**8. Differential diagnosis**

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 therapeutic options and to provide an accurate genetic counselling.

Although each LSD presents with specific sings and symptoms, some general features should prompt the physician to suspect the presence of a LSD in a patient with PME:

1- a familiar history suggestive of a genetic disease, 2- association with other signs of neurological impairment, 3- the presence of visceral involvement.

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

Myoclonic Epilepsy in Lysosomal Storage Disorders 241

with sialidosis type II ( Caciotti et al., 2009). However, about 30% of individual from various genetic origins carry a chitotriosidase gene with a 24 bp duplication that prevents the production of the enzyme. Therefore, about 6% of the population is homozygous for this

Patients affected with sialidosis excrete increased amount of several oligosaccharides and sialylglycopeptides derived from glycoproteins. Since the metabolic defect in these patients results in the inability to cleave sialic acid, the accumulated oligosaccharides are rich in sialic acid. Thus, a first screening test that may be performed when a sialidosis is suspected is the analysis of oligosaccharides in urine by thin layer chromatography (TLC). Staining of oligosaccharides resolved by TLC reveals a abnormal pattern in affected patients. However, abnormal patters of urine oligosaccharides are also found in patients affected with other disorders of glycoprotein degradation. In addition it is also possible to analyze the presence of sialic acid containing oligosaccharides by staining the TLC plates with resorcinol (Holmes

Minor elevation of liver enzymes

Elevation serum chitotriosidase acivity

Abnormal pattern of urin oligosaccharides

enzyme (ACE) and ferritin

Table 4. Non specific laboratory findings in patients affected with LSDs that may present

The presence of glycolipid-laden macrophages in various tissues is a hallmark of GD. In particular the presence of these "Gaucher cells" in bone marrow aspirates provide a strong support for this diagnosis. However, these cells have to be distinguished from those present in other disorders that exhibit pathological macrophages as a hallmark, such as the sea blue histiocyte syndrome or NPC disease. In addition, foam cells my also be present in bone marrow samples of patients affected with sialidosis. Although the examination of bone marrow aspiration may be useful for the diagnosis of GD, NPC disease and sialidosis, it should not be necessarily the initial diagnostic test considering the invasiveness of the

A schematic approach to the laboratory diagnosis of the specific LSDs discussed in this

The suspect of **GD** can be confirmed by the assessment of GBA activity in peripheral blood leukocytes or cultured fibrobalsts. A residual activity below 15 % of the mean normal

However, it is important to keep in mind that **AMRF i**s caused by a mistargeting of GBA enzyme due to a defect in its receptor LIMP-2 and therefore patients affected by this disorder

Elevation of acid phosphatase, angiotensin converting

Moderate elevation of serum chitotriosidase acivity

mutant allele and completely lack chitotriosidase activity.

**LSD Non specific laboratory findings** Gaucher Anemia, thrombocytopenia,

Niemann Pick type C Reduced plasma levels of HDL-cholesterol

Sialidosis Elevation of serum chitotriosidase.

AMRF Proteinuria

& O'Brien, 1979).

with PME.

procedure.

**8.1.1 Specific test** 

activity is diagnostic.

chapter is represented in figure 1.

functional and tissue storage levels. Supranuclar gaze palsy is patognomonic in NPC, but is also present in GD3 and AMFR, while blindness affects particularly infantile TSD and NCL (infantile NCL1 and late-infantile NCL Finnish variant, CLN5); green-red and nocturnal visual loss may be present in type 1 sialidosis. Signs of retinal storage are detectable in form of cherry-red spot (in Sialidosis and GM2), pigmentary degeneration (diagnostic sign in NCL) and optic atrophy (NCL and GM2 gangliosidosis). Degeneration of mental capacities with different grade of severity are constantly present in all these pathologies, while skeleton is severely involved by in sialidosis and usually mildly affected in GD3. Finaly, renal failure characterized the late phase of AMFR, but in form of nephritic syndrome may affect sialidosis type 2.


Table 3. visceral and neurologic signs most frequently associated to PME in LSD

#### **8.1 Laboratory diagnosis**

Routine laboratory tests result usually normal in patients with LSDs, with just few exceptions summarized in table 4.

