Brain & Development 37 (2015) 442–445 www.elsevier.com/locate/braindev

Case Report

Seizure recurrence following pyridoxine withdrawal in a patient with pyridoxine-dependent epilepsy Moe Tamaura a, Hiroko Shimbo a, Mizue Iai a, Sumimasa Yamashita a, Hitoshi Osaka a,b,⇑ a

Division of Neurology, Kanagawa Children’s Medical Center, Yokohama, Japan b Department of Pediatrics, Jichi Medical School, Shimotsuke, Tochigi, Japan

Received 8 March 2014; received in revised form 11 July 2014; accepted 19 July 2014

Abstract Pyridoxine-dependent epilepsy (PDE) is an autosomal recessive disorder characterized by early onset and recurrent seizures that can be controlled by a high dose of pyridoxine. PDE is caused by mutations in ALDH7A1, which encodes antiquitin. Antiquitin converts a-aminoadipic semialdehyde to a-aminoadipic acid. Seizure recurrence after pyridoxine withdrawal is a criterion for diagnosis, but PDE can be diagnosed conclusively by genetic testing for mutations in the ALDH7A1 gene. In this case study, we report the long-term follow-up of a patient suspected with PDE. She experienced prolonged generalized tonic seizures and was hospitalized in an intensive care unit following pyridoxine withdrawal. Later, we identified a compound heterozygous mutation, c.1216G>A, p.Gly406Arg, and a novel splice donor site mutation, IVS9+5G>A. Confirmation of these mutations would have prevented an unsafe withdrawal test. This case suggests the importance of the genetic determination of PDE to avoid the diagnostic withdrawal of pyridoxine. Ó 2014 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.

Keywords: Pyridoxine-dependent epilepsy; Pyridoxine; ALDH7A1 gene

1. Introduction Pyridoxine-dependent epilepsy (PDE; OMIM 266100) is an autosomal recessive disorder characterized by recurrent seizures in the neonatal or early infantile period [1]. Patients with PDE cannot be controlled with conventional anticonvulsants; instead, they respond clinically and electroencephalographically to high doses of pyridoxine [2]. PDE is caused by the deficient enzymatic activity of a-aminoadipic semialdehyde ⇑ Corresponding author at: Department of Pediatrics, Jichi Medical School, 3311-1 Yakushiji, Shimotsuke-shi, Tochigi 329-0498, Japan. Tel.: +81 285 58 7366; fax: +81 285 44 6123. E-mail address: [email protected] (H. Osaka).

dehydrogenase (antiquitin), which is encoded by the ALDH7A1 [3], with the accumulation of a-aminoadipic semialdehyde (a-AASA) and D1-piperideine 6-carboxylate (P6C; Supplemental Fig. 1). Approximately 90 mutations of the ALDH7A1 gene have been reported (Human Gene Mutation Database (HGMD) Professional release 2013.12), most of which are missense, nonsense, and splice site mutations, and 5 patients have been diagnosed in Japan [4]. PDE can be diagnosed conclusively by molecular genetic testing of the ALDH7A1 gene; however, the diagnosis of PDE can also be established by a good response to pyridoxine and seizure recurrence after pyridoxine withdrawal [5]. Here, we report the long-term follow-up of an individual with PDE. She experienced prolonged

http://dx.doi.org/10.1016/j.braindev.2014.07.008 0387-7604/Ó 2014 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.

M. Tamaura et al. / Brain & Development 37 (2015) 442–445

generalized tonic seizures that recurred following pyridoxine withdrawal, implying the importance of the genetic confirmation of PDE. 2. Case report The patient was a 23-year-old female born at term to healthy non-consanguineous Japanese parents. She was born at 41 weeks gestation, with Apgar scores of 5 and 9 at 1 and 5 min, respectively. At birth, her body weight was 2600 g. Limb clonic seizures occurred at 5 h after birth. Laboratory data, including plasma ammonia and amino acids, were all normal. Electroencephalographic recordings (EEGs) showed a burst-suppression pattern. Cerebral ultrasound and brain imaging by computed tomography were unremarkable. She was treated unsuccessfully with phenobarbital, valproic acid (VPA), and phenytoin, and required mechanical ventilation. At day 24 after birth, an intravenous injection of pyridoxine (70 mg) stopped the seizures and normalized the EEGs (Fig. 1A and B). We diagnosed her as PDE from the clinical course and started with oral pyridoxine of 10 mg/kg daily in two divided doses. The patient displayed developmental delay with rolling over at 8 months of age and walking at 16 months. At 21 months of age, she started to show complex partial seizures, especially in association with febrile

