IDENTIFICATION OF THE DYSTONIA GENE

Massachusetts General Hospital, Boston MA

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Abstract

The goal of this research is to clone and characterize the DYT1 gene responsible for onset of torsion dystonia in childhood. This is a severely debilitating movement disorder inherited as an autosomal dominant condition with reduced penetrance. Disregulation of movement is believed to originate in the brain without apparent neuronal degeneration. Identification of this disease gene should help elucidate the pathogenesis of this and other forms of dystonia, which afflict 100,000 people in Northern America, and help illuminate molecular mechanisms of movement control in the basal ganglia. At this time all the genomic DNA and a number of cDNAs encoded in the critical region (350-10(X) kb) on chromosome 9q32 have been cloned. Cloned genomic sequences will be assembled into a contiguous stretch and transcribed sequences will be identified within it by "exon amplification". Exons will be used to isolate cDNAs, which will be sequenced and evaluated for mutations. The search for incriminating mutations in the DYT1 gene will be carried out initially by PCR amplification and search for single strand conformational polymorphism (SSCPs) in exons from genomic DNA and in cDNAs generated by reverse transcriptase from patient's cells. Northern blot analysis will be undertaken to evaluate the distribution and size of the corresponding mRNAs in rat tissues and cultured cells from patients and controls. Genomic DNA in the critical region will also be searched for di-, tri- and tetranucleotide repeats which may expand in the disease state. Identification of a mutation will be followed by isolation of full length cDNAs, evaluation of exon structure and sequencing of candidate genes in affected individuals. Once the DYT1 gene identity has been confirmed, antibodies will be generated to deduced amino acid sequences and used in immunoblots and immunocytochemistry to evaluate the size and cellular localization of the encoded protein, dystonin. Our collaborator, Dr. Ann Graybiel (MIT), will evaluate the neuronal localization of this protein in basal ganglia in newborn and adult rat brains by immunocytochemistry an in situ hybridization. These studies should provide us with insights into the possible function of dystonin in the basal ganglia.