Address correspondence to Vincenzo Leuzzi, Department of Paediatrics and Child Neurology and Psychiatry, Via dei Sabelli 108, 00185 Roma, Italy. E-mail: email@example.com
Creatine metabolism disorders include guanidinoacetate methyltransferase (GAMT) deficiency, arginine:glycine amidinotransferase (AGAT) deficiency, and the creatine transporter (CT1-encoded by SLC6A8 gene) deficiency. Epilepsy is one of the main symptoms in GAMT and CT1 deficiency, whereas the occurrence of febrile convulsions in infancy is a relatively common presenting symptom in all the three above-mentioned diseases. GAMT deficiency results in a severe early onset epileptic encephalopathy with development arrest, neurologic deterioration, drug-resistant seizures, movement disorders, mental disability, and autistic-like behavior. In this disorder, epilepsy and associated abnormalities on electroencephalography (EEG) are more responsive to substitutive treatment with creatine monohydrate than to conventional antiepileptic drugs. AGAT deficiency is mainly characterized by mental retardation and severe language disorder without epilepsy. In CT1 deficiency epilepsy is generally less severe than in GAMT deficiency. All creatine disorders can be investigated through measurement of creatine metabolites in body fluids, brain proton magnetic resonance spectroscopy (1H-MRS), and molecular genetic techniques. Blood guanidinoacetic acid (GAA) assessment and brain H-MRS examination should be part of diagnostic workup for all patients presenting with epileptic encephalopathy of unknown origin. In girls with learning and/or intellectual disabilities with or without epilepsy, SLC6A8 gene assessment should be part of the diagnostic procedures. The aims of this review are the following: (1) to describe the electroclinical features of epilepsy occurring in inborn errors of creatine metabolism; and (2) to delineate the metabolic alterations associated with GAMT, AGAT, and CT1 deficiency and the role of a substitutive therapeutic approach on their clinical and electroencephalographic epileptic patterns.
Creatine metabolism disorders are a relatively young group of diseases in which the intracellular transfer of energy is impaired (Stöckler et al., 2007). Figure 1 summarizes the three biochemical steps involved in the biosynthesis of creatine.
Guanidinoacetate methyltransferase (GAMT), arginine:glycine amidinotransferase (AGAT), and creatine transporter 1 (CT1) deficiency share a cluster of common symptoms reflecting the derangement of the higher cortical functions: intellectual disability, language delay, and behavioral disorders (Fig. 2A,C). Brain creatine depletion can be detected noninvasively through measurement of creatine metabolites in body fluids and brain proton magnetic resonance spectroscopy (1H-MRS) (Stöckler et al., 2007). Patients with biochemical and/or spectroscopic abnormalities compatible with the diagnostic suspect of inborn errors of creatine metabolism must undergo the specific molecular genetic tests (Almeida et al., 2007). Epilepsy is one of the main symptoms in two of these conditions, GAMT and CT1 deficiency, whereas the occurrence of febrile convulsions in infancy is a relatively common presenting symptom in all (Fig. 2B,D).
The aims of this review are the following: (1) to describe the electroclinical features of epilepsy occurring in inborn errors of creatine metabolism; and (2) to delineate the metabolic alterations associated with GAMT, AGAT, and CT1 deficiency and the role of a substitutive therapeutic approach on their clinical and electroencephalographic epileptic patterns.
Clinical presentation of GAMT deficiency is usually characterized by normal developmental milestones in the first months of life, which can be abruptly discontinued by arrest/regression of psychomotor development with or without seizures (Stöckler et al., 2007).
