old university student is currently admitted to hospital with life-threatening pneumonia in addition to inflammatory lung and bowel disease. He has had many infections in his life. He does not make a normal amount of two types of antibody called IgA and IgG.  Most of his doctors have concluded he probably has a very rare genetic disorder called hyper IgM syndrome, the curative treatment for which is a bone marrow transplant. A transplant is a risky option, with a substantial mortality rate and risk for complications. There are four genes known to cause hyper IgM syndrome, and a fifth gene is strongly suspected but has not yet been pinpointed. His testing of the four known genes in a clinical laboratory was negative. Showing that he has a mutation in a gene akin to the other hyper IgM genes will give his specialists confidence in potentially recommending a risky course of treatment.

Identification of genes predisposing to idiopathic ventricular fibrillation

Ventricular fibrillation (uncoordinated contraction of the cardiac muscle of the ventricles in the heart) is the most commonly identified arrhythmia in cardiac arrest patients. This arrhythmia is a severe derangement of the heartbeat that usually ends in death within minutes unless corrective measures are taken. In the absence of identifiable structural heart disease or known 'electrical' abnormalities, it is referred to as idiopathic (cause unknown) ventricular fibrillation (IVF), which accounts for ~10% of sudden deaths. A family history of sudden cardiac death is present in 20% of IVF cases, suggesting that at least a subset of IVF is hereditary. However, in only a few cases of IVF a genetic cause (mutation) has been found. The present lack of insight in the genetics of this disorder hinders the understanding of the pathophysiology of the disease and consequently hinders the developments of new therapies for the disorder. In this context, we care for a patient who suffered from IVF while watching TV. He was successfully resuscitated and received an Implantable Cardioverter Defibrillator (ICD), the only current treatment available. Genetic screening uncovered no mutation in the IVF associated gene SCN5A. The patient's uncle and his maternal grandfather's brother had both died suddenly at a young age. Both teenage sons have abnormal ECGs and showed decreased cardiac function on MRI. We have DNA samples from the parents and the 2 children, with research consents for use in this study. We propose to use a method to look at all genes in retained DNA from the 3 patients with IVF/ ECG abnormalities, and compare identified variants between all 4 individuals: putative causal variants should be present in all the affected individuals and absent in the mother.

Results - 

We thought we had found the culprit in this family, in that the whole exome sequencing identified two rare deleterious variants in a gene called SNX13. We were very excited in that a recent Nature paper identified heart failure with cardiomyocyte apoptosis in a zebrafish model of SNX13 knockout. However, despite very good read depth and quality of the exome sequences, the variant could not be confirmed by Sanger sequencing. We even repeated the Sanger sequencing. We may be back to square one with this family, which is unfortunate, as they have 3-generation disease. The next move with them will be to switch to whole genome sequencing.

A comparison of quality of sleep and quality of life in patients with glycogen storage disease on standard and modified uncooked cornstarch

Patients with Glycogen Storage Diseases (GSDs) are often unable to fast for longer than 4 hours and they therefore require overnight feeding with uncooked corn starch.  This represents a significant burden for both the children affected and their families.  They experience anxiety and fatigue related to the use of uncooked cornstarch (UCCS) to prevent nocturnal low blood sugars (hypoglycaemia). Recently, a new modified UCCS appeared on the market allowing a longer fasting period. To date, there is no study looking at the impact of this latest therapy on quality of life and quality of sleep of children with GSDs and their family.

Pathological cell death in chronic granulomatous disease in response to Burkholderia cenocepacia

Chronic granulomatous disease (CGD) is a rare genetic disorder that results in the inability of immune cells to produce bleach-like products crucial for killing invasive bacteria and fungi. As such, patients with CGD develop multiple life-threatening lung infections, which are the ultimate (but potentially preventable) cause of death. Furthermore, these patients suffer from debilitating inflammatory disease, due to other poorly understood abnormalities in immune system functioning. Burkholderia species are the leading bacterial cause of death in CGD and cause increased immune cell death in CGD compared to healthy controls. I intend to investigate pathway of cell death with the goal of identifying key biological mechanisms that can be inhibited to decrease lung inflammation and associated severe illness for patients with CGD. These observations could lead to simple and highly effective strategies for improving the health and longevity of patients with CGD.

