Summary of the genetic, biochemical and clinical features of B6EEs.
Vitamin B6 (vitB6) is a generic term that comprises six interconvertible pyridine compounds. These vitB6 compounds (also called vitamers) are pyridoxine (PN), pyridoxamine (PM), pyridoxal (PL) and their 5′-phosphorylated forms pyridoxine 5′-phosphate (PNP), pyridoxamine 5′-phosphate (PMP) and pyridoxal 5′-phosphate (PLP). VitB6 is an essential nutrient for all living organisms, but only microorganisms and plants can carry out de novo synthesis of this vitamin. Other organisms obtain vitB6 from dietary sources and interconvert its different forms according to their needs via a biochemical pathway known as the salvage pathway. PLP is the biologically active form of vitB6 which is important for maintaining the biochemical homeostasis of the body. In the human body, PLP serves as a cofactor for more than 140 enzymatic reactions, mainly associated with synthesis, degradation and interconversion of amino acids and neurotransmitter metabolism. PLP-dependent enzymes are also involved in various physiological processes, including biologically active amine biosynthesis, lipid metabolism, heme synthesis, nucleic acid synthesis, protein and polyamine synthesis and several other metabolic pathways. PLP is an important vitamer for normal brain function since it is required as a coenzyme for the synthesis of several neurotransmitters including D-serine, D-aspartate, L-glutamate, glycine, γ-aminobutyric acid (GABA), serotonin, epinephrine, norepinephrine, histamine and dopamine. Intracellular levels of PLP are tightly regulated and conditions that disrupt this homeostatic regulation can cause disease. In humans, genetic and dietary (intake of high doses of vitB6) conditions leading to increase in PLP levels is known to cause motor and sensory neuropathies. Deficiency of PLP in the cell is also implicated in several diseases, the most notable example of which are the vitB6-dependent epileptic encephalopathies. VitB6-dependent epileptic encephalopathies (B6EEs) are a clinically and genetically heterogeneous group of rare inherited metabolic disorders. These debilitating conditions are characterized by recurrent seizures in the prenatal, neonatal, or postnatal period, which are typically resistant to conventional anticonvulsant treatment but are well-controlled by the administration of PN or PLP. In addition to seizures, children affected with B6EEs may also suffer from developmental and/or intellectual disabilities, along with structural brain abnormalities. Five main types of B6EEs are known to date, these are: PN-dependent epilepsy due to ALDH7A1 (antiquitin) deficiency (PDE-ALDH7A1) (MIM: 266100), hyperprolinemia type 2 (MIM: 239500), PLP-dependent epilepsy due to PNPO deficiency (MIM: 610090), hypophosphatasia (MIM: 241500) and PLPBP deficiency (MIM: 617290). This chapter provides a review of vitB6 and its different vitamers, their absorption and metabolic pathways in the human body, the diverse physiological roles of vitB6, PLP homeostasis and its importance for human health. Finally, the chapter reviews the inherited neurological disorders affecting PLP homeostasis with a special focus on vitB6-dependent epileptic encephalopathies (B6EEs), their different subtypes, the pathophysiological mechanism underlying each type, clinical and biochemical features and current treatment strategies.
- vitamin B6 (vitB6)
- Salvage pathway
- PLP-dependent enzymes
- inherited vitB6-dependent epilepsies
Vitamin B6 (vitB6) is a generic term that refers to a group of six interconvertible chemical compounds that share a pyridine ring in their centre. These vitB6 compounds (also called vitamers) are pyridoxine (PN), pyridoxamine (PM), pyridoxal (PL) and their 5′-phosphorylated forms pyridoxine 5′-phosphate (PNP), pyridoxamine 5′-phosphate (PMP) and pyridoxal 5′-phosphate PLP)  (Figure 1). VitB6 is required by all living organisms for their survival, but only microorganisms and plants can carry out
1.1 Metabolism of vitB6
Among the six vitB6 compounds, PLP is the biologically active and most important vitamer since it is required as a cofactor for a multitude of enzymes in the body. Humans and other mammals obtain PLP directly from diet or through synthesis from other vitameric forms ingested with food or recycled from degraded PLP-dependent enzymes via the salvage pathway [1, 4] (Figure 2). The central enzyme in this pathway is PNP oxidase (PNPO), a flavin mononucleotide (FMN)-dependent enzyme that is capable of converting PNP or PMP to the active cofactor PLP . Other important enzymes in the salvage pathway are PL kinase (PLK) and a number of different phosphatases .
VitB6 vitamers are widely available in animal and plant food sources. PLP and in a lesser amount, PMP are present as such in animal-derived foods, mainly associated with muscle glycogen phosphorylase, while plant foods are more enriched in PN, PNP and PN glucosides [1, 4, 8].
