**6. Niemann pick type C (NPC) disease**

Niemann Pick type C (NPC) disease (NPC1, MIM 257220; NPC2, MIM 607625) is an autosomal recessive neurodegenerative lysosomal storage disorder, caused by the abnormal function of NPC1 or NPC2 protein. Both proteins are involved in the intracellular trafficking of cholesterol and other lipids. The deficiency of either of them leads to the accumulation of the endocytosed unesterified cholesterol within the lysosomes (Patterson et al., 2001).

Endocyted low density lipoproteins are delivered to the late endosomes/lysosomes where they are hydrolized. In normal cells, free cholesterol is transported to the plasma membrane or to the endoplasmic reticulum through the action of NPC1 and NPC2 proteins. In NPC cells cholesterol accumulate within the lysosomes and the subsequent induction of all lowdensity lipoprotein cholesterol-mediated homeostatic responses, including cholesterol esterification, is compromised.

In addition NPC-deficient cells also accumulate gangliosides and other GSLs. These findings show that the defect in NPC cells encompasses a global transport error. In fact, while unesterified cholesterol is the main lipid accumulated in peripheral tissues, GM3, GM2 and glucosylceramide are the mayor lipids accumulated in brain of NPC patients (Zervas et al., 2001a).

Myoclonic Epilepsy in Lysosomal Storage Disorders 235

pyramidal tract involvment, hearing loss (Wraith et al., 2009). Seizures are uncommon in

In late infantile forms (2 to <6 years), hepatosplenomegaly is usually present. Language delay is frequent and these children often present gait problems, frequent falls and clumsiness. Cataplexy is quite frequent and vertical supranuclear gaze palsy (VSGP) is usually present but it may not be recognized at this early stage. Progressive ataxia is followed by dystonia, dysphagia, dysarthria and central hypotonia. Hearing loss has been described (Wraith et al., 2009; Vanier 2010). A significant proportion of patients develop seizures, partial, generalized or both. In general these patients respond to standard antiepileptic treatment but some cases may be refractory to therapy. Severe epilepsy has a bad prognosis and shortens the lifespan of patients. As disease progress patients develop pyramidal signs, spasticity and swallowing problems. In most cases patients die between 7

The juvenile form (6 to 15 years is in many countries the most frequent form of the disease. Moderate splenomegaly or hepatosplenomegaly is frequently present and may have been detected at early time. However, in at least 10% of the cases organomegaly is not present. School failure, learning disability and behavioral problems are the most common signs. VSGP is almost invariabile present and may be the first sign. As the disease progress the children present frequent falls, clumsiness and develop progressive ataxia, dysarthria, dystonia, dysphagia. Cataplexy and myoclonus are other common symptoms. About half of the patients with this form develop seizures (partial and/or generalized). At late stage patients develop Pyramidal signs, spasticity and swollowing problems (Wraith et al., 2009;

Even if during the last years many patients affected with the adult form (>15 years) of the disease have been reported, this diagnosis has been probably underestimated. Organomegaly or isolated splenomegaly are rare in adult patients and VSGP is usually present. The most common clinical presentation is similar to that of a juvenile form but attenuated. However, it is worth of note that about one third of patients present with psychiatric signs that may appear several years before the onset neurological symptoms. During this period the neurological examination may be normal. Among the psychiatric signs, paranoid delusions and auditory or visual hallucinations are the most commonly described. Other psychiatric signs that may be present in these patients are depressive syndrome, behavioral problems with aggressiveness, social isolation, bipolar disorders, obsessive compulsive disorders. Epilepsy is not very common in this group of patients

As mentioned above, two disease-causing genes, *NPC1 (*NM000271) and *NPC2* (NM006432) have been identified (Steinberg et al., 1994; Vanier et al., 1996 ; Cartsea et al., 1997). About 95% of human NPC disease is caused by mutations in the *NPC1* gene (Naureckiene et al., 2000). *NPC1* gene, located on chromosome 18q11-q12, encodes a large membrane glycoprotein of 1278 aminoacids containing 13 transmembrane domains and located predominantly in late endosomes (Davies & Ioannou, 2000). It presents a sterol sensing domain (SSD), which shows extensive homology with the sterol sensing domains (SSD) found in SREBP cleavage activating protein (SCAP) and 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase, two cholesterol regulated proteins. The SSD domain appears to have important functional

(15%) and the course is similar to that in the juvenile form (Vanier 2010).

these patients and they usually die during the first 5 years of age (Vanier 2010).

to 12 years of age (Vanier 2010).

