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1 Available online at Annals of Clinical & Laboratory Science, vol. 46, no. 5, Creatine Deficiency Syndrome could be Missed Easily: A Case Report of Guanidinoacetate Methyltransferase Deficiency Presented with Neurodevelopmental Delay, Seizures, and Behavioral Changes, but Normal Structural MRI Iliyana Pacheva 1, Ivan Ivanov 1, Marin Penkov 2, Daliya Kancheva 3, Albena Jordanova 3, and Mariya Ivanova 4 1 Department of Pediatrics and Medical Genetics, Medical University Plovdiv, 2 Department of Imaging Diagnosis, University Hospital "St. Ivan Rilski" Sofia, 3 Molecular Neurogenomics Group, VIB Department of Molecular Genetics, University of Antwerp, Antwerpen, Holland; Molecular Medicine Center, Department of Medical Chemistry and Biochemistry, Medical University-Sofia, Sofia, and 4 National Genetic Laboratory, University Hospital of Obstetrics and Gynecology, Sofia; Department of Analytical Chemistry, Sofia University St. Kl. Ohridsky, Sofia, Bulgaria Abstract. A case with GAMT deficiency (homozygous c.64dupg mutation) presented with neurodevelopmental delay, rare seizures, behavioral disturbances, and mild hypotonia, posing diagnostic challenges. Metabolic investigations showed low creatinine in plasma and urine (guanidinoacetate couldn t be investigated) and slightly elevated lactate. MRI was normal. Correct diagnosis was possible only after MR spectroscopy was performed at age 5½ years. A homozygous c.64dupg mutation of the GAMT gene was identified in the proband. In conclusion, every case with neurodevelopmental delay or arrest, especially when accompanied by seizures, behavioral impairment, muscle hypotonia or extrapyramidal symptoms should undergo MRI with MR spectroscopy. Normal structural MRI doesn t exclude a creatine deficiency syndrome. Biochemical investigations of guanidinoacetate, creatine, and creatinine in body fluid should be done to diagnose cerebral creatine deficiency syndromes and to specify the deficient enzyme. Thus, a treatable disease will not be missed. Key words: GAMT deficiency, creatine deficiency, MRI, MR spectroscopy, neurodevelopmental delay, behavioral impairment. Abbreviations and Acronyms: AGAT-arginine glycine amidinotransferase, GAMT-guanidinoacetate methyltransferase, GAA-guanidinoacetate, ccdss-cerebral creatine deficiency syndromes, CrTr-creatine transporter. Introduction Cerebral creatine deficiency syndromes (ccdss) are a relatively new group of metabolic disorders resulting from an impaired creatine metabolism and leads to a creatine deficit in the brain. Creatine deficiency impairs high cortical functions by destroying the intracellular transfer of energy. ccdss consist of three diseases: autosomal recessive guanidinoacetate methyltransferase (GAMT) deficiency, autosomal recessive arginine:glycine amidinotransferase (AGAT) deficiency, and X-linked creatine transporter (CrTr) deficiency. GAMT Address correspondence to Iliyana Pacheva, MD, PhD; Department of Pediatrics and Medical Genetics, Medical University Plovdiv, 15A Vasil Aprilov str, 4000 Plovdiv, Bulgaria; phone: ; fax: ; e mail: inapatcheva@hotmail.com deficiency was the first described ccds in 1994 [1], and about 100 cases have been reported in the literature so far [2-6]. ccdss are characterised by mental retardation, speech delay, seizures, behavioral changes, and movement disorders. The severity of symptoms could vary from lacking to severe [2-7]. The disease usually starts after normal neurodevelopment in the first months. Seizures are usually febrile and start at age 3-6 months. They might evolve into epilepsy with multiple seizure types: generalized tonic or tonic-clonic, astatic, absence, or complex partial with/without secondary generalization. In half of the cases, seizures are resistant to conventional antiepileptic drugs [8,9]. Behavioral changes present with hyperactivity, autistic signs, aggression, and /16/ by the Association of Clinical Scientists, Inc.