After being ingested, phosphorylated vitamers (PLP, PNP and PMP) undergo dephosphorylation by the ecto-enzyme tissue-specific intestinal phosphatase (IP) , whereas PN glucoside (PNG) vitamers from plants are hydrolyzed by a glucosidase before absorption [1, 10]. Absorbed vitamers are carried by the portal circulation to the liver where they are phosphorylated by PLK . Inside liver cells, PNP and PMP are oxidized by PNPO to form PLP, which is then released to the circulation bound to lysine-190 residue of albumin (Figure 3) [9, 10, 11]. Binding of PLP to albumin is thought to protect the cofactor from hydrolysis and other reactions . About 60% of circulating vitB6 is in the form of albumin-bound PLP, while PN, PM and PL constitutes the remaining proportion .
Prior to delivering the circulating PLP to different tissues, it is dephosphorylated to PL by the ecto-enzyme tissue nonspecific alkaline phosphatase (TNSALP) to enable entry into the cells and through the blood–brain barrier. Inside the cell, PL is re-converted by PLK to PLP, which now can be used as a cofactor in many biochemical reactions (Figure 3) [1, 5, 9]. Degradation of PLP-bound enzymes (holo-B6 enzymes) can generate PMP, which is then oxidized back to PLP by the action of PNPO  (Figures 2 and 3).
Besides the liver, it has been shown that the intestine also contributes an important role in vitB6 metabolism.
1.2 Catabolism of vitamin B6
At the other end of vitB6 metabolism, little is known about the catabolic pathways in humans or other mammals. In contrast, these mechanisms are well established in microorganisms [3, 11, 15]. In humans and other mammals, the primary product of the degradation of PLP (and all other vitB6 vitamers) is 4-pyridoxic acid (4-PA). This compound, which is excreted in urine, is generated in two steps. In the first one, PLP is hydrolyzed to PL by the action of an intracellular enzyme known as PLP phosphatase (PLPase). In the following step, PL is oxidized to 4-PA by a non-specific aldehyde oxidase (AOX) or aldehyde dehydrogenase (Figure 3) [3, 6, 12, 15, 16]. In microorganisms, 4-PA is further degraded to other metabolites that can be utilized by the cell in various biochemical processes . Some microbial vitB6 catabolic products such as 5-pyridoxic acid (5-PA), 5-pyridoxolactone  and 4-pyridoxolactone [17, 18] have been also discovered in human individuals under consumption of high amounts of vitB6. Several other PN derivatives have been identified in humans and/or other mammalian species, but their biochemical pathways and precise functions have not yet been unraveled.
For example, Coburn and Mahuren  detected pyridoxine 3-sulfate, pyridoxal 3-sulfate and
Oxidation of PN at the 5′ position, followed by sequential dehydrogenation to form 5-PA, is known to exist only in the PN catabolic pathway of some bacterial species like
1.3 Vitamin B6 transportation across cellular membrane
Multiple experimental evidence suggests that, as with most water-soluble vitamins , the transportation of vitB6 across mammalian cell membrane is carrier-mediated. Studies in cultured human intestinal [12, 25], colonic , and renal cells  and animal-derived renal proximal tubular cells  demonstrated the presence of an efficient and specific carrier-facilitated mechanism for cellular uptake of vitB6. Such a specific transporting membrane carrier was employed to produce a high affinity gene delivery system into cancer cells using a vitB6-coupled vector . However, the molecular identity of vitB6 transporter protein in mammals has remained elusive [12, 30]. Among eukaryotes, the only vitB6 transporters identified so far are the yeast transporters, Tpn1p  and Bsu1 , and, recently, PUP1 in plant species
1.4 Physiological roles of vitamin B6
PLP, the coenzymatically active form of vitamin B6, plays an important role in maintaining the biochemical homeostasis of the body . In the human body, PLP is an essential cofactor for more than 140 distinct enzymatic activities, mainly associated with synthesis, degradation and interconversion of amino acids as well as with neurotransmitter metabolism [35, 36, 37, 38]. PLP-dependent enzymes are also involved in a multitude of other cellular processes, including biologically active amine biosynthesis, lipid metabolism, heme synthesis, nucleic acid synthesis, protein and polyamine synthesis and several other metabolic pathways (Figure 4) [5, 6]. Furthermore, PLP is important in energy homeostasis through glycogen degradation and gluconeogenesis, since PLP is a cofactor for glycogen phosphorylase and gluconeogenic transaminases [36, 41]. In folate-mediated one-carbon metabolism (FOCM), PLP is required as a cofactor for the enzyme serine hydroxymethyltransferase, both its cytoplasmic (SHMT1) and mitochondrial (SHMT2) isoforms. FOCM is an important pathway that is involved in a number of physiological processes such as DNA methylation, redox homeostasis and purines and thymidine biosynthesis [36, 42].
