PNPO Deficiency

Clinical characteristics.

Untreated pyridox(am)ine 5'-phosphate oxidase (PNPO) deficiency, characterized by a range of seizure types, is "classic" (i.e., seizure onset in the neonatal period) in about 90% of affected individuals and "late onset" (seizure onset after the neonatal period) in about 10%. In classic PNPO deficiency, seizures (including status epilepticus) often begin on the first day of life and typically before age two weeks. In both classic and late-onset untreated PNPO deficiency, seizure semiology varies from myoclonic to clonic or tonic seizures, and seizures are typically resistant to common anti-seizure medications. Independent of age of onset, seizures respond to life-long treatment with a B₆ vitamer: pyridoxal 5'-phosphate (PLP) in about 60% of affected individuals and pyridoxine (PN) in about 40%.

About 60% of individuals with PNPO deficiency have developmental impairment, affecting speech, cognition, and behavior; some individuals have neurologic impairment such as muscular hypotonia or dystonia. Severe neurodevelopmental impairment is more likely to occur in individuals with PNPO deficiency who experienced diagnostic delay and prolonged periods of uncontrolled seizures.

Diagnosis/testing.

The diagnosis of PNPO deficiency is established in a proband with suggestive findings (i.e., infantile onset of a wide range of seizure types resistant to common anti-seizure medications and a positive standardized vitamin B₆ trial) and (most commonly) biallelic pathogenic variants in PNPO identified by molecular genetic testing or deficient PNPO enzyme activity.

Management.

Targeted therapy: Pharmacologic treatment: PLP, the active form of vitamin B₆, is typically given orally, divided into 4-6 single daily doses. The lowest effective PLP dose should be used to avoid possible liver toxicity. PN is typically given orally, divided into 3-4 single daily doses. PN side effects can include sensory (or motor) neuropathy, usually reversible with dose reduction.

Supportive care: Supportive care often includes specialists from multiple disciplines such as neurology, developmental pediatrics, speech-language therapy, physical therapy, occupational therapy, and mental health.

Surveillance: Routine monitoring of seizure control and possible adverse effects of PLP therapy; neurologic examination for emergence of new findings and/or response to medications used in symptomatic treatment; and assessment of development/educational needs.

Agents/circumstances to avoid: Avoid anti-seizure medications (such as carbamazepine, valproate, phenytoin, and phenobarbital) that can reduce plasma PLP concentration. Attention to PLP dose requirements when hydrazines or isoniazid are given to treat other conditions.

Evaluation of relatives at risk: When prenatal testing has not been performed on a pregnancy at risk, prompt evaluation of a newborn sib is essential to determine if treatment with either PN or PLP is necessary.

Pregnancy management: Although empiric data on the utility of maternal PN supplementation during pregnancy for a fetus known to have or be at risk for PNPO deficiency are limited, consider giving 50-100 mg/day of PN to the mother during the last 20 weeks of pregnancy. PN (not PLP) supplementation is recommended for two reasons: the mother has the capacity to metabolize PN into PLP, and the risk of PLP hepatotoxicity in pregnancy is unknown.

Genetic counseling.

PNPO deficiency is inherited in an autosomal recessive manner. If both parents are known to be heterozygous for a PNPO pathogenic variant, each sib of an affected individual, irrespective of sex, has at conception a 25% chance of being affected, a 50% chance of being an asymptomatic carrier, and a 25% chance of being unaffected and not a carrier. Once both PNPO pathogenic variants have been identified in an affected family member, carrier testing for at-risk relatives, prenatal testing for a pregnancy at increased risk, and preimplantation genetic testing for PNPO deficiency are possible.

Suggestive Findings

PNPO deficiency should be suspected in individuals with the following suggestive clinical phenotypes, positive response to a standardized vitamin B₆ trial, supportive laboratory findings, and family history.