Identification of genes predisposing to familial arrhythmia syndrome

Normal contraction of the human heart depends on the proper movement of charged ions through special cardiac ion channels across the surface membrane of millions of cardiac cells. Disorders of the conductive ion flow lead to cardiac arrhythmias, which may predispose to sudden cardiac death (SCD) at a young age. SCD accounts for up to half a million of deaths yearly in the US. Although many of these cardiac arrhythmia disorders leading to SCD are rare, its understanding is essential to unravel the mechanisms and pathogenesis of arrhythmias in the general population. Also the availability of genetic diagnostic tests adds an important diagnostic tool, providing new opportunities for patient management such as early (presymptomatic) identification and treatment of individuals at risk for developing fatal arrhythmias.

Characterization of mitochondrial Acetyl CoA Carboxylase 2 enzyme mutation in a new disease

We are following a 4 year old boy with language delay, and two episodes of metabolic crises characterized by acidosis (increased acid in blood), elevated ketones, and urine organic acids that were suggestive of multiple carboxylase deficiency, a metabolic defect affecting an enzyme that is biotin dependent and responds well to biotin treatment. We have started treatment with biotin and he has not had any further metabolic decompensations with acidosis. We have ruled out multiple carboxylase deficiency with genetic testing and also with enzyme analysis in skin fibroblasts. We also tested some the individual enzymes that make up this complex of carboxylases. There is one enzyme Acetyl CoA carboxylase (ACC) that has not been checked because it is not a routine clinical test, and isolated deficiency of this enzyme has never been described in human disease before. We have done full exome sequencing on the family and the gene that has novel variants is the ACC2 gene. In order to prove that this is the gene causing this child's disease, we would like to perform the ACC2 enzyme analysis done by an expert lab in Switzerland. We would also do an RNA expression study to see how well is the enzyme protein produced and the protein quantitative measurements on our patient's fibroblasts (skin biopsy already performed for other enzyme analysis) investigating this new metabolic pathway, with a laboratory method called Western blot. These latter two experiments can be done in Dr. Marion-Coulter Mackie's lab and I am quite familiar performing the methods. If we find that an enzyme deficiency is the cause of his disease, we would understand better the role of this enzyme in the biotin responsive carboxylase deficiencies and could offer prenatal diagnosis for this young couple who would like to have more children.

RUNX2, Cleidocranial Dysplasia, and Cancer

Cleidocranial dysplasia is a rare disorder affecting the development of bones and teeth. According to our yet-unpublished research, it appears there may be a 30-fold increased risk in pediatric cancer in patients with this disorder. The causative gene, RUNX2, may serve a role to suppress the development of cancer. Partial loss of the gene causes the bone disorder, and we will look for total loss of RUNX2 within two tumors of a patient with this disorder. We will also look for RUNX2 mutations in sporadic cancer.

Results -

Six of 1242 individuals with CCD had pediatric cancer. One would expect only 1 in 6250 individuals to have cancer prior to age 20 (SEER CDC data). The estimated relative risk is 30-fold based on this data.  If, however, a co-occurrence of CCD and pediatric cancer is 15 times more likely to be reported in the literature than an occurence without cancer, then the observation of increased relative risk is spurious. Our data suggests a role for RUNX2 in some pediatric cancers and a possible increased relative risk, but low absolute risk, for cancer in CCD. A CCD registry with prospective data collection may be the best means to determine actual risk of pediatric cancer in CCD.

Understanding Mounier-Kuhn syndrome

Mounier Kuhn syndrome, also known as tracheobronchomegaly, is a very rare disorder of loss of elastic tissue of the large airways, resulting in breathing problems and infections. The lack of recurrence in families suggests that most cases are not due to a mutation in a single gene and are probably acquired from destructive inflammation. This project will assess primary production of elastin from fibroblasts from two affected individuals to rule out a primary disorder of elastic fiber assembly, as would be seen in genetic connective tissue diseases such as Familial Cutis Laxa or Marfan Syndrome.