As a coenzyme for the synthesis of several neurotransmitters including D-serine, D-aspartate, L-glutamate, glycine, γ-aminobutyric acid (GABA), serotonin, epinephrine, norepinephrine, histamine and dopamine, PLP is an important vitamer for normal brain function [5, 43]. For example, GABA, the major inhibitory neurotransmitter in the central nervous system (CNS), is synthesized from L-glutamate by the PLP-dependent enzyme glutamate decarboxylase (GAD). Moreover, PLP is a cofactor for branched-chain amino acid aminotransferase (BCAT) which catalyzes the synthesis of L-glutamate, the major excitatory neurotransmitter, from branched-chain amino acids like leucine and valine .
Another important PLP-dependent enzyme in the brain is aromatic L-amino acid decarboxylase (AADC), which catalyzes the final steps in the biosynthetic pathways of serotonin and dopamine (Figure 5) [5, 36]. These neurotransmitters also serve as precursors for other important compounds in the brain, specifically melatonin, norepinephrine and epinephrine (Figure 5) [5, 46].
In addition to its role as an enzymatic cofactor, PLP has been shown to play a role in preventing DNA damage  and in modulating the activity and expression of steroid hormone receptors [6, 48]. vitB6 has also been described as an efficient antioxidant in plants and fungi, with the ability of its different vitamers to quench reactive oxygen species [1, 49, 50].
1.5 PLP homeostasis and its importance for human health
PLP is a highly reactive compound because of its aldehyde group at the 4′ position which can undergo spontaneous complexation with other molecules within the cell [1, 9]. It may bind with amino groups in proteins and disrupt their structure . For example, it has been shown that PLP can react with the lysine residue in the active site of human DNA topoisomerase I, causing its inhibition [51, 52]. Through a chemical reaction known as Knoevenagel condensation, PLP can also react with intermediate metabolites like Δ1-pyrroline 5-carboxylate and Δ1-piperideine 6-carboxylate, which form the molecular basis of PLP depletion in the neurometabolic diseases ALDH7A1 deficiency and hyperprolinaemia type II, respectively . Because of its high reactivity and to prevent toxic accumulation of this cofactor, the intracellular pool of free PLP is maintained at very low concentration (about 1 μM in eukaryotic cells) [1, 5, 6]. It is therefore likely that PLP production in the cell is tightly regulated , and experimental work indicates the presence of an efficient mechanism that maintains intracellular PLP levels within optimum levels . However, how the concentration of PLP is controlled in mammalian tissues is not entirely understood [3, 34].
A number of mechanisms have been proposed that help in PLP homeostasis. First, both enzymes that produce PLP, PLK and PNPO, are inhibited by their product PLP and its rate of synthesis can, therefore, be controlled by this feedback inhibition [1, 5, 6]. Enzymes that degrade PLP and PL, like PLPase and AOX, respectively, have also been proposed as a mechanism that keeps free PLP at low level within the cell [1, 5, 6]. Proteins that are known to naturally bind PLP, like muscle glycogen phosphorylase, plasma albumin and hemoglobin in red blood cells, contribute to reducing the amount of free reactive PLP . In addition to its catalytic role in PLP synthesis, a recent study  demonstrated that PNPO forms a tight a binding with PLP at a noncatalytic site
Conditions that disrupt cellular PLP homeostasis can cause disease. For example, inactivation of PLPP in mice led to increase in PLP levels, anxiety and motor deficits . In humans, intake of high doses of vitB6 is known to cause motor and sensory neuropathies [1, 5]. Deficiency of PLP in the cell is also implicated in several pathologies, most notably the so-called vitB6-dependent epileptic encephalopathies [1, 5, 9, 37].
2. VitB6-dependent epileptic encephalopathies
VitB6-dependent epileptic encephalopathies (B6EEs) represent a clinically and genetically heterogeneous group of rare inherited metabolic diseases [55, 56]. These debilitating conditions are characterized by recurrent seizures in the prenatal, neonatal, or postnatal period, which are typically resistant to conventional anticonvulsant treatment but well-controlled by the administration of PN or PLP [56, 57, 58, 59]. In addition to seizures, children affected with B6EEs may also suffer from developmental and/or intellectual disabilities, along with structural brain abnormalities . The 5 principal types of B6EEs: PN-dependent epilepsy due to ALDH7A1 (antiquitin) deficiency (PDE-ALDH7A1) (MIM: 266100), hyperprolinemia type 2 (MIM: 239500), PLP-dependent epilepsy due to PNPO deficiency (MIM: 610090), hypophosphatasia (MIM: 241500) and PLPBP deficiency (MIM: 617290) [6, 9, 60, 61] (Table 1). According to the underlying pathobiochemical mechanism, these forms of B6EEs can be categorized into: 1) defects in amino acid catabolic pathways causing buildup of byproducts that react with PLP (PDE-ALDH7A1 and hyperprolinemia type 2), 2) defects in the vitB6 salvage pathway (PNPO deficiency), and 3) defects in cellular uptake of PLP (hypophosphatasia) [6, 9] (Table 1). In the most recently discovered type, PLPBP deficiency, the exact mechanism that disrupts PLP homeostasis is not fully understood .