Standardized Vitamin B₆ Trial

A standardized vitamin B₆ trial [Wilson et al 2019] may suggest a diagnosis of PNPO deficiency (Figure 1).

• In the 40% of individuals with PNPO deficiency who are pyridoxine (PN) responsive, the majority show cessation of seizures in one to three days; in others amelioration of clinically evident seizures may take several days or may initially be evident on EEG only.
• In the 60% of individuals with PNPO deficiency who are pyridoxal 5'-phosphate (PLP) responsive, the majority show cessation of seizures in one to three days, accompanied by improvement of abnormal EEG findings.
• Note: PN- and/or PLP-responsive seizures can also be indicative of other vitamin B₆-dependent or vitamin B₆-responsive seizures.
  • Measurement of biomarkers can help to distinguish PNPO deficiency from some of these disorders (e.g., increased plasma and urinary alpha-aminoadipic semialdehyde is indicative of pyridoxine-dependent epilepsy – ALDH7A1, and sulfocysteine is indicative of molybdenum cofactor deficiency or isolated sulfite oxidase deficiency).
  • There is no biomarker for PLPHP deficiency (also referred to as PLPBP or PROSC deficiency).
• While simultaneous EEG recording is informative, it may not be practical as improvement may take hours to days.

Note: In the event of a favorable response to either PLP or PN, administration should be continued until results of biomarkers and genetic testing are available (see Establishing the Diagnosis).

Figure 1.

Stages of a standardized vitamin B₆ trial

Establishing the Diagnosis

The diagnosis of PNPO deficiency is established in a proband with suggestive findings typically by molecular genetic testing or, where available, by enzyme analysis.

Molecular Genetic Testing

The molecular diagnosis of PNPO deficiency is established in a proband with biallelic pathogenic (or likely pathogenic) variants in PNPO identified by molecular genetic testing (see Table 2).

Note: (1) Per ACMG/AMP variant interpretation guidelines, the terms "pathogenic variant" and "likely pathogenic variant" are synonymous in a clinical setting, meaning that both are considered diagnostic and can be used for clinical decision making [Richards et al 2015]. Reference to "pathogenic variants" in this GeneReview is understood to include any likely pathogenic variants. (2) Identification of biallelic PNPO variants of uncertain significance (or of one known PNPO pathogenic variant and one PNPO variant of uncertain significance) does not itself establish or rule out the diagnosis. (See Molecular Pathogenesis, PNPO-specific laboratory technical considerations for more information.)

Molecular genetic testing approaches can include a combination of gene-targeted testing (multigene panel) and comprehensive genomic testing (exome sequencing, genome sequencing).

Gene-targeted testing requires that the clinician determine which gene(s) are likely involved, whereas genomic testing does not. Individuals with the distinctive findings described in Suggestive Findings may be more likely to be diagnosed using gene-targeted testing (see Option 1), whereas those in whom the diagnosis of a seizure disorder caused by an inborn error of metabolism has not been considered are more likely to be diagnosed using genomic testing (see Option 2).

Option 1

A multigene panel (e.g., comprehensive epilepsy panel, infantile epilepsy panel, or epileptic encephalopathy panel) that includes PNPO and other genes of interest (see Differential Diagnosis) is most likely to identify the genetic cause of the condition while limiting identification of variants of uncertain significance and pathogenic variants in genes that do not explain the underlying phenotype. Note: (1) The genes included in the panel and the diagnostic sensitivity of the testing used for each gene vary by laboratory and are likely to change over time. (2) Some multigene panels may include genes not associated with the condition discussed in this GeneReview. (3) In some laboratories, panel options may include a custom laboratory-designed panel and/or custom phenotype-focused exome analysis that includes genes specified by the clinician. (4) Methods used in a panel may include sequence analysis, deletion/duplication analysis, and/or other non-sequencing-based tests.

For an introduction to multigene panels click here. More detailed information for clinicians ordering genetic tests can be found here.

