Stephan Zuchner - US grants

DESCRIPTION (provided by applicant): Hereditary neuropathies of the Charcot-Marie-Tooth (CMT) type comprise the most common inherited neurological disorders and are genetically heterogeneous. The principal investigator and 1 co-investigator on this application have recently identified the mitochondrial fusion factor Mitofusin 2 (MFN2) as a cause for CMT type 2A (CMT2A), the most frequent (=20%) axonal form of hereditary peripheral neuropathies. MFN2 plays a significant role in maintaining the fusion/fission balance for mitochondria. However, how MFN2 mutations lead to a human disease is unknown. In MFN2 knock-out mice the -/- mice died in utero, while the mice showed no signs of neuromuscular disease. These results may indicate loss of function effect for this autosomal dominant disorder. In the light of the success of the PMP22 mouse for demyelinating neuropathies, we think it is important but apparently not easy, to have a mouse model available for future studies of axonal neuropathies. This application aims to combine human genetics with cell biology in order to develop a transgenic mouse model mimicking the human disease. Such a mouse model, based on mutations found in CMT patients, could potentially gain high importance for several reasons: 1) The pathophysiology of MFN2 dysfunction in relation to neuropathies is unknown, although involvement of mitochondrial dysfunction in neuromuscular diseases is well recognized., 2) Axonal neuropathies in general are more frequent then demyelinating forms, but a mouse model for the most common CMT2 form, CMT2A, is missing. 3) There is no treatment available for axonal CMT patients, but recent studies based on mouse models for demyelinating neuropathies revealed for the first time promising results for future treatment.

DESCRIPTION (provided by applicant): Hereditary Spastic Paraplegia (HSP) comprises a group of neurodegenerative disorders characterized by progressive spasticity of the lower limbs and has fascinating clinical and pathophysiological overlap with amyotrophic lateral sclerosis (ALS), hereditary motor neuropathies, and axonal neuropathies. HSP is genetically heterogeneous with autosomal dominant, autosomal recessive, and X-linked forms. Genes have been identified for only 14 of the 30 reported HSP chromosomal loci. The identification and molecular characterization of additional HSP genes is key to improve our understanding of the underlying pathophysiology. Thus, we will strive to identify the underlying gene of the well-defined HSP locus on chromosome 19q (SPG12). We will have access to DNA samples from all known linked SPG12 families through collaborations with investigators in France (Dr Alexandra Durr), Italy (Dr. Antonio Orlacchio), and the UK (Dr. Evan Reid). An identified SPG12 gene will be included in our mutation screening and functional studies. The indispensable basis of our successful HSP genetic program has been the collection of HSP families over more than 15 years. This program has recently been moved with the principal investigators to the University of Miami. The expansion and clinical improvement of this collection is the basis of our genetic and molecular HSP research. Recently we have identified the underlying gene for the SPG31 locus, receptor expression enhancing protein 1 (REEP1), which appears to represent 6.5% of all HSP patients making it the third most common HSP gene after spastin and atlastin. We will clinically study the REEP1 families in more detail and screen for additional REEP1 mutations in a sample of 370 HSP patients collected by Dr. de Jonghe, University of Antwerp. Finally, we propose the molecular characterization of the mutations identified in the novel mitochondrial protein REEP1 and possibly in the SPG12 gene. Dr. Moraes is a world-renowned specialist for mitochondrial disease mechanisms and is a new investigator on this grant specifically to address the third aim. We strongly believe that only such an integrated approach - clinical, genetic, and molecular/functional - will yield significant progress in the understanding of HSP.

ABSTRACT: The various rare forms of hereditary peripheral neuropathies are known as Charcot-Marie-Tooth (CMT) disease and comprise a clinically and genetically heterogeneous set of neurological disorders. Traditionally, the disease is divided into demyelinating CMT1 forms with decreased nerve conduction velocities (NCV) and axonal CMT2 types with normal NCVs. More than 35 different genes have been identified for CMT and include autosomal dominant, recessive and X-linked forms. Still, only 40% of axonal (CMT2) cases currently have a mutation in one of the known genes. Importantly, the degree of phenotypic variation of severity, age-at-onset, and other measures within families is quite remarkable, yet genetic modifying factors have not been identified. Modifying factors in CMT families are likely targets for intervention and may well be important for other non-hereditary peripheral neuropathies, such as diabetic neuropathy. These studies have proven difficult however, primarily due to a lack of collections of patients with consistent clinical evaluations and their DNA. The proposed Rare Diseases Clinical Research Consortia (RDCRC) will substantially enhance our resources and quickly provide a large number of CMT patients and families evaluated with the same clinical severity score. A new generation of genetic tools is now available to tackle these important questions, such as genome-wide association studies and next-generation sequencing technology. These will also be applied to identify additional CMT2 genes in small pedigrees using innovative approaches.

