**7. GM2 gangliosidosis**

236 Novel Aspects on Epilepsy

significance (Watari, et al., 1999). Two luminal functional important domains have been identified: a cysteine-rich loop with a ring-finger motif which harbours about 1/3 of the mutations described in patients and a highly conserved N-terminal domain with a leucine zipper motif which has been shown to possess a cholesterol-binding domain (Davies & Ioannou, 2000). In fact, it has recently been demonstrated that a water soluble fragment of NPC1 is able to bind cholesterol and oxysterols (Infante et al., 2008a; Infante et al., 2008b). The mature NPC1 protein has 14 potential glycosilation sites and shows a size of 170 and 190 kDa *NPC2* gene is mapped to chromosome 14q24.3 and encodes a small soluble protein present

It possess a hydrophobic pocket that has the property to bind cholesterol (Vanier & Millat,

Although it is well known that NPC1 and NPC2 participate together in mediating the egress of cholesterol from endo/lysosomes, the precise mechanism by which these proteins function is not fully understood. It has been demonstrated that a water soluble fragment of NPC1 binds cholesterol in an orientation opposite to NPC2. Based on these results, the following working model was proposed to explain the egress of cholesterol derived from receptor mediated endocytosis of LDL from lysosomes: after liberation from LDL, cholesterol is bound by NPC2 which carries it to the lysosomal membrane, where it transfers

The mutational spectrum of *NPC1* gene is very heterogeneous and to date more than 290 mutations have been reported (http://npc.fzk.de/; Runz et al., 2008). Among them, the mutant allele I1061T is quite frequent in Western Europe and US (Millat et al. 1999, Sun et al. 2001, Park et al. 2003) where it accounts from 20-25% of the alleles. However, it seems to be much less frequent in Italy and Spain (Fernandez- Valero et al., 2005; Fancello et al., 2009; Macias-Vidal et al., 2010), suggesting that there is a gradient of increasing frequency of the

Two other relatively frequent mutations, p.P1007A and p.G992W, have been reported to be associated to the biochemical "variant phenotype" (see section 9), characterized by a milder cholesterol trafficking impairment. The p.G992W mutation is typical of patients from Nova-Scotia but it has been found in patients from other origins (Millat et al., 2001; Ribeiro et al.,

Phenotype-genotype correlation studies are quite difficult to perform due to the very limited number of patients carrying the same genotype. However, some general consideration can be made. It has been shown that the genotype correlates with the neurological form of the disease and not with the systemic manifestations. While a good correlation has been found between the nonsense or frameshift mutations and the more severe infantile form of the disease, the phenotype is more variable in patients carrying missense mutation. However, the presence of missense mutations in the sterol sensing domain of the protein correlates

 It has been proposed that in the homoallelic state mutation I1061T is associated with a severe impairmet of cholesterol trafficking and correlates with the juvenile neurologic form of the disease, while in the heteroallelic state, the final phenotype depends on the mutation present in the second allele but until recently it had never been found in the severe infantile neurologic form. However, a study performed in a Spanish cohort of 30 patients affected with NPC has demonstrated the presence of the p.I1061T mutation in homozygosis in a

in the lumen of the lysosomes (Naureckiene et al., 2000, Vanier & Millat 2004).

to the N-terminal domain of the membrane bound *NPC1* (Kwon et al., 2009).

p.I1061T mutation from southeast to northwest Europe.

2001; Fernandez- Valero et al., 2005; Fancello et al., 2009 ).

patient affected with the severe infantile form (Macias-Vidal, 2010).

with the more severe form of the disease.

2004).

GM2 gangliosidoses are a group of recessive disorders characterized by accumulation of GM2 ganglioside in neuronal cells due to the deficient activity of human β-hexosaminidases (β-N-acetylhexosaminidase, EC3.2.1.52, Hex), ysosomal hydorlases that cleave the terminal N-acetylhexosamine residues from GM2 gangliosides bound to the GM2 activator protein. Two major isoenzymes exist: Hex A consisting of one α and one β subunit encoded by *HEXA* and *HEXB* genes, respectively, and Hex B consisting of two β subunits. In vivo, the GM2/GM2 activator complex is a substrate only for the Hex A isoenzyme. Mutations in either *HEXA* or *HEXB* genes or in the *GM2A* gene (that encodes for the GM2 activator protein) result in GM2 gangliosidosis.

