**Myoclonic Epilepsy in Lysosomal Storage Disorders**

#### Andrea Dardis and Bruno Bembi

*Regional Coorddinator Centre for Rare Diseases University Hospital "Santa Maria della Misericordia" of Udine Piazzale, Santa Maria della Misericordia, Udine, Italy* 

#### **1. Introduction**

220 Novel Aspects on Epilepsy

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Progressive myoclonic epilepsy (PME) constitutes an heterogeneous group of diseases, usually of genetic origin, which begins in childhood and adolescence and presents a variable evolution, ranging from slowly to rapidly progressive forms with refractory seizures and dead within few years (Marseille Consensus Group, 1990). Despite its broad spectrum of manifestations, patients affected with PME share some common specific clinical and electrophysiological features, such as: myoclonus, multiple type of seizures, delay or regression of psychomotor development, cerebellar ataxia, slow background activity on electroencephalogram (EEG), spikes and waves induced by intermittent photo-stimulation and sensory evoked giant potentials (Marseille Consensus Group, 1990).

From the genetic point of view, PME occurs in disorders presenting different genetic inheritance, including: the dentatorubralpallidolusyian atrophy (DRPLA), a disease of trinucleotide repeats, the myoclonic epilepsy with ragged red fibers (MERRF), a mitochondrial disease and autosomal recessive disorders, which may be divided in two main categories: non-lysosomal-related diseases such as Lafora disease and lysosomalrelated-disease such as lysosomal storage disorders (LSDs).

Lysosomal storage disorders are severe genetic diseases caused by the defective activity of lysosomal proteins, cofactors or integral membrane proteins, which result in the intralysosomal accumulation of undegraded metabolites such as sphingolipids, cholesterol, glycoproteins, mucopolysaccharides or glycogen. Even if they are individually rare, the combined frequency of LSDs is estimated to be approximately 1 in 8000 live births (Meilke et al., 1999; Poorthuis et al., 1999; Applegarth et al., 2000; Dionisi-Vici et al., 2002; Pinto et al., 2004; Poupetova et al., 2010).

More than 50 LSDs have been described to date (Staretz-Chacham et al., 2009). Although they are characterized by a wide spectrum of clinical phenotypes, many of these disorders present with severe progressive neurological impairment. Among the neurological symptoms, the presence of PME has been reported in different LSDs, including Gaucher disease, action myoclonus-renal failure syndrome, neuronal ceroid lipofuscinoses, sialidosis, Niemann Pick type C disease, and GM2 gangliosidosis. Each of these LSDs is characterized by a series of specific sings and symptoms. However, many of them share some clinical and biochemical features, such as the presence of signs of neurological impairments other than PME or organ disorders, which may be useful in the diagnosis of patients presenting with PME due to LSD.

Myoclonic Epilepsy in Lysosomal Storage Disorders 223

showed the presence of PME in 2 (Kraoua et al., 2010). Similarly, unpublished data collected

The analysis of the clinical phenotype in a group of 16 GD3 patients presenting with PME , showed that this is not an homogeneous phenotype. In fact, many clinical features found among this group were quite variable including age, sex, ethnic background, degree of visceral, skeletal, cognitive and cerebellar involvement and MRI findings. However, a clinical finding shared by all patients was the slowing of the horizontal saccadic eye movements, a feature present in GD3 patients that was independent of the extent of nonneurological manifestations. In addition, another finding shared among these patients was the abnormal EEG, often with generalized seizures. As disease progressed many of them

Human GBA is a peripheral membrane glycoprotein. The mature non-glycosylated polypeptide is composed of 497 aminoacids with a molecular weight of about 56 kD while the glycosylated enzyme has a molecular weight of 63 kD (Leonova and Grabowski, 2000). The human *GBA1* gene (GBA; MIM# 606463; GenBank accession no. J03059.1) of approximately 7.5 kb is located on chromosome 1q21 and contains 11 exons. A highly homologous 5.5 kb-pseudogene (GBAP; MIM# 606463; GenBank accession no. J03060.1) is located 16 kb downstream from the active gene (Horowitz et al.,1989). The *GBA* mRNA has two in-frame ATG translational sites located in exons 1 and 2 (Sorge et al., 1985). Both are efficiently translated and produce two polypeptides with signal peptides of 39 and 19