On the other hand, in patients with PME in whom the presence of a LSD is suspected, some relative simple tests may be performed (Table 4). The assessment of chitotriosidase activity in serum, a marker of macrophage activation, is substantially elevated in patients affected with GD and may be slightly elevated in patients with NPC disease. In addition, a recent report described the presence of high levels of chitotriosidase activity in 2 patients affected

functional and tissue storage levels. Supranuclar gaze palsy is patognomonic in NPC, but is also present in GD3 and AMFR, while blindness affects particularly infantile TSD and NCL (infantile NCL1 and late-infantile NCL Finnish variant, CLN5); green-red and nocturnal visual loss may be present in type 1 sialidosis. Signs of retinal storage are detectable in form of cherry-red spot (in Sialidosis and GM2), pigmentary degeneration (diagnostic sign in NCL) and optic atrophy (NCL and GM2 gangliosidosis). Degeneration of mental capacities with different grade of severity are constantly present in all these pathologies, while skeleton is severely involved by in sialidosis and usually mildly affected in GD3. Finaly, renal failure characterized the late phase of AMFR, but in form of nephritic syndrome may

dysmorphisms - - - + visceral storage + - +- + -

joundice - - + - - macrocephaly - - -- - + ataxia + + + + + + dystonia - - + + - + dysarthria - + + - - + parkinsonism - - - + - gaze palsy + + +- - blindness - - -+ + + cherry red spot - - - - + +

optic atrophy - - -+ - +

involvement + - - - + -

involvement - + - - + -

Routine laboratory tests result usually normal in patients with LSDs, with just few

On the other hand, in patients with PME in whom the presence of a LSD is suspected, some relative simple tests may be performed (Table 4). The assessment of chitotriosidase activity in serum, a marker of macrophage activation, is substantially elevated in patients affected with GD and may be slightly elevated in patients with NPC disease. In addition, a recent report described the presence of high levels of chitotriosidase activity in 2 patients affected

Table 3. visceral and neurologic signs most frequently associated to PME in LSD

**GD3 AMRF NPC NCL Sialidosi GM2** 


+ + + + + +

**gangliosidosis** 

affect sialidosis type 2.

protracted

retinal degeneration

mental deterioration

skeletal

**8.1 Laboratory diagnosis** 

exceptions summarized in table 4.

renal

with sialidosis type II ( Caciotti et al., 2009). However, about 30% of individual from various genetic origins carry a chitotriosidase gene with a 24 bp duplication that prevents the production of the enzyme. Therefore, about 6% of the population is homozygous for this mutant allele and completely lack chitotriosidase activity.

Patients affected with sialidosis excrete increased amount of several oligosaccharides and sialylglycopeptides derived from glycoproteins. Since the metabolic defect in these patients results in the inability to cleave sialic acid, the accumulated oligosaccharides are rich in sialic acid. Thus, a first screening test that may be performed when a sialidosis is suspected is the analysis of oligosaccharides in urine by thin layer chromatography (TLC). Staining of oligosaccharides resolved by TLC reveals a abnormal pattern in affected patients. However, abnormal patters of urine oligosaccharides are also found in patients affected with other disorders of glycoprotein degradation. In addition it is also possible to analyze the presence of sialic acid containing oligosaccharides by staining the TLC plates with resorcinol (Holmes & O'Brien, 1979).


Table 4. Non specific laboratory findings in patients affected with LSDs that may present with PME.

The presence of glycolipid-laden macrophages in various tissues is a hallmark of GD. In particular the presence of these "Gaucher cells" in bone marrow aspirates provide a strong support for this diagnosis. However, these cells have to be distinguished from those present in other disorders that exhibit pathological macrophages as a hallmark, such as the sea blue histiocyte syndrome or NPC disease. In addition, foam cells my also be present in bone marrow samples of patients affected with sialidosis. Although the examination of bone marrow aspiration may be useful for the diagnosis of GD, NPC disease and sialidosis, it should not be necessarily the initial diagnostic test considering the invasiveness of the procedure.