Epilepsy is the second most frequent symptom in GAMT deficiency after intellectual disability. Febrile seizures have often been reported in the early phase of the disease occurring during the first 24 months of life (mainly 3–6 months) (Patient 1 Caldeira Araujo et al., 2005; Schulze et al., 1997; Patient 2 Van der Knaap et al., 2003; Ensenauer et al., 2004). Only in a few subjects onset of epilepsy was delayed (Leuzzi et al., 2006; Vodopiutz et al., 2007; Dhar et al., 2009 case 1). A description of seizures type is available for almost half of the cases described. The pattern of seizures is not consistent, and more than one type of seizures can occur in the same patient at different ages. Life-threatening tonic seizures with apnea or myoclonic seizures can be observed in the first months of life, whereas myoclonic astatic seizures, generalized tonic–clonic seizures, partial seizures with secondary generalization, drop attacks, absences, and staring episodes appear in early infancy or in adolescence (Fig. 2B). Febrile seizures, generalized tonic–clonic seizures, and myoclonic astatic seizures are the most commonly reported seizure types (Fig. 2B and Table S1).
No typical electroencephalography (EEG) pattern can be defined in GAMT deficiency. A description of EEG features has been provided for one third of the published cases (Table S1). An early derangement of background organization and interictal multifocal spikes and slow wave discharges are frequently recorded (Caldeira Araujo et al., 2005). Focal EEG abnormalities, with a prominent involvement of frontal regions, have also been reported (Leuzzi et al., 2000; Schulze et al., 2003; Leuzzi et al., 2006).
Severe epilepsy has been reported in almost all cases. Epilepsy was partially responsive to conventional antiepileptic drugs in 29 of 44 patients and completely refractory in 12 of 44. The occurrence of the seizures has always resulted in a further derangement of mental functions, and no improvements in intellectual performances were observed when seizure control was obtained (Mercimek-Mahmutoglu et al., 2006).
Movement disorders, such as athetosis, chorea, choreoathetosis, ballismus, and dystonia have been reported in 22 of 44 patients; in a single case dystonia was associated with ataxia, which was the only motor disorder in three subjects (Dhar et al., 2009). Although usually precocious, movement disorders can emerge as late as 17 years or later (O’Rourke et al., 2009; Hinnell et al., 2011 , Patient 2).
The most typical neuroimaging alteration in GAMT deficiency is represented by bilateral pallidal lesions (hypointensity in T1-weighted and hyperintensity in T2-weighted magnetic resonance imaging [MRI] images). These lesions have been reported in 8 of 33 patients, and movement disorders were evident in 5 of them. These data confirm the vulnerability of pallidum to neurotoxins resulting from several metabolic disorders other than GAMT deficiency such as methylmalonic aciduria, Wilson disease, mitochondrial encephalopathies, or succinic semialdehyde dehydrogenase deficiency (Zimmerman, 2011).
Epilepsy, movement disorders, and pallidal alteration on MRI may occur independently: Case 8 by Dhar et al., 2009 had, at the onset, a generalized dystonia and intellectual disability without epilepsy or MRI alterations (Dhar et al., 2009).
The aim of treatment is to correct both the depletion of creatine/creatine-phosphate pools and the accumulation of GAA. GAA shares the same transporter system as creatine; therefore, a high GAA level in biologic fluids potentially affects transport and restoration of cerebral creatine (Leuzzi et al., 2000). In GAMT deficiency, a lifelong oral supplementation with high doses of creatine monohydrate (350 mg/kg/day to 2 g/kg/day) has been shown, by plasma creatine assessment (muscle creatine pool) and brain 1H-MRS (brain creatine pool), to replenish body creatine pools (Caldeira Araujo et al., 2005; Bianchi et al., 2007). The high dosage required to replenish brain creatine in GAMT deficiency is probably due to the competitive effect of blood GAA at level of CT1 (Wyss & Kaddurah-Daouk, 2000) as well as to the low efficiency of creatine transport throughout the blood–brain barrier (Braissant, 2012). Although creatine supplementation should theoretically lower GAA levels through the inhibition of AGAT transcription (Wyss & Kaddurah-Daouk, 2000), it results in only a partial reduction of GAA in plasma and brain of patients with GAMT deficiency (Schulze et al., 2001). A further abating effect on AGAT activity can be obtained through a dietary restriction of arginine (15 mg/kg/day) coupled with ornithine supplementation (ornithine aspartate 350–800 mg/kg/day) (Schulze et al., 2001; Schulze et al., 2003; Schulze et al., 2006; Dhar et al., 2009). Medicaments such as sodium benzoate and phenylbutyrate, which remove arginine and glycine, respectively, have also been proposed according to a similar substrate inhibition approach (Schulze et al., 2006).