More than 300 mutations in the *GBA* gene have been reported to date, including all kinds of defects such as single base changes, splicing alterations, insertions, partial and total

Mutations N370S, 84GG, L444P, IVS2+1G>A account for 90% of mutant alleles in the Jewish population while they represent fewer than 75% of alleles among non-Jewish Caucasian patients with some differences in defined subpopulations (Beutler & Gelbart, 1993; Grabowski & Horowitz, 1997). In any case N370S and L444P alleles are the most prevalent

Although, no consistent correlation between the genotype and phenotype has been found, some general conclusions can be drawn regarding the neuroprotective nature of the N370S

The molecular study of the *GBA1* gene in a cohort of 16 GD3 patients with PME showed also within this subgroup a remarkable genotype heterogeneity even among patients with similar clinical presentation. However, an interesting finding of this study was the fact that while 72% of 122 GD3 patients included in the International Gaucher Registry carry the p.L444P/p.L444P genotype, only one out of 16 GD3 patients with PME presented this genotype, suggesting that the most frequent genotype found in GD3 patients would be underrepresented among GD3 patients with PME. In contrast, some rare mutants were encountered among GD3 patients with PME. In particular three point mutations seems to be associated with this phenotype, the V394L, N188S and G377S, suggesting that GD3 patients carrying one of these mutations in the absence of the N370S mutation should be carefully

deletions, gene-pseudogene rearrangements (www.hgmd.org; Stenson et al., 2003).

mutation and the association between the L444P allele and the severe phenotype.

from GD3 patients followed in our Center showed that 3 out of 13 developed PME.

developed ataxia, dementia and spasticity (Park et al., 2003).

residues, respectively (Sorge et al., 1987; Pasmanik-Chor et al., 1996).

**2.2 Molecular aspects** 

throughout most population.

evaluated for PME (Park et al., 2003).

Although LSDs are the main cause of the inherited form of PME, lysosomal defects are poorly known as a cause of PME and the differential diagnosis might be challenging, particularly in adult patients who may present a milder form of the diseases.

Therefore, the aim of this review is to overview the clinical and molecular findings in patients with PME affected with LSDs and their therapeutic options.

#### **2. Gaucher disease (GD)**

GD, the most frequent LSD, is an autosomal recessive inherited disease due to the deficiency in the lysosomal hydrolase, acid -glucosidase (GBA). The enzyme is present in the lysosomes of all nucleated cells and cleaves the −glucosidic linkage of glucosylceramide (GlcCer) yielding glucose and ceramide. GBA deficiency leads to the progressive lysosomal accumulation of GlcCer and other glycosphingolipids (GSLs) and subsequent multi-organ dysfunction. The storage predominantly occurs in cells of the monocyte-macrophage lineage, but an increase in GlcCer concentration is detectable in most of the body tissues (Beutler & Grabowski, 2001).

GD is panethnic (Beutler & Grabowski, 2001; Zimran et al., 1992; Cox & Shofield, 1997; Erikson, 1986) and presents an incidence of one case per 60,000 live births in the general population (Meikle et al., 1999). However, it is the most frequent genetic disease in the Ashkenazi Jewish population where it shows a incidence of one case per 850 live births (Beutler et al., 1993).

