Clinical Features

EPM1 is characterized by onset at 6–16 years, progressive stimulus-sensitive, action-activated myoclonus and tonic-clonic epileptic seizures³. As EPM1 progresses, patients develop ataxia, dysarthria, intentional tremor, and decreased coordination, which reflect widespread neuronal degeneration in the brain. The EPM1 patients show mild intellectual decline over time, but overall their cognitive functions are less impaired than their motor functions. The EEG in EPM1 patients is abnormal with spontaneous spike-wave discharges, photosensitivity, polyspike discharges during REM sleep and background slowing^3,4. The EEG abnormalities are more pronounced at initial diagnosis, but in general diminish as the disease stabilizes. At the time of diagnosis the MRI is usually normal. At later stages, atrophy in cortical motor areas, and in pons, medulla, and cerebellar hemispheres has been reported^5,6. The limited histopathological findings consist of widespread degenerative changes with no evidence for storage material.

Symptomatic pharmacologic and rehabilitative management including psychosocial support are the mainstay of EPM1 patients’ care³. Valproic acid is the first drug of choice and diminishes both myoclonus and the frequency of tonic-clonic seizures. Clonazepam and piracetam are effective as add-on therapy for myoclonus. Anecdotal evidence exists of the efficacy of levetiracetam, topiramate and zonisamide, but sodium channel blockers and GABAergic drugs should be avoided. With modern antiepileptic medication the life expectancy has gradually increased and is nowadays most probably normal. The relative intensity of symptoms and the speed of disease progression vary from one case to another, even within one family.

The Cystatin B Gene and Protein

The CSTB gene underlying EPM1 was identified by positional cloning². CSTB is alternatively spliced with at least five isoforms of unknown physiological significance, some of which show tissue specificity. The gene has two potential transcription start sites which are located 67 and 78 nucleotides downstream of the dodecamer repeat element in the promoter.

CSTB encodes Cystatin B, a ubiquitously expressed 98 amino acid protein comprising a molecular weight of approximately 11 kDa. CSTB is a protease inhibitor that inhibits in vitro several lysosomal cysteine proteases, cathepsins, by tight, reversible binding⁷. The main function of cathepsins is non-selective degradation of intracellular proteins, but they also participate in antigen processing and apoptosis.

Cytoplasmic, lysosomal and nuclear localization has been reported for wild-type CSTB protein in proliferating cells, and mainly cytoplasmic localization in differentiated cells⁸ with somewhat controversial results between different studies, possibly due to the different cellular models and antibodies used. Available data suggest that CSTB is attached to the outer side of the lysosomal membrane rather than within the lysosome⁸.

EPM1-associated Cystatin B Gene Mutations

Eleven mutations have been reported to underlie EPM1. The most common mutation is an unstable expansion of a 12-nucleotide, dodecamer, repeat (5′-CCCCGCCCCGCG-3′), in the promoter region of CSTB^9,10. This is normally polymorphic with two or three copies present. EPM1-associated alleles have been reported to contain at least 30 repeat copies. The expansion mutation is found in approximately 90% of the disease alleles worldwide and in homozygous form in the majority of the patients, especially in populations with a founder effect. No significant correlation between the length of the expanded repeat and the age of onset or disease severity has been reported.

The other EPM1-associated CSTB mutations change single amino acids, affect splice sites or predict truncated proteins. The three reported missense mutations, p.Gly4Arg, p.Gly50Glu and p.Gln71Pro are likely to affect the interaction of the CSTB protein with its target cysteine proteases.^8,11. With the exception of the p.Gly4Arg substitution mutation, these mutations have been reported to occur in compound heterozygous form with the repeat expansion. The expansion mutation causes significant down-regulation of CSTB mRNA expression with less than 10% of expression from that in controls¹¹. Consequently, the CSTB protein expression and its inhibitory activity are also significantly reduced in cells of EPM1 patients. Decreased inhibitory activity of CSTB correlates with enhanced activity of cathepsins B, L, and S in EPM1 patients’ cells, providing in vivo evidence for cathepsins being regulated by CSTB¹². Available data suggest that reduced CSTB expression is the primary pathological consequence in the majority of EPM1 mutations, with possible exclusion of the amino acid substitution mutations. When transiently over-expressed in cells, these missense mutant proteins fail to associate with lysosomes^8,11 implying an essential role of lysosomal localization for the physiological function of CSTB.