2.1 PN-dependent epilepsy (ALDH7A1 deficiency)
2.1.1 Disease mechanism
PN-dependent epilepsy (PDE-ALDH7A1) is caused by homozygous or compound heterozygous mutations in the
2.1.2 Clinical features
The main clinical manifestation of PDE-ALDH7A1 is recurrent perinatal-onset seizures that are resistant to conventional anticonvulsant treatment, but which show remarkable response to the administration of high doses of PN [60, 72]. Seizures usually relapse when PN treatment is discontinued, either incidentally or for diagnostic purposes . In some cases, the mother of an affected child has described abnormal fetal movements during pregnancy, suggestive of pre-natal onset of seizures [55, 73, 74, 75]. In atypical cases, seizure onset can be delayed to up to 3 years of age , and in one exceptional case, Srinivasaraghavan et al.  reported an Indian female with genetically proven PDE-ALDH7A1 in whom seizures did not start until the age of 17 years (juvenile onset).
In addition to seizures, most PDE-ALDH7A1 patients (about 75%) also suffer from developmental delay and moderate to severe intellectual disability [60, 72, 77]. In addition, as revealed by neuroimaging analysis, a spectrum of structural brain defects have been described in affected children with anomalies of corpus callosum (agenesis/hypoplasia/dysplasia) and white matter being common features [75, 77, 78, 79]. Motor deficits (hypotonia/hypertonia/dystonia), irritability, autism spectrum disorder (ASD), attention-deficit hyperactivity disorder (ADHD) and anxiety are additional features reported in patients [75, 77, 80].
The phenotypic spectrum of PDE-ALDH7A1 may also include non-neuronal features, but these are less frequently observed in patients. Reported examples are ocular problems, hypoglycemia, hypothyroidism, lactic acidosis, profound electrolyte disturbances, diabetes insipidus, coagulopathy, anemia, respiratory distress and hypotension [60, 77, 79, 81, 82].
2.1.3 Biochemical features and diagnostic biomarkers
In PDE-ALDH7A1, blockade of the ATQ-catalyzed step in the lysine catabolism pathway leads to accumulation of 3 upstream metabolites, P6C, α-AASA and PIP, as discovered by screening of patients’ body fluids. Presence of these metabolites in supraphysiological levels is considered the hallmark biochemical feature of ATQ deficiency and have been utilized as diagnostic biomarkers . Recently, two additional lysine metabolites discovered to accumulate in patients have been suggested as novel biomarkers. The first one is 6-oxopipecolate (6-oxo-PIP), which was found to be present in large concentrations in plasma, urine, and CSF of ATQ deficiency patients [83, 84]. By means of an untargeted metabolomics approach, Engelke et al.  identified another novel metabolite, 6-(2-oxopropyl)piperidine-2-carboxylic acid (2-OPP), that accumulated in biofluids of affected individuals.
Because P6C inactivates PLP and causes cellular depletion of this enzymatic cofactor, a number of biochemical abnormalities occur that are associated with secondary deficiencies of PLP-dependent enzymes, mainly affecting amino acid metabolism. Table 2 lists some amino acid changes reported in PDE-ALDH7A1 patients and possible links to PLP-dependent enzymes in their metabolic pathways.