#### **2.1 Clinical aspects**

The disease has been classically classified in three major clinical variants based on the presence and progression of central nervous system involvement. Type 1 GD (MIM# 230800), the most common phenotype, is characterized by enlargement and dysfunction of liver and spleen, displacement of normal bone marrow by storage cells and bone damage leading to infarctions and fractures. Although type 1 GD is considered a non-neuropathic form, there is increasing evidence that neurological involvement (i.e. Parkinson syndrome, seizures, oligophrenia, perceptive deafness) can occur. Type 2 GD (MIM# 230900) is a rare phenotype associated with an acute neurodegenerative course and death at a very early age. These patients commonly present during the first month of life with evidence of brainstem dysfunction consisting in supranuclear gaze palsy and hepatosplenomegaly followed by progressive deterioration, opisthotonus dysphagia, pyramidal signs, failure to thrive and cachexia. They may also have intersititial lung disease and repeated respiratory infections. Type 3, the chronic neuronopathic GD (MIM# 231000), comprises an extremely heterogeneous group of patients who present with either mild or severe systemic disease associated with some form of neurological involvement and with an onset of symptoms that might range from childhood to early adulthood (Beutler & Grabowski, 2001). A most consistent finding in patients affected with this form of GD is an abnormality of the horizontal gaze. Among GD3 patients it has been widely demonstrated the existence of a subgroup of patients sharing the rare finding of PME (Rapin et al., 1986; Seeman et al., 1996; Garvey et al., 2001; Park et al., 2003; Kraoua et al., 2010, Tylki-Szymanska et al., 2010). Published data from the International Collaborative Gaucher Group showed the presence of myoclonic epilepsy in 3 out of 121 patients who had suffered from seizures when first assessed. However, a study performed in a French cohort of 10 patients affected with GD3 showed the presence of PME in 2 (Kraoua et al., 2010). Similarly, unpublished data collected from GD3 patients followed in our Center showed that 3 out of 13 developed PME.

The analysis of the clinical phenotype in a group of 16 GD3 patients presenting with PME , showed that this is not an homogeneous phenotype. In fact, many clinical features found among this group were quite variable including age, sex, ethnic background, degree of visceral, skeletal, cognitive and cerebellar involvement and MRI findings. However, a clinical finding shared by all patients was the slowing of the horizontal saccadic eye movements, a feature present in GD3 patients that was independent of the extent of nonneurological manifestations. In addition, another finding shared among these patients was the abnormal EEG, often with generalized seizures. As disease progressed many of them developed ataxia, dementia and spasticity (Park et al., 2003).

#### **2.2 Molecular aspects**

222 Novel Aspects on Epilepsy

Although LSDs are the main cause of the inherited form of PME, lysosomal defects are poorly known as a cause of PME and the differential diagnosis might be challenging,

Therefore, the aim of this review is to overview the clinical and molecular findings in

GD, the most frequent LSD, is an autosomal recessive inherited disease due to the deficiency in the lysosomal hydrolase, acid -glucosidase (GBA). The enzyme is present in the lysosomes of all nucleated cells and cleaves the −glucosidic linkage of glucosylceramide (GlcCer) yielding glucose and ceramide. GBA deficiency leads to the progressive lysosomal accumulation of GlcCer and other glycosphingolipids (GSLs) and subsequent multi-organ dysfunction. The storage predominantly occurs in cells of the monocyte-macrophage lineage, but an increase in GlcCer concentration is detectable in most of the body tissues

GD is panethnic (Beutler & Grabowski, 2001; Zimran et al., 1992; Cox & Shofield, 1997; Erikson, 1986) and presents an incidence of one case per 60,000 live births in the general population (Meikle et al., 1999). However, it is the most frequent genetic disease in the Ashkenazi Jewish population where it shows a incidence of one case per 850 live births

