Pyridoxal 5ꞌ-phosphate-responsive epilepsy
with novel mutations in the PNPO gene:
a case report
M. Veeravigrom1,2, P. Damrongphol1,2, R. Ittiwut3,4, C. Ittiwut3,4,
K. Suphapeetiporn3,4 and V. Shotelersuk3,4
Division of Neurology, Department of Pediatrics, Faculty of Medicine,
Chulalongkorn University, Thailand
2
Division of Neurology, Department of Pediatrics,
King Chulalongkorn Memorial Hospital, The Thai Red Cross Society,
Bangkok, Thailand
3
Center of Excellence for Medical Genetics, Department of Pediatrics,
Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand
4
Excellence Center for Medical Genetics, King Chulalongkorn Memorial Hospital,
The Thai Red Cross Society, Bangkok, Thailand
1

Corresponding author: K. Suphapeetiporn
E-mail: kanya.su@chula.ac.th
Genet. Mol. Res. 14 (4): 14130-14135 (2015)
Received June 2, 2015
Accepted September 1, 2015
Published October 29, 2015
DOI http://dx.doi.org/10.4238/2015.October.29.34

ABSTRACT. Pyridoxal 5'-phosphate (PLP)-responsive epilepsy is a rare
autosomal recessive epileptic disorder caused by deficiency of pyridox(am)ne 5'-phosphate oxidase (PNPO). Neonatal onset seizures in PLP
responsive epilepsy are usually resistant to common anticonvulsants and
pyridoxine, but respond to PLP. Various PNPO mutations are associated
with this disorder. In this report, we have described a case of a female
baby with neonatal onset seizures responding to PLP. Exome sequencing
revealed that the patient was compound heterozygous for pathogenic
mutations [c.546+1G>A (IVS5+1 G>A) and c.620delG (p.G207VfsX215)]
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in the PNPO gene. The c.546+1G>A was inherited from the mother while
the c.620delG was inherited from the father. Both mutations were absent in
122 unrelated Thai controls. The results of this study indicated the presence
of two newly identified mutations in this Thai patient with PLP-responsive
epilepsy for the first time, expanding the mutational spectrum of PNPO.
Key words: Epilepsy; Pyridoxal 5'-phosphate; Mutation;
Pyridox(am)ine 5'-phosphate oxidase

INTRODUCTION
Pyridoxal 5'-phosphate (PLP)-responsive epilepsy or pyridox(am)ine 5'-phosphate
oxidase (PNPO) deficiency is a rare autosomal recessive disorder caused by deficiency of PNPO
(OMIM 610090). The PNPO enzyme is a key factor for conversion of pyridoxine and pyridoxamine
into PLP. PLP is an important vitamin B6 cofactor for synthesis of neurotransmitters, especially
gamma-aminobutyric acid (GABA). Seizures in PLP-responsive epilepsy are resistant to common
anticonvulsants and pyridoxine (Mills et al., 2005).
The PNPO gene contains seven exons, and encodes a 261-amino acid protein. At least
24 different disease-causing mutations scattered throughout the PNPO gene have been reported,
with the most common being missense/nonsense mutations (Human Gene Mutation Database,
http://www.hgmd.cf.ac.uk, accessed April 2015). In this study, we have reported a Thai female
infant with PLP-responsive epilepsy. Two novel mutations in the PNPO gene, including a splicing
mutation and a single-base pair deletion, were identified.