Vanier 2010).

**6.2 Molecular aspects** 

Approximately 95% of NPC patients present mutations in *NPC1* gene (MIM 607623) (Carstea et al., 1993; Vanier et al., 1996) , while the other 5% of patients present mutations in *NPC2* gene (MIM 601015) (Naureckiene et al., 2000)

The incidence of NPC disease has been difficult to assess. Estimates of incidences ranging from 0,66 to 0,83 per 100000 were proposed for France, UK and Germany based on the diagnoses made over a period 1988-2002. This incidence is probably underestimated since the wide clinical spectrum of NPC disease was not recognized until the early 90's and no specific laboratory testing was available until the mid 80s. A probably more realistic incidence of 0,96/100000 was recently calculated considering the total amount of cases diagnosed in France from 2000-2009 (including prenatal cases from terminated pregnancies) vs the number of birth during the same period. However, this data is likely to be still underestimated due to the presence of atypical phenotypes that may not be recognized, in particular among adult patients (Patterson, 2001; Vanier and Millat, 2003).

#### **6.1 Clinical aspects**

Clinically, NPC disease presents a highly variable phenotype ranging from fetal to adult age. It is classically a neurovisceral condition, characterized by liver and/or spleen enlargement, and neurological or psychiatric manifestations. Systemic disease, when present, always precedes the neurological symptoms. However, it is absent in about 15% of patients and in about half of the adult onset patients (Vanier 2010).

It is important to point out that the course of the systemic signs is independent of that of the course of the neurological symptoms and that disease progression and lifespan are always correlated with the age at onset of the neurological symptoms.

Even if initial manifestations may be systemic, neurological, or psychiatric, the disease has been classified according to the age at onset of neurological symptoms. Although the neurological forms of the disease may be considered as a continuous of phenotypes, the disease has been classically classified in a severe infantile form (onset before 2 y of age), a late infantile form (onset between 3-5 y of age), a juvenile form (onset between 5 and 16 y) and an adult form (onset at age>16 y) (Patterson et al., 2001; Vanier & Millat, 2003).

A perinatal form of NPC has also been described. This form is characterized by the presence of prolonged neonatal cholestatic icterus, appearing within the first weeks of life and often associated with progressive hepatosplenomegay (Kelly et al., 1993; Vanier et al., 1998; Yerushalmi et al., 2002). In most cases, the icterus spontaneously resolves at 2-4 month of age while the hepatosplenomegaly remains for a variable period. In about 10% of patients the icterus worsens leading to liver failure and dead within the first 6 month of age (Vanier et al., 1998). Some patients, in particular those presenting mutations in *NPC2* gene, may present with hepatosplenomegay in association with a severe respiratory insufficiency, which in most cases is fatal. It is important to note that NPC patients do not present neurological symptoms during the neonatal period. However, an important observation to consider during the genetic counseling is the fact that in many cases patients who die during the perinatal period have siblings affected with the infantile or juvenile neurological form (Vanier & Susuki, 1998; Vanier and Millat, 2003).