Results - 

We identified 141 publications describing 336 occurrences of tracheobronchomegaly. We observed that affected individuals could be grouped into subgroups according to clinical features. Type 1A (105 individuals) consists of infants who developed TBM after having undergone fetoscopic tracheal occlusion, and Type 1B patients (24 individuals) are infants and children who developed TBM after prolonged intubation. Type 2 individuals developed TBM following recurrent pulmonary infections (2A) (14 individuals) or pulmonary fibrosis (2B) (10 individuals). Type 3 represents TBM with evidence of extra-pulmonary elastolysis (18 individuals), and Type 4 denotes the development of TBM with no clear pre-disposing factors (167 individuals). Both of our patients had TBM and evidence of extra-pulmonary elastolysis. As well, one patient had a mildly dilated aortic root, which is a previously unreported co-occurrence. In conclusion, we introduced a novel classification scheme, which may sort patients into etiologically distinct groups, furthering our understanding of its pathogenesis and potentially, prevention or therapy. We also hypothesize that TBM and generalized elastolysis may have etiological commonalities, suggesting a need for further study.

A Clinical Classification Scheme for Tracheobronchomegaly (Mounier-Kuhn Syndrome).  Payandeh J, McGillivray B, McCauley G, Wilcox P, Swiston JR, Lehman A.  Lung. 2015 Oct;193(5):815-22.

Long Term Outcomes among Adult Fontan Patients

The normal heart has a right sided pump that sends blue blood to the lungs to get oxygen and a left sided pump that sends red blood with oxygen to the body. One in 3000 live babies born has only one heart pump and there is mixing of blue and red blood leading to low oxygen levels and eventual death. A Fontan surgery is the creation of tunnels that direct blue blood to the lungs without the need for a heart pump. The functioning single heart pump is used to send red blood to the body. Although this is lifesaving and children do well, many problems occur in adulthood that limit life span to mid-late 30's. Our group at St.Paul's Hospital follows these patients and has published research showing that clot formation is a major problem in adults. But there are many more questions that need to be answered to prevent early death in these patients. We are writing a grant proposal to establish a Fontan database across the country (approximately 1500 adult Fontan patients are followed in Canada) so that we can answer some of these questions in a larger group of patients. This rare disease grant will allow us to perform a pilot study looking at factors causing Fontan failure at our center, and this pilot study is needed to get the bigger grant funded.

Characterization of the SRRM2 splicing activator's function in alternative splicing and gene expression profiling in a neurodegenerative disease

I have been following a family with three affected siblings, two sisters and one brother. One of the sisters presented with progressive neurodegenerative course, with lower limb weakness, spasticity, hemiplegia, spinal cord atrophy, and white matter changes on brain imaging, which lead to a prolonged coma, then seizures, and death after one year being in and out of the hospital. The other sister has progressive lower limb weakness, spasticity, spinal cord atrophy, and episodes of hemiplegia. The youngest brother has no weakness yet, but has spinal cord atrophy. The overall characterization of this family's disease resembles Amyotrophic lateral sclerosis (ALS), but it also has similarities with Parkinson's and Alzheimer's disease, as we found intracellular aggregates in the deceased sibling's brain tissue. We performed a series of biochemical and molecular tests that show evidence of multiple gene and protein involvement, which led to identification of the dysregulated biological pathway through full exome sequencing. The gene that would be most responsible for the familial disease is one of the mRNA splicing factors, SRRM2. Alternative splicing can generate many different types of the same protein which would explain the several biochemical differences that we found with previous testing. In order to prove that this gene is causing this family's neurodegeneration we need to perform full transcriptome analysis (testing all the types and forms of proteins in the body, but mainly interested in the brain proteins). This would have a major effect on the treatment of the two living siblings, as there were recent reports of treatment of splicing defects with a dietary supplement called kinetin. This could potentially halt the progression of the disease in the sister, and prevent neurodegeneration in the brother.