2 558 Annals of Clinical & Laboratory Science, vol. 46, no. 5, 2016 autoaggression (including self mutilation) [2-4]. The movement disorder is characterized by extrapyramidal symptoms like chorea and dystonia [5,8]. Muscular hypotonia, mild spasticity, and coordination disturbances could also occur [8]. We present an illustrative case of GAMT deficiency diagnosed by MR spectroscopy after 5 years of laborious investigations. Case Report History. The patient is a 7-year-old Bulgarian boy. He was born at full term to healthy nonconsanguineous parents after an uneventful pregnancy. His birth weight, length, and head circumference were all within the normal range. His family history was positive for syndromic autism (a second degree relative suffered from Williams syndrome). The child had normal neurodevelopment until the age of 3 months, and then he showed a mild delay: at the age of 6 months, he occasionaly grasped objects; at 7 months of age, he started babbling and moving objects from hand to hand; at the age of 8 months, he could sit, recognize and differentiate between his relatives and unknown people, and follow objects and people with gaze; at 10 months of age, he started to roll over; at 13 months of age, he could already stand upright. He only occaisionally fulfilled commands, but he would look at the person that was calling his name. At 15 months of age, he could climb steps with support and pronounce only a few syllables. During the next years, there were periods of neurodevelopmental arrest or regression as well as periods of mild progress. He started walking at age 2 ½ years. There was no expressive language, nor any practical life skills. He followed elementary commands rarely. Hyperactivity, aggressiveness, self-injuries, and autistic features with poor eye contact were noticed. Seizures debuted at 10 months of age with a complex, febrile, generalized tonic-clonic convulsion with lateralization to the left, which lasted 25 minutes. His next seizures were also complex febrile and occurred at the ages of 4 years 10 months, 5 years 9 months, and 5 years 11 months. Generalized myoclonic-like events had also been reported repeatedly since 2 ½ years of age, but they were considered as shuddering attacks. Physical and neurologic examination. His first examination at 6 months of age revealed mild muscle tone abnormalities - axial hypotonia, hypertonia of arm adductors, and occasionally dystonic postures. At the age of 1 year 4 months, mild lower extremity hypotonia was found. At 2 years 8 months, clumsy gait was registered. At the age of diagnosis (5 years 10 months), the physical examination was normal except for slender muscle mass, mild bilateral ptosis, and mild muscular hypotonia with exaggerated tendon reflexes. There was also gross and fine motor delay. He could not run, stand on one leg, or climb stairs unsupported. Neuropsychological evaluation. His neurodevelopmental quotient was within normal range at 6 months of age. After 10 months of age, it decreased fast, reaching 58% at 16 months and 23% at 5 years 10 months. Thus, severe mental retardation was diagnosed. At 5 years 10 months, behavioral signs were also observed: hyperactivity, aggressiveness, and autistic features. Features would include impaired social communication and interactions, poor eye contact, lack of spontaneous make-believe play, abnormal play with children that consisted only of gazing at them, no finger pointing at interesting or desired objects, and rare imitation of actions. His score on Childhood Autism Rating Scale was 36, and that suggested mild to moderate autism. Laboratory, functional and imaging investigations. The patient s electroencephalograms (EEG) deteriorated with age. The first EEG during sleep stages I and II at age 1 year 4 months showed only few epochs of dysrhythmic activity. At age 2 years 9 months, the EEG on awakening already displayed pronounced slowed and dysrhythmic background activity. It remained the same until 5 years 9 months when EEG in stage II sleep revealed epochs of disorganized, high amplitude slow wave 1-2 Hz activity with rare multifocal spikes or sharp waves among delta and theta background activity (i.e. suggestive of modified hypsarrhythmia). Brain CT at 2 years of age, MRI at 5 years of age, and brainstem auditory evoked potentials at 3 years of age were normal. Metabolic investigations revealed normal amino acids in plasma and organic acids in urine, but increased lactate in urine. Serum lactate was slightly elevated (3 mmol/l). Creatinine level in both serum and urine was low. MR spectroscopy confirmed ccds based on the lack of creatine peak (Figure 1), despite normal structural MRI. Levels of guanidinoacetate (GAA) and creatine in serum and urine are not available technically for investigation in Bulgaria nowadays. In differential diagnosis. Diseases with secondary creatine deficiency such as urea cycle disorders, gyrate atrophy of the choroid and retina caused by ornithine delta-aminotransferase deficiency, HHH (hyperammonemia, hyperornithinemia, homocitrullinuria) were excluded by metabolic investigations.

