**2. Genetics**

Mitochondrial disorders derive from mutations of mitochondrial (mtDNA) or nuclear DNA (nDNA), which lead to the impairment of the mitochondrial respiratory chain activity or mitochondrial ATP synthesis. Mitochondrial disorders typically involve tissues or organs with high energy demand including peripheral nervous system (PNS), central nervous system (CNS), eyes, ears, heart, endocrine system, kidney, guts, and liver. Fever, infection and stress may aggravate the neurological symptoms.

Generally, the genotype-phenotype correlation in mitochondrial disorders is poor, since the clinical phenotype is not only dependent on the type and pathogenicity of the DNA mutation, but it may also derive from other genetic and environmental factors.

A possible mechanism of inheritance is maternal transmission of the mutations located in the mtDNA. It is noteworthy that variable amounts of the mutated mtDNA (mutation load) usually coexist with wild-type molecules in the different tissues, resulting in a "heteroplasmic state". Moreover, the phenotypic expression of mtDNA mutations may be dependent on a threshold effect, which can be dissimilar in different tissues, in relation to

Epilepsy in Mitochondrial Disorders 287

Myoclonus epilepsy may manifest at variable age and can associate with other symptoms including general weakness, muscle wasting, deafness, dementia, short stature, optic atrophy, peripheral neuropathy, cardiomyopathy, myoglobinuria and renal tubular

As an example of the heterogeneity affecting also well defined ME with epilepsy, we report here some features of a MERRF family, bearing the common MTTK mutation, including four affected siblings with similar symptoms, but clinically different severity. The proband, previously described by Roger et al. (1982), had typical and severe progressive myoclonus epilepsy associated with optic atrophy. His brother showed occasional seizures and photoparoxysmal EEG response. His sister had late-onset action myoclonus and

A: EEG recording of a 58 years-old female patient with MERRF syndrome showing slow background activity and diffuse slow waves; EMG recording includes positive and negative myoclonic jerks. B: EEG recording of the 24 years-old son of the previous patient showing a sequence of diffuse slow

His nephew showed decreased visual acuity, associated with photosensitive (but not action-

*MELAS* (MIM ID #540000) syndrome is characterized by mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes. The disorder is characterized by symptoms and signs of central nervous system involvement, including seizures, migraine, hemiparesis, hemianopsia, cortical blindness, and episodic vomiting. Other common symptoms are: hearing loss, reduced statural growth, diabetes (Ciafaloni et al, 1992).

Fig. 1. Heterogeneous EEG-EMG features in the same family with MERRF syndrome

waves; in this case, EMG shows regular muscular contraction.

induced) myoclonus (figure 2).

dysfunction (Wu et al, 2010).

photoparoxysmal EEG response (figure 1).

specific energy demand. Nuclear "modifier" genes, environmental factors, mtDNA haplotypes (polymorphisms) or clusters of mtDNA variants could also influence the expression of mtDNA.

The mtDNA contains 37 genes: 13 encoding for subunits of the respiratory chain complexes I (ND1–6, ND4L), III (cytochrome b), IV (COX I-III) and V (ATPase6, ATPase8) (oxidative phosphorylation system, OXPHOS); 22 encoding for transfer RNAs (tRNAs); 2 for ribosomial RNAs (rRNAs). The mitochondrial genetic code differs from the universal genetic code since mtDNA is not protected by any repair mechanism, thus resulting prone to mutations; indeed mutation rates of mtDNA are 10 times higher than those of nDNA.

MtDNA mutations are classified as either large-scale rearrangements (partial deletions or duplications) or point mutations. Rearrangements are frequently sporadic, while point mutations are commonly inherited.

The nuclear genome encodes more than 95% of all proteins located in the mitochondria. An increasing number of clinical conditions have been associated with nDNA mutations, which may involve different genes. These genes have been divided into four groups, according to their function. First group includes genes encoding for structural components of the respiratory chain; second group genes encoding for assembly factors of the respiratory chain complexes; third group genes responsible for factors of mtDNA stability; fourth group genes involved in biogenesis of mitochondria (Finsterer, 2006). Mutations of the nDNA are inherited by autosomal mechanism.