Results - 

This study led to a better understanding of the mechanism of how SRRM2 mutation is causing disease, and to an application for developing a mouse model, characterizing a new disease, and ultimately to use this knowledge for better management of the patients with this mutation.

Identification genes underlying an infant form of lethal cardiac fibrosis

We care for a family with a unique infant form of lethal scarring of the heart. The parents are unaffected first cousins, which guides us to certain kinds of mutations. They have one healthy teenager and one apparently healthy infant. The intervening 3 children each died within the first 2 years of life, with autopsies demonstrating marked 'scarring of the pumping chambers of the heart'. We have DNA samples from two of these victims, as well as living children and parents, with research consents for use in this study. We will use a method to look at all genes in retained DNA from the 2 infant victims, and compare it to the surviving parents and children. Identifying the gene for this condition in this family will allow for a more definitive diagnosis in the remaining infant, and contribute to our understanding of the control of spontaneous scarring of the heart. In addition, a form of scarring of the heart is endemic to rural regions of Mozambique: endomyocardial fibrosis. A recent report of endomyocardial fibrosis in a 22 month old child born to a father originating from Mozambique, but who had not himself travelled there, suggesting a genetic link. The previously hypothesized relationship of endomyocardial fibrosis to the Malaria pathogen in this region has recently been disproven. Gene diagnosis in our family may contribute to understanding endemic endomyocardial fibrosis.

Results - 

In this family with recurrent SCD with fibrosis in infancy in 3 children, in the face of parental consanguinity, we have identified a homozygous rare variant in one of 3 isoforms of a Krebs cycle/electron transport enzyme. A different Krebs cycle enzyme has been identified to cause a cardiomyopathy with fibrosis in an Amish family, so we think we are on the right track. However, the variant we see in this family is present in the reference sequence of the dog genome. Thus we are not completely certain that it is the culprit. We have frozen tissue available from the heart of one of the affected infants, and we are working with two metabolic expert groups to evaluate the enzyme function in their assays. However, we are a bit held up at present in terms of finding some normal frozen ventricular tissue for control assay.

Diagnosis of focal overgrowth disorders caused by gene abnormalities in the PI3K/AKT/mTOR signalling pathway using a targeted sequencing platform

This project studies rare congenital syndromes of overgrowth resulting in progressive distortion of normal body form as seen in Proteus syndrome, CLOVES, polymicrogyria-polydactyly-hemimegalencephaly (PPMG) and megalencephaly-cutis marmorata (M-CM) syndromes.  In these disorders, an affected newborn has mild asymmetry of body structures; however, as they grow, there is progressive, and at times 'malignant', overgrowth of the affected body segment(s). Those affected develop debilitating and even life-threatening medical problems.   Recently published research has shown that these conditions are caused by specific mutations in genes of the PI3K/AKT/mTOR signalling pathway, occurring early in the developing embryo, result in a mixture, or 'mosaicism', of normal cells and mutated cells.  This leads to 'focal' dysregulated tissue growth involving one or more segments of the body, such as the lower limbs. These identified genes are implicated in many human cancers, highlighting the critical role of this signalling pathway in preventing uncontrolled cell division. 

Treatment for these rare "focal overgrowth syndromes" may now be possible because of current research developments for treatment of cancer targeting mutations in the PI3K/AKT/mTOR pathway.    Making a correct molecular diagnosis will be essential prior to initiating treatment; however, molecular testing is not available clinically for these syndromes.  The causative gene changes are typically present in only a small proportion of cells, and diagnosis by standard techniques is not possible.