DESCRIPTION (provided by applicant): Next-generation sequencing technology is opening up new opportunities to rethink the way we identify disease causing genetic variation. An early application, whole exome sequencing, has now been established by a small number of research labs, including ours. Exome sequencing allows obtaining a near complete set of protein coding genomic variation in single individuals for less than $5,000. Promising targets for exome sequencing studies are Mendelian diseases, such as hereditary spastic paraplegias (HSP). HSP comprise a genetically very heterogeneous set of neurological disorders with currently 39 different HSP chromosomal loci being reported; yet, the identified genes explain only 60% of the genetic effect at best. Traditional methods of gene identification require linkage analysis of large families, but face increasing difficulties to identify such extended pedigrees for rare HSP forms. However, the innovative approach described in this application will overcome some of these limitations and utilize relatively small pedigrees for highly effective gene identification. We will apply exome sequencing, which will characterize all coding changes and flanking exonic variation in two individuals of a family. We have developed a multi-tiered strategy to reduce the number of identified novel variants to the very causative change in an individual family. We propose to study at least 60 HSP families, which are too small to yield conclusive results with linkage analysis. If the developing technology permits we will consider a larger sample or perform whole genome sequencing. Beyond the important benefit to genetics of HSP, this study will allow us to further establish this new method, which will benefit a large range of additional disease studies.

DESCRIPTION (provided by applicant): Approximately 20 million Americans develop peripheral neuropathy with annual costs to Medicare alone in excess of $3.5 billion. These diseases are poorly understood and therapeutic options are limited to non- existent. The hereditary forms of peripheral neuropathies present a unique window of opportunity to identify and dissect the key genes and pathways. In fact, these hereditary neuropathies, known as Charcot-Marie- Tooth disease (CMT), represent the most common inherited disorders in Neurology with 1/2500 individuals affected. Despite impressive success in gene identification in CMT, only ~30% of the genetic causes have been identified for the arguably most important, axonal subtype. Classic methods for gene identification, which depend on large pedigrees, become increasingly ineffective to resolve this problem. New high- throughput sequencing technology is now available that permits for the efficient analysis of whole genomes or highly informative proxies thereof, such as the exome, the entire collection of coding exons. Under the lead of Dr. Zuchner, Director of the Center for Human Molecular Genomics at the state-of-the-art Hussman Institute for Human Genomics, we have recently published the first exome sequencing study of a multigenerational pedigree and the first exome study on CMT and have now published several new genes identified with this method. We fully expect that this technology will add tremendously to resolving causative genes in relatively small CMT families not suitable for classic linkage analysis. The second PI of this proposal, Dr. Michael Shy, is Director of the largest CMT Clinic in the country as well as the PI of the NINDS/ORD funded Rare Disease Clinical Research Center (RDCRC) for genetic neuropathies and the MDA/CMTA funded North American Database and the North American CMT Network. Consequently, we have access to CMT patients throughout the world and propose in this grant a bold approach involving the application of high- throughput genomic technologies that will lead to the discovery of a considerable number of genes in a few years time. In addition we are pursuing genetic results with an innovative functional design in multiple biological systems, yeast, zebrafish, mammalian cell culture, that lend themselves to higher throughput studies. We have assembled an interdisciplinary team of clinicians, molecular and statistical geneticists, bioinformaticians, and molecular biologists to successfully apply these highly complex technologies. All data will be made available to publicly accessible databases laying the foundation for a future genomic repository for peripheral neuropathies. It is only by identifying the genetic causes of CMT that we will be able to study the function of encoded proteins and develop rational approaches to therapeutic intervention. Importantly, related axonal neuropathies, such as diabetic neuropathy, drug-induced neuropathies and degenerative diseases of motor and sensory neurons will greatly benefit from the results of such studies.