The disease has been classically classified in three major clinical variants based on the presence and progression of central nervous system involvement. Type 1 GD (MIM# 230800), the most common phenotype, is characterized by enlargement and dysfunction of liver and spleen, displacement of normal bone marrow by storage cells and bone damage leading to infarctions and fractures. Although type 1 GD is considered a non-neuropathic form, there is increasing evidence that neurological involvement (i.e. Parkinson syndrome, seizures, oligophrenia, perceptive deafness) can occur. Type 2 GD (MIM# 230900) is a rare phenotype associated with an acute neurodegenerative course and death at a very early age. These patients commonly present during the first month of life with evidence of brainstem dysfunction consisting in supranuclear gaze palsy and hepatosplenomegaly followed by progressive deterioration, opisthotonus dysphagia, pyramidal signs, failure to thrive and cachexia. They may also have intersititial lung disease and repeated respiratory infections. Type 3, the chronic neuronopathic GD (MIM# 231000), comprises an extremely heterogeneous group of patients who present with either mild or severe systemic disease associated with some form of neurological involvement and with an onset of symptoms that might range from childhood to early adulthood (Beutler & Grabowski, 2001). A most consistent finding in patients affected with this form of GD is an abnormality of the horizontal gaze. Among GD3 patients it has been widely demonstrated the existence of a subgroup of patients sharing the rare finding of PME (Rapin et al., 1986; Seeman et al., 1996; Garvey et al., 2001; Park et al., 2003; Kraoua et al., 2010, Tylki-Szymanska et al., 2010). Published data from the International Collaborative Gaucher Group showed the presence of myoclonic epilepsy in 3 out of 121 patients who had suffered from seizures when first assessed. However, a study performed in a French cohort of 10 patients affected with GD3

particularly in adult patients who may present a milder form of the diseases.

patients with PME affected with LSDs and their therapeutic options.

**2. Gaucher disease (GD)** 

(Beutler & Grabowski, 2001).

(Beutler et al., 1993).

**2.1 Clinical aspects** 

Human GBA is a peripheral membrane glycoprotein. The mature non-glycosylated polypeptide is composed of 497 aminoacids with a molecular weight of about 56 kD while the glycosylated enzyme has a molecular weight of 63 kD (Leonova and Grabowski, 2000).

The human *GBA1* gene (GBA; MIM# 606463; GenBank accession no. J03059.1) of approximately 7.5 kb is located on chromosome 1q21 and contains 11 exons. A highly homologous 5.5 kb-pseudogene (GBAP; MIM# 606463; GenBank accession no. J03060.1) is located 16 kb downstream from the active gene (Horowitz et al.,1989). The *GBA* mRNA has two in-frame ATG translational sites located in exons 1 and 2 (Sorge et al., 1985). Both are efficiently translated and produce two polypeptides with signal peptides of 39 and 19 residues, respectively (Sorge et al., 1987; Pasmanik-Chor et al., 1996).

More than 300 mutations in the *GBA* gene have been reported to date, including all kinds of defects such as single base changes, splicing alterations, insertions, partial and total deletions, gene-pseudogene rearrangements (www.hgmd.org; Stenson et al., 2003).

Mutations N370S, 84GG, L444P, IVS2+1G>A account for 90% of mutant alleles in the Jewish population while they represent fewer than 75% of alleles among non-Jewish Caucasian patients with some differences in defined subpopulations (Beutler & Gelbart, 1993; Grabowski & Horowitz, 1997). In any case N370S and L444P alleles are the most prevalent throughout most population.

Although, no consistent correlation between the genotype and phenotype has been found, some general conclusions can be drawn regarding the neuroprotective nature of the N370S mutation and the association between the L444P allele and the severe phenotype.

The molecular study of the *GBA1* gene in a cohort of 16 GD3 patients with PME showed also within this subgroup a remarkable genotype heterogeneity even among patients with similar clinical presentation. However, an interesting finding of this study was the fact that while 72% of 122 GD3 patients included in the International Gaucher Registry carry the p.L444P/p.L444P genotype, only one out of 16 GD3 patients with PME presented this genotype, suggesting that the most frequent genotype found in GD3 patients would be underrepresented among GD3 patients with PME. In contrast, some rare mutants were encountered among GD3 patients with PME. In particular three point mutations seems to be associated with this phenotype, the V394L, N188S and G377S, suggesting that GD3 patients carrying one of these mutations in the absence of the N370S mutation should be carefully evaluated for PME (Park et al., 2003).

