Laura Pérez-Gay

The Influence of Valproic Acid and Carbamazepine Treatment on Serum Biotin and Zinc Levels and on Biotinidase Activity

We determined the serum concentration of biotin, zinc, antiepileptic drugs, and biotinidase enzyme activity in 20 children treated with valproic acid, in 10 children treated with carbamazepine, and in 75 age- and sex-matched healthy controls. There were no significant differences in the serum levels of biotin, and biotinidase enzyme activity between the patients treated with valproic acid, the patients treated with carbamazepine, and the control group. Zinc serum levels were lower in the patients treated with valproic acid and with carbamazepine than in the control group, but within the normal range. Hair loss was observed in 3 patients treated with valproic acid, with normal serum levels of biotin, zinc, and biotinidase activity, and the alopecia disappeared with the oral administration of biotin (10 mg/d) in 3 months. These results suggest that the treatment with valproic acid does not alter the serum levels of biotin, zinc, and biotinidase enzyme activity.

Copy number variation analysis of patients with intellectual disability from North-West Spain

Intellectual disability (ID) is a complex and phenotypically heterogeneous neurodevelopmental disorder characterized by significant deficits in cognitive and adaptive skills, debuting during the developmental period. In the last decade, microarray-based copy number variation (CNV) analysis has been proved as a strategy particularly useful in the discovery of loci and candidate genes associated with these phenotypes and is widely used in the clinics with a diagnostic purpose. In this study, we evaluated the usefulness of two genome-wide high density SNP microarrays -Cytogenetics Whole-Genome 2.7M SNP array (n=126 patients; Group 1) and CytoScan High-Density SNP array (n=447 patients; Group 2)- in the detection of clinically relevant CNVs in a cohort of ID patients from Galicia (NW Spain). In 159 (27.7%) patients, we detected 186 rare exonic chromosomal imbalances, that were grouped into the following classes: Clinically relevant (67/186; 36.0%), of unknown clinical significance (93/186; 50.0%) and benign (26/186; 14.0%). The 67 pathogenic CNVs were identified in 64 patients, which means an overall diagnostic yield of 11.2%. Overall, we confirmed that ID is a genetically heterogeneous condition and emphasized the importance of using genome-wide high density SNP microarrays in the detection of its genetic causes. Additionally, we provided clinical and molecular data of patients with pathogenic or likely pathogenic CNVs and discussed the potential implication in neurodevelopmental disorders of genes located within these variants.