Option 2

When the diagnosis of PNPO deficiency has not been considered because an individual has atypical clinical findings, comprehensive genomic testing (which does not require the clinician to determine which gene is likely involved) can be considered. Exome sequencing is most commonly used; genome sequencing is also possible.

For an introduction to comprehensive genomic testing click here. More detailed information for clinicians ordering genomic testing can be found here.

Table 2.

Molecular Genetic Testing Used in PNPO Deficiency

Clinical Description

As of 2022, approximately 90 individuals have been identified with pyridox(am)ine 5'-phosphate oxidase (PNPO) deficiency based on the identification of biallelic pathogenic variants in PNPO.

The following description of the phenotypic features of PNPO deficiency is based on these reports [Levtova et al 2015, Mercimek-Mahmutoglu et al 2015, Raimondi et al 2015, Veeravigrom et al 2015, Jaeger et al 2016, di Salvo et al 2017, Fung et al 2017, Guerriero et al 2017, Olson et al 2017, Xue et al 2017, Borst & Tchapyjnikov 2018, Fathalla et al 2018, Kvarnung et al 2018, Wilson et al 2019, Mohanlal et al 2020, Alghamdi et al 2021, Jiao et al 2022]. Levtova et al [2015] includes a review of all publications up to 2015. Alghamdi et al [2021] also includes a review of all publications up to 2021; some affected individuals included in subsequent follow-up reports may have been counted more than once.

Note that the clinical presentations of PNPO deficiency, ALDH7A1 deficiency, and PLPHP deficiency (also referred to as PLPBP or PROSC deficiency) are indistinguishable (see Differential Diagnosis).

The spectrum of the PNPO deficiency phenotype ranges from classic (i.e., neonatal-onset seizures) to late-onset seizures (i.e., manifesting after the neonatal period), which can be distinguished by age of onset and seizure patterns.

Classic PNPO deficiency (89% of affected individuals) is characterized by high seizure frequency and status epilepticus. The vast majority of these infants have seizures before age two weeks, with 30% presenting on day one of life.

In about 10% of pregnancies, rhythmic fetal movements have been reported in the last trimester, suggesting intrauterine seizure onset.

In newborns, seizures caused by PNPO deficiency can be confused with seizures associated with birth asphyxia as a result of prematurity (reported in about 64% of infants) and fetal distress (reported in about 15% of infants).

Seizures are typically resistant to common anti-seizure medications. Seizure semiology varies from myoclonic to clonic or tonic seizures.

To date, epileptic spasms (ES) have been reported in seven infants with classic PNPO deficiency. In four infants ES was the presenting seizure type (within the first 24 hours of life); three infants who had onset of focal seizures before age one month subsequently developed ES and other seizure types (generalized tonic/clonic/tonic-clonic and focal seizures). EEG records on six individuals showed hypsarrhythmia in three, atypical hypsarrhythmia in two, and burst suppression in one; see Fung et al [2017] (Patient 55); Jiao et al [2022].

Affected individuals may show episodes of roving eye, head movements without ictal EEG patterns, and signs of encephalopathy such as hyperalertness and inconsolable crying [Schmitt et al 2010] (full text; see especially supporting information Video S4).

About 60% of individuals with PNPO deficiency have developmental impairment, affecting speech, cognition, and behavior; some have neurologic impairment such as muscular hypotonia or dystonia. Severe neurodevelopmental impairment is more likely to occur in individuals with PNPO deficiency who experienced diagnostic delay and prolonged periods of uncontrolled seizures.

Other organ involvement. Systemic findings may include respiratory distress, vomiting, abdominal distention, hepatomegaly, and failure to gain weight. Routine laboratory investigations may show anemia, hypoglycemia, elevated plasma lactate, metabolic acidosis, and/or coagulopathy.

The observation of older deceased sibs (with manifestations consistent with PNPO deficiency) in families of probands with PNPO deficiency suggests that the mortality rate of untreated PNPO deficiency is high.