|Disease name||PN-dependent epilepsy (PDE-ALDH7A1)||PLP-dependent epilepsy||Hyperprolinemia type 2||Hypophosphatasia||PLPBP deficiency|
|Affected enzyme or protein/pathway(s)||α-AASA dehydrogenase/lysine catabolism pathway||PNP oxidase/vitB6 salvage pathway||P5C dehydrogenase/Proline catabolism pathway||TNSALP/Extracellular dephosphorylation of PLP, Bone mineralization||PLPHP/PLP homeostasis|
|Pathophysiological mechanism of PLP deficiency||Accumulating lysine metabolite, P6C, reacts with and inactivates PLP||PNPO is required for intracellular production of PLP from PNP/PMP||Accumulating proline metabolite, P5C, reacts with and inactivates PLP||TNSALP is required for extracellular conversion of PLP to PL to enable its cellular uptake||PLPHP is required for maintaining cellular PLP homeostasis|
|Main clinical features||Neonatal seizures, DD/ID||Neonatal seizures, DD/ID||Infantile seizures, DD/ID, ataxia||Rickets, Osteomalacia, Neonatal seizures||Neonatal seizures, DD/ID|
|Biomarkers (biofluid)||High α-AASA (U/P), P6C (P), PIP (P)||High PM, PM/PA ratio (P)||High proline (P), P5C (U)||Low ALP (P), high PLP (P), high PEA (U)||No specific biomarker|
|Commonly used vitB6 treatment||PN||PLP||PN||PN||PN|
|[9, 60, 62]||[9, 57, 63]||[6, 60, 63, 64]||[9, 60, 65, 66]||[7, 67, 68]|
|Amino acid (tissue/fluid, change)*||Implicated PLP-dependent enzyme(s)**||Enzyme’s function**|
|Glycine (CSF & plasma, ↑)||Glycine dehydrogenase (decarboxylating)||Important component of the glycine cleavage system|
|Threonine (CSF, ↑)||Glycine C-acetyltransferase||Catalyzes the second step in the pathway that converts threonine to glycine|
|Threonine deaminase||Catalyzes the first step in the catabolic pathway of threonine |
|Serine (plasma, ↑)||Involved in breakdown/conversion of serine to other metabolites|
|Alanine (CSF & plasma, ↑)||Involved in breakdown/conversion of alanine to other metabolites|
|Phenylalanine (CSF, ↑)||Aromatic L-amino acid decarboxylase||Converts phenylalanine to phenethylamine|
|Arginine (CSF, ↓)||Ornithine δ-aminotransferase||Catalyzes the formation of ornithine, an indirect precursor for arginine synthesis [57, 87]|
|Histidine (CSF, ↑)||Histidine decarboxylase||Converts histidine to histamine|
2.1.4 Treatment and its outcome
In patients with PDE-ALDH7A1, seizures are effectively controlled by PN treatment in about 90% of cases . Patients require life-long intake of pharmacological doses of PN for seizure control as PN withdrawal leads to seizure recurrence . In a subset of patients with ATQ deficiency, better seizure control is achieved when folinic acid is added to the PN regimen (known as folinic acid-responsive seizures or FARS) . The subset of FARS patients can be distinguished by the appearance of a characteristic peak (Peak X) on CSF biogenic amine neurotransmitter analysis [60, 89].
Despite effective control of seizures with PN, treatment outcome is usually still poor, and a large proportion of children with PDE-ALDH7A1 have neurodevelopmental impairments . It has been suggested that PN treatment alone cannot prevent the accumulation of high levels of lysine metabolites (P6C, α-AASA and PIP) in the brain which may have neurotoxic effects .
To limit the accumulation of these metabolites, substrate (lysine) reduction therapies have been implemented. These consisted of lysine-restricted diet , arginine supplementation  and triple therapy . Arginine is a natural antagonist of lysine because the two amino acids use the same transporter (known as the y + system) for their transportation across the BBB. Therefore, it was suggested that arginine could compete with lysine and limit its entry to the brain [72, 92]. Triple therapy refers to a combination therapy of lysine-restriction and arginine supplementation (in addition to PN treatment, therefore it was termed “triple therapy”) . Clinical trials using these dietary therapies reported reduction in lysine metabolite levels and improvements in the neurodevelopmental outcome in most treated patients [79, 85, 91, 93, 94, 95, 96].
2.2 PLP-dependent epilepsy (PNPO deficiency)
2.2.1 Disease mechanism
PNPO catalyzes the rate-limiting step in the biosynthetic pathway of PLP from other vitB6 vitamers (salvage pathway, Figure 2). Patients affected with pathogenic variants in its encoding gene,
2.2.2 Clinical features
Similar to PDE-ALDH7A1, PNPO deficiency is characterized by early onset, drug-resistant epileptic encephalopathy . Since the disease gene discovery in 2005 , about 90 cases of PNPO deficiency have been reported in the medical literature with a phenotypic spectrum that extends from early postnatal lethality to milder forms with well-controlled seizures and normal neurodevelopmental outcome [88, 97, 98, 99]. Prematurity is observed in about 50% of the PNPO deficiency cases . Seizures usually start very early after birth (within the first day of life in about 60% of the cases), but can also have a later onset within the first 6 months of life [86, 88].
2.2.3 Biochemical features and diagnostic biomarkers
PNPO deficiency is associated with a number of biochemical alterations most commonly affecting biogenic amine neurotransmitters. The PLP-dependent enzyme, AADC, plays a central role in the biosynthetic pathway of these neurotransmitters (Figure 5). A number of amine neurotransmitter metabolites in this pathway were found to be present at abnormal levels in PNPO-deficient patients, suggesting an impaired flux through the AADC catalyzed step. For example, elevated levels of 3-
The biochemical spectrum of PNPO deficiency also includes amino acid and vitB6 vitamer perturbations. Elevated concentrations of threonine, glycine, histidine and taurine and low concentrations of arginine in CSF and/or plasma have been all reported in patients [6, 10, 88]. Unlike PDE-ALDH7A1, systemic PLP deficiency is a typical finding in PNPO deficiency as evidenced by the detection of low PLP levels in pre-treatment patient samples (CSF and/or plasma) [37, 88, 101]. Another common vitamer finding is the accumulation of PM, the precursor of PNPO substrate, detected in both pre- and post-treatment plasma samples [101, 102].