Nuclear-mitochondrial interactions play a fundamental role in cellular homeostasis. The optimal interaction between nuclear and mitochondrial encoded factors is essential for transcription and translation of mtDNA and also for the correct assembly and function of the OXPHOS system.

In both maternal and autosomal inherited MEs, single or multiple defects of the respiratory chain can be detected in the blood cells or muscular specimens. However, biochemical analysis can also identify defective activity of the respiratory chain complexes (in particular complex I and IV) in case of mitochondrial DNA depletion (see for example the mtDNA depletion syndrome 4A, due to mutation in the nuclear gene encoding for mitochondrial polymerase gamma) or multiple deletions.

### **3. Maternally inherited syndromes with epilepsy, associated with mtDNA mutations**

Among MEs characterized by maternal inheritance, generalized seizures and myoclonus are the main symptoms of *MERRF* (myoclonus epilepsy with ragged-red fibers) syndrome (MIM ID #545000). This syndrome, initially described by Fukuhara et al (1980), is characterized by myoclonus, seizures, progressive cerebellar syndrome, and ragged-red fibers in muscle biopsy. It can be due to mutations in more than one mitochondrial gene, e.g. MTTK, MTTL1, MTTH, MTTS1, MTTS2, MTTF and MTND5. However, a specific mtDNA mutation of the tRNA(Lys) gene (MTTK), implying an A-to-G transition at nucleotide 8344, accounts for 80 to 90% of MERRF cases (Shoffner and Wallace, 1992). Biochemically, this mutation produces multiple deficiencies in the enzyme complexes of the respiratory chain (typically complexes I and IV), consistent with a defect in translation of all mtDNA-encoded genes.

A typical MERRF picture was observed in a patient bearing a mutation of the MTTF gene which codes tRNA(Phe) (Mancuso et al, 2004).

specific energy demand. Nuclear "modifier" genes, environmental factors, mtDNA haplotypes (polymorphisms) or clusters of mtDNA variants could also influence the

The mtDNA contains 37 genes: 13 encoding for subunits of the respiratory chain complexes I (ND1–6, ND4L), III (cytochrome b), IV (COX I-III) and V (ATPase6, ATPase8) (oxidative phosphorylation system, OXPHOS); 22 encoding for transfer RNAs (tRNAs); 2 for ribosomial RNAs (rRNAs). The mitochondrial genetic code differs from the universal genetic code since mtDNA is not protected by any repair mechanism, thus resulting prone to mutations; indeed mutation rates of mtDNA are 10 times higher than those of nDNA. MtDNA mutations are classified as either large-scale rearrangements (partial deletions or duplications) or point mutations. Rearrangements are frequently sporadic, while point

The nuclear genome encodes more than 95% of all proteins located in the mitochondria. An increasing number of clinical conditions have been associated with nDNA mutations, which may involve different genes. These genes have been divided into four groups, according to their function. First group includes genes encoding for structural components of the respiratory chain; second group genes encoding for assembly factors of the respiratory chain complexes; third group genes responsible for factors of mtDNA stability; fourth group genes involved in biogenesis of mitochondria (Finsterer, 2006). Mutations of the nDNA are

Nuclear-mitochondrial interactions play a fundamental role in cellular homeostasis. The optimal interaction between nuclear and mitochondrial encoded factors is essential for transcription and translation of mtDNA and also for the correct assembly and function of

In both maternal and autosomal inherited MEs, single or multiple defects of the respiratory chain can be detected in the blood cells or muscular specimens. However, biochemical analysis can also identify defective activity of the respiratory chain complexes (in particular complex I and IV) in case of mitochondrial DNA depletion (see for example the mtDNA depletion syndrome 4A, due to mutation in the nuclear gene encoding for mitochondrial

**3. Maternally inherited syndromes with epilepsy, associated with mtDNA** 

consistent with a defect in translation of all mtDNA-encoded genes.

which codes tRNA(Phe) (Mancuso et al, 2004).