Myoclonic Epilepsy in Lysosomal Storage Disorders 225

LIMP-2 is a 478 residue type III transmembrane protein (Fujita et al., 1991) comprised of about 400 aminoacid luminal domain, two transmembrane domais and a cytosolic domain of 20 residues. It presents a highly glycosylated loop within the lysosomal lumen (Eskelinen et al., 2003). It has been recently demonstrated that the binding region to the GBA protein is located between amonoacids 145 and 288 within the luminal domain of LIMP-2 , which probably mediates the binding in a carbohydrate independent manner (Blanz et al., 2010). In humans LIMP-2 is encoded by the *SCARB2* gene (NM\_005506) located on chromosome 4q13-21 (Reczek et al., 2007). To date, 12 mutations in the *SCARB2* gene have been reported in 11 patients affected by AMRF (Berkovic et al., 2008; Balreira at al., 2008; Dardis et al., 2009; Dibbens et al., 2009). Among these mutations, five are located in intronic regions and may affect the mRNA splicing process, three are non sense, three are small deletions or

The impact of two nonsense mutations, W178X (c.533G.A) and Q288X (c.862C.T), one frameshift mutation, W146SfsX16 (c.435\_436insAG), and the missense mutation H363N, on the LIMP-2 traffiking and binding properties was analyzed in vitro. Both nonsense mutations and the frameshift mutation led to the synthesis of truncated proteins that were retained in the endoplasmic reticulum. When the interaction between these LIMP-2 mutants and the GBA was analyzed, it was found that while the Q288X mutant retained its binding capacity, the mutants W146SfsX16 and W178X, lost their ability to bind the GBA almost

The H363N mutant protein was retained in the ER and its expression level was reduced with respect to wild-type. Unexpectedly, the H363N mutant seems to bind GBA even more

Although the number of patients affected by AMRF studied to date is quite limited it seems that there is no correlation between the genotype and the clinical presentation of the disease. Studies in large series of patients as well as longer periods of clinical follow up are needed to better understand the molecular bases and the phenotypic expression of this disease.

The neuronal ceroid lipofuscinosis (NCLs) are a group of severe progressive neurodegenerative diseases, which present an incidence in Scandinavian countries of 1:12000 live births while the worldwide incidence is 1:100000 (Santavuori, 1988). NCLs are caused by mutations in at least ten human genes, eight of which have been characterized (*CLN1*, *CLN2* , *CLN3* , *CLN5* , *CLN6* , *CLN7* , *CLN8* , *CLN10* ) (Jalanko et al., 2009). Although they constitute a genetically heterogeneous group, they share some clinical and histopatological characteristics. All NCLs present a degeneration of nerve cells mainly in the cerebral and cerebelar cortex and the accumulation of autofluorescent ceroid lipopigments

NCLs are considered lysosomal diseases since the ceroid lipopigments accumulate within the lysosomes and many proteins that are deficient in the NCLs are localized within the lysosomes (Futerman et al., 2004; Kyttala et al., 2006). However, the accumulated material is not a disease specific substrate and the main storage material is the c subunit of the mitochondrial ATP synthase or the sphingolipid activator proteins A and D (saposine A and

insertions that cause a shift in the reading frame and one is missense.

efficiently than wild-type LIMP-2 (Blanz et al., 2010).

**4. Neuronal ceroid lipofuscinoses** 

both in the neural and peripheral tissues.

D) (Tyynela et al., 1993; Elleder et al., 1997).

**3.2 Molecular aspects** 

entirely.

The correlation between the presence of N188S and the occurrence of PME in GD3 patients has been further supported by the work of Kowarz et al. showing a high frequency of the N188S mutation in a series of 17 GD3 patients with PME (Kowarz et al., 2005). In addition, the N188S/S107L genotype was also found in a GD3 patient with visual seizures and PME (Filocamo et al., 2004).