In particular, mutations in the *HEXA* gene cause Tay Sachs disease (TSD; MIM 272800), characterized by deficiency of Hex A activity, while mutations in the *HEXB* gene lead to Sandhoff disease (SD; MIM 26880), characterized by combined deficiency of Hex A and Hex B activities. On the other hand, mutations in the *GM2A* gene cause GM2 activator deficiency, characterized by normal Hex A and Hex B activities but the inability to form a functional GM2/GM2 activator complex. Only few patients with a defect in the *GM2A* gene have been reported whereas most patients affected by GM2 gangliosidosis present mutations in *HEXA* or *HEXB* genes.

While SD disease is panethnic, the incidence of TSD is about one in 3600 Ashkenazi Jewish, corresponding to a carrier frequency of 1 in 30. Among Sephardic Jews and all non-Jews, the disease incidence has been observed to be about 100 times less common, corresponding to a tenfold lower carrier frequency (between 1/250 and 1/300).

#### **7.1 Clinical aspects**

The clinical phenotypes associated with each biochemical variant vary widely from the infantile onset of rapidly progressive neurodegenerative forms, leading to death before the fourth year of life, to the later onset forms, a progressive neurological condition compatible with survival into childhood or long survival (Gravel et al., 2001)

For TSD, three main phenotypes have been identified: classic infantile, juvenile and chronic or adult forms. Signs of the classic infantile TSD are generally evident within the first semester of life. In general noise hypersensitivity with startle response precedes psychomotor retardation, generalized hypotonia, growing of head circumference leading to macrocephalia, amurosis and myoclonic epilepsy. Cherry red spots may be present at funduscopic examination. The peripheral organs are spared from storage process. Disease progression leads to a very severe neurological degeneration until decerebration state. The juvenile form has a later onset, generally between the age of 2-6 years, presenting with behavior modifications and progressive cognitive impairment. Ataxia become evident and

Myoclonic Epilepsy in Lysosomal Storage Disorders 239

mutation (Kaback et al., 1993). By contrast, the abnormal splicing mutation c.1073+1G>A (IVS9+1G>A), absent among the Jewish population, is found in about 15% of the non-Jewish carriers (Akerman et al., 1992). There are mutations in the *HEXA* gene causing the B1 Variant, associated with the late onset form of TSD. This biochemical phenotype is characterized by a Hex A isoenzyme catalytically inactive against the physiological substrate, GM2 ganglioside, but active towards commonly used synthetic substrate (4 methylumbelliferyl β-Nacetylglucosaminide) (Tutor, 2004). Concerning the *HEXA*  mutations associated with the B1 Variant, the most common is the c.533G>A (p.R178H) that was first found predominantly in Portuguese patients (dos Santos et al., 1991; Gravel et al., 2001) and which has been subsequently detected in individuals with different European

Human *HEXB* gene has been located to chromosome 5q13 (MIM 26880) and contains 14 exons distributed over about 40 kb of DNA. To date, about 40 different mutations have been identified to cause SD, most of the have been identified in individual families (http://www.hexdb.mcgill.ca/hexadb; http://www.hgmd.org/). However, a common mutation found in patients with different ethnic backgrounds is a deletion at the 5′ end of the gene that removes 16 kb of DNA including the HEXB promoter, exons 1–5, and part of intron , which account for about 27% of SD alleles (Neote et al., 1988; Bolhuis & Bikker, 1992) . This mutation seems to be quite unfrequent in Italian SD patients. Among this population the most frequent mutation is the c.850C>T (p.R284X) present in 27% of the affected alleles. The high

frequency of this mutation is probably due to a founder effect (Zampieri et al., 2009).

patients with a rare juvenile SD variant (Wang et al., 2008).

therapeutic options and to provide an accurate genetic counselling.

neurological impairment, 3- the presence of visceral involvement.

Although the number of SD patients characterized to date is quite small to perform an analysis of phenotype/genotype correlation, it is of note that missense mutations p.P504S, p.R505Q and p.R533H, seem to be associated to the late onset form of the disease (Maegawa et al., 2006). In addition, the missense mutation p. D459A has been recently discovered in six

The diagnosis of the specific LSD present in patients affected with PME may be challenging. However, the correct diagnosis is crucial in order to implement the best available

Although each LSD presents with specific sings and symptoms, some general features

1- a familiar history suggestive of a genetic disease, 2- association with other signs of

The visceral and neurologic signs most frequently associated to PME in LSD are shown in table 3. At physical examination, dysmorphism is a constant feature of sialidosis type II. Visceral storage represents a major sign of GD and sialidosis, while is generally less evident in NPC, where protracted jaundice is a highly suggestive sign that must be searched during patient anamnesis. Macrocephaly is a diagnostic sign in the infantile TSD, where abnormal growing of head circumference becomes evident with disease progression. With disease progression, ataxic motor impairment is generally detected in all of them, with dystonic movements evident in NPC, NCL and GM2 gangliosidosis, while dysarthria is detectable in AMRF, NPC and GM2 gangliosidosis. Parkinsonian syndrome may be present in adult patients with NCL. Involvement of ocular system is widely described in many LSD, both at

should prompt the physician to suspect the presence of a LSD in a patient with PME:

backgrounds (Montalvo et al., 2006).