Among MEs characterized by maternal inheritance, generalized seizures and myoclonus are the main symptoms of *MERRF* (myoclonus epilepsy with ragged-red fibers) syndrome (MIM ID #545000). This syndrome, initially described by Fukuhara et al (1980), is characterized by myoclonus, seizures, progressive cerebellar syndrome, and ragged-red fibers in muscle biopsy. It can be due to mutations in more than one mitochondrial gene, e.g. MTTK, MTTL1, MTTH, MTTS1, MTTS2, MTTF and MTND5. However, a specific mtDNA mutation of the tRNA(Lys) gene (MTTK), implying an A-to-G transition at nucleotide 8344, accounts for 80 to 90% of MERRF cases (Shoffner and Wallace, 1992). Biochemically, this mutation produces multiple deficiencies in the enzyme complexes of the respiratory chain (typically complexes I and IV),

A typical MERRF picture was observed in a patient bearing a mutation of the MTTF gene

expression of mtDNA.

mutations are commonly inherited.

inherited by autosomal mechanism.

polymerase gamma) or multiple deletions.

the OXPHOS system.

**mutations** 

Myoclonus epilepsy may manifest at variable age and can associate with other symptoms including general weakness, muscle wasting, deafness, dementia, short stature, optic atrophy, peripheral neuropathy, cardiomyopathy, myoglobinuria and renal tubular dysfunction (Wu et al, 2010).

As an example of the heterogeneity affecting also well defined ME with epilepsy, we report here some features of a MERRF family, bearing the common MTTK mutation, including four affected siblings with similar symptoms, but clinically different severity. The proband, previously described by Roger et al. (1982), had typical and severe progressive myoclonus epilepsy associated with optic atrophy. His brother showed occasional seizures and photoparoxysmal EEG response. His sister had late-onset action myoclonus and photoparoxysmal EEG response (figure 1).

A: EEG recording of a 58 years-old female patient with MERRF syndrome showing slow background activity and diffuse slow waves; EMG recording includes positive and negative myoclonic jerks. B: EEG recording of the 24 years-old son of the previous patient showing a sequence of diffuse slow waves; in this case, EMG shows regular muscular contraction.

Fig. 1. Heterogeneous EEG-EMG features in the same family with MERRF syndrome

His nephew showed decreased visual acuity, associated with photosensitive (but not actioninduced) myoclonus (figure 2).

*MELAS* (MIM ID #540000) syndrome is characterized by mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes. The disorder is characterized by symptoms and signs of central nervous system involvement, including seizures, migraine, hemiparesis, hemianopsia, cortical blindness, and episodic vomiting. Other common symptoms are: hearing loss, reduced statural growth, diabetes (Ciafaloni et al, 1992).

gene.

Epilepsy in Mitochondrial Disorders 289

or rarely by the association of seizures with other symptoms. In other families with an overlap MERFF-MELAS, Nakamura et al. (1995) identified a heteroplasmic mutation in the MTTS1 gene, while Melone et al. (2004) reported a heteroplasmic mutation in the MTTH

A: EEG recording of a patient with MELAS syndrome showing asymmetric background activity and

In some patients, NARP may clinically overlap with Leigh syndrome (NARP/MILS overlap) due to point mutations in the mitochondrial ATPase6 gene, presumably resulting in impaired ATP synthesis. In this syndrome usually associated with T8993G mutation, have

Other mitochondrial syndromes causing epilepsy are associated with multiple mtDNA deletions or tissue-specific depletions. These conditions are due to nuclear gene defects

**4. Autosomal inherited syndromes with epilepsy, associated with nDNA** 

been described infantile spasms with hypsarrithmia (Desguerre at al, 2003)