We are requesting funding to pilot a project for a novel and custom Next Generation Sequencing (NGS) diagnostic platform targeting all known genes identified to cause focal overgrowth syndromes.  Utilizing the latest NGS technologies, we are able to generate thousands of copies of DNA sequences from a small amount of tissue.  This approach makes feasible the finding of mutations that are 'a needle in a haystack'.  Using these NGS methods, we can correctly and rapidly diagnose patients, even with small amounts of affected tissue obtained from biopsies or surgical specimens.  Preliminary data has shown these methods are successful at detecting mutations to levels of mosaicism as low as 2.5% (personal communication, Dr. Stephen Yip).  We have successfully used this technology to identify a novel mutation in a PI3K-pathway gene in tissue from a biopsy of a hemangioma in an infant with a unique, previously undescribed, focal overgrowth disorder.  Our custom platform builds on the services and methods currently implemented at the Centre for Translational and Applied Genomics (CTAG) at the B.C. Cancer Agency.  With the funding from the Rare Disease Foundation we will test the platform on two additional children with focal overgrowth syndromes.  If we demonstrate that this methodology is effective, we will expand this project to diagnose and potentially treat other affected children referred to our centre.

Impact of Missed or Neglected Sleep Problems in Children with Rare Diseases: A Patient and Care Giver Perspective

Sleep is an essential function for physical and emotional wellbeing, and a pre-requisite for child development (brain maturation).  Amongst children with genetic conditions and rare diseases (RD), sleep problems (SPs) are often missed or neglected, incurring a significant personal suffering for patients and their families.  Some SPs in RD have been investigated extensively (e.g. Mucopolysaccharidosis), and knowledge about the importance of sleep is growing, but the social and emotional impact of missed or neglected SPs on parents (caregivers) and the economic burden (out of pocket costs, productivity loss etc.) has not yet been investigated.  The goal of this study is to investigate the burden of missed/neglected SPs in children with RD from the patient and family perspective. This study runs parallel to an ongoing research project that is investigating the direct economic care costs of missed SPs from a social service use perspective.  The suggested proposal will support parent advocacy initiatives recommending measures to reduce fragmentation of care.

Results - 

Up to 80% of children and adolescents with neurodevelopmental conditions suffer from sleep problems. Treatment strategies for their challenging/disruptive behaviours are typically daytime-focused, and heavily reliant on the prescription of psychostimulants and atypical antipsychotics. Consequently, sleep problems might be missed, misdiagnosed and/or exacerbated by the medications. In order to understand the impact of sleep problems on patients and their caregivers, we interviewed representatives from non-governmental organizations providing services to affected families, as well as a sleep health researcher. Interviewees shared their experiences with sleep problems and their associated symptoms, and offered suggestions on how to improve the current situation. All interviewees highlighted the significance of knowledge dissemination, increased awareness and advocacy, and came up with four starting points for improvement, which are based on the concept of personalized care: (a) medical partnerships; (b) active listening; (c) medical homes; and (d) care guidelines. Furthermore, we suggest the implementation of an outcome-oriented database, which will enable comparisons and ensure the strategic use of quality-of-care benchmarks, thus offering the potential to overcome the current standstill and systemic neglect of sleep. A poster and publication were created out of the research - the publication was unfortunately rejected by the first journal, but after reworking will be resubmitted.

Identifying the cause of congenital myasthenic syndrome and implications for therapy

Congenital Myasthenic Syndromes (CMS) are rare genetic diseases that affect muscular function. Patients are usually newborns or young children who can have devastating effects produced by generalized weakness affecting breathing, motor development, exercise tolerance, and leading to crises caused by infections, fever, or exercise. It is essential to correctly diagnose these conditions in order to avoid life-threatening events, and try to direct appropriate therapy. With the present proposal, we would like to investigate the genetic basis of CMS in a consanguineous Canadian family with two children affected by this devastating disease. The first child has died because of CMS complications. By identifying the cause of this disease in our family, we may be able to offer more clues for targeted therapy and improve the life-expectancy of the second affected child currently under generic intensive-care therapy. Our findings will also help the family with prenatal diagnosis in a future pregnancy.

Results - 

We have identified a strong candidate gene involved in neurotransmitter activity, and at the moment we are working on obtaining further funds to do functional studies in collaboration with two basic researchers here at the University of Alberta. I hope by next year to have results and complete the story, and maybe participate in your Research Day.

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