Currently there is no specific diagnostic biomarker for PNPO deficiency and genetic testing of the
2.2.4 Treatment and its outcome
Seizures are usually controlled by supplementation of pharmacological doses of PLP or PN. Based on early reports [57, 104, 105], PNPO deficiency has for some time been viewed as a disease that is only treatable by PLP but not PN (and hence was given the name “PLP-dependent epilepsy”). This was also consistent with the notion that the defective enzyme, PNPO, in these patients is unable to convert supplemented PN to PLP which explains the lack of response to PN treatment. However, it was later found that a subset of affected children (about 40% of cases ) show better clinical response to PN while PLP may in fact exacerbate their seizures [87, 106]. Mills et al.  suggested that certain genotypes (namely R225H/C and D33V) seem to be more likely to benefit from PN treatment. This was attributed to possible residual enzyme activity that is associated with these
In some patients, better seizure control was achieved by adjunct treatments like anti-seizure drugs  and/or riboflavin  in combination with vitB6 therapy. Riboflavin is a precursor of flavin mononucleotide (FMN), the cofactor of PNPO, and therefore may enhance residual enzyme activity . There were multiple reports of liver problems in patients receiving PLP treatment, and these were linked to possible toxic effects of chronic PLP administration, an observation that warrants careful mentoring of PLP-treated patients [109, 110, 111].
Neurodevelopmental outcome is still poor in a large proportion of affected children. A recent literature survey of 87 cases of PNPO deficiency  found that 56% of patients suffered developmental and/or intellectual deficits in spite of adequate seizure control with vitB6 therapy. Other reports suggested that early diagnosis and initiation of treatment could lead to normal developmental outcome [4, 109, 112].
2.3 Hyperprolinemia type 2
2.3.1 Disease mechanism
The genetic cause of hyperprolinemia type 2 (HP2), first identified in an Irish traveler family , was found to be due to recessive mutations in
2.3.2 Clinical features
The clinical manifestation of HP2 is variable  and asymptomatic cases have been described . Seizures are the most common clinical fining in HP2 which occur in about 50% of the cases [6, 114]. They are often triggered by febrile illness and have variable age of onset; commonly occurring during infancy or childhood but can also be up to late adulthood (63 years in one HP2 case ) [6, 114, 117, 118]. Intellectual and neuropsychiatric abnormalities have also been described in some HP2 patients. In the original HP2 Irish traveler family, 9 out of the 13 affected individuals developed seizures and two of them had intellectual disability . Van de Ven  reported 5 HP2 patients; all presented with seizures, 3 had intellectual disability and 4 suffered behavioral problems.
2.3.3 Biochemical features and diagnostic biomarkers
The key biochemical features of HP2 are elevated plasma and urinary levels of proline (about 10–15 folds higher in plasma) and P5C. A combination of both biomarkers is diagnostic of HP2 and distinguishes it from hyperprolinemia type 1 [119, 120]. Walker and Mills  identified a new metabolite, N-(pyrrole-2-carboxyl) glycine, that accumulated in urine of HP2 subject. They subsequently confirmed the presence of this compound in another 4 patients and suggested its use as a diagnostic biomarker for HP2. Other metabolic alterations reported in HP2 patients include increased plasma concentrations of lactate [116, 119], glycine [115, 120], ornithine , and alanine  and urinary xanthurenic acid; probably secondary to PLP deficiency . VitB6 was previously analyzed in 5 HP2 patients [59, 116, 119] and found to be decreased in 3 patients and at low normal levels in the other two.
2.3.4 Treatment and its outcome
VitB6 supplementation has been used to treat HP2 associated seizures with variable response. Most of the case studies reported effective control of seizures with vitB6, either alone or in conjugation with anti-seizure medications [59, 114, 116], while few described irresponsiveness to vitB6 therapy . Van de Ven et al.  assessed the long-term clinical outcome in 4 HP2 patients treated with vitB6 and/or anti-seizure medications. Seizures resolved spontaneously in 3 patients by the age of 12–18 years, however, neurobehavioral problems were persistent in most patients despite therapy. The clinical course was non-progressive and did not correlate with the vitB6 dose and vitB6 therapy .
