Louis Kunkel, PhD - US grants

The region Xp21 of the human genome will reduced to an ordered array of cloned DNA segments by chromosome "walking" from multiple start points from within the region. The locus responsible for Duchenne muscular dystrophy known to lie within Xp21 will be identified and the product encoded by this locus characterized. The manner in which the product of the DMD locus is altered in patients will be determined by direct analysis of the DNA of the affected individuals. Mutations which disrupt normal function will be used as indicators of where the normal product from the locus functions. Structural characteristics of the Xp21 region which might affect normal expression from the locus will be studied with special emphasis given to possible mutational instablity of the region. The genetic distance of the entire region will be precisely determined and meiotic exchange points identified. Accurate prediction of individuals at risk for the disease will result by either direct analysis of the locus or indirect analysis with tightly linked markers. Ultimately, the pathways in neuromuscular development will be identified which are altered in Duchenne muscular dystrophy patients and possibly patients with other neuromuscular diseases.

We propose a program of research on the localization and analysis of genetic determinants on human chromosomes, as well as subchromosomal structural changes underlying diseases, with emphasis on the human X, #15, #21, and other regions of the genome in which abnormalities can be associated with mental retardation and/or developmental defects. The research planned utilizes techniques which allow intense study of subchromosomal regions, such as fluorescence activated chromosome sorting and phenol-enhanced differential hybridization, recombinant DNA methodology, cytogenetics, somatic cell genetics and pedigree analysis. The goal is to obtain, localize, and characterize cloned DNA segments, as well as deleted, duplicated, rearranged, or labile subchromosomal regions associated with specific diseases. Using Xp21 deletions in DMD as a prototype and starting point, we will explore adjacent regions and deletions responsible for glycerol kinase deficiency, the McCloud phenotype, as well as X- linked eye diseases such as retinitis pigmentosa, retinoschisis, and choroideremia, the last associated with a deletion in proximal Xq. Structural analysis of Xp21, as well as distal Xp and Xp will be used to study normal and abnormal meiosis and the role of the latter in deletion-related diseases, insertion of a Yp segment into Xp in 46,XX males, and aneusomy. Recombination based screening will be used to obtain molecular probes near the fragile site in distal Xq to study its structural features and unusual mode of mutation. Existing and newly sought molecular probes will be used to analyze MR-associated diseases in 12p and proximal 15q1, in the case to understand better the nature and causes of structural changes associated with the Prader-Willi syndrome, as well as to refine diagnosis of this disease. Other genomic probes, as well as antibodies, will be used to isolate cDNA sequences mapping to chromosome #21 to investigate abnormalities, related to Down syndrome, of tissue-specific #21 DNA expression, e.g. as regards neuronal organization and cell surface changes underlying developmental defects. Of particular interest will be #21-linked amyloid beta protein cDNA and the relationship of amyloid to premature aging in Down syndrome and to Alzheimer disease. The information sought should both be of basic mechanistic interest, and it should translate readily into practical molecular genetic diagnostic applications.

Over the last few years there have been a few commercial DNK sequencers available but each has had problems in operation or accuracy. The ABI sequencer has been rated the best overall by those who have used it. In addition the manufacturer has developed the greatest improvements in technology. During this same period the need for rapid and accurate generation of DNA sequence has become more apparent. Each of the listed NIH funded research proposals have a DNA sequencing component which might be handled manually but would be greatly facilitated by a central automated facility. Not only will combined use make efficient utilization of the high volume capacity of the automated sequencer but by having an automated facility each investigator will be assured of constant and accurate sequence information. By keeping one individual in charge of the facility the performance can be kept constant and accuracy maintained. This shared major equipment purchase will enable a facility to exist for multiple lisers where it would be impossible to justify based on the use by one investigator.

Dystrophin is a large molecular weight component of the muscle and nerve cell cytoskeleton. Disruption of normal function leads to the genetic disorders Duchenne and Becker muscular dystrophy. Deletion mutations are the most common means to disrupt function but other mutations are known to exist. The proposed characterization of these other mutations will greatly increase the accuracy of diagnosis for at risk individuals and should shed additional light on how normal function can be affected. Dystrophin has been shown to be a member of a family of related proteins with overlapping yet discreet functions. Some of these related proteins may partially compensate for absent dystrophin and hence represent potential candidates for therapeutic intervention to strengthen the compensatory ability. These related proteins are themselves prime candidates to be disrupted in other neuromuscular diseases which, like Duchenne and Becker dystrophy, are slowly progressive. New members of the family will be cloned, mapped to chromosomal locations, and tested for disruption in candidate neuromuscular diseases. Functional analysis of these proteins should shed light on their role in the membrane cytoskeleton and how this role is altered in disease. This should result not only in the identification of other disease causing genes, but also proteins with the potential to mitigate the absence of dystrophin in Duchenne dystrophy.