Late-onset seizures (i.e., manifesting after the neonatal period) have been reported to date in eight individuals from seven unrelated families [Mills et al 2014, di Salvo et al 2017, Xue et al 2017, Kvarnung et al 2018].

In the report by Mills et al [2014], Patient 7 had onset of infantile spasms at age five months with hypsarrhythmia on EEG.

In the report of Xue et al [2017]:

• Patient 3 had an afebrile seizure at age five months and gradually developed therapy-resistant epilepsy and developmental delay;
• Patient 4 had episodic stiffening at age 40 days that increased to daily seizures by age ten months, controlled by a combination of valproate and topiramate.

In the report of di Salvo et al [2017]:

• Patient 1 had focal clonic status epilepticus at age 20 months;
• Patient 2 had myoclonic atonic as well as generalized tonic-clonic seizures starting at age three years and two months (diagnosed as myoclonic atonic epilepsy with developmental decline);
• Patient 3 had episodic stiffening (clustering during febrile episodes) starting at age eight months, subsequent mild cognitive impairment, and occasional seizures while on valproic acid monotherapy.

In the report of Kvarnung et al [2018], two brothers had seizure onset at age eight months; no other details were provided.

Prognosis. In the absence of specific treatment with vitamin B_6, individuals with classic PNPO deficiency may experience early death. Preterm babies are at higher risk for developmental delay (84%) compared to term babies (50%). In the presence of neonatal-onset seizures, shorter treatment delay (<4 weeks) is clearly correlated with better outcomes. Seizure onset beyond the neonatal period appears to have a better prognosis. About 40% of all individuals with PNPO deficiency reported to date have a sustained favorable response to pyridoxine (PN). PN response has been observed in individuals with neonatal-onset seizures and is even more likely in individuals with late-onset seizures [Mills et al 2014, Plecko et al 2014].

Detailed EEG records available for 41 individuals with PNPO deficiency (classic and late onset) revealed the following:

• Seventeen individuals had burst suppression patterns prior to the administration of PLP or PN.
• Seventeen individuals demonstrated a pattern of multifocal or bilateral epileptic discharges.
• Three individuals had hypsarrhythmia.
• Four individuals had a normal EEG.

Cranial imaging reports, available for 40% of all individuals with PNPO deficiency (including 28 MRI results, one CT, and one cranial ultrasound) revealed the following:

• Fifteen individuals had normal initial imaging studies; on follow up, three individuals had brain atrophy or delayed myelination.
• Fifteen individuals had abnormal initial MRIs ranging from increased brain edema, signal intensity of basal ganglia or white matter, delayed myelination, intraventricular hemorrhage or middle cerebral artery infarction to simplified gyral patterns with shallow sulci.

Alghamdi et al [2021] reviewed MRI data of 55 individuals with classic and late-onset PNPO deficiency, some of whom were counted twice, with follow-up reports also included. Of these, 34 had a normal brain MRI, eight had brain atrophy, three had ischemic changes or leukomalacia, and three had delayed myelination.

Genotype-Phenotype Correlations

No genotype-phenotype correlations have been reported.

Nonetheless, the following observations about PN responsiveness may be helpful in guiding clinical management. See Table 5 for details.

• Seizures in individuals homozygous for the variant c.674G>A;p.Arg225His may respond to treatment with PN [Mills et al 2014, Plecko et al 2014].
• The seizures of four of the seven individuals reported to be homozygous or compound heterozygous for the variant c.98A>T;p.Asp33Val responded to treatment with PN [Wilson et al 2019].
• The majority of individuals who are homozygous for the variant c.347G>A;p.Arg116Gln, which may be present alongside other homozygous or compound heterozygous PNPO pathogenic variants (i.e., have a total of two or three pathogenic variants), have late-onset seizures that responded to treatment with PN [Mills et al 2014, di Salvo et al 2017]. See also Molecular Pathogenesis, PNPO-specific laboratory technical considerations.

