CLONING AND FUNCTIONAL CHARACTERIZATION OF INHERITED NEURODEGENERATIVE DISORDERS

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This project entails the identification of genes responsible for a variety of human inherited disorders. These include development and refinement of methodologies for gene mapping and isolation, and the characterization and functional analyses of these genes for a better understanding of their pathogenic mechanisms that will lead to therapy and intervention. Using positional cloning strategies, our laboratory participated in the identification the Huntington Disease (HD) gene and the ataxia-telangiectasia gene (A-T). HD is a progressive, neurodegenerative disorder with onset in midlife. and the gene contains a polymorphic (CAG)n trinucleotide repeat which is pathogenic when the repeats expand to 42 or more. Subcellular expression and localization of the normal and expanded versions of the full length cDNA constructs are being examined in baculovirus and mammalian expression systems. In addition, mouse YAC transgenic lines are being developed by microinjecting a YAC containing the entire HD gene that has been retrofitted with an expanded CAG repeat into the pronucleus of fertilized eggs. A-T is an autosomal recessive disorder characterized by progressive cerebellar degeneration, oculocutaneous telangiectasia, immunodeficiency, radiosensitivity, susceptibility to cancer and chromosomal instability. Current research efforts for A-T involves the function of homologous sequences in S. cerevisae; generation of antisera specific for the A-T protein; characterization of the expression pattern and localization of the protein product; and generation of knockout mouse models. A current positional cloning project involves the identification of the defective gene for Niemann-Pick type C. NPC is an autosomal-recessive, neurovisceral lipid storage disorder marked by neurological decline in patients which is highlighted by supranuclear gaze palsy and is typically accompanied by progressive cognitive impairment, ataxia, dystonia, dysphagia, dysarthria, seizures, cataplexy and dementia. Recombinant studies point to a localization at 18q11-q12, more specifically in a 2 cM interval between markers D18S44 and D18S480. We are currently in the process of refining the genetic map by searching for additional polymorphic markers and assembling YAC and cosmid contigs for the region. Candidate genes are being isolated by cDNA selection and exon trapping. Additionally, entire YACs are being introduced into NPC cells to check for correction of the cellular defect. Taking advantage of an NPC mouse model, backcrosses are being performed to further delimeate the affected locus in mice and identify the synthenic region in humans. A novel method for gene identification is also underway and this involves development of a YAC fragmentation vector that includes 3' exon trapping cassette, yeast selectable marker, Alu sequences for targeting, yeast telomere sequences. Acentric and centric versions on the vector are being made with Alu sequences also found in both orientations.