ABSTRACT: IDENTIFYING GENETIC FACTORS THAT CAUSE AND MODIFY CMT More than 80 different genes have been identified to cause the various forms of CMT. For the demyelinating forms of CMT, a genetic cause can be found most of the time, with CMT1A (PMP22 duplication) explaining ~70% of these cases. In contrast, a mutation in one of the currently known genes can be found in less than 40% of axonal (CMT2) cases, mostly for the severe, early onset cases. Genetic studies have fundamentally transformed our knowledge on CMT and have catalyzed much of the research in neuropathies in the past 20 years. We fully expect that by taking advantage of new technologies, this progress will continue to a point where (1) >90% of CMT1 and CMT2 patients can receive a genetic diagnosis; (2) a sizable number of important genetic modifiers that account for a significant portion of the phenotypic variability in some forms of CMT will be identified; (3) a proportion of the heretofore idiopathic/sporadic neuropathies will be found to have a genetic cause; (4) genetic risk factors for developing neuropathy to diabetes and various medications will be identified. The members of the INC consortium work in a collaborative manner with multiple sources of funding to achieve these goals. In particular, the INC has allowed us to collect high-quality samples for novel gene identification (15 new CMT genes) and also for reliable gene modifier studies, as demonstrated by our results in a CMT1A study. In this renewal, we propose to expand our efforts to find new genes that cause CMT and genetic modifiers of CMT. Novel genes will expand our understanding of pathogenic mechanisms and demyelination. They will also provide new therapeutic targets. Modifiers will also be important targets for intervention, and may well be important in the manifestations of acquired peripheral neuropathies, and even other diseases, such as amyotrophic lateral sclerosis (ALS) and multiple sclerosis, in which axonal degeneration has been implicated in the pathogenesis. Finally, as our collaborative group and others move rapidly towards genomic approaches, we will establish a unified, secure, and accessible resource for all genomic data of the INC that will be open to all CMT and other genetic researchers, that can also serve as a blueprint for other RDCRN groups interested in inherited diseases.

Advancing Genomics Of Recessive Ataxias Project summary Hereditary spastic paraplegias have a heterogeneous genetic etiology. While ~50% of all patients of dominant HSP are explained by three major genes (spastin, atlastin, REEP1), the remaining genetic causes are rare and explain no more then another 15% of patients. For recessive HSP only about 40-50% of patients receive a genetic diagnosis. Recent progress is tremendous and will likely identify the genetic basis for another 20% of patients in the coming five years. Especially interesting is that less than 50% of cases are clarified in select exome sequencing approaches, implaying that whole genome studies will be necessary. In addition, HSPs often show clinical overlap with related neurological diseases, such as ataxias, leukodystrophies, metabolic disorders, and mitochondrial disorders. Thus, there is a two-fold need: 1) to standardize the clinical classification in a collaborative fashion and 2) to expand the knowledge of underlying causative genes and molecular pathways. This will be beneficial beyond HSP and contribute to our understanding of a number of related neurological diseases. For this study, we will create a collaborative structure between investigators in the USA, Germany, France, Belgium, Brazil, Austarlia, and indirectly Middle East and Northern Africa. We will be able to systematically study THE largest clinical sample of HSP in the world led by world-renowned clinical and genetic experts. Importantly, existing genomic data will be contributed in kind by our collaborators and jointly used in the analysis. Our recent success in identifying dozens of genes for familial neurological disorders, including HSP validates the suggested approach. Data will be shared with dbGAP. We expect to identify and publish 2 ? 4 novel genes per year and create a lasting resource of clinical and genomic data.

PROJECT SUMMARY Recent advances in science and technology are now making tackling of rare and undiagnosed disorders possible. By utilizing these advances, along with scientific and clinical expertise, the Undiagnosed Disease Network (UDN) promises improvement in the lives of individuals and their families affected with undiagnosed disorders. To benefit from this effort, the nation as a whole, especially the rich and rapidly increasing diversity of the US population must be taken into account. With its longstanding excellence in clinical care and research in South Florida, University of Miami is in an unsurpassed position to be a part of the UDN. We propose to create the Clinical Site-Miami (CS-Miami), which will recruit, evaluate, and provide data from participants with undiagnosed disorders. While we will be open to all participants from any location, we will specifically focus on South Florida. The diverse population of South Florida includes Hispanics from the Caribbean (e.g. Cuba, Puerto Rico and the Dominican Republic), Mexico, South and Central America, Caribbean born blacks, Jews and others from all over the world. Many of these minorities are recent immigrants whose original populations are ideal for the occurrence of rare diseases due to isolation, inbreeding, and founder effects. Our Clinical and Translational Science Institute (CTSI) is the only member of the national CTSA consortium co-funded by the National Institute on Minority Health and Health Disparities (NIMHD) to specifically focus on the health of underrepresented minorities. To represent the minority populations, we are one of the recruitment sites for NIH`s AllofUs Precision Medicine Initiative, again, with a focus of enrolling >50% of minority participants. We will build CS-Miami upon existing expertise in community engagement, electronic medical records, biorepositories, phenotypic delineation of rare diseases, characterization of their underlying biology, and access to a large collaboration network. Specific aims will be to 1) recruitment, enroll, and engage minorities into CS-Miami; 2) characterize rare and undiagnosed disease phenotypes; 3) to reach a diagnosis via collaborative analysis of standard data. Our team's combined expertise in clinical assessment, patient engagement, informatics, ethics and genomics will provide the requisite oversight and structure to ensure we meet our stated goals.