B: After repeated seizures, the EEG recording of the same patient shows diffuse attenuation of the background activity and the appearance of subcontinuous slow waves on posterior derivations. Fig. 3. EEG changes in a patient with MELAS syndrome after repeated seizure occurrence *Neurogenic muscle weakness, ataxia, and retinitis pigmentosa (NARP)* syndrome typically presents with proximal motor neuropathy, sensory disturbances, cerebellar ataxia, and retinitis pigmentosa. More rare features include developmental delay, mental retardation,

occasional diffuse spike and waves with posterior prevalence.

dementia, epilepsy, or cardiomyopathy.

**mutations** 

MELAS syndrome can be caused by mutation in several genes of the mtDNA coding for tRNA or polypeptides, including MTTL1, MTTQ, MTTH, MTTK, MTTS1, MTND1, MTND5, MTND6, and MTTS2. The 3243A-G transition in the MTTL1 gene can be found in about 80% of the patients (Goto et al, 1992).

A and B: EEG recording of a female patient with MERRF syndrome showing diffuse spike and slow waves prevalent on posterior derivations (boxes) with occasional spontaneous occurrence. C: Intermittent photic stimulation (IPS) induces an increase of the epileptic discharges.

Fig. 2. Spontaneous and photically-induced epileptiform abnormalities in MERRF syndrome

In MELAS syndrome, epilepsy is a frequent symptom; seizures are often of motor type and can be grouped in clusters (until the extreme picture of *epilepsia partialis continua*) even without the evidence of concomitant new acute brain insult (Canafoglia et al, 2004). However, a relationship between clusters of seizures and stroke-like episodes is common: focal neuronal hyperexcitability or epileptic activity may cause an increase of ATP demand which can be followed by depletion of ATP and consecutive vasogenic edema (Iizuka et al, 2002 ; 2003). Finally, the vasogenic edema turns into laminar necrosis indicated by T1 hyperintensity at cerebral MRI. Another explanation suggests that stroke like episodes are due to metabolic derangement, spreading beyond an ischemic focus. Figure 3 shows the dramatic change of EEG activity after repeated seizures, indicated by the appearance of subcontinuous focal slow waves, in a patient with MELAS syndrome.

A syndrome characterized by features of both MERRF and MELAS (*overlap MERRF-MELAS*) has been described by Zeviani et al. (1993) in association with a point mutation at nucleotide 8356 (T-to-C transition) in the MTTK gene. The phenotype was characterized by myoclonic epilepsy, neural deafness, ataxia, stroke-like episodes in the majority of the affected siblings

MELAS syndrome can be caused by mutation in several genes of the mtDNA coding for tRNA or polypeptides, including MTTL1, MTTQ, MTTH, MTTK, MTTS1, MTND1, MTND5, MTND6, and MTTS2. The 3243A-G transition in the MTTL1 gene can be found in about 80%

A and B: EEG recording of a female patient with MERRF syndrome showing diffuse spike and slow waves prevalent on posterior derivations (boxes) with occasional spontaneous occurrence.

Fig. 2. Spontaneous and photically-induced epileptiform abnormalities in MERRF syndrome In MELAS syndrome, epilepsy is a frequent symptom; seizures are often of motor type and can be grouped in clusters (until the extreme picture of *epilepsia partialis continua*) even without the evidence of concomitant new acute brain insult (Canafoglia et al, 2004). However, a relationship between clusters of seizures and stroke-like episodes is common: focal neuronal hyperexcitability or epileptic activity may cause an increase of ATP demand which can be followed by depletion of ATP and consecutive vasogenic edema (Iizuka et al, 2002 ; 2003). Finally, the vasogenic edema turns into laminar necrosis indicated by T1 hyperintensity at cerebral MRI. Another explanation suggests that stroke like episodes are due to metabolic derangement, spreading beyond an ischemic focus. Figure 3 shows the dramatic change of EEG activity after repeated seizures, indicated by the appearance of