**8. Differential diagnosis** 

the disease progresses to decerebrate rigidity. Unlike classic form, blindness is not obligatory. Death occurred between ages 5 and 15 years. Finally, in the adult phenotype the disease may be silent for a prolonged period, becoming evident during school-age. However, the diagnosis may be delayed until adulthood. Clinical presentation is variegated, some patients present with symptoms of atypical Friedreich ataxia, while in others a clinical picture suggestive of Kugelberg-Welander phenotype (progressive leg weakness and fasciculations) was described. A different pattern of motor impairment (including: ataxia, progressive gait disturbance, clumsiness, generalized weakness, mild spasticity, dystonia, dysarthria, tremor involuntary jerks) and cognitive deterioration (loss of memory and comprehension, dementia) has been detected. In some patients mental capacity and behaviour are normal (Neudorfer et al., 2005; Maegawa et al., 2006).

Imaging studies on TSD patients showed different findings in the three different forms, an involvement of basal ganglia and thalamus with cortical atrophy has been detected in classic infantile form, while both juvenile and adult phenotypes do not present basal ganglia abnormalities but show a cortical and cerebellar atrophy, the later characteristic of adult form (Grosso et al., 2003; Inglese et al., 2005; Aydin et al., 2005; Maegawa et al. 2006).

Neurophysiological studies showed a variable pattern of EEG abnormalities with an early progressive loss of the VEP in infantile form. Saccadic abnormalities and impairment of smooth pursuit have also been observed at the evaluation of eye movements in some patients **(**Rapin 1986; Rucker et al. 2004).

In SD clinical findings are indistinguishable from those of TSD. In infantile onset, startle reaction, psychomotor deterioration, early blindness, macrocephaly, cherry red spots are all present. The course of the disease is rapidly fatal, with death within the third year of life. In late-onset forms, cognitive and mental involvement (school difficulties, emotional lability, intermittent psychosis, confusional state) as well as neurological deterioration (muscle weakness, muscle atrophy, fasciculations, supranuclear gaze palsy, muscular atrophy, hyperreflexia, myoclonic jerks, seizures) have been described. Imaging and neurophysiological studies are similar to TSD (Yüksel et al., 1999; Alkan et al., 2003; Hendriksz et al., 2004; Jain et al., 2010)

#### **7.2 Molecular aspets**

The human *HEXA* gene (MIM# 606869) is located on chromosome 15q23-q24 and contains 14 exons. More than 100 mutations have been identified to cause TSD disease, including single base substitutions, small deletions, small duplications/insertions, partial gene deletions, splicing alterations and complex gene rearrangements (http://www.hexdb.mcgill.ca/hexadb; http://www.hgmd.org/; Stenson et al., 2003). Most of these alterations are "private" mutations and have been detected in single or very few families. Others are present in small isolated populations and only a few have been frequently found in diverse populations. In the Ashkenazi Jewish population three distinct *HEXA* mutations are responsible for 98% of all mutant alleles: the most common four-bases duplication c.1274\_1277dupTATC and the splicing mutation c.1421+1G>C (IVS12+1G>C) account for 81% and 15% of alleles, respectively; the alteration in exon 7 c.805G>A (p.G269S), associated with the late onset form of the disease, has been found in approximately 2% of alleles (Kaback et al., 1993). Among the non-Jewish populations the mutation pattern is completely different. Only 30% of the alleles are due to the duplication c.1274\_1277dupTATC, none present the IVS12+1G>C and about 5% carry the G269S

the disease progresses to decerebrate rigidity. Unlike classic form, blindness is not obligatory. Death occurred between ages 5 and 15 years. Finally, in the adult phenotype the disease may be silent for a prolonged period, becoming evident during school-age. However, the diagnosis may be delayed until adulthood. Clinical presentation is variegated, some patients present with symptoms of atypical Friedreich ataxia, while in others a clinical picture suggestive of Kugelberg-Welander phenotype (progressive leg weakness and fasciculations) was described. A different pattern of motor impairment (including: ataxia, progressive gait disturbance, clumsiness, generalized weakness, mild spasticity, dystonia, dysarthria, tremor involuntary jerks) and cognitive deterioration (loss of memory and comprehension, dementia) has been detected. In some patients mental capacity and