Cystatin B-deficient Mouse Model for EPM1

A mouse model for EPM1 has been generated by targeted disruption of the mouse Cstb gene¹³. Cstb−/− mice develop myoclonic seizures by one month of age and progressive ataxia by six months of age and thus recapitulate key clinical features of EPM1. The myoclonic seizures occur during sleep and progress from twitching of isolated muscles to spasms affecting the entire body. Electrocorticogram recordings reveal bilaterally synchronous 4–6-Hz repetitive spiking commencing with the myoclonus. No tonic-clonic seizures, photosensitivity or spike-wave complexes in EEG have been reported. The genetic background has an impact on the clinical outcome implying that genetic factors influence the phenotype.

The neuropathological hallmark in the Cstb−/− mice is progressive apoptotic loss of cerebellar granule neurons¹³. There is less marked neuronal apoptosis in the hippocampal formation and entorhinal cortex in young animals and widespread cortical and white matter gliosis in older mice^13,14. In addition, the superficial neurons of the prosubiculum in the cerebral cortex display prominent cellular atrophy, suggesting that neuronal dysfunction may also contribute to the phenotype. Mutant mice in both “seizure-prone” and “seizure-resistant” genetic backgrounds display similar neuropathological changes indicating that neuronal degeneration and loss is a consequence of CSTB deficiency independent of seizure events. These data suggest that CSTB has an endogenous neuroprotective role and that EPM1 should be classified as a primary neurodegenerative disorder, with specific neuronal populations affected and with both neuronal death and dysfunction contributing to the phenotype.

Studies in hippocampal slice preparations have revealed hyperexcitability in Cstb−/− mice, as they responded to afferent stimuli in the CA1 region with multiple population spikes and as kainate perfusion provoked the appearance of epileptic-like activity earlier than in wild-type mice¹⁵. The density of GABA-positive cells in hippocampus is reduced in Cstb−/− mice and the hyperexcitability may thu be due to loss of inhibition. Moreover, Cstb−/− mice have increased susceptibility to kainate-induced seizures with a shorter latency to seizure onset and more severe seizures compared to wild-type control mice. They also display increased sensitivity to seizure-induced cell death. It has been hypothesized that CSTB acts as a physiological safeguard and that loss of its activity not only triggers hyperexcitability and neurodegeneration, but also makes neurons more susceptible to prolonged seizure-induced cell death contributing to disease progression in EPM1¹⁵.

Oxidative stress in EPM1

A novel function for CSTB in defending cerebellar granule neurons from oxidative stress has been identified¹⁶. It implies impaired redox homeostasis as a key mechanism by which CSTB deficiency triggers neurodegeneration. Oxidative stress induces CSTB expression in cerebellar granule neurons, and both Cstb knockdown in rats and knockout in mice sensitize granule neurons to oxidative stress-induced cell death. Predisposition to oxidative stress in neurons, induced by CSTB deficiency, is mediated by the lysosomal protease Cathepsin B. CSTB deficiency triggers oxidative injury specifically in the cerebellum, leading to diminished antioxidant capacity and elevated lipid peroxidation¹⁶. Consistent with these findings, cerebellar granule neuron degeneration is reduced in mice deficient for both CSTB and Cathepsin B compared to mice deficient for CSTB only¹⁷. The double-knockout mice retain myoclonic seizures and ataxia suggesting that oxidative stress and CSTB may regulate neuronal excitability in EPM1 independently of deregulated Cathepsin B activity.

PRICKLE1 and SCARB2 Mutations in EPM1-like Patients

Mutations in two genes have been reported in CSTB mutation negative patients presenting with symptoms closely resembling EPM1. A p.Arg104Gln mutation in the PRICKLE1 gene encoding a protein in the non-canonical WNT signaling pathway has been described in three possibly related families of Middle Eastern descent¹⁸. Onset of symptoms is between five and ten years of age, i.e. a slightly younger age than EPM1 patients. The presenting symptom is usually ataxia, with action myoclonus and seizures developing later. Intellect is generally preserved. This disorder was originally named EPM1B because of its similarity to EPM1, but is now referred to as Progressive Myoclonus Epilepsy with Ataxia.

The second gene harboring mutations in EPM1-like patients is SCARB2 that encodes a lysosomal membrane protein. Mutations in SCARB2 were originally reported in action myoclonus-renal failure syndrome (AMRF)¹⁹. AMRF patients present typically at 15–25 years of age with either neurological symptoms including tremor, action myoclonus, seizures, and ataxia, or with proteinuria that progresses to renal failure. In a subset of patients harboring mutations in SCARB2 no evidence of renal failure during up to 15 years of follow-up have been observed, implying that mutations in SCARB2 are an important cause of PME cases resembling EPM1 at onset²⁰. A missense mutation and five mutations predicting either aberrant splicing or a truncated protein due to a frameshift alteration have been identified in EPM1-like patients. The nature or the location of the mutation in SCARB2 does not explain the absence of renal features in these patients.