Normalization of GAA levels and complete restoration of brain creatine or creatine-phosphate pool are not always obtained through the abovementioned therapeutic approaches. (Leuzzi et al., 2000; Van der Knaap et al., 2003; Bianchi et al., 2007). When compared with pretreatment values, the reduction of GAA in biologic fluids was relevant in plasma (57–99%) and CSF (50–84%), whereas it was more variable in urine (0–83%) (Mercimek-Mahmutoglu et al., 2006). Figure 3A,B show the trend of blood GAA in two GAMT-deficient patients treated along several years according to different therapeutic approaches. The treatment is generally well tolerated. However, patient 1 experienced hyperammonemia after 11 years of treatment, probably as a cumulative effect of dipropylacetate treatment and arginine depletion (Fig. 3A).
Bianchi et al. (2000) reported the first cases of AGAT deficiency in 2000: two sisters with mild-to-moderate mental retardation and severe language delay. Other than an isolated episode of febrile seizures at age 18 months in one of the sisters, neither epilepsy nor movement disorders were present (Bianchi et al., 2000; Battini et al., 2002). 1H-MRS demonstrated lack of the creatine peak in both patients. Serum GAA levels were low in one patient and within the low normal range in the other (Carducci et al., 2002). Both patients were homozygous for a point mutation, resulting in a stop codon on exon 3 of the AGAT gene (9093A>G) (Item et al., 2001). Creatine monohydrate therapy resulted in remarkable clinical improvement and restoration of creatine peak on H-MRS (Bianchi et al., 2007).
To date, AGAT deficiency has been reported in seven patients across four families, and its clinical presentation seems rather nonspecific, being characterized by psychomotor delay during the first years of life, autistic-like behavior, severe language delay, and mild to moderate mental disability. EEG recordings while awake and asleep were normal in all patients (Bianchi et al., 2000; Battini et al., 2002; Battini et al., 2006; Edvardson et al., 2010).
Under creatine supplementation, clinical outcome is favorable, ranging from normal mental and neurologic functioning to mild learning difficulties (Ndika et al., 2012; Battini R, unpublished data).
A single patient, from the index family, was diagnosed during the first weeks of life and treated with creatine (100 mg/kg/day) before the emergence of the disease (Battini et al., 2006). Although the restoration of brain creatine was partial, his psychomotor development under creatine supplementation was normal. He presented an occasional febrile convulsion at the age of 3. At present, at the age of 7, he is a mentally normal boy with mild learning disabilities. Creatine/N-acetyl-aspartate (NAA) ratio at brain 1H-MRS is about 70% of that detected in age-matched controls (Battini R, Cioni G, unpublished data).
The index patient was a 6-year-old Caucasian boy who was diagnosed at 7 months with developmental delay and central hypotonia. At the age of 2 years, he had partial status epilepticus, multifocal epileptiform discharges at interictal EEG, and small T2 hyperintense focus in the right posterior periventricular white matter on brain MRI. At the age of 6 years, his speech and language functions were severely retarded, and he had short attention span and mild hypotonia. Blood creatinine and GAA in serum and urine were normal, whereas serum and urine creatine levels were higher than normal. Oral creatine monohydrate supplementation for 3 months resulted in a 10-fold increase in urinary creatine and an increase in CSF creatine, without concurrent appearance of creatine/creatine-phosphate peak on H-MRS or improvement of clinical conditions (Cecil et al., 2001; Salomons et al., 2001).
Apart from a few severely affected cases presenting with developmental delay and paroxysmal and/or persistent movement disorders, clinical onset of CT1 deficiency seems to be more delayed than GAMT deficiency (Anselm et al., 2006).
