**9. Therapeutic options**

244 Novel Aspects on Epilepsy

patients may be difficult. Measurement of the LDL-induced rate of cholesterol esterification may be used as a secondary test. However, while very low levels rates of esterification are detected in cell lines with a "classical" biochemical phenotype only a mild or non-significant impairment is detected in those with a "variant" phenotype. Therefore, in these cases mutational analysis of *NPC1* and *NPC2* are necessary in order to provide a definitive diagnosis. Since some mutations of *NPC1* gene have been associated to the variant biochemical phenotype (see 6.2), it is advisable to screen the presence of these mutations in

Finally, the molecular analysis should be performed in all newly diagnosed patients since molecular genetic studies are the highly preferred strategy for prenatal diagnosis, and the

The suspect of **GM2 gangliosidosis** can be confirmed by the measurement of βhexosaminidases activities in blood leukocytes or cultured fibroblasts. From the biochemical point of view, the differential diagnosis between SD and TSD, the most common causes of GM2 gangliosidosis, can be performed by the assessment of total Hex activity, the HEX activity after heat inactivation and the specific assay of the HexA isoenzyme in leukocytes or fibroblasts. The synthetic substrate usually used is the 4-methylumbelliferyl N- acetyl glucosaminide which can be digested by both HexA (heterodimeroand HexB (/ homodimero) isoenzymes and it is used to determine the total Hex activity. Since the HexA is thermolabile, it can be inactivated by heating the sample at 50°. The activity against the 4 methylumbelliferyl N- acetyl-glucosaminide after heat inactivation is represented only by HexB. This value is used to determine the % of HexA and HexB activity The specific activity of Hex A isoenzyme can be measured using the synthetic substrate, 4-methylumbelliferyl Nacetyl -glucosamine 6-sulfate (MUGS) (Bayleran, et al., 1984 ). Sandhoff disease is characterized by the impairment of both HexA and HexB activities and therefore total Hex activity is very low. A residual Hex A activity may be detected in these patients due the presence of HexS, consisting in two subunits, which is not deficient in SD and is also

Tay Sachs disease is confirmed by the presence of reduced levels of total Hex and very low levels of HexA. It is important to keep in mind that the B1 variant of Tay Sachs is characterized by the presence of an Hex A isoenzyme catalytically inactive against the physiological substrate, GM2 ganglioside, but active towards commonly used synthetic substrate 4-methylumbelliferyl –N-acetyl glucosaminide (Tutor, 2004). Biochemical identification of these patients requires always the use of the specific substrate MUGS.

In the case of normal Hex activities a deficiency of the GM2 activator protein should be suspected. In this case, the definitive diagnosis is achieved by the molecular analysis of the

In patients with a biochemical diagnosis of SD and TSD it is advisable to perform the molecular analysis of *HEXA* or *HEXB* genes, respectively, in order to confirm the diagnosis and to provide genetic counseling. In addition in patients with a biochemical pattern compatible with a diagnosis of TSD disease it is important to exclude the presence of a pseudodeficiency due to specific mutations (p.R247W and p.R249W) in the *HEXA* gene. These protein variants are inactive towards the synthetic substrates but active towards the

natural substrate, GM2 ganglioside (Triggs-Raine et al., 1992; Cao et al., 1993).

only reliable one for identification of carriers in blood relatives (Vanier et al., 2010).

patients presenting a variant phenotype.

active towards the synthetic substrate.

(Bayleran et al., 1984 ).

GM2A gene.

Twenty years ago the availability of enzyme replacement therapy (ERT) for GD opened a new era for the treatment of LSDs, giving to the patients a concrete hope for recovering (Brady, 2006; Connock et al. 2006). However, clinical history of GD demonstrated the limited effect of ERT on neurological phenotypes. The difficulty to cross the blood-brain barrier for macromolecule such glycoproteins prevent the neuronal access to the intravenous infused enzyme. Despite the good efficacy in correcting the visceral and hematological alterations of the disease also in neurological phenotypes, only a very limited number of patients seem to benefit from ERT, showing an improvement of EEG pattern and a stabilization of neurological conditions. Quite all of them carried the L444P mutation in homozygosis. On the contrary very few are GD3 patients presenting with myoclonic epilepsy that carry these mutation in homozygosis or heterozygosis with other rare mutations. Therefore, myoclonic epilepsy represents a unfavorable prognostic factor in GD3 (Altarescu et al., 2001). Despite the negative results obtained by Schiffmann et al. (2008), Capablo at al. (2007) showed an improvement of neurologic conditions and EEG pattern as well as a decrease of the epileptic crisis in patients who presented with myoclonic sezures and the L444P/E326K+N188S phenotype, after 12 month treatment with combined ERT and substrate reduction therapy (SRT). Recently, Accardo et al. (2010), demonstrated the recovery of saccades in two GD3 sisters in course of SRT. The availability of small molecules capable to cross the blood-brain barrier might widening therapeutic prospective in neuronopathic GD.

Glycosphingolipids reduction therapy may represent a strategy also for other glycosphingolipidosis, like NPC and GM2 gangliosidosis (Platt et al., 2005; Platt & Lachmann, 2009).

Different clinical experiences have been reported in literature concerning SRT in NPC patients. The results of clinical trials performed both in pediatric and adult patients showed a significant improvement of swallowing and saccades, as well as an overall stabilization of neurological conditions (Patterson et al. 2007; Galanaud et al. 2009). Substrate reduction therapy has also been used to reduce glicosphingolipids synthesis in GM2 gangliosidosis patients (Bembi et al., 2006;Shapiro et al., 2009), both in infantile and late-onset forms, without any evidence of measurable benefits.

Very recently a Clarke JT et al. (2011) have demonstrated an in vivo enhancement of Hex A activity in a group of late-onset GM2 patients (TSD and Sandhoff) treated with pyrimethamine for a period of 16 weeks. The study was aimed to analyze drug safety and no data on clinical results are available at present.

Apart from symptomatic and supportive therapy, no specific treatments are at present available for NCL and sialidosis, even if preclinical therapeutic programs are ongoing, based on enzyme and gene therapy, stem cell replacement and immunotherapy (Wang et al, 2005; Hobert & Dawson, 2006).