Mutation N188S was first described in Korean and Chinese Type I GD patients (Kim et al., 1996). Later, it was demonstrated by in vitro expression experiments that the GBA protein carrying the N188S mutation retained a high residual enzymatic activity (67% of control, Montfort et al., 2004). Furthermore, the residual GBA activity found in cultured fibroblasts obtained from a GD3 patient with PME who presented the N188S mutation was 24% of control (Park et al., 2003). The reasons for this apparent discordance between the residual activity and the clinical phenotype are not fully understood. However, the association between the presence of N188S mutation and PME in GD suggests that despite the high residual activity the mutation might alter the protein structure, binding, post-translational processing or might modify the role of other proteins involved in the ethiology of the PME.

#### **3. Action myoclonus-renal failure syndrome (AMRF)**

AMRF (MIM 254900) is a lethal inherited form of PME associated with renal failure. It was initially described in French- Canadians but it has been reported in patients with various ethnic origins (Andermann et al., 1986; Badhwar et al., 2004). It is caused by the deficiency of the lysosomal integral membrane protein type 2 (LIMP-2) (Berkovic et al., 2008, Balreira et al., 2008), an ubiquitously expressed transmembrane protein (Fujita et al. , 1992) mainly found in the lysosomes and late endosomes (Fukuda, 1991), that mediates the mannose 6 phosphate-independent targeting of GBA to the lysosomes (Reczek et al., 2007). The deficient activity of LIMP-2 leads to the mistarget of the GBA protein, which can not reach the lysosome. In fact, this condition is characterized by pathological levels of GBA activity in fibroblasts, normal or slightly reduced levels in leukocytes, but increased levels in plasma (Balreira et al., 2008; Dardis et al., 2009).

#### **3.1 Clinical aspects**

Clinically it presents at the age of 15-25 years with proteinuria evolving to renal failure and/or with neurological symptoms.

The renal pathology is characterized by focal glomerulosclerosis and sometimes with features of glomerular collapse, while the main neurological symptoms are tremor, action myoclonus, seizures and later ataxia without intellectual impairment.

In most ARMF patients reported until recently, the neurological and renal features developed simultaneously or the renal symptoms appeared first. However, mutations in the *SCARB2* gene (encoded LIMP-2 protein) have been demonstrated in a group of five AMRF patients who developed neurological symptoms before the appearance of the renal symptoms. When neurological symptoms develop first, the renal disease begun after 3 to 11 years and always by the age of 30 years (Dibbens et al., 2009, Dardis et al., 2009). These findings stressed the concept that a sorting defect of the GBA enzyme should be always considered in patients with PME of unknown etiology even in the absence of renal impairment (Dibbens et al., 2009, Dardis et al., 2009)

### **3.2 Molecular aspects**

224 Novel Aspects on Epilepsy

The correlation between the presence of N188S and the occurrence of PME in GD3 patients has been further supported by the work of Kowarz et al. showing a high frequency of the N188S mutation in a series of 17 GD3 patients with PME (Kowarz et al., 2005). In addition, the N188S/S107L genotype was also found in a GD3 patient with visual seizures and PME

Mutation N188S was first described in Korean and Chinese Type I GD patients (Kim et al., 1996). Later, it was demonstrated by in vitro expression experiments that the GBA protein carrying the N188S mutation retained a high residual enzymatic activity (67% of control, Montfort et al., 2004). Furthermore, the residual GBA activity found in cultured fibroblasts obtained from a GD3 patient with PME who presented the N188S mutation was 24% of control (Park et al., 2003). The reasons for this apparent discordance between the residual activity and the clinical phenotype are not fully understood. However, the association between the presence of N188S mutation and PME in GD suggests that despite the high residual activity the mutation might alter the protein structure, binding, post-translational processing or might modify the role of other proteins involved in the ethiology of the PME.

