Phenotypic and molecular spectrum of pyridoxamine‐5′‐phosphate oxidase deficiency: A scoping review of 87 cases of pyridoxamine‐5′‐phosphate oxidase deficiency

Abstract Pyridoxamine‐5′‐phosphate oxidase (PNPO) deficiency is an autosomal recessive pyridoxal 5′‐phosphate (PLP)‐vitamin‐responsive epileptic encephalopathy. The emerging feature of PNPO deficiency is the occurrence of refractory seizures in the first year of life. Pre‐maturity and fetal distress, combined with neonatal seizures, are other associated key characteristics. The phenotype results from a dependency of PLP which regulates several enzymes in the body. We present the phenotypic and genotypic spectrum of (PNPO) deficiency based on a literature review (2002‐2020) of reports (n = 33) of patients with confirmed PNPO deficiency (n = 87). All patients who received PLP (n = 36) showed a clinical response, with a complete dramatic PLP response with seizure cessation observed in 61% of patients. In spite of effective seizure control with PLP, approximately 56% of patients affected with PLP‐dependent epilepsy suffer developmental delay/intellectual disability. There is no diagnostic biomarker, and molecular testing required for diagnosis. However, we noted that cerebrospinal fluid (CSF) PLP was low in 81%, CSF glycine was high in 80% and urinary vanillactic acid was high in 91% of the cases. We observed only a weak correlation between the severity of PNPO protein disruption and disease outcomes, indicating the importance of other factors, including seizure onset and time of therapy initiation. We found that pre‐maturity, the delay in initiation of PLP therapy and early onset of seizures correlate with a poor neurocognitive outcome. Given the amenability of PNPO to PLP therapy for seizure control, early diagnosis is essential.

between the severity of PNPO protein disruption and disease outcomes, indicating the importance of other factors, including seizure onset and time of therapy initiation.
We found that pre-maturity, the delay in initiation of PLP therapy and early onset of seizures correlate with a poor neurocognitive outcome. Given the amenability of PNPO to PLP therapy for seizure control, early diagnosis is essential.  Table S1). PLP-dependent enzymes have essential roles in a variety of biochemical processes, including amino acid metabolism, glycolysis, gluconeogenesis, glycogenolysis, transsulfuration, polyamine biosynthesis, and synthesis of sphingoid bases, and the heme precursor δ-aminolevulinic acid. 4,5 Hence, PLP is one of the most central molecules for the general cellular metabolism.
Its metabolism in the liver requires many enzymes, including (1) pyridoxal kinase, which is responsible for vitamin B6 phosphorylation, an important step for the transfer of vitamin B6 to pyridoxal-5-phosphate; (2) pyridoxal phosphate phosphatase, the enzyme that is involved in the preferred degradation route of PLP by aldehyde oxidase to 4-pyridoxic acid through PLP dephosphorylation; and (3) PNPO then catalyzes the last step in PLP synthesis 6 (Figure 1).
Given the variety of PLP-dependent enzymes, PLP deficiency might be expected to have diverse clinical presentations. However, the neurological phenotype is the predominant phenotype of PNPO deficiency, sometimes co-occurring with non-neurological manifestations such as impaired growth and hypochromic microcytic anemia that responds dramatically to treatment with PLP. Epileptiform convulsions in infants are a common presentation due to defective conversion of glutamic acid into γ-aminobutyric acid (GABA). Other neurological manifestations including irritability and peripheral neuritis arise due to improper production of serotonin, epinephrine, norepinephrine, and GABA. Defects in the synthesis of sphingolipids lead to nerve demyelination, which is manifested as neuropathy. 4,5 This article reviews 87 cases of PNPO deficiency describing the spectrum of the neurological and non-neurological phenotypes of PNPO deficiency as well as its diagnostic biochemical profile, genotypic basis, and therapeutic response to PLP and PN. F I G U R E 1 Vitamin B6 metabolism as it travels from the intestine to the portal circulation, crosses the blood-brain barrier, and enters the brain cells. A, In the intestine, the dietary phosphorylated form is hydrolyzed to the free form by intestinal hydrolase (IH)/tissue-specific intestinal phosphatase (TSIP) prior to absorption. This is followed by its uptake by intestinal cells, which is believed to occur through simple diffusion. Through portal circulation, the free B6 forms reach the liver, where metabolism in the liver is catalyzed by many enzymes. (1) Pyridoxal kinase (PK), (2) pyridoxal phosphate phosphatase, (3) pyridox(am)ine-5 0 -phosphate oxidase. B, The unphosphorylated forms of vitamin B6 are able to cross the blood-brain barrier, probably by facilitated diffusion, mostly at the choroid plexus (CP). The CP traps PLP via pyridoxal kinase and can release PLP to a remarkable extent (and pyridoxal to a lesser extent). C, Excessive PLP in the CSF and extracellular space enters brain cells, and the B6 vitamers must be dephosphorylated so that they can enter brain cells and then metabolically trapped by being rephosphorylated by pyridoxal kinase. Pyridoxine phosphate and pyridoxamine phosphate are then oxidized by PNPO to form the active cofactor, PLP. PLP, pyridoxal 5 0 -phosphate