Patients affected with early infantile form (3 month to < 2 years**)** almost invariable present with isolated hepatosplenomagaly during the first month of age followed by delay of development motor milestones, which presents at around 8-9 month of age, and central hypotonia. Subsequent clinical course includes loss of acquired motor skills, spasticity with

Approximately 95% of NPC patients present mutations in *NPC1* gene (MIM 607623) (Carstea et al., 1993; Vanier et al., 1996) , while the other 5% of patients present mutations in

The incidence of NPC disease has been difficult to assess. Estimates of incidences ranging from 0,66 to 0,83 per 100000 were proposed for France, UK and Germany based on the diagnoses made over a period 1988-2002. This incidence is probably underestimated since the wide clinical spectrum of NPC disease was not recognized until the early 90's and no specific laboratory testing was available until the mid 80s. A probably more realistic incidence of 0,96/100000 was recently calculated considering the total amount of cases diagnosed in France from 2000-2009 (including prenatal cases from terminated pregnancies) vs the number of birth during the same period. However, this data is likely to be still underestimated due to the presence of atypical phenotypes that may not be recognized, in

Clinically, NPC disease presents a highly variable phenotype ranging from fetal to adult age. It is classically a neurovisceral condition, characterized by liver and/or spleen enlargement, and neurological or psychiatric manifestations. Systemic disease, when present, always precedes the neurological symptoms. However, it is absent in about 15% of

It is important to point out that the course of the systemic signs is independent of that of the course of the neurological symptoms and that disease progression and lifespan are always

Even if initial manifestations may be systemic, neurological, or psychiatric, the disease has been classified according to the age at onset of neurological symptoms. Although the neurological forms of the disease may be considered as a continuous of phenotypes, the disease has been classically classified in a severe infantile form (onset before 2 y of age), a late infantile form (onset between 3-5 y of age), a juvenile form (onset between 5 and 16 y)

A perinatal form of NPC has also been described. This form is characterized by the presence of prolonged neonatal cholestatic icterus, appearing within the first weeks of life and often associated with progressive hepatosplenomegay (Kelly et al., 1993; Vanier et al., 1998; Yerushalmi et al., 2002). In most cases, the icterus spontaneously resolves at 2-4 month of age while the hepatosplenomegaly remains for a variable period. In about 10% of patients the icterus worsens leading to liver failure and dead within the first 6 month of age (Vanier et al., 1998). Some patients, in particular those presenting mutations in *NPC2* gene, may present with hepatosplenomegay in association with a severe respiratory insufficiency, which in most cases is fatal. It is important to note that NPC patients do not present neurological symptoms during the neonatal period. However, an important observation to consider during the genetic counseling is the fact that in many cases patients who die during the perinatal period have siblings affected with the infantile or juvenile neurological form

Patients affected with early infantile form (3 month to < 2 years**)** almost invariable present with isolated hepatosplenomagaly during the first month of age followed by delay of development motor milestones, which presents at around 8-9 month of age, and central hypotonia. Subsequent clinical course includes loss of acquired motor skills, spasticity with

and an adult form (onset at age>16 y) (Patterson et al., 2001; Vanier & Millat, 2003).

particular among adult patients (Patterson, 2001; Vanier and Millat, 2003).

patients and in about half of the adult onset patients (Vanier 2010).

correlated with the age at onset of the neurological symptoms.

(Vanier & Susuki, 1998; Vanier and Millat, 2003).

*NPC2* gene (MIM 601015) (Naureckiene et al., 2000)

**6.1 Clinical aspects** 

pyramidal tract involvment, hearing loss (Wraith et al., 2009). Seizures are uncommon in these patients and they usually die during the first 5 years of age (Vanier 2010).

In late infantile forms (2 to <6 years), hepatosplenomegaly is usually present. Language delay is frequent and these children often present gait problems, frequent falls and clumsiness. Cataplexy is quite frequent and vertical supranuclear gaze palsy (VSGP) is usually present but it may not be recognized at this early stage. Progressive ataxia is followed by dystonia, dysphagia, dysarthria and central hypotonia. Hearing loss has been described (Wraith et al., 2009; Vanier 2010). A significant proportion of patients develop seizures, partial, generalized or both. In general these patients respond to standard antiepileptic treatment but some cases may be refractory to therapy. Severe epilepsy has a bad prognosis and shortens the lifespan of patients. As disease progress patients develop pyramidal signs, spasticity and swallowing problems. In most cases patients die between 7 to 12 years of age (Vanier 2010).