A syndrome characterized by features of both MERRF and MELAS (*overlap MERRF-MELAS*) has been described by Zeviani et al. (1993) in association with a point mutation at nucleotide 8356 (T-to-C transition) in the MTTK gene. The phenotype was characterized by myoclonic epilepsy, neural deafness, ataxia, stroke-like episodes in the majority of the affected siblings

C: Intermittent photic stimulation (IPS) induces an increase of the epileptic discharges.

subcontinuous focal slow waves, in a patient with MELAS syndrome.

of the patients (Goto et al, 1992).

or rarely by the association of seizures with other symptoms. In other families with an overlap MERFF-MELAS, Nakamura et al. (1995) identified a heteroplasmic mutation in the MTTS1 gene, while Melone et al. (2004) reported a heteroplasmic mutation in the MTTH gene.

A: EEG recording of a patient with MELAS syndrome showing asymmetric background activity and occasional diffuse spike and waves with posterior prevalence.

B: After repeated seizures, the EEG recording of the same patient shows diffuse attenuation of the background activity and the appearance of subcontinuous slow waves on posterior derivations.

Fig. 3. EEG changes in a patient with MELAS syndrome after repeated seizure occurrence

*Neurogenic muscle weakness, ataxia, and retinitis pigmentosa (NARP)* syndrome typically presents with proximal motor neuropathy, sensory disturbances, cerebellar ataxia, and retinitis pigmentosa. More rare features include developmental delay, mental retardation, dementia, epilepsy, or cardiomyopathy.

In some patients, NARP may clinically overlap with Leigh syndrome (NARP/MILS overlap) due to point mutations in the mitochondrial ATPase6 gene, presumably resulting in impaired ATP synthesis. In this syndrome usually associated with T8993G mutation, have been described infantile spasms with hypsarrithmia (Desguerre at al, 2003)

## **4. Autosomal inherited syndromes with epilepsy, associated with nDNA mutations**

Other mitochondrial syndromes causing epilepsy are associated with multiple mtDNA deletions or tissue-specific depletions. These conditions are due to nuclear gene defects

Epilepsy in Mitochondrial Disorders 291

Molecular exams could identify heterogeneous mutations in various mitochondrial and nuclear genes coding for complex I, complex III and complex IV and complex V gene. Mutations have been found also in genes encoding mitochondrial tRNA proteins (MTTV, MTTK, MTTW, and MTTL1) and in components of the pyruvate dehydrogenase complex (e.g. PDHA1: X-linked Leigh syndrome). The French-Canadian (or Saguenay-Lac Saint Jean) type of Leigh syndrome with COX deficiency (LSFC) is caused by mutation in the LRPPRC

In the Leigh syndrome, both generalized and focal seizures have been described, according

Seizures can manifest in many patients with infantile MEs, who were diagnosed on the bases of their biochemical defects but still not classified genetically. Epilepsy may manifest as catastrophic neonatal forms, neonatal myoclonic encephalopathies, infantile spasms, refractory status epilepticus, epilepsia partialis continua, myoclonic epilepsy (El Sabbagh et al , 2010), Landau Kleffner, Lennox-Gastaut syndromes, unclassified generalized epilepsy or partial epilepsy (Canafoglia et al, 2001; Lee et al, 2008). Thus, epilepsy may be either focal or generalized and its severity varies in different case series, though the appearance of drugresistant seizures possibly marks a severe turn in the disease with high risk of neurological

Among various biochemical defects, it's worth noting complex I deficiency. Complex I deficiency, due to mutations in mtDNA genes coding for ND subunits, has been described in patients with heterogeneous syndromic (MELAS, Leigh) and non-syndromic MEs,

Mitochondrial dysfunctions may be implicated also in sporadic forms of partial epilepsy such is temporal lobe epilepsy, since severe impairment of the respiratory chain activity has been detected *in vitro* on hippocampus samples from patients with drug resistant epilepsy. This observation was also supported by various evidences obtained *in vivo* using

