A Brief Description of Currently Funded Research Grants
2010-2011
Candidate gene analysis of genes located within regions of chromosomes linked to Mabry Syndrome in patients/families (hyperphosphatasia with neurologic deficit and seizures - OMIM # 239300)
Dr. Danielle Andrade
Dr. David Cole
Dr. W. Burnham
Dr. Miles Thompson
University of Toronto
Toronto, Ontario
Summary of Proposed Research
The purpose of the research is to understand the genetics, pathology and interventions possible for the intellectual disability Mabry syndrome characterized by serum hyperphosphatasia. Hyperphosphatasia is the result of a measurable increase in the enzyme alkaline phosphatase in the blood. It occurs during puberty but also during liver disease, cancer and bone abnormalities. We became interested in understanding the cause of hyperphosphatasia that accompanies developmental delay and seizures because it lacked the bone abnormalities we expected. While the disorder was originally reported by Mabry in 1970, it was not until our work, that the bone abnormality, brachytelephelangy (improper growth in the terminal portions of the digits), was noticed. This clinical observation allowed us to revise our definition of the syndrome (also consisting of a characteristic facial dysmorphology, growth retardation, and generalized seizures).
Of relevance to genetics is the fact the hyperphosphatasia trait in Mabry syndrome appears to be dimorphic. Type I Mabry syndrome has an extreme (20-fold) elevation of ALP, whereas type II has a 2-fold ALP increase. Furthermore, due to sibling recurrence and evidence of consanguinity – we conclude that ALP elevation is inherited in an autosomal recessive manner (requiring the inheritance of one mutation from each parent). Our work with the Dutch group lead by Han Brunner and a German group lead by Stefan Mundlos is – submitted to Nature Genetics – has already identified the gene for type I. The proposed work on type II will be a self contained effort, however, focusing on the type II cases we have recruited and published with collaboration from Dr. Brunner and Dr. Mabry himself.
The present study will: 1) identify the biochemical and clinical consequences of the gene mutated in type I Mabry syndrome that we have already identified, and 2) identify the gene or genes for the type II syndrome in the remaining 80% of cases.
The research plan is to identify mutations that cause elevated ALP in Mabry type II. We anticipate that the gene responsible for type II in our patients will belong to the pathway of ALP regulation disrupted in type I. Type I Mabry’s results from a mutation in the PIGV gene that disrupts the production of the phosphoinositol glycan (PIG) tether for ALP – the component that regulates ALP secretion into blood (Appendix 6). Mutations in an unknown gene or genes for type II Mabry syndrome, therefore, may disrupt others among the 10 genes that encode enzymes critical to PIG biosynthesis. These genes will be investigated in a clinical & biochemical context.
Proof of principle. Other neurological disorders such as paroxysmal nocturnal hemoglobinurea (PNH) and inherited GPI deficiency are caused by mutations in genes related to PIG (PIGA and PIGM, respectively). Since these conditions also result in blood cell abnormalities, we will also study blood cells for the presence and absence of PIG anchored proteins such as CD59 and CD25.
The anticipated outcome is that we will identify mutations in at least two of the biosynthesis enzymes for the PIG anchor. This will allow correlation of the phenotypic variability present in Mabry syndrome with the mutations we identify in the 10 gene pathway. Current data suggests that type II Mabry syndrome may map to two chromosomal regions that contain genes of interest because they are integral to PIG anchor biosynthesis. One gene, on chromosome 1p31, is close to the PIGV gene implicated in type I. This region is of interest since it encodes the PIGK enzyme. Another ‘hot spot’ we identified is on chromosome 2p22 – a region that includes another PIG biosynthesis enzyme: PIGF. This project will result in better knowledge of the function of the genes encoding the PIG biosynthesis enzymes. We argue that identifying the genes mutated in Mabry’s will yield insight into the biochemistry of the PIG anchor as resulted from identifying PIGA & PIGM mutations in PNH & GPI deficiency.
The projected benefits and application of findings include identifying the consequences of elevated alkaline phosphatase (ALP) or hyperphosphatasia. We believe that this work will have clinical relevance since we will map the consequences of disrupting two or more of the ten genes that control the biosynthesis pathway of the PIG anchor. Once the causal genes are known, potential treatments will be possible that consider the gene disrupted or the cell damage observed. For example, we will be able to extend our previous finding that vitamin B6 can relieve seizures in some Mabry cases – with respect to the genetics of types I & II. More effective counseling regarding parental & childhood therapeutic choices will be possible.
