Excessive Seizure Clusters in an Otherwise Well-Controlled Epilepsy as a Possible Hallmark of Untreated Vitamin B6-Responsive Epilepsy due to a Homozygous PLPBP Missense Variant

Abstract

Recently, mutations in the PLPBP gene were described as a novel cause for vitamin B6-responsive epilepsy. We report the outcome in case of a male adolescent with a novel homozygous missense variant in PLPBP who was never treated with pyridoxine until the age of 16 years. He presented with only mild cognitive impairment and an early-onset, well-controlled epilepsy. In our patient, excessive seizure clusters and anxiety states occurred intermittently, suggesting that the combination might be a hallmark in untreated patients. Thus, mutations in PLPBP should be addressed even in adolescent patients with only mild learning disabilities and relatively good seizure control over the years.

Introduction

Biallelic variants in the PLPBP (previously PROSC ) gene have recently been described as a novel cause of vitamin B6 responsive epilepsy in a total of 16 patients. ^{1 2 3 4 5} PLPBP (pyridoxal phosphate-binding protein) encodes a pyridoxal 5′-phosphate (PLP) containing protein that has been proposed to maintain intracellular PLP homeostasis, but the exact function and underlying mechanisms are still unknown. ¹ Being the active cofactor of pyridoxine, PLP has an important function in many enzymatic reactions of the amino acid and neurotransmitter metabolism. So far, four additional vitamin B6 responsive epilepsies (namely pyridoxine-dependent epilepsy [PDE], pyridoxal 5′-phosphate dependent epilepsy, hyperprolinemia type II, hypophosphatasia) with different genetic causes have been described, leading to impaired production or inactivation of PLP. They can be distinguished by specific biomarkers such as alpha-aminoadipic semialdehyde (AASA), pipecolic acid (PA), amino acids, and PLP in urine, plasma, or cerebrospinal fluid (CSF). ⁶ Diagnosis is confirmed by molecular genetic testing. The phenotypic spectrum in early treated patients with PLPBP mutations is similar to that in PDE and other vitamin B6 responsive seizures ranging from microcephalic and severely retarded children with magnetic resonance imaging (MRI) abnormalities as in pyridoxine and pyridoxal 5′-phosphate dependent epilepsy to normocephalic and only mildly intellectually disabled patients with normal MRI scans as in patients with hyperprolinemia type II and congenital hypophosphatasia. ^{1 2} Biochemical analyses in patients with PLPBP mutations prior to treatment showed low PLP concentrations in CSF and alterations in PLP-dependent pathways in the patients reported in the study by Darin et al. ¹ However, as low PLP concentrations in CSF are also seen in PLP-dependent epilepsy and other metabolic disorders (e.g., molybdenum cofactor deficiency), ⁷ this parameter cannot be used as a specific marker for PLPBP -associated epilepsy. Additionally, no specific other vitamin B6 or amino acid profiles in plasma and CSF, respectively, were found in other patients with PLPBP mutations under pyridoxine treatment. ² This is in contrast to the four genetically heterogeneous vitamin B6 responsive epilepsies that can be detected by specific findings in urine, plasma, and CSF and confirmed by molecular genetic testing. ⁶

To date, outcomes in patients with PLPBP gene mutations who never or only very late in childhood received vitamin B6 treatment have not been reported. Here, we report the case of a male adolescent with an early-onset but relatively well-controlled epilepsy and mild learning disability who had never been treated with vitamin B6. A novel homozygous missense variant in the PLPBP gene was confirmed, and vitamin B6 treatment was initiated after he showed a severe deterioration of seizures at the age of 16 years.

Methods

The patient chart was reviewed for the clinical history and the laboratory (including metabolic and genetic) and radiological investigations. DNA samples from whole blood were isolated by standard procedures. Trio whole-exome sequencing (trio-WES) was performed with DNA samples of both healthy parents and the index patient, as described previously. ⁸ For validation, polymerase chain reaction and Sanger sequencing were performed as described previously. ⁹ Primer pairs are available upon request. WES was performed after written informed consent was obtained from the parents according to national genetic regulations (Hamburg Medical Chamber, PV3802).

The functional impact of the identified variants was predicted using CADD, REVEL, M-CAP, and Polyphen2 in silico tools. ^{10 11 12 13}