Studies suggest that affected individuals with some variants (e.g., c.685C>T;p.Arg229Trp) that affect the flavin-binding site may benefit from riboflavin therapy in addition to PN and/or PLP therapy [Musayev et al 2009].

Penetrance

Not all individuals homozygous for the variant p.Arg116Gln develop seizures [Wilson et al 2019].

Nomenclature

PN- and PLP-responsive seizures (see Differential Diagnosis) are diagnosed in children who have not had molecular confirmation of PNPO deficiency, ALDH7A1 deficiency, or PLPHP deficiency * and who have either:

• Intractable seizures for which seizure control is only partially improved with the addition of PN or PLP; or
• Seizures that do not recur after PN or PLP is withdrawn.

* Pyridoxine-dependent epilepsy caused by pathologic variants in PLPBP is also referred to as PLPHP deficiency (PLPHP is the abbreviation for the protein encoded by PLPBP, the pyridoxal phosphate homeostasis protein).

Prevalence

As of 2022, approximately 90 individuals have been identified with biallelic pathogenic variants in PNPO [Levtova et al 2015, Mercimek-Mahmutoglu et al 2015, Raimondi et al 2015, Veeravigrom et al 2015, Jaeger et al 2016, di Salvo et al 2017, Fung et al 2017, Guerriero et al 2017, Olson et al 2017, Xue et al 2017, Kvarnung et al 2018, Mohanlal et al 2020, Alghamdi et al 2021, Jiao et al 2022]. Of note, some individuals have been reported more than once.

Variants found commonly in specific populations include the following (see Table 5 for details):

• The variant c.674G>A;p.Arg225His has been reported in several individuals from the former Yugoslavia and additional individuals of southeastern European ancestry [Plecko et al 2014, Lugli et al 2019].
• Individuals from the United Arab Emirates and one individual from Qatar were homozygous for c.674G>T;p.Arg225Leu [Guerriero et al 2017].
• The c.448_451delCCTG variant may be of high prevalence in individuals from China [Xue et al 2017, Jiao et al 2022].

Other Considerations in the Differential Diagnosis of PNPO Deficiency

Inborn pyridoxine dependency states. While other inborn pyridoxine dependency states have been described – for example, pyridoxine-dependent anemia and pyridoxine-dependent forms of homocystinuria (see Homocystinuria Caused by Cystathionine Beta-Synthase Deficiency), xanthurenic aciduria (OMIM 236800), and cystathioninuria (OMIM 219500) – these conditions are not genetically related to pyridoxine-dependent epilepsy and are phenotypically distinct disorders.

Nonketotic hyperglycinemia (NKH). Similar to seizures in infants with NKH, seizures in infants with PNPO deficiency commence within the first few days of life and may be associated with a burst suppression pattern alongside elevated plasma and cerebrospinal fluid glycine. However, seizures associated with NKH do not respond to vitamin B₆, and it appears that the CSF glycine / plasma glycine ratio in PNPO deficiency is not as high as in NKH [Mills et al 2005].

Mitochondrial disorders. PNPO deficiency may present with lactic acidosis. Lactate acid elevation can be an important, albeit nonspecific, marker of mitochondrial disease and mislead clinicians toward other less treatable inborn errors of metabolism.

Hypoxic ischemic encephalopathy (HIE). About 15% of individuals with PNPO deficiency have fetal distress followed by poor adaptation and low APGAR scores. This presentation can mislead clinicians toward symptomatic seizures as a sign of HIE. Resistance to common anticonvulsants, a history of prematurity, and severely pathologic or persistent EEG changes should lead to reevaluation for genetic causes of neonatal seizures.

Acquired epilepsy as a result of impaired PLP availability or synthesis. Certain drugs (see Possible Drug Interactions) can bind to and inactivate PLP. Other compounds that can affect PLP synthesis include pyridoxal kinase inhibitors such as 4-O-methyl pyridoxine in Ginkgo nuts [Hasegawa et al 2006] and aminophylline [Glenn et al 1995].