| Neurological manifestations of PNPO deficiency
The described neurological changes varied widely among the literature. The most prominent neurological manifestation is a seizure within the first day of life, occurring in 59% of the reported cases.
Abnormal fetal movement was noticed by the mother in the third tri-   gene who had normal brain imaging. These patients had residual enzyme activity accompanied by later onset and a milder seizure phenotype than patients with total loss of PNPO function. 7 One patient reported with homozygous for a c.283C > T; (p. R95C) mutation at PNPO gene who had a normal brain MRI initially at 1 month of age but his follow-up imaging at 3 years showed severe diffuse atrophy. 11 The most common abnormality revealed on brain imaging was diffuse atrophy, which was observed in eight patients (14.5%), 8,9,16,20 followed by ischemic changes and encephalomalacia in five patients (5.5%), 1,13,16,17 and delayed myelination and atrophy in another three patients. 18

| Non-neurological manifestations of PNPO deficiency
Most of the reported cases showed pre-and perinatal complications including pre-maturity, fetal distress and intrauterine growth restriction (IUGR) with oligohydramnios.
Pre-maturity was commonly observed in 50% of PNPO-deficient patients and 58% of the pre-mature cases suffered fetal distress.
There were two detected cases of oligohydramnios 11

| Biochemical profiles in PNPO deficiency
The biochemical analysis protocols varied among the included studies.
Notably, there was no available panel with specific biomarkers in the reviewed literature. The analytical testing in the majority of reports   Table S4). The biochemical tests used for diagnosis and their reference ranges vary among these studies. To allow a significant comparison, we only considered tests that were available for at least 10 patients. CSF 5-HIAA and PLP were most frequently performed (>20 patients; Supplementary Figure S1). Elevation of urine VLA and decrease of CSF PLP were significant in the majority of patients, which make these measurements potential suggestive biomarkers for PNPO deficiency. Other tests are summarized in Table 2. 3.3 | Molecular aspect of PNPO deficiency

| Structural analysis and predicted effect of variants
Based on their position in the protein structure (Protein Data Bank (PDB) accession number 1nrg 37 and type of mutation, PNPO variants can be separated into four categories. The largest category (here termed category I) contains mutations that directly affect the catalytic site and its capacity for ligand and cofactor binding (colored magenta in Figure 3 and Table 3 The second category, category II, contains mutations that affect the fold and stability of the protein because of a non-conservative side chain substitution (blue in Figure 3 and Table 3). Their effects range from a mild loss of stabilizing surface interactions (E50K) and  Figure 3).

| Correlation between molecular effects and clinical outcomes
Our overview reveals a striking lack of correlation between the reported clinical effects of PNPO variants and the molecular effects they have on the protein structure and function (

| PNPO deficiency outcome
Finally, we assessed factors that impact the outcomes of PNPO deficiency based on cognitive function and/or developmental assessment.
Pre-maturity, fetal distress, seizure onset and initiation of PLP therapy have been documented and correlated with outcome variables including death, developmental delay and normal development (Supplementary Table S2).
To evaluate the impact of pre-maturity, we studied all 87 cases based on gestational age at birth. Pre-maturity of birth was observed in the majority of patients (n = 44); 24 of them had shown fetal distress during the neonatal period. Only seven patients developed normally given their pre-maturity, while 37 patients either died or showed variable degrees of developmental delay. We have categorized patients based on the maturity and outcome ( Figure 4A). Four cases of PNPO deficiency were presented with liver cirrhosis or abnormal liver function test after receiving PLP treatment.
Although abnormal liver function might expand the phenotypic spectrum of PNPO deficiency, the PLP administration is probably the cause of liver impairment. Schmitt et al. reported a 2 years and 6 months old boy with PNPO deficiency who developed an abnormal liver function test after escalating a PLP dose to 100 mg/kg/ day and it had to be reduced to 53 mg/kg/day. 22 Sudarsanam et al have postulated that liver dysfunction in their patient was due to a high dose of administered PLP (100 mg/kg/day). 23 Although reduction of the PLP dose and frequency resulted in substantial reduction in the liver transaminases, episodes of uncontrolled seizures and encephalopathy required high doses of PLP, which negatively affected the liver function. 23 Porri et al has also reported a mild elevation in alanine aminotransferase and aspartate aminotransferase levels on a PNPO-deficient patient treated with 50 mg/kg/day of PLP. 25 Later, Coman et al described a 4 years old boy with PNPO deficiency in whom liver cirrhosis has been showed while receiving a 50 mg/kg/day of PLP. 24 Last two cases have never received "high dose" of PLP, rather a dosage range of 30 to 50 mg/kg/day. 24,25 In all PNPO-deficient patients, liver derangement occurred after longterm administration of PLP with substantial improvement after dose adjustment which indicates that liver toxicity is probably related to PLP administration and should be carefully monitored. [22][23][24][25] In addition, a previously reported liver toxicity case secondary to high-dose PLP for treating homocystinuria was documented by Yoshida et al supported further this hypothesis. 42 Collectively, these reports highlight the possibility of PLP-related liver dysfunction in PNPO-deficient patient, and hence surveillance for evidence of liver cirrhosis should be part of management of PNPO-deficient patients receiving PLP.
We conclude that early detection of PNPO deficiency combined with early PLP treatment is key to optimizing the clinical outcome.
While newborn screening is useful for the early detection of some diseases, it might not be feasible in PNPO deficiency due to the absence of sensitive biomarkers. However, we identified suggestive biochemical profiles in the literature that should motivate a definitive molecular diagnosis of PNPO gene variants, especially in cases of a suspected family history indicating PNPO deficiency.