The juvenile form (6 to 15 years is in many countries the most frequent form of the disease. Moderate splenomegaly or hepatosplenomegaly is frequently present and may have been detected at early time. However, in at least 10% of the cases organomegaly is not present. School failure, learning disability and behavioral problems are the most common signs. VSGP is almost invariabile present and may be the first sign. As the disease progress the children present frequent falls, clumsiness and develop progressive ataxia, dysarthria, dystonia, dysphagia. Cataplexy and myoclonus are other common symptoms. About half of the patients with this form develop seizures (partial and/or generalized). At late stage patients develop Pyramidal signs, spasticity and swollowing problems (Wraith et al., 2009; Vanier 2010).

Even if during the last years many patients affected with the adult form (>15 years) of the disease have been reported, this diagnosis has been probably underestimated. Organomegaly or isolated splenomegaly are rare in adult patients and VSGP is usually present. The most common clinical presentation is similar to that of a juvenile form but attenuated. However, it is worth of note that about one third of patients present with psychiatric signs that may appear several years before the onset neurological symptoms. During this period the neurological examination may be normal. Among the psychiatric signs, paranoid delusions and auditory or visual hallucinations are the most commonly described. Other psychiatric signs that may be present in these patients are depressive syndrome, behavioral problems with aggressiveness, social isolation, bipolar disorders, obsessive compulsive disorders. Epilepsy is not very common in this group of patients (15%) and the course is similar to that in the juvenile form (Vanier 2010).

#### **6.2 Molecular aspects**

As mentioned above, two disease-causing genes, *NPC1 (*NM000271) and *NPC2* (NM006432) have been identified (Steinberg et al., 1994; Vanier et al., 1996 ; Cartsea et al., 1997). About 95% of human NPC disease is caused by mutations in the *NPC1* gene (Naureckiene et al., 2000). *NPC1* gene, located on chromosome 18q11-q12, encodes a large membrane glycoprotein of 1278 aminoacids containing 13 transmembrane domains and located predominantly in late endosomes (Davies & Ioannou, 2000). It presents a sterol sensing domain (SSD), which shows extensive homology with the sterol sensing domains (SSD) found in SREBP cleavage activating protein (SCAP) and 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase, two cholesterol regulated proteins. The SSD domain appears to have important functional

Myoclonic Epilepsy in Lysosomal Storage Disorders 237

So far, only 19 mutations have been reported in the *NPC2* gene. Among them the most frequent mutation is the p.E20X. A good correlation between the severity of the mutation

Most reported patients affected with mutations in the *NPC2* gene presented a very severe fatal form of the disease leading to dead within the first years of life. Only few patients presenting a slower disease progression and a longer survival have been described so far

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

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*

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

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

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

and the clinical course of the disease has been found.

**7. GM2 gangliosidosis** 

or *HEXB* genes.

**7.1 Clinical aspects** 

protein) result in GM2 gangliosidosis.

(Klunemann et al., 2002; Millat et al., 2001; Millat et al., 2005).

tenfold lower carrier frequency (between 1/250 and 1/300).

with survival into childhood or long survival (Gravel et al., 2001)

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

It possess a hydrophobic pocket that has the property to bind cholesterol (Vanier & Millat, 2004).

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

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 p.I1061T mutation from southeast to northwest Europe.

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

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 with the more severe form of the disease.

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

So far, only 19 mutations have been reported in the *NPC2* gene. Among them the most frequent mutation is the p.E20X. A good correlation between the severity of the mutation and the clinical course of the disease has been found.

Most reported patients affected with mutations in the *NPC2* gene presented a very severe fatal form of the disease leading to dead within the first years of life. Only few patients presenting a slower disease progression and a longer survival have been described so far (Klunemann et al., 2002; Millat et al., 2001; Millat et al., 2005).