Evaluations Following Initial Diagnosis

To establish the extent of disease and needs in an individual diagnosed with PNPO deficiency, the evaluations summarized in this section (if not performed as part of the evaluation that led to the diagnosis) are recommended.

For classic and late-onset PNPO deficiency

• Neurologic examination to evaluate eye movement and muscle tone (for hypotonia or rigidity), and to describe seizure semiology
• EEG including sleep and wake cycles (preferably with a recording time of two hours) beginning at the time of seizure onset
• Physical examination including measurement of weight, length, and head circumferenceand evaluation of liver involvement by determination of transaminases, liver function tests, and abdominal ultrasound
• Consultation with a medical geneticist, certified genetic counselor, or certified advanced genetic nurse to inform affected individuals and their families about the nature, mode of inheritance, and implications of PNPO deficiency in order to facilitate medical and personal decision making

For late-onset PNPO deficiency

• Developmental/educational assessment using age-appropriate standardized testing
• Brain MRI in individuals with abnormal development

Treatment of Manifestations

Surveillance

Table 4.

Recommended Surveillance for Individuals with PNPO Deficiency

Agents/Circumstances to Avoid

Several anti-seizure medications (such as carbamazepine, valproate, phenytoin, and phenobarbital) can cause a low plasma concentration of PLP [Footitt et al 2011].

Evaluation of Relatives at Risk

Prenatal testing of a fetus at risk. If the PNPO pathogenic variants have been identified in an affected family member, prenatal molecular genetic testing may be performed via amniocentesis or chorionic villus sampling on future pregnancies at risk (see Pregnancy Management). This will allow precise counseling for maternal pyridoxine supplementation during pregnancy and postnatal treatment of the newborn.

When prenatal testing has not been performed on a pregnancy at risk, prompt evaluation of the newborn is essential to determine if treatment with PN or PLP is necessary. While pending results of molecular genetic testing, two options for management of an at-risk newborn are:

• Prophylactic treatment with either PN or PLP (whichever was effective in the affected sib) until molecular genetic testing clarifies whether or not the newborn is affected. A note of caution: at least one newborn at risk for ALDH7A1-related pyridoxine-dependent epilepsy developed status epilepticus after being given high-dose PN treatment before molecular genetic testing determined that the child was not affected [Hartmann et al 2011].
• Clinical and EEG monitoring with initiation of treatment with PN or PLP (whichever was effective in the affected sib) at the first sign of seizures or encephalopathy.

Symptomatic younger sib. If a younger sib (without prior molecular genetic testing) of a proband presents with encephalopathy or a non-febrile seizure, PN or PLP (depending on the drug to which the proband responded) should be administered acutely (ideally under EEG monitoring) for both diagnostic and therapeutic purposes. Note: It would be unlikely for the proband's older sibs who have not experienced seizures to have PNPO deficiency.

Pregnancy Management

As recurrence risk for couples who have had a child with PNPO deficiency is 25%, there has been discussion about the utility of empiric supplementation of PN during pregnancies in women carrying an at-risk fetus. In contrast to reports on PN supplementation in pregnancies at risk for pyridoxine-dependent epilepsy – ALDH7A1, the literature on vitamin B₆ supplementation in pregnancies at risk for PNPO deficiency is limited. In one case report, the mother of a fetus with molecularly confirmed PNPO deficiency took a multivitamin containing 2.6 mg/day of PN throughout pregnancy (recommended daily allowance for PN for women during pregnancy is 1.9 mg/day), and PLP (dose not specified) for the last three days of pregnancy. At birth the infant was immediately started on PLP therapy; neurodevelopment was reported as normal at age 2.5 years [Hatch et al 2016]. In two pregnancies, mothers were treated with 10 mg/day of PN [Peter Clayton, personal communication]; no details on timing of therapy or fetal outcome are available.