Behavioral and physiological assessment of lateral neural interactions in autism
Dr. Armando Bertone
Dr. Dave Saint-Amour
Hôpital Rivière-des-Prairies
Montreal, Quebec
Purpose of research
Autism spectrum disorder is one of the most prevalent disorders of childhood. It is diagnosed based on the absence of behaviors usually present during social interaction and communication, as well as atypically present restricted interests and repetitive behaviors. Although the origin of autism is unknown, it is believed that its causal origins are genetic, pervasively affecting different aspects of development. Relative to other neurodevelopmental conditions, many characteristic behaviors manifested by persons with autism are related to the visual perception, exemplified by either atypically absent (i.e., lack of interpersonal eye contact) or present (i.e., preoccupation with flickering or spinning objects) mannerisms. The purpose of the proposed research is to better understand and define underlying differences in the autistic visual brain that may explain some of the behaviors often presented in this condition.
Proposed research plan
Although there is much evidence suggesting that the perception of cognitive and social (i.e., faces) information is atypical in autism, there is little knowledge about whether these difficulties are the result of differences at early levels (i.e., visual) of brain functioning. The proposed research aims at defining whether the visual brain of high-functioning persons with autism is wired the same way as persons without autism. There is reason to suggest that the higher-level, cognitive and behavioral characteristics that define autism may at least in part be the due to the different manner with which autistic persons perceive visual information. In the proposed studies, we assess a specific hypothesis that suggests that connectivity within early visual areas in autism is different, consequently resulting in an atypical internal representation of the external visual world that ultimately affects how persons with autism interact with their social environment. We propose to assess this hypothesis by measuring the ability to detect simple visual patterns, and consequent brain activity to such patterns that are processed by early visual areas of high-functioning adolescent and adult persons with autism.
Anticipated outcome
It is expected that the functioning of early visual brain areas in autism will differ from neurotypical participants at both behavioral (sensitivity) and brain (brain activity) levels. This hypothesis is based on indirect evidence suggesting that the lateral connections within early visual brain areas may be responsible for atypical visually-related cognitive abilities and social behavior. The expected results may also have implications regarding the etiology of autism as the mechanisms expected to be wired differently in autism are dependent on the transmission of important chemicals involved in several aspects of brain functioning.
Projected benefits and applications of findings
Recent studies have suggested that the prevalence of autism spectrum disorders is rising. Although this notion needs validation, the fact remains that individuals with autism are among the most difficult and costly to treat, and the impact their families in terms of quality of life is enormous and sometimes devastating. If effective intervention strategies are to be improved and/or developed, an accurate and objective characterization of sensory and perceptual processing in autism is clearly needed. This is because many current intervention approaches assume that perceptual abilities and strategies in autism are intact. Understanding the nature of perceptual deficit and strengths in autism is therefore crucial for the improvement of such approaches.
High Resolution Study of MeCP2 Isoforms in Neurons for Therapy Applications in Rett Syndrome
Dr. Mojgan Rastegar
University of Manitoba
Winnipeg, Manitoba
Introduction: One in every 150 individuals suffers from autism, a neurological disease with an early childhood onset. Autism Spectrum Disorders (ASD) refer to different forms of autism characterized by harmful changes that take place in the brain as it grows and develops. Rett Syndrome (RTT) is the best studied example of autism and the primary cause of mental retardation in females (1:10,000). RTT patients are born and develop normally up to 6-18 months of age when they start to show symptoms including loss of speech and purposeful hand movements, mental retardation, learning disabilities, seizures, respiratory abnormalities, anxiety and autism. RTT results from mutations in the Methyl CpG Binding Protein 2 (*MECP2) gene and does not have any treatment. However, in RTT mice, reactivation of the *Mecp2 gene after the onset of disease rescues RTT phenotype. This demonstrates the possibility of RTT gene therapy strategies in neurons, where delivering MECP2 into the affected neurons may indeed improve RTT symptoms. Alternatively, drug treatment strategies can be designed to target proteins, capable of compensating for MeCP2 loss in neurons. Two *MeCP2 protein variants exist; MeCP2E1 and E2, which are both widely expressed. However, RTT symptoms are mainly neuronal, and MeCP2 isoform-specific expression and function in neurons has not been determined.
*MECP2: human gene; Mecp2: murine gene; MeCP2: human/ murine protein
Purpose of the Research: We aim to perform a comprehensive study of MeCP2E1 and E2 expression, sub-cellular localization and function in neurons. We will compare the rescue effect of MECP2E1 and E2 lentiviruses through gene therapy delivery into Mecp2 deficient neurons of a RTT mouse. We are generating novel tools specific to study sub-cellular localization of MeCP2E1/E2 and will compare them to selected functional competitors, aiming to identify proteins with potential to compensate for MeCP2 loss in neurons. Our data will ultimately help to develop novel RTT therapeutic strategies.