CLN1 Disease: CLN1 (PPT1)

Infantile NCL is caused by mutations in CLN1, the gene encoding the enzyme palmitoyl protein thioesterase 1 (PPT1)²³. PPT1 is an enzyme that removes fatty acids in covalent linkage to cysteine residues in proteins, ensuring the eventual metabolic disposal of S-acylated cysteine residues during lysosomal degradation. In non-neuronal cells PPT1 is a soluble lysosomal protein that is targeted to lysosomes through the mannose-6-phosphate pathway. In neuronal cells it is localized to synaptosomes and synaptic vesicles. Mutations in CLN1 are associated with granular osmiophilic storage deposits (GRODs) in the cells, where the main components of accumulated protein are the sphingolipid activator proteins (SAPs) A and D.

Over 40 disease-causing mutations distributed throughout the gene have been described in CLN1. The most common mutation (c.364A>T) is associated with a founder effect in the Finnish population. This causes onset in infancy as do the majority of the mutations identified. Early development is normal until 6–18 months when decreased head growth and developmental retardation appear. Epileptic seizures, hypotonia, ataxia, and visual failure occur associated with severe brain atrophy and death at 8–15 years. Less severe mutations (missense in peripheral sites) in CLN1 also cause NCL forms with later onset and protracted progression.

Neurophysiologic findings are non-specific. There is decreased reactivity in EEG to passive eye opening and closing around the age of 1 year. Sleep spindles are lost by the age of 2 years and by 3 years the EEG becomes isoelectric. Thalami show markedly decreased signal intensity to the basal ganglia on T2-weighted MR images and increased signal intensity on T1-weighted images even before the clinical disease is apparent. Generalized cerebral and cerebellar atrophy is found by the age of 13 months. Enzyme assay of PPT1 in white cells is the mainstay of diagnosis and has been used for prenatal diagnosis.

CLN2 Disease: CLN2 (TPP1)

Classical late infantile NCL is caused by mutations in the CLN2 gene encoding the lysosomal enzyme tripeptidyl peptidase (TPP1)²⁴. This gene was identified using a proteomics approach to identify the mannose-phosphorylated protein that was missing in brain samples from patients with the disease. TPP1 is a pepstatin-insensitive protease that cleaves tripeptides from the amino-termini of polypeptides undergoing degradation. Subunit c of mitochondrial ATP synthase, the major protein component of the storage material, particularly in this subtype, is likely to be a substrate. Ultrastructural changes are pure curvilinear membrane bound lysosmal aggregates.

Over 60 different disease-causing mutations have now been identified in the CLN2 gene. Two are most common: c.509-1G>C affecting the splicing of the transcript, and c.622C>T creating a stop codon (p.Arg208X).

Most mutations in CLN2 cause classical late infantile INCL. The clinical features usually begin around the third year of life with delay of psychomotor development or sudden onset of seizures, which may be generalized tonic–clonic, partial, or often of a severe myoclonic type. Blindness eventually develops due to retinal atrophy. Death usually occurs in mid-childhood. A few of the mutations cause a more protracted or juvenile onset form of NCL.

The EEG shows characteristic occipital spike responses to slow flash (1–2 Hz) stimulation before the onset of seizures. The electroretinogram is diminished or absent early in the disease even before noticeable visual loss. Severe cerebellar atrophy on MRI is seen at the time of diagnosis. Enzyme assay of TPP1 in white cells is the mainstay of diagnosis.

CLN3 Disease: CLN3

Juvenile NCL, the most frequent subtype of the NCLs, is caused by mutations in CLN3, which was localized to chromosome 16 by demonstration of linkage to the haptoglobin locus and subsequently identified by a positional cloning strategy²⁵. CLN3 encodes a 438 amino acid transmembrane protein which has been located to various cellular organelles such as lysosomes, Golgi, and mitochondria.

The function of CLN3 is still unknown but it is evolutionary conserved down to yeast which suggests an important function in eukaryotic cells. Possible roles include maintenance of lysosomal pH homeostasis, inward lysosomal arginine transport, a role in membrane tracking and an antiapoptotic function. The typical ultrastructural feature is fingerprint profiles. At least 40 mutations are currently known in CLN3. The most common of these is a 1-kb deletion (c.462–677del) causing a frameshift after the cysteine residue at position 153 and premature termination. It occurs world-wide but especially in European and North American populations. This mutation is so predominant that most patients are homozygous or at least heterozygous for it. A wide variety of other mutations exist, some of which are associated with a later onset or a more protracted disease course.