nucleic acid sequence; genetic disorder diagnosis; biomedical facility; tissue resource /registry; developmental neurobiology; mental retardation; gene mutation; molecular genetics; linkage mapping; genotype; computer assisted sequence analysis; human genetic material tag; human tissue;

photography; information dissemination; biomedical facility; computer graphics /printing; digital imaging; publications; developmental neurobiology; information display; informatics; videotape /videodisc; mental retardation; behavioral /social science research tag; human data;

PROGRAM DESCRIPTION (provided by applicant): The last decade has witnessed remarkable progress in defining primary defects that cause inherited muscle disorders. The genetic heterogeneity of these diseases is enormous; mutations in more than 40 different genes are implicated. Many critical questions remain concerning the pathogenesis of muscle cell degeneration in these diseases and strategies for their treatment. This Program Project will use classical methods of gene and protein analysis and state-of-the-art gene expression array technology to study these questions. The investigators in this program have contributed importantly to the muscular dystrophy field. The proposed 4 projects have unique features but overlapping concepts and methodologies. Project 1 will study the dystrophin-associated complex of proteins, emphasizing the sarcoglycans and the newly described filamin-C. Project 2 will investigate the biology of dysferlin, its potential protein partners, and how these are altered by dysferlin gene mutations. Project 3 will examine the function of myotubularin in normal muscle development and the mechanisms by which its mutations cause developmental myopathies. Project 4 will study the biological and therapeutic properties of muscle stem cells. Three Cores will provide administrative oversight and services essential to the smooth progression of this program. Core B will coordinate sample acquisition and muscle RNA preparation for each project. Core C will perform the microarray analysis of gene expression and provide expertise in bioinformatics and data interpretation. The aim is to identify patterns of gene expression that are global in all dystrophies or distinct to specific sets of dystrophies and myopathies; this will provide insight into the molecular basis of normal muscle development and its dysfunction in these disease states. The long-term goal is to use this information in conjunction with the insights from studies of stem cell biology to devise new approaches to the treatment of the muscular dystrophies and related myopathies.

DESCRIPTION: The Cell Sorter Core offers Center investigators access to high quality flow cytometry and cell sorting. It also provides expertise to investigators concerning the best approaches to sample preparation and staining techniques. In addition, Core personnel can assist investigators with data interpretation and presentation.

DESCRIPTION: The Molecular Genetics Core provides basic genomic services of DNA sequencing and genotyping to Center investigators, with the new addition of expression profiling using microarray technologies. Economies of scale and consolidation of expertise would be utilized to provide fundamental genome-based information to the Center investigators.

DESCRIPTION: The Multimedia Core provides conventional photographic support, digital imaging services, and advanced digital multimedia and web-based services. The Core is directed by Dr. Louis Kunkel, who provides almost daily input, and the Director of Operations, Mr. Dixon Yun, who has supervised this facility for 20 years. There is quarterly input from the Internal Advisory Board. The Core operates on a fee for service basis with fees proportional to the amount of time or item (e.g., slide or print) requested.

Over the last 5 years, we have been working to establish the zebrafish as a model for muscular dystrophy. In this capacity, we have published the phenotype of zebrafish lacking dystrophin and 8- sarcoglycan, completed a large genetic screen to isolate additional dystrophic mutants, and identified the mutant gene in the runzelmutant. Using our experiences in muscle research and in establishing the zebrafish as a disease model, we now propose to use the fish to investigate the pathogenesis of muscular dystrophy and evaluate cell therapy as a potential treatment option. The first aim in this project proposes to fully characterize two of our available dystrophic zebrafish models (emz and sof) with the goal of better understanding the pathogenesis of muscular dystrophy. These mutants show a muscle degeneration phenotype very similar to the dystrophin mutant (sapje) suggesting that this phenotype is symptomatic of muscular dystrophy. We propose to identify the genetic mutations in these mutants using a traditional mapping approach and then sequencing candidate genes to identify the specific mutation. If the orthologous human genes are not currently associated with muscular dystrophy, these genes will be considered disease candidate genes and sequenced in human patients for which the cause of muscular dystrophy is unknown. Since mutations in seemingly unrelated proteins can manifest as muscular dystrophy, the identification of additional genes would be helpful for establishing disease pathways. Secondly, we have established methods to transplant cell populations in zebrafish at all developmental stages and now propose using this system to identify the cell population most capable of engrafting into and correcting the diseased muscle. Gene expression profiles of muscle engrafting cell populations will be compared with non-engrafting cells to identify genes expressed predominantly in the engrafting cells. Differentially expressed genes will be considered potential markers and used to purify analogous cell populations in mammals for future experimentation and therapy. Finally, we plan to dissect the lineage relationship of various stem cell populations by assaying the developmental potential of zebrafish muscle progenitor cells. This will be accomplished by transplanting limited populations of labeled cells early in development and then following their fate as the fish matures.