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events. VPA, carbamazepine, and zonisamide were started, in addition to pyridoxine. A neuropsychological evaluation at 5 years of age revealed mental retardation with an IQ of 55 with Tanaka-Binet Intelligence Scale. She has not had a seizure since 6 years of age with VPA and pyridoxine treatment. At 16 years of age, when she was living outside of Japan, VPA was reduced to 100 mg/day without seizures. Pyridoxine was discontinued, and generalized tonic-clonic seizures occurred at 1 month after cessation. She was hospitalized to an intensive care unit and intravenous diazepam and phenytoin were used to terminate status convultics. Pyridoxine and VPA were restarted. Currently, she is 23 years old and seizure-free with 480 mg/day pyridoxine and 800 mg/day VPA. 3. Materials and methods Genomic DNA was extracted from peripheral white blood cells using a QuickGene DNA Whole Blood Kit S (Fujifilm, Tokyo, Japan) according to the manufacturer’s instructions. PCR of all exons and exon–intron boundaries of the ALDH7A1 gene was performed with specific primers using an Ex Taq PCR Version 1.0 Kit (Takara, Shiga, Japan) according to the manufacturer’s instructions (Supplemental Table 1). Total RNA was extracted from leukocytes using the ISOGEN reagent

A Fp1−Fp2 F7−C3 C3−C4 C4−F8 T3−P3 P3−P4 P4−T4 O1−O2 Fp1−O1 Fp2−O2 Fp7−F8 T3−T4 ECG 1s

50 mv

B Fp1−Fp2 F7−C3 C3−C4 C4−F8 T3−P3 P3−P4 P4−T4 O1−O2 Fp1−O1 Fp2−O2 Fp7−F8 T3−T4 50 mv 1s

Fig. 1. EEGs of the patient before (A) and after intravenous pyridoxine injection, showing rapid EEG normalization (B).

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(Wako, Osaka, Japan). RT-PCR was performed with primers that covered from exon 8 to exon 10 using a PrimeScript One Step RT-PCR Kit, Version 2.0 (Takara, Shiga, Japan). The PCR fragments were sequenced using a BigDye Terminator Cycle Sequence Kit (v1.1; Applied Biosystems, Foster City, CA, USA). The patient and their family provided informed consent to undergo genetic testing, which was approved by the ethics committee of Kanagawa Children’s Medical Center. 4. Results We identified a maternally inherited compound heterozygous mutation, c.1216G>A, p.Gly406Arg, which has been reported previously [4]. We also identified a novel paternally inherited splice donor site mutation, IVS9+5G>A (c.871+5G>A). The c.871+5G>A mutation was not identified in 100 normal control chromosomes and was considered pathogenic. This mutation resulted in an exon 9 deletion (98 bases) by exon skipping (Fig. 2). Frameshift mutations in the ALDH7A1 gene can create an early stop codon and result in a truncated protein (p.Thr259Glufs*16). We could not found these mutations, c.1216G>A and c.871+5G>A, in Japanese population using Human Genetic Variation Database. 5. Discussion Our patient had been treated with pyridoxine and no further seizures occurred; however, following pyridoxine

A

c.1216G>A, p.Gly406Arg

withdrawal, she exhibited generalized tonic seizures and required treatment in an intensive care unit. There are two biochemical pathways for lysine catabolism: (1) P6C is produced via the pipecolic acid pathway; and (2) a-AASA is produced via the saccharopine pathway (Supplemental Fig. 1) [6]. Antiquitin, which is encoded by the ALDH7A1 gene, converts a-AASA to a-AAA. In antiquitin deficiency, a-AASA and P6C accumulate. P6C condenses pyridoxal 50 -phosphate (PLP), resulting in the depletion of PLP and the accumulation of pipecolic acid. Depletion of PLP, a cofactor for various enzymes, results in the depletion of the inhibitory neurotransmitter GABA and causes seizures [7]. In addition, the increased levels of pipecolic acid, which modulate GABA levels, also contribute to seizure generation [8]. Patients with PDE require lifelong supplementation of pyridoxine. In the past, seizure recurrence after pyridoxine withdrawal was a criterion for diagnosis [1,9]. In one report, patients with PDE, diagnosed by ALDH7A1 deficiency, were withdrawn from pyridoxine and seizures recurred in 61% of patients (14/23 patients; range 1–51 days; mean = 13.5 days; median = 9 days) [10]. In the present study, genetic testing of the ALDH7A1 gene was used to confirm the diagnosis of PDE. An early seizure control could not prevent developmental delay and intellectual disability in our patient, as reported in most patients [8]. However, our results indicate that, if there is genetic confirmation of PDE, the patient should continue pyridoxine without the need to undergo a withdrawal test that may add further disabilities.

B

exon14

IVS9+5 G>A (c.871+5G>A)

exon9

intron9

exon8

exon10

gDNA

C

D exon8

exon9

cDNA

Fig. 2. Chromatogram of genomic DNA (gDNA) analysis of the patient showing the heterozygous c.1216G>A (A) and IVS9+5G>A (c.871+5G>A) (B) mutations. cDNA analysis of the patient shows two spliced transcription products: normal (C) and exon 9 skipping (D).

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6. Conflict of interest statement We have no conflict of interest to disclose.

[5] [6]

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/ 10.1016/j.braindev.2014.07.008.

[7] [8]

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Seizure recurrence following pyridoxine withdrawal in a patient with pyridoxine-dependent epilepsy.

Pyridoxine-dependent epilepsy (PDE) is an autosomal recessive disorder characterized by early onset and recurrent seizures that can be controlled by a...
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