Based on the Hatch et al [2016] case report and the data on prenatal vitamin B₆ supplementation of women carrying a fetus with pyridoxine-dependent epilepsy – ALDH7A1, the authors suggest considering empiric supplementation with PN (50-100 mg/day) during the last 20 weeks of a pregnancy with a fetus at risk for PNPO deficiency or known to have PNPO deficiency based on prenatal testing for the family-specific PNPO pathogenic variants. PN supplementation is recommended rather than PLP, as the mother, who is heterozygous for PNPO deficiency, has a 50% capacity to form PLP out of PN, and the risk of hepatotoxicity of PLP in pregnancy is unknown.

Therapies Under Investigation

Search ClinicalTrials.gov in the US and EU Clinical Trials Register in Europe for access to information on clinical studies for a wide range of diseases and conditions. Note: There may not be clinical trials for this disorder.

Mode of Inheritance

Pyridox(am)ine 5'-phosphate oxidase (PNPO) deficiency is inherited in an autosomal recessive manner.

Risk to Family Members

Parents of a proband

• The parents of an affected child are presumed to be heterozygous for a PNPO pathogenic variant.
• Molecular genetic testing is recommended for the parents of a proband to confirm that both parents are heterozygous for a PNPO pathogenic variant and to allow reliable recurrence risk assessment.
• If a pathogenic variant is detected in only one parent and parental identity testing has confirmed biological maternity and paternity, it is possible that one of the pathogenic variants identified in the proband occurred as a de novo event in the proband or as a postzygotic de novo event in a mosaic parent [Jónsson et al 2017]. If the proband appears to have homozygous pathogenic variants (i.e., the same two pathogenic variants), additional possibilities to consider include:
  • A single- or multiexon deletion in the proband that was not detected by sequence analysis and that resulted in the artifactual appearance of homozygosity;
  • Uniparental isodisomy for the parental chromosome with the pathogenic variant that resulted in homozygosity for the pathogenic variant in the proband.
• Heterozygotes (carriers) are asymptomatic and are not at risk of developing the disorder.

Sibs of a proband

• If both parents are known to be heterozygous for a PNPO pathogenic variant, each sib of an affected individual, irrespective of sex, has at conception a 25% chance of being affected, a 50% chance of being an asymptomatic carrier, and a 25% chance of being unaffected and not a carrier.
• Heterozygotes (carriers) are asymptomatic and are not at risk of developing the disorder.

Offspring of a proband

• The offspring of an individual with PNPO deficiency will be obligate heterozygotes (carriers) for a pathogenic variant in PNPO.
• If an individual with PNPO deficiency has children with an individual who is heterozygous for a PNPO pathogenic variant, offspring are at a 50% risk of being affected by PNPO deficiency. The carrier frequency of PNPO deficiency in the general population is unknown. Genetic counseling and PNPO molecular genetic testing of the unaffected reproductive partner may be warranted.
• Note: The fertility status of adults with PNPO deficiency is not known and there have been no published reports about offspring of affected individuals.

Other family members. Each sib of the proband's parents is at a 50% risk of being a carrier of a PNPO pathogenic variant.

Carrier Detection

Carrier testing for at-risk relatives requires prior identification of the PNPO pathogenic variants in the family.

Related Genetic Counseling Issues

See Management, Evaluation of Relatives at Risk for information on evaluating at-risk relatives for the purpose of early diagnosis and treatment.

Family planning

• The optimal time for determination of genetic risk and discussion of the availability of prenatal/preimplantation genetic testing is before pregnancy.
• As the recurrence risk for couples who have a child with PNPO deficiency is 25%, the parents should receive counseling regarding maternal vitamin B₆ supplementation in future pregnancies (see Pregnancy Management).
• It is appropriate to offer genetic counseling (including discussion of potential risks to offspring and reproductive options) to young adults who are affected, are carriers, or are at risk of being carriers.