#### **8.1.1 Specific test**

A schematic approach to the laboratory diagnosis of the specific LSDs discussed in this chapter is represented in figure 1.

The suspect of **GD** can be confirmed by the assessment of GBA activity in peripheral blood leukocytes or cultured fibrobalsts. A residual activity below 15 % of the mean normal activity is diagnostic.

However, it is important to keep in mind that **AMRF i**s caused by a mistargeting of GBA enzyme due to a defect in its receptor LIMP-2 and therefore patients affected by this disorder

Myoclonic Epilepsy in Lysosomal Storage Disorders 243

Fig. 1. Schematic representation of the laboratory diagnostic strategy in suspected cases of

PME due to LSD

also show low levels of GBA activity in fibroblasts but slightly reduced or normal in peripheral blood leukocytes. AMRF should always be considered in patients with reduced intracellular GBA activity in the absence of other markers of Gaucher disease, such as elevated serum chitotriosidase activity or the presence of "Gaucher" cells in bone marrow.

The determination of GBA activity in serum should be perform in order to provide a differential diagnosis since it is elevated only in patients affected with AMRF (Dardis et al., 2009).

In both cases the molecular analysis of *GBA* or *SCARB*-*2* gene should be carried out in order to confirm the diagnosis and to provide a genetic counseling.

The diagnostic approach of **NCL** depends on the type of defect that is suspected. As shown in figure 1, the diagnosis of **NCL 1, NCL2 and NCL10** can be achieved by the assessment of PPT1, TPP1 or Cathepsin D activity in leukocytes or cultured fibroblasts. If **NCL3** is suspected, the diagnosis can be confirmed by the presence of typical vacuoles in the cytoplasm of the patient lymphocytes, which are detectable on a regular blood smear (Kohlschütter & Schulz, 2009).

In the case of **NCL5, NCL6, NCL7 and NCL8** it is advisable to investigate the presence of storage material by electron microscopic examination of skin biopsy material or isolated lymphocytes as a first approach and then procede to the molecular genetic studies. The definitive diagnosis in all cases is reached by the molecular analysis of the corresponding genes (Kohlschütter & Schulz, 2009).

The definitive diagnosis of **sialidosis** is achieved by measuring the NEU1 activity is fresh samples of blood leukocytes or cultured fibroblasts. Special care should be taken to ensure that the tissue to be examined has not been frozen or exposed to prolonged sonication since the neuraminidase is quite unstable (Den Tandt & Brossemer, 1984). The residual enzymatic activity is extremely low or absent in patients affected with sialidosis independently of the severity of the clinical phenotype. On the contrary, a good correlation between the genotype and the phenotype has been found, therefore the molecular analysis of the *NEU1* may provide useful information about disease severity and progression, which is particularly relevant to provide a better genetic counseling.

It is important to keep in mind that also the Galactiosialidosis, a LSD associated with combined deficiency of NEU1 and galactosidase due to the defect of the proteictive protein /cathepsin A (PPCA), results in reduced levels of NEU1 activity. However, in this case the levels of NEU1 are not as low as in sialidosis and they are associated with low levels of galactosidase activity.

The diagnosis of NPC disease may be quite challenging. It is time consuming and should be performed by specialized centers with the required experience.

The biochemical diagnosis is based on the demonstration of the impaired intracellular cholesterol transport and homeostasis in fibroblasts in culture. The filipin test is considered the more specific and sensitive assay. Cells are cultured in the presence of LDL enriched medium and then fixed and stained with filipin, a molecule that has a high affinity for unesterified cholesterol (Blanchette-Mackie et al., 1988). In patients with NPC disease, fluorescence microscopic examination of stained cells shows in most of them, the presence of strong fluorescent perinuclear vesicles evidencing the intralysosomal accumulation of cholesterol. The majority of NPC patients present this "classical" biochemical pattern. However, about 20% of NPC patients present a milder level of unesterified cholesterol storage, presenting the so called "variant" biochemical phenotype.The diagnosis in these

