Progressive myoclonus epilepsy type 1 (EPM1), also known as Unverricht-Lundborg disease, is one of the rare forms of epilepsy that shows a clear pattern of autosomal recessive inheritance. The gene defective in this disease was linked to the distal tip of chromosome 21, in band q22.3. In this study, we have collected 93 samples from 15 EPM1 families and 5 affected individuals as the basis for identifying the EPM1 gene. We have also constructed a 770 kb cosmid and bacterial artificial chromosome contig covering the candidate EPM1 region, and have isolated expressed sequences from this contig. For three of the genes that we isolated (GT335, GT334, and PWP2), we have identified and sequenced a full-length cDNA, identified the putative protein, assessed the expression pattern of the gene by Northern blot, determined the exon/intron structure of the gene, characterized basepair polymorphisms within each gene, and finally excluded each of these genes as the one defective in EPM1 patients. Using these new polymorphisms, and others that were available and that we had identified, we were able to construct detailed haplotypes on each of the affected EPM1 chromosomes, to help pinpoint, the location of the EPM1 gene, and help estimate the number of different mutations we might have in our collection. / While these studies were underway, another group identified the cystatin B (STFB) gene as that defective in EPM1. This allowed us to directly test this gene for mutations in our collection of EPM1 patients. We could identify four different mutations in the STFB gene, the most common of which consisted of a variable length insertion in the 5 ' flanking region of the gene, and which was previously undescribed. This mutation, which is found in 78% of unrelated EPM1 chromosomes we studied, showed some level of meiotic instability, and mapped to a polymorphic 12-bp GC-rich repeat. Using a combination of PCR and Southern blotting assays, we could accurately diagnose nearly 100% of all EPM1 patients. This represents a significant step forward in our ability to diagnose this disease at the molecular level, and should allow a more precise definition of the progressive myoclonus epilepsies, as a whole.

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Indiana University-Purdue University Indianapolis (IUPUI) / Lafora disease is a neurodegenerative disorder caused by mutations in either the EPM2A or the EPM2B gene that encode a glycogen phosphatase, laforin and an E3 ubiquitin ligase, malin, respectively. A hallmark of the disease is accumulation of insoluble, poorly branched, hyperphosphorylated glycogen in brain, muscle and heart. The laforin-malin complex has been proposed to play a role in the regulation of glycogen metabolism and protein degradation/quality control. We evaluated three arms of protein quality control (the autophagolysosomal pathway, the ubiquitin-proteasomal pathway, and ER stress response) in embryonic fibroblasts from Epm2a-/-, Epm2b-/- and Epm2a-/- Epm2b-/- mice. There was an mTOR-dependent impairment in autophagy, decreased proteasomal activity but an uncompromised ER stress response in the knockout cells. These defects may be secondary to the glycogen overaccumulation. The absence of malin, but not laforin, decreased the level of LAMP1, a marker of lysosomes, suggesting a malin function independent of laforin, possibly in lysosomal biogenesis and/or lysosomal glycogen disposal. To understand the physiological role of malin, an unbiased diGly proteomics approach was developed to search for malin substrates. Ubiquitin forms an isopeptide bond with lysine of the protein upon ubiquitination. Proteolysis by trypsin cleaves the C-terminal Arg-Gly-Gly residues in ubiquitin and yields a diGly remnant on the peptides. These diGly peptides were immunoaffinity purified using anti-diGly antibody and then analyzed by mass spectrometry. The mouse skeletal muscle ubiquitylome was studied using diGly proteomics and we identified 244 nonredundant ubiquitination sites in 142 proteins. An approach for differential dimethyl labeling of proteins with diGly immunoaffinity purification was also developed. diGly peptides from skeletal muscle of wild type and Epm2b-/- mice were immunoaffinity purified followed by differential dimethyl labeling and analyzed by mass spectrometry. About 70 proteins were identified that were present in the wild type and absent in the Epm2b-/- muscle tissue. The initial results identified 14 proteins as potential malin substrates, which would need validation in future studies.