Description (provided by applicant): Over the last 5 years, we have been working to establish the zebrafish as a model for muscular dystrophy. In this capacity, we have published the phenotype of zebrafish lacking dystrophin and 5- sarcoglycan, completed a large genetic screen to isolate additional dystrophic mutants, and identified the mutant gene in the runzel mutant. Using our experiences in muscle research and in establishing the zebrafish as a disease model, we now propose to use the fish to investigate the pathogenesis of muscular dystrophy and evaluate cell therapy as a potential treatment option. The first aim in this project proposes to fully characterize one of our available dystrophic zebrafish models with the goal of better understanding the pathogenesis of muscular dystrophy. We have selected the emz mutant for further analysis since its mutation rough maps to a genomic interval void of any genes orthologous to those currently associated with muscular dystrophy. This mutant shows a phenotype very similar to the dystrophin mutant (sapje) suggesting that the emz phenotype of muscle degeneration is symptomatic of muscular dystrophy. We propose to identify the genetic mutation in this mutant using a traditional mapping approach and then sequencing candidate genes to identify the specific mutation. If the orthologous human gene is not currently associated with muscular dystrophy, the gene will be considered a disease candidate and sequenced in human patients for which the cause of muscular dystrophy is unknown. Since mutations in seemingly unrelated proteins can manifest as muscular dystrophy, the identification of additional genes would be helpful for establishing disease pathways. Secondly, we have established, methods to transplant cell populations in zebrafish at all developmental stages and now propose using this system to identify the cell population most capable of engrafting into and correcting the diseased muscle. Gene expression profiles of muscle engrafting cell populations will be compared with non-engrafting cells to identify genes expressed predominantly in the engrafting cells. Differentially expressed genes will be considered potential markers and used to purify analogous cell populations in mammals for future experimentation and therapy. Finally, we plan to dissect the lineage relationship of various stem cell populations by assaying the developmental potential of zebrafish muscle progenitor cells. This will be accomplished by transplanting limited populations of labeled cells early in development and then following their fate as the fish matures.

Cell Cycle; Cell Division Cycle; Cell Isolation; Cell Segregation; Cell Separation; Cell Separation Technology; Cell surface; Cells; Cytofluorometry, Flow; DNA Content; DNA Index; DNA Ploidy; Data; Flow Cytofluorometries; Flow Cytometry; Flow Microfluorimetry; Fluorescence; GFP; Green Fluorescent Proteins; Human Resources; Investigators; Life; Manpower; Measurement; Methods and Techniques; Methods, Other; Microfluorometry, Flow; Ploidies; Preparation; Research Personnel; Researchers; Sampling; Services; Sorting - Cell Movement; Staining method; Stainings; Stains; Techniques; Training; base; cell sorting; chromosome complement; flow cytophotometry; personnel; sorting; stem