3 Creatine deficiency syndrome could be missed: case report 559 Figure 1. MR spectroscopy of the brain demonstrates an abnormal spectrum with a creatine peak virtually absent. Figure 2. MR spectroscopy demonstrates normal cerebral creatine peak. Diagnosis of ccds was reached with difficulty after several hospital admissions. The diagnoses applied or suspected previously in this case were mental retardation, atactic cerebral palsy, complex febrile seizures, mitochondrial disease, or another genetic syndrome. Considering the clinical features and the low level of creatinine in plasma and in urine, we suggested GAMT deficiency as the most probable diagnosis in this case. Sanger sequencing of the coding exons and exon-intron boundaries of the GAMT gene identified a homozygous c.64dupg mutation in the proband and heterozygosity for the same mutation in both parents. Treatment and follow up. At the age of 6 years, 6g (300mg/kg) of creatine monohydrate per day was added to levetiracetam. One year after onset of creatine monohydrate supplementation, second MRI with MR spectroscopy was done, which revealed normal brain MRI and normalization of cerebral creatine level (Figure 2). No epileptic seizures occurred in the last year, and there was progress in motor development: the gait became more stable, and running and climbing with no support were achieved. Behavior slightly improved: decreased hyperactivity, better attention, improved eye contact, and better following of speech commands were noticed. Unfortunately, there is still no expressive language, and neurodevelopment came to a standstill in the last year. His EEG showed improvement in background activity, but there were still epochs of disorganized high amplitude slow and sharp waves. Restriction of arginine in the diet and supplementation of ornithine 800 mg/kg was recommended to the parents, but they couldn't afford that treatment. Discussion Here we report the first diagnosed case of ccds in Bulgaria. Despite the fact that CrTr deficiency is the most frequent type of ccds, the first registered patient in Bulgaria had GAMT deficiency. We suspect underdiagnosis of ccdss in Bulgaria for several reasons: low awareness of neuropediatricians and professionals about ccdss; not performing laboratory investigations of GAA and creatine that are important for diagnosis; and rare use of MR spectroscopy in clinical practice, which is crucial for diagnosis of ccdss. All of these disadvantages could be easily overcome without substantial resources. Moreover, GAMT deficiency could be a treatable disease, especially if the treatment starts early in life [10,11]. There may be other countries with the same problems. This case had a typical clinical picture of GAMT deficiency, characterized by severe mental retardation registered after normal neurodevelopment during first months of life. Intellectual disability is the most common clinical feature of GAMT deficiency, and it is severe in 80% of the cases, although it might also be mild [5-8]. Unlike most cases with severe and commonly pharmacoresistant epilepsy, this patient had rare febrile seizures and suspected myoclonias [8,9]. The onset of seizures in this case was at 10 months of age, although it is typical to be earlier - during the first months of life or even in

4 560 Annals of Clinical & Laboratory Science, vol. 46, no. 5, 2016 the neonatal period [9]. The behavioral problems observed were also typical - hyperactivity, aggressiveness, and autistic features. However, there was not self-mutilation [2-3]. Movement disorders were the mildest signs in this patient. There were only slight muscular hypotonia and some dystonic postures in infancy. They disappeared gradually in the next years. Movement disorders, such as extrapyramidal signs, occur in about 40% of patients. They usually occur before 12 years of age, and they are associated with basal ganglia lesions visualized on brain MRI [2,7,8]. An interesting and challenging fact in this case was the normal brain MRI, which could be an exception of the disease (especially when lacking extrapyramidal symptoms). However, it made the diagnosis of ccdss even more difficult to establish where no MR spectroscopy was applied. Muscle tissue is another place where creatine is necessary for energy production, but myopathy is described rarely in ccdss [2,8,12]. The slight muscular hypotonia and scarce muscle bulk, combined with increased lactate in plasma in this case were motives to suggest a mitochondrial disease. After proving GAMT deficiency, secondary defects in respiratory transport chains could be speculated. The recommended screening tests for ccdss and for their differentiation are levels of GAA, creatine, and creatinine in urine and plasma [8]. Plasma GAA may discriminate between the GAMT (high concentration), AGAT (low concentration) and CrTr (normal concentration) deficiencies. The creatine to creatinine concentration ratio in urine is elevated in CrTr deficiencies. However, in countries like Bulgaria where the measuring of levels of GAA and creatine is unavailable, we recommend MR spectroscopy for diagnosis. For the following differentiation, we could rely only on the creatinine level in plasma and urine. If it is decreased, the most probable type is GAMT deficiency, and we should consider GAMT gene sequencing. Gene sequencing will be more expensive than these biochemical investigations, and all such countries should initiate introducing them into practice. Recently, MR spectroscopy has started to become part of MRI protocol. So as not to miss cases of ccds, all