Epilepsy is frequent, but is never the presenting symptom in CT1 deficiency. It is rarely severe and it is usually responsive to conventional antiepileptic drugs (Schiaffino et al., 2005; Mancardi et al., 2007). Its onset ranges between 16 months and 12 years. Febrile convulsions represent the first seizure-type in a number of subjects (Battini et al., 2007; Battini et al., 2011; Valayannopoulos et al., 2012), and in a single case they led to subcontinuous generalized tonic–clonic seizures (Mancardi, 2007). Seizure pattern and EEG alterations can be extremely variable (Schiaffino et al., 2005; Mancardi et al., 2007). Seizure-types include myoclonic seizures, generalized tonic–clonic seizures, convulsive status epilepticus, and partial seizures with secondary generalization (Schiaffino et al., 2005; Mancardi et al., 2007; Fig. 2D). EEG recordings include normal tracing, diffuse slowing, aspecific sharp abnormalities, and focal/generalized paroxysmal or slow abnormalities, with or without sleep activation (Table S2; see also Fig. 4A,B). However, according to our experience, paroxysmal abnormalities are generally less severe as the child grows older. SLC6A8 genotype is not associated with epilepsy, as exemplified by personal observations and cases from the literature: for example the c.1631C>T mutation resulted in severe epilepsy in one case (Mancardi et al., 2007), but it was not associated with seizures in others (Mancini et al., 2005; Fons et al., 2008).
Neuroimaging and clinical features suggest in some patients a possible perinatal ischemic insult (see for example Fig. 5A,B). This aspect may be confounding from the diagnostic point of view because clinical history rarely justifies this suspect. However, these lesions are congruent with the concept of creatine as a protective factor against potentially ischemic damage. Their possible role in the determinism of epilepsy needs to be elucidated.
Some adult cases suggest CT1 defect as a nonstationary disorder, including corticobasal dementia associated with signs of muscle and visceral involvement, such as chronic constipation, megacolon, gastric and duodenal ulcer disease, ileus, and bowel perforation (Kleefstra et al., 2005). A progressive cerebral atrophy on brain MRI was also described (DeGrauw et al., 2002).
There are a few clinical reports on females carrying SLC8A6 gene mutations. When symptomatic, they express a milder phenotype, including mild intellectual disability, behavioral disorders, problems of language development, learning difficulties, impairment of visual-constructional and fine motor skills, mild cerebellar symptoms, and constipation (Salomons et al., 2001; Mancini et al., 2005; Póo-Argüelles et al., 2006; Van de Kamp et al., 2011; Valayannopoulos et al., 2012). Late occasional epileptic seizures have been described but not systematically studied (Ardon et al., 2010; Van de Kamp et al., 2011). An abrupt onset of severe epilepsy was recently described in a 3 year and 7-month-old girl with intellectual disability carrying c.1067G>T (Gly356Val) mutation (Mercimek-Mahmutoglu et al., 2010). She presented with extremely frequent seizures with psychomotor arrest, humming, breath-holding, eye rolling, stiffening of arms and legs, head drops, and falls. Interictal EEG at the age of 6 showed dysrhythmic background (6–7 Hz posterior dominant rhythm), excessive generalized paroxysmal theta and delta, maximally seen in the frontal areas, and frequent spikes and sharp waves in the central areas. Cerebral white matter alterations were detected by MRI. Her seizures were resistant to antiepileptic drugs and ketogenic diet but were controlled by a combined therapy with creatine plus arginine and glycine (see below) (Mercimek-Mahmutoglu et al., 2010).
No key clinical and/or neuropsychological cues have been identified to suggest the diagnosis of CT1 deficiency in girls with epilepsy and intellectual disability or learning difficulties. Apart from a single exception (Mercimek-Mahmutoglu et al., 2010), all the reported female patients affected by SLC6A8 gene defects were relatives of symptomatic males. Creatine depletion in the brain, as assessed by 1H-MRS, is found in symptomatic females. In a single case it was detected as early as 9 days of life (Cecil et al., 2003). However, 1H-MRS usually shows a variable reduction of creatine peak with a wide range between 13% and 50% (Van de Kamp et al., 2011; Valayannopoulos et al., 2012). These values overlap with normal controls in some affected females (Van de Kamp et al., 2011; Valayannopoulos et al., 2012). For these reasons gene sequencing seems to be the best diagnostic tool for females with a clinical suspect of CT1.