MATERIAL AND METHODS
Patient information
The female infant (24 days old) was referred to King Chulalongkorn Memorial Hospital.
The patient was the second child of non-consanguineous Thai parents. The pregnancy was
complicated, with intrauterine growth restriction and oligohydramnios. The patient was born
prematurely at 32 weeks gestation; the birth weight of the patient was 2305 g, and her Apgar
scores at 1 and 5 min were 9 and 9, respectively. She developed respiratory distress and
was diagnosed with respiratory distress syndrome. She exhibited symptoms of myoclonic
seizures 6 h after birth. A full-septic work-up and metabolic investigations were performed with
unremarkable results. The patient was treated with phenobarbital, which was slowly titrated to
9 mg·kg-1·day-1.
At the age of 10 days, the patient underwent several episodes of myoclonic seizures.
Phenytoin was added at a maintenance dose of 7 mg·kg-1·day-1. Subsequently, the frequency
of myoclonic seizures was reduced to 1-2 times per day. The patient was then discharged at
the age of 21 days. Two days after the hospital discharge, the patient experienced multiple
clusters of multifocal and generalized myoclonic seizures, intermixed with eye deviation and
inconsolable crying, which developed into myoclonic status epilepticus. A computed tomography
(CT) scan of the brain showed cerebral edema with diffused, non-specific, poor gray-white
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matter differentiation; subsequently, assisted ventilation, and a regimen of cefotaxime and
acyclovir was initiated. The epilepticus status of the patient was refractory to multiple-antiepileptic medications, including phenytoin, phenobarbital, levetiracetam, and sodium valproate.
A trial of intravenous pyridoxine did not stop the clinical seizure. Continuous midazolam
infusion was administered and titrated up to 40 µg·kg-1·min-1. The patient was then referred
to our hospital. Cerebral function monitoring during high-dose midazolam infusion showed a
burst-suppression pattern. A 10 mg/kg PLP trial resulted in an increased suppression ratio and
flattening of the electroencephalogram (EEG). A brain CT scan revealed no other causes of
electrocerebral silence. The patient brain showed normal results when subjected to magnetic
resonance imaging and magnetic resonance spectrometry; in addition, her urine organic acid
screening was unremarkable. Discontinuation of the midazolam treatment resulted in a gradual
return of continuous EEG pattern and the patient regaining consciousness. Because of her
dramatic response to PLP, PLP-responsive epilepsy was suspected. The maintenance of a PLP
dose of 30 mg·kg-1·day-1 helped control the seizures. Sodium valproate was tapered off by the
time the patient turned 2 months old. The patient remained seizure-free when subjected to a
constant dosage of 6 mg·kg -1·day -1 phenytoin, 4 mg·kg -1·day -1 phenobarbital, and 20 mg·
kg-1·day-1 levetiracetam, in addition to 30 mg·kg-1·day-1 PLP.

Mutation analysis
Mutation analysis was performed by whole exome sequencing (WES). This study was
considered to be exempt from review by the Institutional Review Board of the Faculty of Medicine,
Chulalongkorn University. Informed consent was obtained from the parents, and peripheral blood
samples were collected from the patient and her parents. Genomic DNA was extracted from the
peripheral blood leukocytes of the patient using the Gentra Puregene Blood Kit (Qiagen, Valencia,
CA, USA), and sent to Macrogen Inc. (Seoul, Korea) for WES.
The primers for mutation validation were designed using the Primer3 software.
The primers used to detect the c.546+1G>A and c.620delG mutations in the PNPO gene
were 5'-CAGAGCCATCCCTGAGCAG-3' and 5'-CACTTTCCCTTCTCACTGTCCT-3', and
5'-CCCTTGTCAGATGCATCCCA-3' and 5'-AGGTCCCAGAGTTAAGGTGC-3', respectively. The
PCR products were treated with ExoSAP-IT (USP Corporation, Cleveland, OH, USA) and sent for
direct sequencing to Macrogen. The sequence data were analyzed using the Mutation Surveyor
software (Softgenetics, State College, PA, USA).

RESULTS
Two different novel pathogenic mutations were discovered in the PNPO gene. The
patient displayed compound heterozygosity for the c.546+1G>A (IVS5+1 G>A) and c.620delG
(p.G207VfsX215) located in exon 7. Both were validated by Sanger sequencing of the genomic
DNA obtained from the patient and her parents (Figure 1). The splicing mutation c.546+1G>A
was inherited from the mother, while the single-base pair deletion causing a frameshift
(c.620delG) was inherited from the father. Both mutations were absent in 122 unrelated Thai
exome controls.
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Figure 1. Electropherograms of the patient and both parents. A. The c.546+1G>A (IVS5+1 G˃A) mutation is inherited from
the mother. B. The c.620delG (p.G207VfsX215) mutation is inherited from the father. Each mutation is indicated by an arrow.

