A PTG Variant Contributes to a Milder Phenotype in Lafora Disease

et al. (2011) A PTG Variant Contributes to a Milder Phenotype in Lafora Disease. PLoS ONE 6(6): e21294. doi:10.1371/journal.pone.0021294 A PTG Variant Contributes to a Milder Phenotype in Lafora Disease Rosa Guerrero 0 Santiago Vernia 0 Rau l Sanz 0 Irene Abreu-Rodrguez 0 Carmen Almaraz 0 Mara 0 Garca-Hoyos 0 Roberto Michelucci 0 Carlo Alberto Tassinari 0 Patrizia Riguzzi 0 Carlo Nobile 0 Pascual 0 Sanz 0 Jose M. Serratosa 0 Pilar Go mez-Garre 0 Francesc Palau, Instituto de Ciencia de Materiales de Madrid - Instituto de Biomedicina de Valencia, Spain 0 1 Laboratorio de Neurolog a-Unidad de Epilepsia, Servicio de Neurolog a, Instituto Investigaci o n Sanitaria Fundacio n Jime nez D az, and Centro de Investigacio n Biome dica en Red de Enfermedades Raras (CIBERER) , Madrid , Spain , 2 Instituto de Biomedicina de Valencia, Consejo Superior de Investigaciones Cient ficas (CSIC), and Centro de Investigacio n Biome dica en Red de Enfermedades Raras (CIBERER) , Valencia , Spain , 3 Laboratorio de Investigaciones Biome dicas, Instituto de Biomedicina de Sevilla (IBiS) , Sevilla , Spain , 4 Unit of Neurology, Department of Neurosciences, Bellaria Hospital , Bologna , Italy , 5 Section of Padua, CNR-Institute of Neurosciences , Padua , Italy , 6 Unidad de Trastornos del Movimiento, Servicio de Neurolog a y Neurofisiolog a Cl nica, Instituto de Biomedicina de Sevilla (IBiS) , Sevilla , Spain , 7 University of Bologna , Bologna , Italy Lafora disease is an autosomal recessive form of progressive myoclonus epilepsy with no effective therapy. Although the outcome is always unfavorable, onset of symptoms and progression of the disease may vary. We aimed to identify modifier genes that may contribute to the clinical course of Lafora disease patients with EPM2A or EPM2B mutations. We established a list of 43 genes coding for proteins related to laforin/malin function and/or glycogen metabolism and tested common polymorphisms for possible associations with phenotypic differences using a collection of Lafora disease families. Genotype and haplotype analysis showed that PPP1R3C may be associated with a slow progression of the disease. The PPP1R3C gene encodes protein targeting to glycogen (PTG). Glycogen targeting subunits play a major role in recruiting type 1 protein phosphatase (PP1) to glycogen-enriched cell compartments and in increasing the specific activity of PP1 toward specific glycogenic substrates (glycogen synthase and glycogen phosphorylase). Here, we report a new mutation (c.746A.G, N249S) in the PPP1R3C gene that results in a decreased capacity to induce glycogen synthesis and a reduced interaction with glycogen phosphorylase and laforin, supporting a key role of this mutation in the glycogenic activity of PTG. This variant was found in one of two affected siblings of a Lafora disease family characterized by a remarkable mild course. Our findings suggest that variations in PTG may condition the course of Lafora disease and establish PTG as a potential target for pharmacogenetic and therapeutic approaches. - Funding: This work was supported by grants from the Fundacio n Mutua Madrilen a to PGG and RG, the Spanish Ministry of Education and Science (SAF200801907, SAF2007-61003) to PS and JMS and from the Generalitat Valenciana (Prometeo 2009/051) to PS. There are no current external funding sources for this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. Lafora disease (LD; MIM#254780) is an autosomal recessive form of progressive myoclonus epilepsy that typically manifests during adolescence and is characterized by epilepsy, progressive neurologic deterioration, myoclonus and epileptic seizures. The disease leads to a vegetative state and death, usually within less than a decade from the onset of the initial symptoms [1]. The hallmark of LD is the presence of polyglucosan intracellular bodies, first described by Lafora and Glueck in 1911 [2]. These inclusions have been found in many tissues, including brain, spinal cord, liver, skin, skeletal muscle, heart and retina [3]. Two genes have been associated with LD: EPM2A [45] and EPM2B [6]. EPM2A (MIM#607566) is a four-exon gene which encodes a protein known as laforin. Initially, laforin was partially characterized and described as a dual-specificity phosphatase [45]. Subsequently, the complete coding human sequence of the gene including the ATG initiation codon region was reported [7]. The complete protein shows an amino-terminal carbohydrate binding module (CBM) that is critical for association with glycogen both in vitro and in vivo [8]. EPM2B (MIM#608072) is a singleexon gene which codes for an E3-ubiquitin ligase, known as malin, that contains a RING finger domain and six NHL-domains involved in protein-protein interactions [7,9]. Malin interacts with and ubiquitinates laforin, leading to its degradation [9]. Laforin and malin appear to be part of a multiprotein complex that may be associated with the formation of intracellular glycogen particles. Within this complex, laforin interacts with protein targeting to glycogen known as PTG (Gene symbol PPP1R3C, MIM#602999), one of the glycogen targeting regulatory subunits of protein phoshatase 1 (PP1) [10]. Recently, it has been shown that the glycogenic activity of PTG is down-regulated by the laforin-malin complex by inducing protesome-dependent degradation [1113]. However, the absence of malin in mice does not affect the levels of glycogen synthase, PTG or debranching enzyme [14]. Lafora disease patients show variability in the age and symptoms at onset, as well as in the duration of disease progression, even among affected siblings with the same mutation [1519]. The striking finding of hepatic disease as the first manifestation of LD in one of two affected siblings, suggests that modifier genes must condition the clinical expression of the disease [20]. To determine if genes involved in the regulation of EPM2A and EPM2B can modify the onset and progression of the disease, we screened a total of 43 genes coding for proteins related to laforin/ malin function and/or glycogen metabolism in a collection of LD families that showed intrafamilial phenotypic differences. We found two heterozygous variations in the PPP1R3C gene. In this work we tested the significance of these variations on the glycogenic capacity of human PTG and their possible role in an exceptionally mild form of the disease. Haplotype analysis reveals PPP1R3C as a candidate gene that modifies disease progression We evaluated if LD patients with intrafamilial phenotypic differences (age at onset or disease progression) presented distinct haplotypes for each of the selected 43 gene loci related to laforin/ malin function or to glycogen metabolism (see Materials and Methods). Genotyping (...truncated)