DE PEDIATRIC COBRE: MOLECULAR MECHANISMS IN PELIZAEUS MERZBACHER DISEASE

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Pelizaeus-Merzbacher disease (PMD), an X-linked leukodystrophy, is caused by a variety of genetic defects of the proteolipid protein 1 gene (PLPI) that encodes the major central nervous system myelin protein. Approximately 60% of PMD families have a duplication of a genomic region that includes PLP1, while 15-20% of families have point mutations in PLP1. Studies in mice have shown that an increased dosage of PLP1 protein can account for disease pathogenesis, the severity of which is related to the amount of PLP1 overexpression. Further, studies in mice and humans have shown that mutations within noncoding regions can alter PLP1 expression levels or the ratio of alternatively spliced forms PLP1 and DM20. The long-term objective of this work is to understand the molecular mechanisms involved in generating the PMD phenotype so that rational treatments and improved diagnostic techniques can be developed. In the first aim, the hypothesis that most PMD-associated gene duplications are caused by a novel mechanism characterized by homologous pairing at high copy repeats followed by nonhomologous end-joining will be tested using cytogenetic, molecular, and in silico approaches to identify sequences at duplication breakpoints that will reveal the mechanism. In the second aim, the hypothesis that the structure of the PMD duplication can affect expression of PLP1 will be tested by engineering duplications with different structures into embryonic stem cell lines and analyzing the structural effects on gene expression before and after differentiation into oligodendrocytes, tn the third aim, the hypothesis that some PMD mutations dysregulate splicing of PLP1 will be tested by using gene transfer into otigodendrocytes and RNA-protein binding assays to investigate the cis-acting elements and trans-acting factors that are involved in PLP1/DM20 alternative splicing. The results of these studies have greatest potential for impact through their generalizabilJty to other genomic and splicing diseases, which have only recently been recognized as important types of genetic disease.