Pyruvate dehydrogenase complex (PDHC) is a mitochondrial matrix enzyme complex that catalyzes the oxidative decarboxylation of pyruvate to acetyl CoA, nicotinamide adenine dinucleotide (the reduced form, NADH), and CO2. This reaction constitutes the bridge between anaerobic and aerobic cerebral energy metabolism. The great majority of PDH complex deficiencies results from mutations in the X-linked pyruvate dehydrogenase (E1)

The clinical severity can vary from early neonatal presentation with severe lactic acidosis to a progressive disease with mental retardation and neurological complications. Some females

Epilepsy has been reported with a high frequency in children with PDH deficiency (Canafoglia et al, 2001; Kang et al, 2007). Epilepsy is frequently severe and may have

are only mildly affected or asymptomatic in relation to the pattern of X-inactivation.

variable characteristics including some forms of epileptic encephalopathy.

frequently associated with severe epilepsy (Antozzi et al, 1995; Malfatti et al, 2007).

**7. Pyruvate dehydrogenase (PDH) Deficiency (MIM ID #312170)** 

gene. Deficiency of coenzyme Q10 can present as Leigh syndrome.

deterioration and fatal outcome (El Sabbagh et al , 2010).

neuroimaging techniques (Zsurka and Kunz, 2010).

alpha subunit gene (PDHA1). Gene map locus: *Xp22.2-p22.1.* 

to genetic and biochemical heterogeneity.

**6. Non-syndromic pictures** 

involved in controlling the nuclear-mitochondrial intergenomic signaling (Spinazzola and Zeviani, 2005).

These conditions include the following syndromes:

Developmental delay or dementia, lactic acidosis, cyclic vomiting, seizures, failure to thrive, hearing loss, myopathy, liver failure, renal tubular acidosis, pancreatitis, manifesting at 1-3 months, characterize childhood myo-cerebro-hepatopathy spectrum (MCHS).

*Myoclonic epilepsy, myopathy, sensory ataxia (MEMSA)* is characterized by epilepsy, myopathy, ataxia without ophthalmoplegia (previously defined as spinocerebellar ataxia with epilepsy: SCAE).

Ataxia neuropathy spectrum (ANS) is characterized by ataxia and neuropathy, seizures (reported in two-thirds of the patients), ophthalmoplegia (one-half), clinical myopathy (rare). This disorder was previously defined as recessive ataxia syndrome: MIRAS and sensory ataxia neuropathy dysarthria and ophthalmoplegia: SANDO.

Autosomal Recessive progressive external ophthalmoplegia (arPEO) and Autosomal Dominant progressive external ophthalmoplegia (adPEO) are characterized by myopathy, variable sensorineural hearing loss, axonal neuropathy, ataxia, depression, parkinsonism, hypogonadism, and cataracts (previously defined as CPEO plus). Seizures are uncommon; however, few patients may have signs reminding those observed in MELAS syndrome, including stroke-like episodes and seizures (Deschauer et al, 2007). These syndromes are typically associated with mutations in the nuclear-encoded DNA polymerase-gamma gene (gene map locus: 15q25).

The syndrome variably defined as Alpers' disease, Alpers' syndrome, Alpers-Huttenlocher's disease, progressive neuronal degeneration of childhood, progressive sclerosing poliodystrophy or progressive infantile poliodystrophy is characterized by neuronal degeneration of the cerebral cortex and elsewhere, caused by recessive mutations in nDNA, coding for the mitochondrial DNA polymerase-gamma. In this syndrome, the onset is usually before age four years and up to age 25-35; often there is pre-existing developmental delays of variable severity. The syndrome is characterized by seizures, episodic psychomotor regression, liver dysfunction or failure, which may follow exposure to certain antiepileptic medication. The electro-clinical pattern is typically characterized by periodic EEG and epilepsia partialis continua.