Abstract Genetics is at the core of CMT diagnoses, pathophysiology, and preparation for trials and gene therapy approaches. The latter requires precise genetic diagnosis for each individual patient and genetics will also be highly informative for long-term outcomes. However, even with 90+ CMT and related genes identified, over 50% of CMT2 patients do not receive a diagnosis today. This gap in heritability has many potential explanations, including additional CMT genes to be discovered, non-coding mutations, unconventional variation (i.e. repeat expansions), oligogenic inheritance, and Variants of Unknown Significance. Our long-term commitment to the genetic modifier study in CMT1A has yielded a genome wide significant association (GWAS) of SIPA1L2 as the first CMT1A modifier gene. Importantly, this discovery promises a novel strategy to correct the increased gene dosage of PMP22. The INC has collected 1,700 samples of CMT1A patients and another 1,000 are expected over the next five years. With new statistical methods and genome sequencing now available we will bring this unparalleled effort to its full potential by combining all these strengths and comprehensively define the genetic architecture of CMT1A. We anticipate that some of these risk factors have effects on other more common types of CMT, such as CMT1B, CMTX, and CMT2A. Over the past funding period, we have discovered 22+ novel CMT and related genes, including a CMT causing repeat expansion disease. We will continue this work, but focus on improving our sample and data resources in size and ancestral diversity. This will be achieved by further expanding data sharing with CMT-ID, AOINC, and the MRC in the UK. We will develop a CMT genetic data archive and utilize the GENESIS genomic software platform that has helped to discover most of the above mentioned CMT genes. Finally, in the previous funding cycle we have created the Inherited Neuropathy Variant Browser (INVB) to collect disease related variation in CMT genes internationally and provide this information openly to the community. Given our specialized interest in CMT and related disorders and our international reach, this CMT-focused effort goes far beyond the reach of NIH ClinVar. We will develop INVB version 2.0 that includes more data, additional correlations, and deeper information on VUS. Specifically, we propose to (1) Perform an Iterative expansion of modifier studies in CMT1A and other relatively common CMT subtypes, (2) Build a sustained infrastructure for data sharing and continued discovery of new CMT genes, and (3) Develop the CMT variant browser 2.0 that will also provide a phenotype bank for CMT enabling patients and investigators to rapidly obtain diagnostic and de-identified clinical information on disease causing mutations.

Project Summary/Abstract Neuromuscular diseases are amongst the most common rare diseases and also are characterized by unparalleled locus and allele heterogeneity. In fact, the full extent of the gene and variant spectrum is still unclear with 30-50% of patients with these diseases no receiving genetic diagnosis after large panel and exome sequencing. A sizable part of this diagnostic gap can be attributed to Variants of Uncertain Significance (VUS). A number of initiatives are aimed at reducing the diagnostic gap; mainly these are research studies to identify new genetic loci and alleles and variant curation efforts provide high quality, standardized guidance on pathogenicity for known genes and variants. Here we describe the goals of the recently established ClinGen Neuromuscular Clinical Domain Working Group (NMD CDWG), its executive committee, and the proposed GCEP and new VCEP for three major NMD disease groups comprising 197 genes and over 10,000 variants. We aim to eventually curate the majority of genes causing inherited peripheral neuropathies, muscular dystrophies, and congenital myopathies. A rigorous curation effort, acknowledges by FDA, will be the basis of upcoming gene therapies targeting neuromuscular and related diseases.