DESCRIPTION (provided by applicant): Painful bladder syndrome/interstitial cystitis (PBS/IC) is a chronic, debilitating clinical syndrome presenting as severe pelvic pain with extreme urinary urgency and frequency in the absence of any known cause. The etiologic mechanisms underlying PBS/IC are unknown, but recurrence risks to siblings of affected individuals, concordance among monozygotic twins, and our own preliminary studies indicate a strong genetic contribution to the cause of PBS/IC. The overall goal of this proposal is to identify the genes containing mutations that result in PBS/IC and determine how the different encoded proteins of these genes interact with one another in a common biological pathway. Ultimately, understanding how mutations in up to five different genes yield the symptoms of PBS/IC should lead to improved diagnosis and possible therapies. We plan to attain our goals via the following specific aims: 1) Accurately characterize PBS/IC patients and their family members and enroll them in our genetic studies. Carefully evaluated cohorts of patients and their families are essential to the discovery of the genetic variation underlying PBS/IC. 2) Map the locations of PBS/IC genes by linkage analysis in families in which PBS/IC is segregating. We have already recruited several families with autosomal dominant inheritance of PBS/IC and identified linkage peaks in the most informative pedigrees. 3) Identify within linked regions of the genome the first genes that control inherited risk of PBS/IC and correlate the types of mutations with clinical symptoms. 4) Determine how proteins encoded by these PBS/IC genes interact with one another. Such knowledge should yield a better understanding of the biochemical or developmental pathways leading to PBS/IC. PUBLIC HEALTH RELEVANCE: Painful bladder syndrome/interstitial cystitis (PBS/IC) is a severe, chronic, debilitative clinical syndrome with pelvic pain, extreme urinary urgency and frequency without a known underlying cause. This proposal should lead to a better understanding of the genetic components of the disorder and the underlying biochemical and developmental pathways leading to PBS/IC. Such knowledge could lead to improved diagnosis and rational design of therapeutic interventions.

MOLECULAR GENETICS CORE (CORE F) ABSTRACT The fields of molecular genetic and genomic technology, instrumentation and data handling/analysis have undergone revolutionary changes in the previous 5 years of the IDDRC grant to Boston Children's Hospital. Many of these changes, including a dramatic reduction in price, has made exome or genome sequencing within reach of individual laboratory investigators and clinicians. It is now possible to obtain exome/genome sequences on families or individuals with intellectual and developmental disabilities and determine the underlying genetic cause of the problem-- leading to more accurate diagnoses and targets for therapeutic intervention. The technology and interpretation of the output data is very complicated, and is often beyond the expertise of individual laboratories or clinicians. The Boston Children's Hospital's IDDRC Molecular Genetics Core laboratory has evolved with these revolutionary changes and represents a central location to which IDDRC investigators can obtain advice on project design and outsourcing to the best and least expensive vendors. Additionally, IDDRC investigators can receive training and assistance in analyzing the resulting data, such that they will have a smooth path to new gene discovery. This benefits both their NIH supported research as well as individual patients and their families. Some investigator needs have not changed over the past 5 years. There is still the need for confirmatory sequence verification, as well as advice on functional studies. The demand for our economical array-based transcriptome studies and PCR analysis continues to grow. Sometimes laboratories or cores can collaborate on projects, saving time and money for all parties, but need assistance on how to find a suitable partner. The Molecular Genomics Core facilitates all of the above and more; all of these functions are combined in one central laboratory available easily to all IDDRC investigators and their lab members. This produces an economy of scale and a place for training and collaboration. The Molecular Genetics Core Laboratory will emphasize the following specific aims: Aim 1: Provision of general design, implantation, and interpretation of IDDRC studies. Aim 2: Obtaining the best available price, quality, and turnaround time for Next Generation Sequencing (NGS) for the study of IDD/Neurodevelopment. Aim 3: Producing high quality Array and other Services on demand. Aim 4: Training and intellectual development for our researchers and clinicians. Aim 5: Assisting in collaborations within and outside the Boston Children's Hospital IDDRC.

The Autism Spectrum Disorders (ASD) encompass a spectrum of disorders which include autism, Pervasive Developmental Disorder - Not Otherwise Specified (PDD-NOS), Asperger"s disorder and Rett syndrome. The working hypothesis of this proposal is that individuals with ASD experience disturbances in the intricate interplay between genetic predisposition, environmental triggers and experience-mediated neuronal activity during a sensitive period of development, and that this leads to an altered program of gene expression. Thus the neurological disabilities that are characteristic of the disorder result from genetic variability in the processes of synaptic development, maturation, refinement, and connectivity that normally shape the brain, and from an environmental insult that perturbs normal experience-dependant synaptic development. This proposal offers a novel approach aimed at unraveling the genetic mechanisms, improving diagnosis, deciphering the interplay between environment and genetics, and developing rational approaches to therapy.