Dravet Syndrome and Mitochondrial Dysfunction

We read with great interest the recent contribution by Frye, in the Journal of Child Neurology. He reported 2 children with Leber hereditary optic neuropathy mutations associated with infantile-onset myoclonic epilepsy, with clinical features consistent with Dravet severe myoclonic epilepsy of infancy (Dravet syndrome) and without mutations in the alpha subunit of the sodium channel (SCN1A). The author concludes that Leber hereditary optic neuropathy mutations can be important to considerer in children with early-onset myoclonic epilepsy similar to Dravet severe myoclonic epilepsy in infancy who are SCN1A negative. The possible relation between the Dravet syndrome and a mitochondrial dysfunction was postulated previously. In this sense, in 1995 we reported in the journal Child’s Nervous System a child with an epileptic syndrome with clinical features consistent with Dravet syndrome who had lactate levels elevated in cerebrospinal fluid and urine. A muscle biopsy performed at the age of 5.5 years showed a slight increase in the number of muscle mitochondria. Quantification of the mitochondrial enzymatic activities demonstrated partial deficiency of the mitochondrial respiratory chain complex III and IV. In 1997, we reported a girl with Dravet syndrome and elevated blood lactate levels. Her first muscle biopsy at the age of 19 months revealed abnormal abundant grouped mitochondria, some with ultrastructural abnormalities, but analysis of the respiratory chain complexes was normal. The patient was treated with a high dose of valproic acid (80 mg/kg/day), clonazepam, and carnitine. At the age of 32 months, she was admitted because of a cluster of seizures during a febrile illness. She developed liver failure and coma and soon deteriorated into multiorgan failure and death. Biochemical analysis of mitochondrial respiratory chain complexes, from a muscle biopsy performed immediately after death, disclosed a partial deficit of complex IV. Analysis of mitochondrial DNA excluded point mutations. Posteriorly, depletion of mitochondrial DNA was demostrated (case 9 of this report). In 1998, Fernández-Jaén et al also reported 1 case with several characteristics of Dravet syndrome, in which they found an elevation of blood lactate, and a muscle biopsy demonstrated an increased number of abnormally structured mitochondria. Enzymatic analysis showed a severe deficiency of complex IV. In 2009, Bolszak et al reported a child with Dravet syndrome and mutations in the SCN1A and in the mitochondrial polymerase gamma 1 (POLG1) genes. Recently, Nishri et al described an 11-month-old boy who developed liver failure after febrile status epilepticus while being treated with valproic acid for myoclonic epilepsy and recurrent partial and generalized seizures. The diagnosis of Alpers-Huttenlocher disease was considered. A muscle biopsy showed mitochondrial dysfunction. Mitochondrial DNA depletion was ruled out, and sequencing of the POLG1 gene did not detect any mutation. The child continued to have episodes of status epilepticus triggered by fever, which were unresponsive to medications. The occurrence of long-lasting seizures triggered by fever, myoclonic jerks, and developmental regression in a previously normal infant led to suspecting the diagnosis of Dravet syndrome. Sequencing of the SCN1A gene revealed a significant novel amino acid substitution (p.Val 1537 Glu), not identified in either parent. In conclusion, in rare cases, Dravet syndrome is associated with mitochondrial dysfunction; however, it is not possible to identify these patients in advance and there is no satisfactory hypothesis to explain the association. One possibility is that the deficit arises at a relatively late stage: in our first case, the deficit was detected in a muscle biopsy obtained at age of 5.5 years, whereas in the second case, no deficit was detected in a biopsy taken at age 19 months, despite apparent abnormalities in mitochondrial morphology, and in this sense, deletions and depletion of mitochondrial DNA can become evident at different ages. Another possibility is a relation with the level of neuronal Naþ channels phosphorylation, because in normal conditions, these channels are less phosphorylated in early age and more excitable compared with the rest of life since phosphorylated Naþ channels are known to have reduced Naþ influx. On the other hand, their association with a mitochondrial dysfunction increases the risk of liver failure because of valproic acid administration, a commonly accepted treatment for Dravet syndrome; and multiorgan Journal of Child Neurology 26(10) 1331-1334 a The Author(s) 2011 Reprints and permission: sagepub.com/journalsPermissions.nav http://jcn.sagepub.com

Primary Adenosine Monophosphate (AMP) Deaminase Deficiency in a Hypotonic Infant

The spectrum of the adenosine monophosphate (AMP) deaminase deficiency ranges from asymptomatic carriers to patients who manifest exercise-induced muscle pain, occasionally rhabdomyolysis, and idiopathic hyperCKemia. However, previous to the introduction of molecular techniques, rare cases with congenital weakness and hypotonia have also been reported. We report a 6-month-old girl with the association of congenital muscle weakness and hypotonia, muscle deficiency of adenosine monophosphate deaminase, and the homozygous C to T mutation at nucleotide 34 of the adenosine monophosphate deaminase-1 gene. This observation indicates the possible existence of a primary adenosine monophosphate deaminase deficiency manifested by congenital muscle weakness and hypotonia.