Juvenile NCL has a usual age of onset of 4–7 years, with progressive visual failure leading to blindness always occurring first. The ocular pathology is initially a pigmentary retinopathy which may be mistaken for retinitis pigmentosa or cone dystrophy.

Seizures (at a mean age of 10 years) and psychomotor deterioration with behavioral problems follow. The main types of seizures are generalized tonic–clonic seizures, but partial seizures and myoclonic jerks also occur. Seizures tend to increase in frequency and severity but there is considerable individual variability. In the EEG, progressive background abnormality and an increase in paroxysmal activity are seen.

Extrapyramidal symptoms and signs are noted in about 50% between the ages of 12 and 15 years. Signs include impaired balance, rigidity, hypokinesia, stooped posture and shuffling gate, whereas tremor is usually mild and inconstant. Death occurs in the second or third decade. MRI demonstrates variable cerebral and cerebellar atrophy after the age of about 14 years. Vacuolated lymphocytes visible in the peripheral blood are unique to this form of NCL and a valuable diagnostic aid. Screening for the 1-kb deletion is now widely available as a diagnostic service. The majority (>80%) of children of white Caucasian origin will be homozygotes for the common 1-kb deletion and almost all of the rest will be compound heterozygotes for the 1-kb deletion and another disease causing mutation which may be unique to the family.

Variant Late Infantile NCLs: CLN5, CLN6, CLN7 (MFSD8), CLN8

CLN5 Disease. The NCL subtype with mutations in the CLN5 gene was originally described in the Finnish population and is often referred to as Finnish variant late-infantile NCL. The CLN5 gene was localized to chromosome 13q22 by a genome-wide scan and later identified by a positional cloning strategy²⁶. The gene is conserved in vertebrates and encodes a lysosomal glycoprotein with no homology to other known proteins. The function of CLN5 is currently unknown.

Over 25 different disease-causing mutations in CLN5 have been described. The most common of these, c.1175delAT (p.Tyr392X), present only in the Finnish population, blocks lysosomal targeting of the mutant protein.

The first manifestations include attention deficit and motor clumsiness and usually occur between the ages of 5 and 7 years, later than the classical form. Thereafter, mental decline and visual failure, myoclonic and tonic-clonic seizures and ataxia are features of the disease. Specific EEG features include posterior spikes to low-frequency photic stimulation from 7–8 years and giant somatosensory evoked potentials (SEPs). Age of the death is between 13 and 21 years. MRI shows cerebellar atrophy between the ages 4 and 7 years.

CLN6 Disease. A variant late infantile form caused by mutations in the CLN6 gene is mainly found in the Czech Republic, Croatia, Portugal, Central and South America, and, more rarely, in Central and Northern Europe, Turkey and the Indian subcontinent. The CLN6 gene was localized to chromosome 15q21–23 by homozygosity mapping and identified by positional cloning^27,28. The CLN6 protein has seven transmembrane domains and localizes to the endoplasmic reticulum (ER). It is highly conserved across vertebrates, has no homology with known proteins and its function is currently unknown.

Over 40 disease-causing mutations have been identified in the CLN6 gene. One of the two most common of these is a nonsense mutation c.214G>T (p.Glu72X) present in patients of Costa Rican origin.

Ultrastructural deposits include fingerprint, curvilinear, as well as rectilinear patterns.

Most mutations in CLN6 cause a clinical phenotype similar to classical LINCL with an age of onset between 3–5 years but the range is from 18 months to 8 years. Early features may include gait and speech disturbance and epileptic seizures.

CLN7 Disease. After a genomewide scan and homozygosity mapping in nine Turkish families and one Indian family, not linked to any of the known NCL loci, a novel variant LINCL locus was mapped to chromosome 4q28.1–q28.2. Six different mutations were identified in the MFSD8 gene, which encodes a 518-amino acid membrane protein that belongs to the major facilitator superfamily of transporter proteins²⁹. Like the majority of the previously identified NCL proteins, MFSD8 localizes mainly to the lysosomal compartment.

A total of 22 mutations have now been identified in CLN7 in patients of various ethnic origins including Italy. A significant group of Roma patients originating from the former Czechoslovakia was shown to bear the c.881C>A (p.Thr294Lys) mutation in CLN7/MFSD8, possibly due to a founder effect. With one exception these patients presented a phenotype indistinguishable from the other variant late-infantile NCL forms. In one patient with an in-frame amino acid substitution mutation in CLN7/MFSD8, the disease onset was later and the disease course slower.