Fig. 1. Schematic representation of the laboratory diagnostic strategy in suspected cases of PME due to LSD

also show low levels of GBA activity in fibroblasts but slightly reduced or normal in peripheral blood leukocytes. AMRF should always be considered in patients with reduced intracellular GBA activity in the absence of other markers of Gaucher disease, such as elevated serum

The determination of GBA activity in serum should be perform in order to provide a differential diagnosis since it is elevated only in patients affected with AMRF (Dardis et al.,

In both cases the molecular analysis of *GBA* or *SCARB*-*2* gene should be carried out in order

The diagnostic approach of **NCL** depends on the type of defect that is suspected. As shown in figure 1, the diagnosis of **NCL 1, NCL2 and NCL10** can be achieved by the assessment of PPT1, TPP1 or Cathepsin D activity in leukocytes or cultured fibroblasts. If **NCL3** is suspected, the diagnosis can be confirmed by the presence of typical vacuoles in the cytoplasm of the patient lymphocytes, which are detectable on a regular blood smear

In the case of **NCL5, NCL6, NCL7 and NCL8** it is advisable to investigate the presence of storage material by electron microscopic examination of skin biopsy material or isolated lymphocytes as a first approach and then procede to the molecular genetic studies. The definitive diagnosis in all cases is reached by the molecular analysis of the corresponding

The definitive diagnosis of **sialidosis** is achieved by measuring the NEU1 activity is fresh samples of blood leukocytes or cultured fibroblasts. Special care should be taken to ensure that the tissue to be examined has not been frozen or exposed to prolonged sonication since the neuraminidase is quite unstable (Den Tandt & Brossemer, 1984). The residual enzymatic activity is extremely low or absent in patients affected with sialidosis independently of the severity of the clinical phenotype. On the contrary, a good correlation between the genotype and the phenotype has been found, therefore the molecular analysis of the *NEU1* may provide useful information about disease severity and progression, which is particularly

It is important to keep in mind that also the Galactiosialidosis, a LSD associated with combined deficiency of NEU1 and galactosidase due to the defect of the proteictive protein /cathepsin A (PPCA), results in reduced levels of NEU1 activity. However, in this case the levels of NEU1 are not as low as in sialidosis and they are associated with low

The diagnosis of NPC disease may be quite challenging. It is time consuming and should be

The biochemical diagnosis is based on the demonstration of the impaired intracellular cholesterol transport and homeostasis in fibroblasts in culture. The filipin test is considered the more specific and sensitive assay. Cells are cultured in the presence of LDL enriched medium and then fixed and stained with filipin, a molecule that has a high affinity for unesterified cholesterol (Blanchette-Mackie et al., 1988). In patients with NPC disease, fluorescence microscopic examination of stained cells shows in most of them, the presence of strong fluorescent perinuclear vesicles evidencing the intralysosomal accumulation of cholesterol. The majority of NPC patients present this "classical" biochemical pattern. However, about 20% of NPC patients present a milder level of unesterified cholesterol storage, presenting the so called "variant" biochemical phenotype.The diagnosis in these

chitotriosidase activity or the presence of "Gaucher" cells in bone marrow.

to confirm the diagnosis and to provide a genetic counseling.

2009).

(Kohlschütter & Schulz, 2009).

genes (Kohlschütter & Schulz, 2009).

levels of galactosidase activity.

relevant to provide a better genetic counseling.

performed by specialized centers with the required experience.

Myoclonic Epilepsy in Lysosomal Storage Disorders 245

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

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

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,

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

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;

LSDs are the main cause of the inherited form of PME. However, they are poorly known as a cause of PME and the differential diagnosis might be challenging. An accurate diagnosis is crucial to provide the best therapeutic approach and an appropriate genetic counselling.

**9. Therapeutic options** 

neuronopathic GD.

Lachmann, 2009).

Hobert & Dawson, 2006).

**10. Conclusions** 

without any evidence of measurable benefits.

no data on clinical results are available at present.

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 patients presenting a variant phenotype.

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

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 active towards the synthetic substrate.

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. (Bayleran et al., 1984 ).

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 GM2A gene.

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).