DISCUSSION
In addition to its rarity, the non-specific clinical features and EEG patterns increase the
difficulty of early recognition of PLP-responsive epilepsy. Our patient experienced neonatal onset
seizures refractory to anticonvulsant medications. The discovery that the seizures responded to
PLP and not pyridoxine prompted the investigation of the genetic defects in the PNPO gene. Exome
sequencing, followed by targeted Sanger sequencing, revealed two novel causative mutations in
the proband, each of which was inherited from each parent.
PLP-responsive epilepsy is highly correlated with the incidence of prematurity (Mills et
al., 2014; Plecko et al., 2014). Seizures resulting from PNPO deficiency typically occur in utero,
or shortly after birth, with various, ictal manifestations predominantly multifocal and generalized
myoclonic seizures that are refractory to conventional anticonvulsants and pyridoxine. Our patient
was born prematurely, and the mother did not report any rhythmic fetal movements. The patient
developed seizures 6 h after birth, exhibited a transient response to phenobarbital and phenytoin,
and subsequently progressed to myoclonic status epilepticus.
EEG findings in PNPO deficiency are not pathognomonic. A previous study has described
a burst suppression pattern in PNPO deficiency mimicking nonketotic hyperglycinemia and
Ohtahara syndrome (Mills et al., 2005). Our patient was not subjected to an EEG at the onset of
the seizures. At 25 days of age, a supramaximal dose of midazolam resulted in a burst suppression
pattern. EEG was transformed into electrocerebral silence after a trial of PLP. In a PNPO deficient
state with accumulation of glutamate, administration of PLP would cause the same result as an
intravenous pyridoxine infusion in pyridoxine-dependent epilepsy, i.e., electrocerebral suppression,
hypotension, and apnea. The exact mechanism of this reaction is unknown; however, it may be
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related to a sudden increase in GABA concentration in the cerebrospinal fluid (CSF) (Bass et
al., 1996). This emphasizes the need for close monitoring and preparation for resuscitation after
administration of PLP in PNPO deficiency, as well as pyridoxine-dependent epilepsy.
The metabolic investigations in PNPO deficiency characteristically demonstrate low PLP
levels in the plasma and CSF, elevated levels of L-3,4-dihydroxyphenylalanine, 3-O-methyldopa,
glycine, and threonine in CSF, and increased urinary excretion of very long chain fatty acids (Bass
et al., 1996; Mills et al., 2005; Clayton, 2006). Biochemical studies were not performed in this
case because of sample unavailability. However, the diagnosis of PNPO deficiency was further
confirmed by mutation analysis, which revealed mutations in both alleles of the PNPO gene.
Various clinical courses and outcomes have been reported in previous cases of PNPO
deficiency (Plecko, 2013; Plecko et al., 2014). If left untreated, the patients can develop failure to
thrive, refractory seizures, and severe encephalopathy, leading to early death. Patients in whom
the disease was recognized early, and who received appropriate treatment are believed to be more
likely to have favorable outcomes (Hoffmann et al., 2007; Porri et al., 2014). This was supported
by the results of a previous study describing a male infant with a homozygous splice-site mutation
in the PNPO gene. The patient had seizures within 12 h after birth, and received PLP treatment
on day 3; a favorable neurological outcome was observed at the age of 3 months (Porri et al.,
2014). Delay in PLP treatment usually leads to motor and intellectual disabilities. However, some
studies have also reported a good outcome in patients with delayed PLP treatment (Hoffmann et
al., 2007; Schmitt et al., 2010; Pearl et al., 2013). One patient developed a seizure on the second
day after birth, and was administered PLP at 27 days when PNPO deficiency was suspected. A
developmental screening test performed at 9 months of age revealed normal findings. This patient
was compound heterozygous for a missense mutation and a single-base pair deletion in the
PNPO gene (Schmitt et al., 2010). Another patient with a homozygous missense PNPO mutation
experienced seizures within 12 h after birth; this patient was administered with PLP at 3.5 months
of age because of breakthrough seizures despite pyridoxine therapy. Subsequent developmental
assessment at the age of 28 months showed mild hypotonia and language delay. Different types of
mutations in the PNPO gene were theorized to affect the disease severity.
Because of the limited number of cases with PLP-responsive epilepsy confirmed by
molecular methods, the genotype-phenotype correlations remain unclear. Our patient was
compound heterozygous for the c.546+1G>A and c.620delG mutations. The c.546+1G>A
(IVS5+1 G>A), located in intron 5, was predicted to disrupt the splice site consensus sequence.
The splice donor site mutation would cause either aberrant splicing involving exon 5, resulting in
a shortened protein product, or nonsense-mediated decay, wherein the entire transcript would
be removed to avoid the production of a truncated protein. The c.620delG located in exon 7
was expected to cause a frameshift mutation, leading to premature termination at codon 215
(p.G207VfsX215). This shortened protein would lead to a loss of several amino acids that play an
important role in facilitating binding of the flavin mononucleotide to the active site of PNPO (Mills
et al., 2005). Previous studies have investigated the effect of the mutations in exon 7 (including
p.R225H and p.R229W) on PNPO, discovering a significant reduction in the PNPO activity (Mills
et al., 2014).
In conclusion, two novel mutations were successfully identified in the PNPO gene in a Thai
family, and the mutational spectrum of PNPO causing PLP-responsive epilepsy was expanded.
This study has emphasized the significant implication of genetic testing in PNPO-deficient patients,
leading to a more accurate genetic diagnosis and effective genetic counseling.
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Conflicts of interest
The authors declare no conflict of interest.

ACKNOWLEDGMENTS
We would like to thank Dr. Tayard Desudchit and Dr. Khemmachart Pongsanon for
providing excellent patient care. Research supported by the Thailand Research Fund, the National
Science and Technology Development Agency, and the Ratchadaphiseksomphot Endowment
Fund of Chulalongkorn University (#RES560530177-HR).

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