pediatricians and pediatric neurologists should require MR spectroscopy for children with global developmental delay or intellectual disability of unknown origin, especially in combination with seizures, movement disorders, and behavioral problems. The c.64dupg mutation in GAMT, as discovered in our patient, was previously reported only in two sisters from Albania [13]. Although the patient has no Albanian relatives, it seems this mutation is common for the populations inhabiting the Balkan peninsula. Therefore, we recommend all ccdss patients of this origin with suspected GAMT deficiency to be tested for this mutation first. Epilepsy in GAMT deficiency, unlike CrTr deficiency, could be managed by creatine monohydrate supplementation mg/kg per day, but there is little improvement in cognitive development [11]. The phenotype of GAMT deficiency is due not only to brain creatine deficiency, but also to toxicity of the accumulated GAA. This could explain the only mild improvement in behavior and the absence of speech development of this child. Addition of ornithine 800 mg/kg and restriction of arginine by reduction of protein intake to g/ kg per day are recommended in such situations in order to decrease the toxic role of GAA [14]. Unfortunately the parents couldn t afford this. If these additional therapeutic measures had been taken, the improvements in behavior and cognition might have been more significant. The suboptimal effect of our treatment could be due to the late stage of the disease when creatine supplementation was started (6 years of age). Recent studies proclaim that the start of treatment early in life may make GAMT disease treatable [10]. That is why some countries have introduced neonatal screening for GAMT deficiency by investigation of GAA in dried blood spots [15]. In conclusion, every case with neurodevelopmental delay or arrest (especially when accompanied by seizures of any type, behavioral signs like hyperactivity, autistic and aggressive features, muscle hypotonia, or extrapyramidal symptoms) should undergo MRI with MR spectroscopy. Normal structural

5 Creatine deficiency syndrome could be missed: case report 561 MRI doesn t exclude ccdss. Occasionally, GAMT deficiency could mimic a mitochondrial disease. Biochemical investigations of GAA, creatine, and creatinine in body fluid should be done to diagnose ccdss and to specify the deficient enzyme. Thus, a treatable disease will not be missed. References 1. Stöckler S, Holzbach U, Hanefeld F, Marquardt I, Helms G, Requart M, et al. Creatine deficiency in the brain: a new, treatable inborn error of metabolism. Pediatr Res. 1994;36(3): Mercimek-Mahmutoglu S, Stoeckler-Ipsiroglu S, Adami A, Appleton R, Araujo HC, Duran M, et al. GAMT deficiency: features, treatment, and outcome in an inborn error of creatine synthesis. Neurology. 2006;67: Stockler-Ipsiroglu S, vankarnebeek CD. Cerebral creatine deficiencies: a group of treatable intellectual developmental disorders. Semin Neurol. 2014;34(3): Mercimek-Mahmutoglu S, Ndika J, Kanhai W, devillemeur TB, Cheillan D, Christensen E, et al. Thirteen new patients with guanidinoacetatemethyltransferase deficiency and functional characterization of nineteen novel missense variants in the GAMT gene. HumMutat. 2014;35(4): O'Rourke DJ, Ryan S, Salomons G, Jakobs C, Monavari A, King MD. Guanidinoacetatemethyltransferase (GAMT) deficiency: late onset of movement disorder and preserved expressive language. DevMedChildNeurol. 2009;51: Caldeira Araújo H, Smit W, Verhoeven NM, Salomons GS, Silva S, Vasconcelos R, Tomás H, TavaresdeAlmeida I, Jakobs C, Duran M. Guanidinoacetate methyltransferase deficiency identified in adults and a child with mental retardation. Am J MedGenet A. 2005;133A: Lion-François L, Cheillan D, Pitelet G, Acquaviva-Bourdain C, Bussy G, Cotton F, et al. High frequency of creatine deficiency syndromes in patients with unexplained mental retardation. Neurology. 2006;67: Mercimek-Mahmutoglu S, Salomons GS. Creatine Deficiency Syndromes Jan 15 [Updated 2015 Dec 10]. In: Pagon RA, Adam MP, Ardinger HH, et al., editors. GeneReviews [Internet]. Seattle (WA): University of Washington, Seattle; Available from: 9. Leuzzi V, Mastrangelo M, Battini R, Cioni G.Inborn errors of creatine metabolism and epilepsy. Epilepsia. 2013;54(2): Schulze A, Hoffmann GF, Bachert P, Kirsch S, Salomons GS, Verhoeven NM, et al. Presymptomatic treatment of neonatal guanidinoacetate methyltransferase deficiency. Neurology. 2006;67: Verbruggen KT, Sijens PE, Schulze A, Lunsing RJ, Jakobs C, Salomons GS, et al. Successful treatment of a guanidinoacetate methyltransferase deficient patient: findings with relevance to treatment strategy and pathophysiology. MolGenetMetab. 2007b;91: Morris AA, Appleton RE, Power B, Isherwood DM, Abernethy LJ, Taylor RW, et al. Guanidinoacetate methyltransferase deficiency masquerading as a mitochondrial encephalopathy. J Inherit Metab Dis. 2007; 30(1): Item CB, Mercimek-Mahmutoglu S, Battini R, Edlinger-Horvat C, Stromberger C, Bodamer O, et al. Characterization of seven novel mutations in seven patients with GAMT deficiency. Hum Mutat. 2004;23: Schulze A, Ebinger F, Rating D, Mayatepek E. Improving treatment of guanidinoacetate methyltransferase deficiency: reduction of guanidinoacetic acid in body fluids by arginine restriction and ornithine supplementation. Mol Genet Metab. 2001;74: Pasquali M, Schwarz E, Jensen M, Yuzyuk T, DeBiase I, Randall H, Longo N. Feasibility of newborn screening for guanidinoacetatemethyltransferase (GAMT) deficiency. J InheritMetabDis. 2014;37(2):231-6.