Treatment and clinical follow-up
No effective treatment is available for males with CT1 defect. The supplementation of creatine, also at high dosages, does not improve 1H-MRS detectable brain creatine pool and/or clinical status (Cecil et al., 2001). In vitro experiments showed that arginine and glycine supplementation can restore intracellular creatine in peripheral cells lacking CT1 (Leuzzi et al., 2008). In vivo trials were less encouraging, since they resulted in only mild or no clinical improvement with scarce or absent variation of brain creatine 1H-MRS signal (Chilosi et al., 2008; Fons et al., 2008; Valayannopoulos et al., 2012; Van de Kamp et al., 2012). However some anecdotic experiences, such as those reported in Fig. 4A,B, where a clinical improvement could be observed under arginine supplementation, suggest that this topic deserves further study.
In contrast, creatine, as well as creatine precursor, supplementation is potentially effective in symptomatic females where the defect of CT1 is partial. In a recently described case, the administration of creatine-monohydrate, l-arginine, and l-glycine resulted in a persistent seizure control and increase of 1H-MRS creatine peak after 12 months of treatment (Mercimek-Mahmutoglu et al., 2010). In this context, the availability of a potential effective therapy makes it mandatory to test all females with intellectual disability for CT1 deficiency (Van de Kamp et al., 2011). A recent study showed that cognitive deficits in SLC6A8−/y mouse can be reversed through nine weeks of treatment with cyclocreatine (Kurosawa et al., 2012).
Pathogenetic and Physiopathologic Aspects
Animal models of inborn errors of creatine metabolism
Two animal models of primary disorders of creatine metabolism have been published to date. The GAMT knockout mouse has a biochemical phenotype overlapping that of affected human beings, but clinical presentation is characterized mainly by muscle involvement, increased neonatal mortality, decreased male fertility, no epilepsy, and other severe neurologic symptoms and minor cognitive defects (Schmidt et al., 2004; Kan et al., 2005; Torremans et al., 2005). In contrast, the recently generated CT1 knockout mouse fits with the mental phenotype found in the affected subjects, but presents with a more generalized creatine depletion that involves striate muscles (included heart) and serum creatine, probably as a consequence of the derangement of intestinal transport of creatine. Some alterations such as those of serotoninergic and dopaminergic system remain to be explored in man (Skelton et al., 2011).
The epileptogenic role of guanidinoacetic acid
Some neurologic symptoms, mainly occurring in patients with GAMT deficiency (such as severe epilepsy, movement disorders, pallidal lesions), strongly point to a pathogenic role of GAA (or related guanidine compounds), which is increased in all biologic fluids and tissues only in this disorder. The epileptogenic effect of GAA is well known. GAA induces convulsions when administered intracisternally to rabbits (De Deyn & Macdonald, 1990). In the striatum of rats, GAA probably interferes with energetic metabolism at the level of complex II, ATPase and creatine kinase activities through lipid peroxidation causing oxidative stress (Zugno et al., 2006; Zugno et al., 2007). Creatine prevents the neurotoxicity of GAA in rat striatum (Kolling & Wyse, 2010). Moreover, GAA (but not creatine) has a specific mimetic effect on GABA A receptors (Neu et al., 2002). This effect probably results in a reduction of convulsive threshold through various possible detrimental adaptive mechanisms on these receptors (Neu et al., 2002).