Results

Patient Data

The now 16-year-old patient was born at term after an uneventful pregnancy by cesarean section as the first child to healthy, nonconsanguineous Bosnian parents. At birth, body weight, length, and head circumference were within the normal range. Motor milestones were normal, but speech development was mildly delayed. Mild learning difficulties became obvious in school, and an intelligence test showed a low normal IQ (intelligence quotient) of 85. After completing school, the boy started his professional education as a hairdresser. At the age of 15 days, first seizures occurred, and burst-suppression pattern was apparent in the initial electroencephalograms (EEGs). Under monotherapy with phenobarbital and short-term valproic acid, seizures appeared occasionally in infancy and childhood and were described as focal and generalized. Later, he developed seizures with anxiety, tachycardia, and vomiting followed by confusion over hours. At the age of 2, 6, and 11 years, clusters of seizures lasting 2 weeks occurred spontaneously without specific triggers. At 11 years of age, anticonvulsive treatment was switched to oxcarbazepine in combination with levetiracetam, leading to seizures only once in a while. At the age of 16 years, the family moved to Germany, and shortly after their arrival, he was admitted to our hospital due to an increase in focal motor seizures with impaired awareness and focal-to-bilateral tonic–clonic seizures. ¹⁴ Additionally, he showed episodes of unspecific visual hallucinations, anxiety, and confusion. On examination, no neurological abnormalities were found, and cognitive presentation was consistent with mild learning disabilities. Initially, seizures were responsive to emergency treatment with phenobarbital, benzodiazepine, or levetiracetam. After increasing the dose of oxcarbazepine and levetiracetam, the patient could be discharged seizure-free after 3 weeks. Five days later, he was admitted again with another and now explosive increase in seizures that were only partially responsive to different antiepileptic drugs, such as phenobarbital, benzodiazepine, levetiracetam, and lacosamide as emergency drugs and under add-on therapy with topiramate, pregabalin, steroids, and benzodiazepine. Several interictal EEGs were unremarkable, but left-sided temporal sharp waves with secondary generalization were recorded during 24-hour EEG monitoring combined with gaze and head deviation followed by bilateral tonic–clonic seizure and sometimes preceded by panic attacks. At day 6 after admission, the boy was referred to the intensive care unit for propofol anesthesia due to refractory status epilepticus. After reduction and cessation of propofol therapy, seizures reappeared immediately, and thiopental in combination with midazolam was engaged shortly after. After thiopental and midazolam were successively stopped, the boy still experienced one to three short-lasting focal seizures daily. Brain MRI at the beginning and before the transfer to the intensive care unit was completely normal. Routine and extensive neurometabolic work-up including pipecolic and amino acids and AASA in plasma, AASA, organic and pipecolic acids in urine, and amino acids in CSF remained normal. Neuronal auto-antibodies in plasma and CSF were not detected. At day 24 after admission, homozygosity for a novel missense variant affecting exon 8 of the PLPBP gene was revealed through WES, and pyridoxine was immediately started. Pyridoxine was given in a dosage of 300 mg intravenously on the first day, and a total dose of 1,200 mg intravenously was administered on the following 2 days. Since then, the patient continued on oral pyridoxine treatment, 300 mg per day, and went seizure-free. At the time of the report, anticonvulsive treatment could be further reduced to oxcarbazepine and levetiracetam. Several EEG during the follow-up showed no abnormalities. Prior to the first vitamin B6 dose, pyridoxine and PLP in plasma were determined and revealed high levels of PLP (PLP: 58.5 µg/L, normal: 7.5–18.5; pyridoxine < 1 µg/L, normal < 2).

Molecular Genetics

Using WES, we identified homozygosity for the novel missense variant c.725T > C, [p.(Ile242Thr)] in PLPBP (RefSeq accession number NM_007198.3). Sanger sequencing and segregation analysis confirmed this variant in a homozygous state in the patient ( Fig. 1 ). The parents were heterozygous carriers of the mutation (data not shown). The detected alteration had not been reported in the dbSNP ( https://www.ncbi.nlm.nih.gov/projects/SNP/ ), ExAC http://exac.broadinstitute.org/ ), or GnomAD ( http://gnomad.broadinstitute.org/ ) databases and was computationally predicted to be functionally relevant with the following scores (CADD: 27.4; REVEL: 0.473; M-CAP: 0.033; Polyphen2: 0.732). The affected Ile242 is within the PLP-binding barrel domain and in the immediate proximity of the Arg241 affected by the already reported missense change determined to be pathogenic. ^{1 2}

Fig. 1.

Part of the sequence electropherograms of the PLPBP exon 8 showing the p.(Ile242Thr) mutation in the homozygous (left, patient) and heterozygous state (right, father/mother).

Discussion

Clinicians often associate the group of vitamin B6-responsive epilepsies with early-onset, therapy-resistant seizures leading to severe epileptic encephalopathy in untreated children who are diagnosed by a specific metabolic marker in plasma, urine, and/or CSF and confirmed by genetic testing. Here, we report on a 16-year-old patient who was never treated with pyridoxine until the age of 16 years. The only mild intellectual deficits and the overall good controlled epilepsy over the years in combination with normal interictal EEG and vitamin B6 profiles with normal levels of AASA, PA, and amino acids in blood and/or urine delayed our diagnosis of vitamin B6 dependent epilepsy. As a possible hallmark of his previous history, until the age of 12 years, the boy experienced three seizure clusters, each lasting 2 weeks, that were refractory to treatment and stopped spontaneously. Moreover, the excessive seizure frequency and the immediate recurrence of seizures even after reduction of propofol anesthesia during the latest hospital stay were remarkable. The seizure type and the seizure onset in early infancy were mainly compatible with structural origin that was not confirmed by repetitive normal MRI scans. Additionally, our patient showed repetitive episodes of hallucinations and anxiety without other specific signs of seizures. Plecko et al reported the case of one patient who experienced hallucinations and panic attacks after pyridoxine withdrawal. ² Therefore, these symptoms may be a typical feature of untreated patients with PLPBP mutations either due to focal seizures or due to the significance of vitamin B6 in neurotransmitter pathways. Low serotonin levels are known in pyridoxin deficiency ¹⁵ and cause symptoms, such as confusion, disorientation, and anxiety. After initiation of oral pyridoxine treatment, the seizures and episodes of psychiatric symptoms stopped immediately. To date, the outcome of untreated patients with PLPBP -associated epilepsy is unknown as previous reports included patients treated with pyridoxine early in the course of the disease. Therefore, our study on a patient who has not been treated with vitamin B6 until his late teens expands the knowledge about this specific type of epilepsy.

In conclusion, mutations in the PLPBP gene should be kept in mind as a novel cause of vitamin B6 dependent epilepsy lacking specific metabolic biomarkers that might, if untreated, lead to only mild learning disabilities and well-controlled epilepsy over many years.

Funding Statement

Funding None.