Chromosomopathy Manifesting as Mitochondrial Disease

1. Kahle KT, Barnett SM, Sassower KC, et al. Decreased seizure activity in a human neonate treated with bumetanide, an inhibitor of the Na(þ)-K(þ)-2Cl(-) cotransporter NKCC1. J Child Neurol. 2009;24:572-576. 2. Kahle KT, Staley KJ. The bumetanide-sensitive Na-K-2Cl cotransporter NKCC1 as a potential target of a novel mechanism-based treatment strategy for neonatal seizures. Neurosurg Focus. 2008; 25:E22. 3. Dzhala VI, Talos DM, Sdrulla DA, et al. NKCC1 transporter facilitates seizures in the developing brain. Nat Med. 2005;11: 1205-1213. 4. Dzhala VI, Brumback AC, Staley KJ. Bumetanide enhances phenobarbital efficacy in a neonatal seizure model. Ann Neurol. 2008;63:222-235. 5. Nardou R, Ben-Ari Y, Khalilov I. Bumetanide, an NKCC1 antagonist, does not prevent formation of epileptogenic focus but blocks epileptic focus seizures in immature rat hippocampus. J Neurophysiol. 2009;101:2878-2888. 6. Mazarati A, Shin D, Sankar R. Bumetanide inhibits rapid kindling in neonatal rats. Epilepsia. 2009;50:2117-2122. 7. Dzhala VI, Kuchibhotla KV, Glykys JC, et al. Progressive NKCC1-dependent neuronal chloride accumulation during neonatal seizures. J Neurosci. 2010;30:11745-11761. 8. Wahab A, Albus K, Heinemann U. Ageand region-specific effects of anticonvulsants and bumetanide on 4-aminopyridineinduced seizure-like events in immature rat hippocampal-entorhinal cortex slices. Epilepsia. 2011;52:94-103. 9. Sullivan JE, Witte MK, Yamashita TS, et al. Pharmacokinetics of bumetanide in critically ill infants. Clin Pharmacol Ther. 1996; 60:405-413. 10. Lopez-Samblas AM, Adams JA, Goldberg RN, et al. The pharmacokinetics of bumetanide in the newborn infant. Biol Neonate. 1997;72:265-272. 11. Lemonnier E, Ben-Ari Y. The diuretic bumetanide decreases autistic behaviour in five infants treated during 3 months with no side effects. Acta Paediatr. 2010;99: 1885-1888. 12. Ben-Ari Y. Excitatory actions of GABA during development: the nature of the nurture. Nat Rev Neurosci. 2002;3:728-739. 13. Wang DD, Kriegstein AR. Blocking early GABA depolarization with bumetanide results in permanent alterations in cortical circuits and sensorimotor gating deficits [published online ahead of print July 19, 2010]. Cereb Cortex. 14. Edwards DA, Shah HP, Cao W, et al. Bumetanide alleviates epileptogenic and neurotoxic effects of sevoflurane in neonatal rat brain. Anesthesiology. 2010;112:567-575. 15. Brandt C, Nozadze M, Heuchert N, et al. Disease-modifying effects of phenobarbital and the NKCC1 inhibitor bumetanide in the pilocarpine model of temporal lobe epilepsy. J Neurosci. 2010;30:8602-8612. 16. Bittigau P, Sifringer M, Genz K, et al. Antiepileptic drugs and apoptotic neurodegeneration in the developing brain. Proc Natl Acad Sci U S A. 2002;99:15089-15094. 17. Ge S, Goh EL, Sailor KA, et al. GABA regulates synaptic integration of newly generated neurons in the adult brain. Nature. 2006; 439:589-593. 18. Cancedda L, Fiumelli H, Chen K, et al. Excitatory GABA action is essential for morphological maturation of cortical neurons in vivo. J Neurosci. 2007;27:5224-5235. 19. Wirrell EC, Armstrong EA, Osman LD, et al. Prolonged seizures exacerbate perinatal hypoxic-ischemic brain damage. Pediatr Res. 2001;50:445-454. 20. Auvin S, Shin D, Mazarati A, et al. Inflammation exacerbates seizure-induced injury in the immature brain. Epilepsia. 2007; 48(suppl 5):27-34. 21. Farwell JR, Lee YJ, Hirtz DG, Sulzbacher SI, et al. Phenobarbital for febrile seizures-effects on intelligence and on seizure recurrence. N Engl J Med. 1990;322:364-369. 22. Reinisch JM, Sanders SA, Mortensen EL, et al. In utero exposure to phenobarbital and intelligence deficits in adult men. JAMA. 1995;274:1518-1525.

Abnormal growth in mitochondrial disease.