Homeostasis of brain creatine: physiologic and physiopathologic mechanisms
Different models have been proposed to explain the complex homeostasis of brain creatine. Experiences in patients with GAMT and AGAT defects underscore the importance of the endogenous synthesis for the normal homeostasis of creatine in brain (Ensenauer et al., 2004). The effectiveness of creatine supplementation in AGAT deficiency suggests that (1) in normal conditions both endogenous and exogenous fonts of creatine contribute to the maintaining of the brain creatine pool, but also that (2) under normal creatine intake, blood supply is not sufficient to balance the failure of cerebral endogenous synthesis (Bianchi et al., 2007). On the contrary, CT1 defect discloses the importance of blood supply for the brain. In fact, lacking CT1, the endogenous synthesis of creatine proves to be an inadequate font of cerebral creatine (also under the supplementation of creatine precursors) (Chilosi et al., 2008; Fons et al., 2008). According to a recent model, the brain creatine pool would result from the mutual vicariance of different nervous cells. GAMT, AGAT, and CT1 expression in these cells is variable: 4% of cortex cells express all three proteins; 8% of them AGAT and CT1; 7% of them GAMT and CT1; and 14%, 15%, and 14% of them only CT1, AGAT, and GAMT, respectively. Twenty-five percent of neurons and 35% of astrocytes (including those of blood–brain barrier) do not express any of the three proteins, whereas no more than 33% are equipped with CT1 (Braissant et al., 2010). This model does not completely fit with the complete restoration of brain creatine pool after 9 months of creatine supplementation and (several months later) the almost complete clinical normalization in patients with AGAT deficiency (Bianchi et al., 2007; Battini et al. unpublished data). The hypothesis that CT1 expression could be induced by high levels of creatine has not been demonstrated so far (Braissant et al., 2011). It has also been suggested that neurons produce creatine from astrocitary GAA (Braissant et al., 2010). If so, in the lack of CT1, GAA (whose efflux from the CSF is also ascribed to CT1) (Tachikawa et al., 2008) should accumulate in extracellular fluids and CSF. Although high GAA was detected by brain 1H-MRS in a CT1-deficient patient, CSF GAA was normal in another subject (Chilosi et al., 2008).
Another dissociated model implies that glial cells express GAMT activity and supply creatine to neurons with a high creatine kinase activity (neuronal networks implied in high energy demanding functions such as motor and sensory processing, learning, memory, and limbic functions) (Tachikawa et al., 2004; Mak et al., 2009). These data well explain the pattern of intellectual disability found in all the three disorders of creatine metabolism. According to this model, in CT1 deficiency, creatine should accumulate in extracellular fluids and CSF. Clinical data did not confirm this accumulation in CSF of CT1-deficient patients (Cecil et al., 2001; Almeida et al., 2004; Chilosi et al., 2008). This model also contrasts with the lack of a clear regional differentiation in creatine depletion in the brains of patients with CT1 defect. Waiting from more consistent data arising from the animal model of SLC6A8 knockout mouse, recent in vitro experimental data suggest the prominence of uptake versus de novo synthesis on the control of creatine homeostasis in nervous tissue (Carducci et al., 2012).
Mental retardation, language derangement, and epilepsy appear to be the specific hallmarks of the primary disorders of creatine metabolism. Why mental functions are selectively affected by creatine deficiency remains to be clarified. Febrile convulsions are a common presenting symptom that anticipates epilepsy. Blood GAA assessment and brain H-MRS examination should be part of diagnostic work-up for all patients presenting with an epileptic encephalopathy of unknown origin. Waiting for a better phenotypic characterization in girls suffering from learning and/or intellectual disabilities with or without epilepsy SLC6A8 gene assessment remains the only reliable tool to diagnose CT1 defect.
We are deeply grateful to all the colleagues of the Italian Group for the Study of Creatine Disorders (Dr MC Bianchi, Dr. Ar. Ferrari, Dr. M. Casarano, Dr M. Tosetti, and Dr MG Alessandrì– IRCSS “Stella Maris”, Pisa; Prof. I. Antonozzi, Dr Cl. Carducci, Prof. Ca. Carducci – Department of Experimental Medicine, “Sapienza”– University of Rome) for their stimulating support on the subject and Dr AM Ferrari, EEG Lab IRCCS Stella Maris, for her help with the illustration and advice on CT1 Fig. 4
None of the authors has any conflict of interest to disclose. We confirm that we have read the Journal’s position on issues involved in ethical publication and affirm that this report is consistent with those guidelines.