2.4.1 Disease mechanism
Hypophosphatasia (HPP) results from autosomal recessive or dominant mutations affecting
2.4.2 Clinical features
There is a remarkable heterogeneity in the clinical presentation of HPP and 5 principal clinical types have been recognized based on skeletal disease features and age of onset. In order of escalating severity, these types are “odonto”, “adult”, “childhood”, “infantile”, and “perinatal” HPP [124, 125]. The severe forms (infantile and perinatal) show autosomal recessive inheritance, while in the milder forms both autosomal dominant or recessive inheritance has been described [124, 126]. Defective mentalization of bone and/or teeth is the clinical hallmark feature of HPP in all of these types . Seizures are the most well described extra-skeletal feature of HPP and are exclusively observed in the infantile and perinatal types . According to a recent metanalysis , seizures occurred in about 20% of patients with pediatric-onset HPP.
Odonto-HPP is the mildest form and can manifest at any age. It involves minor dental problems like premature shedding of deciduous teeth without any other symptoms [123, 124]. Adult HPP typically manifest during middle age or later and can cause debilitating symptoms like osteomalacia leading to bone fractures, chondrocalcinosis, musculoskeletal pain and loss of dentition. Some patients also suffer from pseudogout due to increased extracellular concentrations of PPi [123, 126]. Childhood HPP presents after the age of 6 months and common features include rickets and premature loss of deciduous teeth. Severe forms are also associated with muscle weakness causing delay in walking and abnormal gait . Infantile HPP is a severe type and can lead to death in about 50% of affected infants . It is diagnosed before 6 months of age and features delayed postnatal development, failure to thrive, hypotonia along with rachitic deformities . Hypercalcemia and hypercalciuria are frequently seen and may lead to renal failure . In rapidly progressive cases, rickets causes thoracic deformity and death may ensue due to respiratory insufficiency [125, 129]. VitB6-dependent seizures may develop, sometimes preceding the skeletal features, and usually predict a fatal outcome [123, 125]. Perinatal HPP is the most severe type in which the symptoms start
2.4.3 Biochemical features and diagnostic biomarkers
HPP can be diagnosed by the presence of pathognomonic skeletal radiographic changes along with characteristic biochemical features. The most commonly used biochemical marker for HPP is low serum alkaline phosphatase activity which consistently observed in all forms of HPP . Other reported biochemical findings in HPP include increased levels of TNSALP substrates PPi and PEA in urine, elevated Pi in plasma, hypercalciuria and/or hypercalcemia and high urinary levels of phosphoserine [6, 123, 124, 126]. These features can only be used to support the diagnosis of HPP because they may not be present in all HPP forms and are sometimes observed in other skeletal diseases. A more sensitive and specific biomarker for HPP is elevated serum levels of PLP, which has been detected even in the mildest form of HPP (odonto-HPP) and the degree of PLP elevation seems to correlate with disease severity [123, 131].
2.4.4 Treatment and its outcome
HPP-related seizures are usually responsive to PN supplementation [56, 129]. Effective treatment against the skeletal manifestations was lacking until the advent of Asfotase alfa, an enzyme-replacement therapy that was approved in 2015 [124, 126]. Asfotase alfa is recombinant, fusion protein consisting of the catalytic ectodomain of human TNSALP, the Fc fragment of human immunoglobulin G1 (IgG1) and a deca-aspartate motif for bone targeting [123, 131, 132]. Clinical trials have demonstrated the long-term safety and efficacy of Asfotase alfa in preventing life-threatening complications of HPP [123, 132, 133]. HPP patients, including those with severe forms, treated with Asfotase alfa showed marked improvements in all clinical aspects (radiography, pulmonary, neurodevelopmental and motor functions) along with resolution of pain and disability [123, 126, 133]. At the biochemical level, Asfotase alfa therapy was associated with normalization of plasma levels of PPi and PLP .
2.5 PLPHP deficiency
2.5.1 Disease mechanism
PLPHP deficiency is the latest addition to B6EEs that is caused by recessive mutations in
2.5.2 Clinical features
The general clinical picture of PLPHP deficiency remarkably overlaps with that of ALDH7A1 deficiency and PNPO deficiency which is dominated by pharmaco-resistant seizures that respond to vitB6 treatment. Seizures typically manifest during the first week of life [7, 67, 68, 135] with possible prenatal onset in some cases  and a recent report of late onset at 14 months of age . Johnstone et al.  reported two patients who presented with fatal mitochondrial encephalopathy and a patient with unique movement disorder who lacked epileptic seizures. Developmental delay, intellectual disability, acquired microcephaly and structural brain abnormalities are common co-morbidities observed in this form of B6EEs [7, 67, 68, 137, 138, 139]. Systemic features like metabolic acidosis, anemia and gastrointestinal problems have been also described in PLPHP-deficient pateints [7, 67].