CLN8 Disease. CLN8 was first identified as a causative gene for Northern epilepsy (also known as progressive epilepsy with mental retardation, EPMR) in Finnish patients. Later, recognition of intraneuronal storage material led to this disease being categorised as an NCL. The CLN8 gene was localized to the short arm of chromosome 8 by linkage analysis and identified by positional cloning³⁰. The function of the CLN8 protein is unknown but it is predicted to be a transmembrane protein with several membrane spanning domains. In non-neuronal cells the CLN8 protein is localized to the ER and partially to the ER–Golgi intermediate compartment (ERGIC). In neurons, CLN8 is localized to the ER, but an additional location outside the ER has been suggested in polarized cells. CLN8 has been connected to the TRAM-Lag1p-CLN8 (TLC) superfamily of proteins suggested to have a role in biosynthesis, metabolism, and sensing of lipids.

A total of 15 mutations have now been identified and it is clear that there is allelic heterogeneity with two distinct phenotypes. The most common mutation is c.70C>G (p.Arg24Gly) underlying the EPMR phenotype. The first symptoms in EPMR are epileptic seizures, observed at the age of 5–10 years. Two to 5 years after disease onset, the patients show progressive mental deterioration, and motor and behavioral problems. Additional mutations associated with a variant late-infantile NCL phenotype have subsequently been identified in Turkish, Israeli, Italian, German and Pakistani patients including a novel large deletion in a Turkish family.

CLN10 Disease: CLN10 (CTSD)

As mutations in the Cathepsin D gene had been observed in two naturally occurring animal models for NCL (Swedish Landrace sheep and American bulldogs), CTSD was a candidate gene for human NCL. Recently, it was recognized as the causative gene for two separate human NCL phenotypes.

A homozygous duplication (c.764dupA) that creates a premature stop codon at position 255 (p.Tyr255X) results in congenital NCL with absence of CTSD immunostaining in brain³¹. Congenital NCL is the earliest-onset and most aggressive subtype. It presents with extreme brain atrophy, microcephaly and epilepsy at birth, with death occurring within hours to weeks. In contrast, compound heterozygosity for two missense mutations with resulting CTSD deficiency, underlies a neurodegenerative phenotype with blindness and psychomotor disability manifesting at earlyschool age, but no seizures. Ultrastructural features include massive accumulation of GRODs.

The human CTSD gene is located on chromosome 11p15.5, contains 9 exons, and encodes a lysosomal aspartic protease of 412 amino acids that belongs to the pepsin family and is conserved at least down to Drosophila melanogaster and Caenorhabditis elegans.

CLN4 Disease

The adult onset form of NCL, Kufs disease, has been assigned the as yet unidentified gene locus CLN4. It is the mildest form of NCL and includes at least two subtypes with an age of onset of about 30 years, (range 10 – 50 years). The main clinical symptom is dementia, and the other clinical features depend on the subtype and include progressive myoclonus epilepsy, ataxia, late pyramidal and extra-pyramidal features, behavioral changes and motor disturbances. There are no ophthalmological abnormalities. The EEG shows generalized fast spike-and-wave discharges with photosensitivity. Diagnosis requires demonstration of ultrastructural changes in skeletal or vascular smooth muscle cells which may be fingerprint profiles, curvilinear profiles or granular osmiophilic deposits. Both autosomal recessive (most commonly) and dominant inheritance has been reported. Adult onset NCL can be caused by mutations in the CLN1 gene and this may account for some cases.

CLN9 Disease

The most recently reported subtype of NCL was described in two Serbian sisters and two German brothers and is referred to as CLN9-deficient as enzyme screening and sequencing of the coding regions of other NCL genes was negative³². Their clinical history was characteristic for juvenile NCL and curvilinear inclusions, fingerprint profiles, and granular osmiophilic deposits were found in neurons, lymphocytes, and conjunctival cells. CLN9-deficient fibroblasts have a distinctive phenotype with rapid growth, increased apoptosis and diminished levels of ceramide, dihydroceramide, and sphingomyelin. Transfection with CLN8 but not other NCL genes corrected growth and apoptosis in CLN9-deficient cells. CLN8 is one of the TRAM-Lag1-CLN8 proteins containing a Lag1 motif. The latter imparts dihydroceramide synthase activity to yeast cells suggesting that the CLN9 protein may be a regulator of dihydroceramide synthase.