AMRF (MIM 254900) is a lethal inherited form of PME associated with renal failure. It was initially described in French- Canadians but it has been reported in patients with various ethnic origins (Andermann et al., 1986; Badhwar et al., 2004). It is caused by the deficiency of the lysosomal integral membrane protein type 2 (LIMP-2) (Berkovic et al., 2008, Balreira et al., 2008), an ubiquitously expressed transmembrane protein (Fujita et al. , 1992) mainly found in the lysosomes and late endosomes (Fukuda, 1991), that mediates the mannose 6 phosphate-independent targeting of GBA to the lysosomes (Reczek et al., 2007). The deficient activity of LIMP-2 leads to the mistarget of the GBA protein, which can not reach the lysosome. In fact, this condition is characterized by pathological levels of GBA activity in fibroblasts, normal or slightly reduced levels in leukocytes, but increased levels in plasma

Clinically it presents at the age of 15-25 years with proteinuria evolving to renal failure

The renal pathology is characterized by focal glomerulosclerosis and sometimes with features of glomerular collapse, while the main neurological symptoms are tremor, action

In most ARMF patients reported until recently, the neurological and renal features developed simultaneously or the renal symptoms appeared first. However, mutations in the *SCARB2* gene (encoded LIMP-2 protein) have been demonstrated in a group of five AMRF patients who developed neurological symptoms before the appearance of the renal symptoms. When neurological symptoms develop first, the renal disease begun after 3 to 11 years and always by the age of 30 years (Dibbens et al., 2009, Dardis et al., 2009). These findings stressed the concept that a sorting defect of the GBA enzyme should be always considered in patients with PME of unknown etiology even in the absence of renal

myoclonus, seizures and later ataxia without intellectual impairment.

impairment (Dibbens et al., 2009, Dardis et al., 2009)

**3. Action myoclonus-renal failure syndrome (AMRF)** 

(Balreira et al., 2008; Dardis et al., 2009).

and/or with neurological symptoms.

**3.1 Clinical aspects** 

(Filocamo et al., 2004).

LIMP-2 is a 478 residue type III transmembrane protein (Fujita et al., 1991) comprised of about 400 aminoacid luminal domain, two transmembrane domais and a cytosolic domain of 20 residues. It presents a highly glycosylated loop within the lysosomal lumen (Eskelinen et al., 2003). It has been recently demonstrated that the binding region to the GBA protein is located between amonoacids 145 and 288 within the luminal domain of LIMP-2 , which probably mediates the binding in a carbohydrate independent manner (Blanz et al., 2010).

In humans LIMP-2 is encoded by the *SCARB2* gene (NM\_005506) located on chromosome 4q13-21 (Reczek et al., 2007). To date, 12 mutations in the *SCARB2* gene have been reported in 11 patients affected by AMRF (Berkovic et al., 2008; Balreira at al., 2008; Dardis et al., 2009; Dibbens et al., 2009). Among these mutations, five are located in intronic regions and may affect the mRNA splicing process, three are non sense, three are small deletions or insertions that cause a shift in the reading frame and one is missense.

The impact of two nonsense mutations, W178X (c.533G.A) and Q288X (c.862C.T), one frameshift mutation, W146SfsX16 (c.435\_436insAG), and the missense mutation H363N, on the LIMP-2 traffiking and binding properties was analyzed in vitro. Both nonsense mutations and the frameshift mutation led to the synthesis of truncated proteins that were retained in the endoplasmic reticulum. When the interaction between these LIMP-2 mutants and the GBA was analyzed, it was found that while the Q288X mutant retained its binding capacity, the mutants W146SfsX16 and W178X, lost their ability to bind the GBA almost entirely.

The H363N mutant protein was retained in the ER and its expression level was reduced with respect to wild-type. Unexpectedly, the H363N mutant seems to bind GBA even more efficiently than wild-type LIMP-2 (Blanz et al., 2010).

Although the number of patients affected by AMRF studied to date is quite limited it seems that there is no correlation between the genotype and the clinical presentation of the disease. Studies in large series of patients as well as longer periods of clinical follow up are needed to better understand the molecular bases and the phenotypic expression of this disease.