Sir, We read with great interest the recent article on abnormal growth in mitochondrial disease published in Acta Paediatrica by Wolny et al. (1). The authors described their retrospective study of 24 children and adolescents (median 7.86 years, range: 1.76–20.5 years) with a proved biochemical diagnosis of mitochondrial disease, and with available height and weight measurements. Forty one per cent of children had a height of less than 2 SD below the mean. Their short stature did not become more pronounced with advancing years. Children also had a low median body mass index (BMI) SDS of )1.07, which decrease with advancing years. They concluded that a short stature and a progressive reduction in BMI are features of mitochondrial disease in childhood, and so mitochondrial disease needs to be included in the differential diagnosis of unexplained short stature in children and young adults. Their conclusions are concordant with the reported in Pediatrics by Ficicioglu and An Haack on the revision about the failure to thrive and inborn errors of metabolism, especially when exist associated multisystem progressive findings (2). In this sense, in the recent revision of our 58 consecutive children and adolescents (35 woman and 23 man, median age of 5.65 years, range: 1.5 months to 18 years) with a proved diagnosis of mitochondrial disease following the criteria published previously (3), and with available height and weight measurements in the most recent clinic visit, data used to calculate BMI and height SD using the 1988 Spanish tables of Hernández et al. (4). We observed a height of less than 2 SD below the mean in 31.03%, and this short stature did not become more pronounced with advancing years; the BMI of less than 2 SD below the mean was observed in 39.65%, without any decrease with advancing years. Considering clinical findings, 75.86% of the patients (44 ⁄ 58) were encephalomyopathic forms, and 24.13% (14 ⁄ 58) were myopathic forms. In the group of encephalomyopathic forms, the height and the BMI of less than 2 SD below the mean were in 34.09% and 43.18% respectively, while in the group of myopathic forms these were in 21.42% and 28.57%. We agree with the conclusion of Wolny et al. (1) that mitochondrial diseases are an important cause of short stature and of reduction in BMI, and these diseases needs to be included in the differential diagnosis of unexplained short stature in children and adolescents, specially the encephalomyopathic forms.

A Xq21.31 duplication without features of Prader-Willi syndrome

We read with great interest the recent contribution by Pramyothin et al. [1], in Endocrine. They reported a 20-year-old man diagnosed with 47, XXY during childhood, who presents an appearance similar to that of Prader– Willi syndrome (PWS) with hypogonadism and gynecomastia, developmental delay, and short stature and obesity. Array-based comparative genome hybridization revealed duplication at Xq21.31 in addition to his abnormal karyotype. This duplication was also found in his mother who appeared normal. The authors hypothesized that the phenotype in this patient is a combination of both extra X chromosome and Xq21 duplication. On the other hand, Gabbertt et al. [2] have reported a 4-year-old male with an interstitial tandem duplication of Xq21.1–q21.31, maternally inherited, who presented with clinical features of PWS, and they conclude that duplication of chromosome Xq should be considered in the differential diagnosis of PWS, especially in males. In this sense, we have been evaluating a 3-year-old (at first time) male with developmental delay, autism, hyperactive behavior, hand stereotypes, large ears, synophrys, excessive hair in back, pes planus/valgus, and body mass index in the 95th percentile (height in 50th percentile and weight in 75th percentile), and without features of PWS. No contributory family history (and non-consanguineous parents), normal pregnancy, and delivery. Previous studies were negative for the findings in brain MRI, karyotype, and fragile X syndrome. 7q11.23, 17p13, 22q11.2, and 22q13.3 microdeletions were tested (FISH) and were negative. Recently, he was diagnosed at the age of 9 years of Xq21.31 duplication (3.9 Mb) by means of Cytogenetics Whole-Genomics 2.7 M array (Affymetrix): ArraysSNPs. In his mother, this genetic study was normal. We hypothesized that the duplication of chromosome Xq21.31 should be considered also in males with the association of developmental delay, autism, and hyperactive behaviour without phenotypic features of PWS. This association may be related with that at least seven loci of mental retardation syndromes encompassing the duplication in the Xq21.31 region [1].

Growth in children and adolescents with mitochondrial diseases

There is evidence that children with mitochondrial diseases tend to have short stature, but the growth of these patients has not been assessed in detail. We calculated the standard deviation (SD) of height, weight and body mass index (BMI) of 58 children and adolescents between the ages of 1.5 months and 18 years with a proven diagnosis of mitochondrial diseases. Overall, 31.03% of recorded heights, 29.31% of weights and 39.65% of BMIs were more than 2SD below the mean. In the group of children with encephalomyopathic forms of mitochondrial diseases, 34.09% of heights and weights, and 43.18% of BMIs were more than 2SD below the mean, while in the group with myopathic forms 21.42% of weights, 14.28% of heights and 28.57% of BMIs were more than 2SD below the mean. These results suggest that mitochondrial diseases are an important cause of short stature and of reduction in BMI in children and adolescents, particularly the encephalomyopathic forms.