Prenatal Testing and Preimplantation Genetic Testing

Once the PNPO pathogenic variants have been identified in an affected family member, prenatal and preimplantation genetic testing are possible.

Differences in perspective may exist among medical professionals and within families regarding the use of prenatal testing. While most centers would consider use of prenatal testing to be a personal decision, discussion of these issues may be helpful.

Molecular Pathogenesis

Vitamin B₆, present in the human diet in various forms, is converted to pyridoxal 5'-phosphate (PLP), which acts as a cofactor for various enzymes, including those involved in the synthesis and degradation of some neurotransmitters present in the brain. This conversion involves several enzymatic steps, one of which oxidizes pyridoxamine phosphate (PMP) and pyridoxine phosphate (PNP) to PLP, catalyzed by pyridox(am)ine 5'-phosphate oxidase (PNPO).* Pathogenic variants in PNPO cause a reduction in PNPO enzyme activity, resulting in insufficient amounts of PLP for the enzymes for which it is a necessary cofactor. Because of the vital role of PLP in neurotransmitter metabolism, PLP deficiency manifests as a vitamin B₆-dependent epilepsy, usually of early onset.

* Note: The enzyme encoded by PNPO may be referred to as "pyridox(am)ine 5'-phosphate oxidase" or "pyridoxine-5'-phosphate oxidase." "Pyridox(am)ine 5'-phosphate oxidase" is preferred because it indicates that the enzyme can also use pyridoxamine 5'-phosphate as a substrate – as well as pyridoxine 5'-phosphate.

Mechanism of disease causation. Loss of function

PNPO-specific laboratory technical considerations. Clinicians and laboratories need to note that some individuals with PNPO deficiency can be homozygous for the variant p.Arg116Gln and homozygous or compound heterozygous for other PNPO pathogenic variants. Thus, these individuals may have two or three PNPO pathogenic variants. When this phenomenon was first observed, p.Arg116Gln was classified as a benign variant in the ClinVar database; however, structural assays by di Salvo et al [2017] and enzyme analysis suggest that it is pathogenic [Wilson et al 2017]. Nevertheless, not all individuals homozygous for the variant p.Arg116Gln develop seizures [Wilson et al 2019], an observation of particular importance when interpreting molecular genetic test results performed in the context of prenatal or preimplantation genetic testing.

Deep intronic variants and intragenic deletions may be missed by sequence analysis that only includes exons and exon/intron boundaries.

The effects of variants of uncertain significance can be assessed by analysis of PNPO enzyme activity in dried blood spots [Wilson et al 2017]. In one individual with only one pathogenic variant identified by Sanger sequencing of genomic DNA and sequencing of cDNA [Raimondi et al 2015], the diagnosis of PNPO deficiency was subsequently confirmed by detection of reduced PNPO enzyme activity in a dried blood spot.

Table 5.

Notable PNPO Pathogenic Variants

Author Notes

Dr Barbara Plecko (ta.zarginudem@okcelp.arabrab) and Dr Philippa Mills (ku.ca.lcu@sllim.p) are actively involved in clinical research regarding individuals with PNPO deficiency and other forms of vitamin B₆-dependent epilepsy. They would be happy to communicate with individuals who have any questions regarding diagnosis or other considerations.

PNPO enzyme analysis is available in the laboratory of Dr Mills. Contact Dr Mills (ku.ca.lcu@sllim.p) to inquire about enzyme analysis testing or to review PNPO variants of uncertain significance.

Dr Plecko and Dr Mills are also interested in hearing from clinicians treating families affected by vitamin B₆-dependent epilepsy in whom no causative variant has been identified through molecular genetic testing of the genes known to be involved in this group of disorders.

Acknowledgments

Dr P Mills is supported by funding from the NIHR Great Ormond Street Hospital Biomedical Research Centre. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR, or the Department of Health.

Revision History

• 23 June 2022 (bp) Review posted live
• 22 November 2021 (bp) Original submission

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