Proposed Research Plan (Hypothesis to be tested and Specific Aims): We will use a combination of advanced stem cell biology and gene therapy technologies to perform a high-resolution study of MeCP2E1/E2 functions and their targets in neurons. Our aim is to validate our gene therapy vectors and further identify candidate proteins to compensate for MeCP2 loss in neurons. We hypothesize that both MeCP2 isoforms are induced during differentiation of neural stem cells and aim to confirm this with our novel tools and antibodies. We hypothesize that upon expression of our existing MECP2 gene therapy vectors similar post-translational modifications occur as compared to the endogenous MeCP2, and that we will find a neuronal-specific signature for MeCP2 sub-cellular localization compared to particular functional competitors and partners. We propose the following three specific aims:
Aim1-To study MeCP2 isoform-specific expression in differentiated neural stem cells (NSC).
Aim2-To examine neuronal-specific MeCP2 localization compared to selected competitors/ partners.
Aims 3-To compare rescue effect of MeCP2E1/E2 gene therapy vectors in Mecp2 deficient neurons
Anticipated Outcome: I expect to a) find both MeCP2E1/E2 are induced during NSC differentiation, b) detect a differential co-localization of MeCP2 protein with its partners/ functional competitors leading to a specific MeCP2 signature in neurons when compared to other differentiated cells, c) confirm restoring the expression of MeCP2 known target genes in Mecp2 deficient neurons using our gene therapy viruses. However, the two isoforms may show redundant or different functions. Our research will reveal the redundant or complementary role of MeCP2 isoforms in neuronal maturation and may serve as a model for therapy strategies of other Autism Spectrum Disorders. I believe that the outcome of our research will have a significant impact on developing novel RTT therapeutic strategies and will lead towards a better understanding of the pathobiology behind Rett Syndrome and autism.
Projected Benefits and Applications of Findings: The proposed studies will address fundamental unanswered questions on the dynamics of MeCP2 expression and role during neuronal differentiation, thereby highlighting the expression signature of MeCP2 isoforms in neurons, with applications in future therapies of autism and other neurological disorders. Our findings, related to the expression pattern of each isoform and our studies on their relation compared to MeCP2 partners/ functional competitors, will contribute to the development of innovative therapeutic approaches, and in finding candidate genes capable of compensating MeCP2 loss and reversing RTT phenotypes in neurons.
Using Phenotypic Information to Identify Novel Pathogenic Alleles Overlapping Rare Inherited Copy Number Variants (CNVs) in Autism Spectrum Disorders
Dr. Marc Woodbury-Smith
St. Joseph's Healthcare Hamilton
Hamilton, Ontario
Autism is a severely disabling disorder of brain development characterised by severe difficulties with interpersonal skills and communication and ritualistic patterns of behaviour. A range of symptoms is seen necessitating the term autism spectrum disorders or ASDs. It has long been known that autism is caused primarily by genetic factors, but despite significant efforts over the last 20 years the autism genes have not been found. The need to identify susceptibility genes has become more urgent than ever now as more individuals are being diagnosed, with as many as 1 in 100 of the population affected. Moreover, being a lifelong condition, with few effective treatments, the burden on healthcare is huge.
Purpose of the research: The purpose of this research is to identify novel genetic alleles significantly associated with the ASDs.
Proposed Research Plan: In order to do this, the study has two important steps. First, we will identify families for which a rare genetic abnormality has been transmitted from an apparently unaffected parent to their child with ASD. We will examine the clinical characteristics of these families in detail in order to identify features that are associated with the genetic abnormality. Next, we will use these characteristics to subgroup a much larger sample of individuals with ASDs, who will then undergo analysis to determine whether they share a genetic abnormality in the same location as those with the rare genetic abnormality.
Anticipated Outcome:
• We expect to find that certain clinical characteristics will be shared between ‘unaffected’ parents and their ASD children,
• We expect that these characteristics will define a subgroup of the autism population more generally who have pathogenic alleles at the same location.
Projected benefits and applications of findings:
• This will further our understanding of the important role genetics plays in Autism Spectrum Disorders;
• By identifying the susceptibility genes and understanding their impact on brain development and function, the capacity to find novel treatments and a cure remains a real possibility;
• This study will afford significant capacity to develop a comprehensive understanding of the pathogenesis of the ASDs, and inform the development of therapeutic algorithms to alleviate the symptoms and/or cure the disorder.