Neuronal Ceroid Lipofuscinosis (NCL) or Batten Syndrome is a heterogeneous collection of lysosomal storage diseases associated with optic atrophy, seizures, cortical degeneration and the accumulation of ceroid lipofuscin in all tissues including lymphocytes and EBV-transformed lymphoblasts. Infantile (I), Late Infantile (LI), Juvenile (J) and Adult (A) forms have been identified and the I and J forms shown to be due to recessive mutations located on chromosomes 1 and 16. In the LI and J forms, about 50% of the storage material is mitochondrial ATP synthase subunit 9 (ATPsyn9) suggesting that some NCL patients may harbor defects in mitochondrial bioenergetics, biogenesis or degradation. To clarify the genetic and biochemical heterogeneity of NCL, we will (1) divide the patients into complementation groups by pairwise somatic cell hybridization, (2) analyze each complementation group for defects in mitochondrial biogenesis or turnover, and (3) attempt to clone the affected genes by gene transfer. To identify the complementation groups, lymphoblast cell lines will be transfected with either pSVNeo or pSVHyg, pairs of cells fused and the hybrids selected in G418 and Hygromycin B. Complementation will be assessed by the loss of ceroid lipofuscin granules and the reduced accumulation of lysosomal lipids and proteins. Each complementation group will be tested for defects in mitochondrial oxidative phosphorylation, in mitochondrial biogenesis through analysis of nuclear and mitochondrial DNA (mtDNA) gene expression, and in mitochondrial degradation through analysis of the turnover of mitochondrial translation products, the ATP synthase and the adenine nucleotide translocator. To clone the affected genes, lymphoblasts from each complementation group will be transfected with a cDNA expression library and the transfectants selected in Hygromycin B. The Hygromycin-resistant clones which express the NCL complementing cDNA will be enriched by selecting for increased growth rate and isolated by FACS sorting for cells which have lost their autofluorescent granules. Positive cDNAs will be recovered by polymerase chain reaction amplification of the inserts, recloning, and testing for increase complementation efficiency. Confirmed clones will be used as probes to search for mutations in patient genes mapped to determine if they are located in chromosomes 1 or 16, sequenced, and tested to determine if they are components of mitochondrial structure or metabolism
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