PROJECT SUMMARY/ABSTRACT Duchenne Muscular Dystrophy (DMD) is a degenerative muscle wasting disease caused by mutations in the dystrophin gene. The absence of dystrophin protein in muscle results in dysregulated secondary signaling pathways, many of which remain poorly understood. The laboratory of Dr. Mayana Zatz has identified two dystrophin-deficient dogs from a litter of Golden Retriever Muscular Dystrophy (GRMD) dogs that are mildly affected and escape the early lethality and dystrophic phenotype (muscle wasting) that normally occur in severely affected GRMD dogs. Our microarray analysis of these dogs and other GRMD and control dogs, revealed a strong reduction of the Pitpna gene, which is a known regulator of the PTEN/AKT signaling pathway. In parallel studies using microRNA microarrays performed on human biopsies from patients with DMD, we identified a microRNA, miR-486, that is exclusively reduced in DMD muscle and has been shown by us and others to alter PTEN/AKT signaling. We hypothesize that either overexpression of miR-486 and/or inhibition of Pitpna results in decreased PTEN levels while subsequently increasing phosphorylated (activated) AKT, which might lead to reduced severity of disease course. The induction of phosphorylated-AKT (resulting from miR-486 and Pitpna modulation), are predicted to affect several downstream target genes of this pathway which will result in an increase in muscle hypertrophy, muscle structural genes vascularization genes, the increase in the neuromuscular junction number of branch points, inhibition of apoptosis genes, and validated PTEN/AKT downstream target muscle genes (such as Utrophin). We plan to approach our long-term goal of understanding how these two alter signaling pathways in DMD by modulating expression of miR-486 and/or Pitpna in the mouse (mdx5cv) model according to the following three specific aims: 1) Characterization of miR- 486 and Pitpna regulation of PTEN/AKT signaling pathway in vitro. 2) Manipulation of miR-486 expression to modulate PTEN/AKT signaling in dystrophin-deficient muscle for modeling of future therapeutics. 3) Generation of mice with reduced Pitpna levels in muscle and characterization of its effects on PTEN/AKT signaling.

PROJECT SUMMARY/ABSTRACT Duchenne Muscular Dystrophy (DMD) is a degenerative muscle wasting disease caused by mutations in the dystrophin gene. The absence of dystrophin protein at the muscle sarcolemma results in increased muscle susceptibility to contraction-induced damage as well as dysregulation of secondary signaling pathways, many of which remain poorly understood. Despite advances in treatment strategies under investigation aimed at restoration of dystrophin expression including viral delivery of mini-dystrophin, read-through of translation stop codons, and exon skipping to restore the reading frame, there is no cure for DMD, and the identification of therapies that improve pathology independent of dystrophin would be of significant value to patients. In this vain, genetic modifiers of DMD are emerging as potential therapeutic targets. Our lab recently identified Jagged1 and Pitpna as genetic modifiers of DMD pathology in a Golden Retriever Muscular Dystrophy (GRMD) dog colony in which two exceptional ?escaper? dogs exhibited a drastically milder phenotype than typical GRMD dogs despite being dystrophin-deficient. Normally, GRMD dogs show a severe phenotype similar to human DMD including early progressive muscle degeneration, fibrosis, and premature death often within the first 2 years of life due to cardiopulmonary failure. Gene expression analyses of the escaper dogs compared to severely affected GRMD dogs and control animals revealed that Jagged1 overexpression (OE) and decreased Pitpna expression were hallmarks of the mild phenotype, which including maintained ambulation and normal lifespan. In subsequent genetic studies of dystrophin-deficient zebrafish, we demonstrated that modulation of Jagged1 and Pitpna prevents manifestation of the dystrophic muscle phenotype, increases long-term survival, and improves swim performance. In primary myoblasts derived from normal and DMD patients, we also showed that Jagged1 overexpression and Pitpna inhibition impact AKT/PTEN signaling and improve myoblast fusion. Given this positive data, we will now extend our research of Jagged1 and Pitpna modulation into the well-characterized mdx5cv mouse model of DMD. We will also investigate genetic and pharmacological inhibition of PDE10A, which we have shown to elicit decreased Pitpna expression and improve dystrophic pathology in dystrophin-deficient zebrafish. The long-term goal in this project is to assess and validate the therapeutic potential of the genetic modifiers Jagged1 and Pitpna to ameliorate DMD pathology, and to identify a viable pharmacologic modulator to advance into clinical studies. To accomplish this, we will implement experiments in accordance to the following three specific aims: 1) Determine the effect of reduced Pitpna expression on dystrophic pathology in mdx5cv mice, 2) Characterize the functional role of Jagged1 overexpression in mdx5cv mice, and 3) Assess the therapeutic potential of PDE10A inhibition on dystrophic pathology in primary human muscle cells, zebrafish, and mouse models of DMD.