2.5.3 Biochemical features and diagnostic biomarkers
Biochemical investigations performed in patient samples revealed amino acid and neurotransmitter abnormalities, reflecting the pleiotropic metabolic effects associated with altered PLP homeostasis. Among amino acids, elevated glycine in plasma and/or CSF was the most frequent alteration identified [7, 67, 68]. The enzyme that breaks down glycine, glycine cleavage system, requires PLP as a cofactor . Abnormal monoamine neurotransmitter profile was detected in some patients, possibly due to suboptimal activity of the PLP-dependent enzyme AADC. Reported changes included low CSF levels of HVA (marker of low dopamine) and raised concentrations of 3-OMD, L-dopa, 5-HTP (CSF) and VLA (urine) indicating accumulation of AADC substrates [7, 67, 86]. Low PLP levels were detected in pre-treatment plasma  and CSF  samples from two patients. Johnstone et al.  described accumulation of high levels of PNP in patient fibroblasts and PLPHP-deficient HEK293 cells. There is currently no established biomarker for this disease.
2.5.4 Treatment and its outcome
Seizures typically respond well to vitB6 treatment (PN in majority of cases). In cases with inadequate response to PN, switching to PLP led to better seizure control . About half of the cases required additional anti-seizure medications for optimal seizure control [7, 67]. The addition of folinic acid resulted in improved seizure control in one patient .
While seizures and secondary metabolic alterations are usually normalized with vitB6 therapy, a major fraction of patients still develop some form of neurodevelopmental disability. A recent review of 45 published PLPHP deficiency cases found that 65% of the patients suffered from intellectual disability . The underlying pathophysiological mechanism is not well understood, and currently there is no effective treatment against the neurodevelopmental phenotype of this disorder.
3. Other vitB6-responsive conditions
The therapeutic effect of vitB6 supplementation have been also described in other disease conditions. The following section outlines some examples.
3.1 Hyperphosphatasia with mental retardation syndrome
Hyperphosphatasia with mental retardation (HPMR) syndrome (OMIM Phenotypic Series: PS239300) refers to a group of congenital disorders caused by defects in the biosynthetic pathway of glycosyl phosphatidylinositol (GPI) anchor. GPI-anchor is a glycolipid that is required for tethering of TNSALP and several other proteins (more than 150 in total) to the cell surface and at the blood–brain barrier (BBB) [6, 61]. Six subtypes of HPMR syndrome have been identified to date with variable phenotypic spectrum that extends from mild nonsyndromic intellectual disability (ID) to more complex forms with severe ID, seizures, increased serum alkaline phosphates and dysmorphic features [141, 142, 143]. Low serum PLP has been detected in some patients which may be ascribed to the elevated serum level of alkaline phosphate . Seizures in some HPMR subtypes like PIGV deficiency  and PIGO deficiency  have been shown to respond to pyridoxine treatment.
3.2 PL kinase deficiency
PL kinase (PLK) is an important enzyme in the vitB6 salvage pathway (Figure 2). It is responsible for phosphorylating different vitameric compounds which is a pre-requisite step for their subsequent conversion to the active cofactor PLP (Figure 2). Biallelic mutations in the gene encoding PLK (
3.3 Molybdenum cofactor deficiency
Molybdenum cofactor (MoCoF) deficiency is a severe inherited metabolic disease that causes intractable seizures, developmental delay and structural brain defects. It is due to recessive mutations in either
3.4 Defects in PLP-dependent enzymes
In addition to its coenzymatic role, binding of PLP to its apo-enzymes may also be required for proper folding and correct subcellular targeting of these enzymes [6, 150]. Several inborn errors affecting PLP-dependent enzymes have been described to benefit from PN therapy. Examples are homocystinuria (cystathionine β-synthase deficiency), X-linked sideroblastic anemia (δ-aminolevulinate synthase deficiency), primary hyperoxaluria type I (alanine: glyoxylate aminotransferase (AGT) deficiency), ornithine aminotransferase deficiency and AADC deficiency [6, 9, 150]. The therapeutic effect of PN supplementation could to be attributed to the chaperone-like, stabilizing action of PLP on these mutated proteins . In primary hyperoxaluria type I, it has been hypothesized that at high concentration, PLP promotes AGT dimerization and inhibit the accumulation of monomeric protein species which are mistargeted to the mitochondria [6, 150]. A recent addition to this category of PN-responsive disorders came from the discovery of
3.5 Other epileptic disorders
High-dose vitB6 treatment has been used for seizure control in several other epileptic disorders not related to vitB6 metabolism or its dependent enzymes; such as channelopathies [152, 153, 154] and West syndrome [155, 156, 157]. The specific mechanism of vitB6-repsosivness in these types of seizure disorders is not well recognized. Some authors [6, 154] suggested that vitB6 may have anticonvulsant effects because of the ability of PLP to block P2 purinoceptor 7 (P2X7 receptors), as demonstrated