DE PEDIATRIC COBRE: MOLECULAR MECHANISMS IN PELIZAEUS MERZBACHER DISEASE

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Pelizaeus-Merzbacher disease (PMD), an X-linked leukodystrophy, is caused by defects of the proteolipid protein 1 gene (PLP1) that encodes the major CNS myelin protein. Approximately 60% of families have a genomic duplication that includes the PLP1 gene, and 15-20% of families have PLP1 point mutations. Studies in mice have shown that an increased dosage of PLP1 can account for disease pathogenesis. Further, studies have shown that mutations in noncoding regions can alter PLP1 expression levels or the ratio of the alternatively spliced forms PLP1 and DM20. Our long-term objective is to understand the molecular mechanisms involved in generating the PMD phenotype so rational treatments and improved diagnostic techniques can be developed. In the first aim, results on location, size, structure and sequence at the junctions of PMD duplications support the hypothesis that most are caused by a novel coupled homologous and nonhomologous mechanism. We will continue a combined cytogenetic, molecular, and in silico approach to refine our understanding of the mechanism. In the second aim, the hypothesis that the duplication structure affects expression of PLP1 is being tested by engineering different duplications into ES cell lines and analyzing the structural effects on gene expression before and after differentiation into oligodendrocytes. In the third aim, the hypothesis that some PMD mutations dysregulate splicing of PLP1 is being tested by using gene transfer and RNA-protein binding assays to investigate cis-acting elements and trans-acting factors involved in PLP1/DM20 alternative splicing. We have determined that exon and intron splicing enhancers and the relative strength of the PLP1 and DM20 donor splice sites play an important role in PLP1 alternative splicing. The results of these studies have greatest impact potential through their generalizability to other genomic and splicing diseases, which have only recently been recognized as important types of genetic disease.