## **4. Neuronal ceroid lipofuscinoses**

The neuronal ceroid lipofuscinosis (NCLs) are a group of severe progressive neurodegenerative diseases, which present an incidence in Scandinavian countries of 1:12000 live births while the worldwide incidence is 1:100000 (Santavuori, 1988). NCLs are caused by mutations in at least ten human genes, eight of which have been characterized (*CLN1*, *CLN2* , *CLN3* , *CLN5* , *CLN6* , *CLN7* , *CLN8* , *CLN10* ) (Jalanko et al., 2009). Although they constitute a genetically heterogeneous group, they share some clinical and histopatological characteristics. All NCLs present a degeneration of nerve cells mainly in the cerebral and cerebelar cortex and the accumulation of autofluorescent ceroid lipopigments both in the neural and peripheral tissues.

NCLs are considered lysosomal diseases since the ceroid lipopigments accumulate within the lysosomes and many proteins that are deficient in the NCLs are localized within the lysosomes (Futerman et al., 2004; Kyttala et al., 2006). However, the accumulated material is not a disease specific substrate and the main storage material is the c subunit of the mitochondrial ATP synthase or the sphingolipid activator proteins A and D (saposine A and D) (Tyynela et al., 1993; Elleder et al., 1997).

Myoclonic Epilepsy in Lysosomal Storage Disorders 227

**Clinical signs** 

loss of strength, tremor, language deterioration, ataxia, myoclonic epilepsy, visual failure, blindness,

axial rigidity, hesitation in movement initiatio, coarse postural tremor, myoclonus, speech impairment, loss of vision, aggressive behaviour, memory impairment, mental deterioration

motor impairment, myoclonus, seizures, speech impairment, loss of vision, behavioral changes,

*Northern epilepsy variant*: progressive epilepsy with

ataxia, loss of motor functions at early school age progressive cognitive decline, loss of speech;, pigmentary retinopathy, retinal atrophy, rigidity, tremor,status epilepticus, apnea, microcephaly,

generalized tonic-clonic seizures, mental deterioration; No visual involvement

language impairment, cognitive decline

behavioral changes, mental retardation.

ataxia, myoclonic jerks, seizures, vision deterioration, mental deterioration

Rapid disease progression

mental deterioration

precocious death

As other neurodegenerative disorders, which manifest during the first year of life, generalized hypotonia and psychomotor regression are the first clinical signs of classic INCL. They are generally accompanied by head growth impairment (leading to microcephaly), seizure and myoclonic jerks. Behavior and sleep disturbance are frequently reported. Disease progression leads to visual and language deterioration. Death usually

The late-infantile forms present with a similar clinic phenotype, showing progressive neurological deterioration during pre-school age. The classical late-infantile form of NCL2, generally begins during the second year of life, with slow cognitive regression and language deterioration. Epilepsy appears later, becoming rapidly intractable and accompanied with cognitive loss, myoclonic jerks and retinopathy. Patient autonomy is completely lost within the age of 6-8 and death occurs within adolescence period (Zhong et al., 2000; Steinfeld et al.,

Two major distinct phenotypes have been described for classical juvenile phenotype of NCL3 (Batten disease), according to the patient's genotype: a. patients carrying the 1-kb deletion in homozygous, (firstly described in Finland and Northern Europe), and b. patients

carrying a compound of 1-kb deletion with other mutations (Munroe et. al., 1997).

**NCL9** JNCL declining vision, ataxia, seizures, motor and

INCL: infantile, LINCL: late infantile, JNCL: juvenile, ANCL: adult, GROD: granular osmiophilic

deposits; CLP: curvilinear profiles; FPP: fingerprint profiles; RLP: rectilinear profiles. Table 1. NCLs classification, age at onset, storage pattern and clinical signs.

**Storage pattern** 

FPP

FPP

FPP

GROD

occurs within the first decade of life (Williams et al., 2006).

**NCL Clinical** 

**NCL5 (Finnish variant)** 

**NCL10** 

**phenotype** 

**NCL6** LINCL RLP,

**NCL 7** LINCL RLP,

**NCL8** LINCL CLP

LINCL

2002; Kohan et al., 2009).

Congenital

LINCL CLP,