Imaging studies on TSD patients showed different findings in the three different forms, an involvement of basal ganglia and thalamus with cortical atrophy has been detected in classic infantile form, while both juvenile and adult phenotypes do not present basal ganglia abnormalities but show a cortical and cerebellar atrophy, the later characteristic of adult

Neurophysiological studies showed a variable pattern of EEG abnormalities with an early progressive loss of the VEP in infantile form. Saccadic abnormalities and impairment of smooth pursuit have also been observed at the evaluation of eye movements in some

In SD clinical findings are indistinguishable from those of TSD. In infantile onset, startle reaction, psychomotor deterioration, early blindness, macrocephaly, cherry red spots are all present. The course of the disease is rapidly fatal, with death within the third year of life. In late-onset forms, cognitive and mental involvement (school difficulties, emotional lability, intermittent psychosis, confusional state) as well as neurological deterioration (muscle weakness, muscle atrophy, fasciculations, supranuclear gaze palsy, muscular atrophy, hyperreflexia, myoclonic jerks, seizures) have been described. Imaging and neurophysiological studies are similar to TSD (Yüksel et al., 1999; Alkan et al., 2003;

The human *HEXA* gene (MIM# 606869) is located on chromosome 15q23-q24 and contains 14 exons. More than 100 mutations have been identified to cause TSD disease, including single base substitutions, small deletions, small duplications/insertions, partial gene deletions, splicing alterations and complex gene rearrangements (http://www.hexdb.mcgill.ca/hexadb; http://www.hgmd.org/; Stenson et al., 2003). Most of these alterations are "private" mutations and have been detected in single or very few families. Others are present in small isolated populations and only a few have been frequently found in diverse populations. In the Ashkenazi Jewish population three distinct *HEXA* mutations are responsible for 98% of all mutant alleles: the most common four-bases duplication c.1274\_1277dupTATC and the splicing mutation c.1421+1G>C (IVS12+1G>C) account for 81% and 15% of alleles, respectively; the alteration in exon 7 c.805G>A (p.G269S), associated with the late onset form of the disease, has been found in approximately 2% of alleles (Kaback et al., 1993). Among the non-Jewish populations the mutation pattern is completely different. Only 30% of the alleles are due to the duplication c.1274\_1277dupTATC, none present the IVS12+1G>C and about 5% carry the G269S

form (Grosso et al., 2003; Inglese et al., 2005; Aydin et al., 2005; Maegawa et al. 2006).

behaviour are normal (Neudorfer et al., 2005; Maegawa et al., 2006).

patients **(**Rapin 1986; Rucker et al. 2004).

Hendriksz et al., 2004; Jain et al., 2010)

**7.2 Molecular aspets** 

mutation (Kaback et al., 1993). By contrast, the abnormal splicing mutation c.1073+1G>A (IVS9+1G>A), absent among the Jewish population, is found in about 15% of the non-Jewish carriers (Akerman et al., 1992). There are mutations in the *HEXA* gene causing the B1 Variant, associated with the late onset form of TSD. This biochemical phenotype is characterized by a Hex A isoenzyme catalytically inactive against the physiological substrate, GM2 ganglioside, but active towards commonly used synthetic substrate (4 methylumbelliferyl β-Nacetylglucosaminide) (Tutor, 2004). Concerning the *HEXA*  mutations associated with the B1 Variant, the most common is the c.533G>A (p.R178H) that was first found predominantly in Portuguese patients (dos Santos et al., 1991; Gravel et al., 2001) and which has been subsequently detected in individuals with different European backgrounds (Montalvo et al., 2006).

Human *HEXB* gene has been located to chromosome 5q13 (MIM 26880) and contains 14 exons distributed over about 40 kb of DNA. To date, about 40 different mutations have been identified to cause SD, most of the have been identified in individual families (http://www.hexdb.mcgill.ca/hexadb; http://www.hgmd.org/). However, a common mutation found in patients with different ethnic backgrounds is a deletion at the 5′ end of the gene that removes 16 kb of DNA including the HEXB promoter, exons 1–5, and part of intron , which account for about 27% of SD alleles (Neote et al., 1988; Bolhuis & Bikker, 1992) . This mutation seems to be quite unfrequent in Italian SD patients. Among this population the most frequent mutation is the c.850C>T (p.R284X) present in 27% of the affected alleles. The high frequency of this mutation is probably due to a founder effect (Zampieri et al., 2009).

Although the number of SD patients characterized to date is quite small to perform an analysis of phenotype/genotype correlation, it is of note that missense mutations p.P504S, p.R505Q and p.R533H, seem to be associated to the late onset form of the disease (Maegawa et al., 2006). In addition, the missense mutation p. D459A has been recently discovered in six patients with a rare juvenile SD variant (Wang et al., 2008).
