PDXK mutations cause polyneuropathy responsive to pyridoxal 5'-phosphate supplementation

Abstract

Objective: To identify disease-causing variants in autosomal recessive axonal polyneuropathy with optic atrophy and provide targeted replacement therapy.

Methods: We performed genome-wide sequencing, homozygosity mapping, and segregation analysis for novel disease-causing gene discovery. We used circular dichroism to show secondary structure changes and isothermal titration calorimetry to investigate the impact of variants on adenosine triphosphate (ATP) binding. Pathogenicity was further supported by enzymatic assays and mass spectroscopy on recombinant protein, patient-derived fibroblasts, plasma, and erythrocytes. Response to supplementation was measured with clinical validated rating scales, electrophysiology, and biochemical quantification.

Results: We identified biallelic mutations in PDXK in 5 individuals from 2 unrelated families with primary axonal polyneuropathy and optic atrophy. The natural history of this disorder suggests that untreated, affected individuals become wheelchair-bound and blind. We identified conformational rearrangement in the mutant enzyme around the ATP-binding pocket. Low PDXK ATP binding resulted in decreased erythrocyte PDXK activity and low pyridoxal 5'-phosphate (PLP) concentrations. We rescued the clinical and biochemical profile with PLP supplementation in 1 family, improvement in power, pain, and fatigue contributing to patients regaining their ability to walk independently during the first year of PLP normalization.

Interpretation: We show that mutations in PDXK cause autosomal recessive axonal peripheral polyneuropathy leading to disease via reduced PDXK enzymatic activity and low PLP. We show that the biochemical profile can be rescued with PLP supplementation associated with clinical improvement. As B₆ is a cofactor in diverse essential biological pathways, our findings may have direct implications for neuropathies of unknown etiology characterized by reduced PLP levels. ANN NEUROL 2019;86:225-240.

Figure 1

Biallelic PDXK mutations are associated with axonal polyneuropathy and optic atrophy. (A) Pedigree of the 2 families with PDXK mutations. * = individuals examined. Arrow = proband. (B) Phenotype of the cases with PDXK mutations presenting with muscle atrophy of the intrinsic muscles of the hands, with clawing of the hands, thin wrists, pes cavus, and muscle atrophy in the feet and calves (B1‐B3, Case F1‐II‐5; B4‐B6, Case F1‐II‐6). (C) Fundoscopy‐confirmed bilateral optic disc atrophy in both cases (C1, F1‐II‐5; C2, F1‐II‐6). (D) Nerve biopsy from Case F1‐II‐6 showing axonopathy. There is diffuse and severe depletion of both small and large myelinated axons with most of the surviving axons being <5μm in diameter. There were no axonal ovoids, but there were regenerating clusters (arrows) consistent with longstanding indolent axonopathy. There was no demyelination process. (E) Sanger sequencing confirming the homozygous c.682G>A mutation in the 2 affected siblings and heterozygous state in an unaffected family member in Family 1, and homozygous c.659G>A in the 2 affected siblings and segregation in Family 2. (F) Vitamin B₆ metabolic pathway. Phosphorylated B₆ vitamers (pyridoxamine 5′‐phosphate [PMP]; pyridoxine 5′‐phosphate [PNP]; pyridoxal 5′‐phosphate [PLP]) present in the diet are hydrolyzed to pyridoxal (PL), pyridoxamine (PM), and pyridoxine (PN) by intestinal phosphatases prior to absorption and then converted to their 5′‐phosphate derivatives in the liver by PL kinase (PDXK). PNP and PMP are then converted to PLP by pyridox(am)ine 5′‐phosphate oxidase (PNPO). PLP re‐enters the circulation bound to a lysine residue of albumin. Homeostatic regulation of tissue levels of PLP is achieved by various mechanisms, including feedback inhibition of PNPO and PL kinase by PLP.62 Albumin in plasma, hemoglobin in erythrocytes, and glycogen phosphorylase in muscle also play a role, binding to PLP and helping to keep concentrations of this very reactive aldehyde low63 so as to avoid any unwanted reactions with biologically important molecules. Subsequent delivery of PLP to the tissues requires hydrolysis of circulating PLP to PL by the ectoenzyme tissue nonspecific alkaline phosphatase (TNSALP). The resulting pyridoxal is able to enter cells prior to being rephosphorylated by PL kinase to produce the active cofactor, PLP, required by B₆‐dependent apoenzymes.64 Within cells, recycling pathways also exist, with PMP being oxidized by PNPO to form PLP.63 AOX = aldehyde oxidase; GPI = glycosylphosphatidylinositol; PA = pyridoxic acid.

Figure 2

PDXK is highly expressed in the peripheral and central nervous systems and in the same regulon with genes already linked to axonal peripheral neuropathies. (A) Expression of PDXK in human tissues. Box and whisker plots show the expression of PDXK across multiple human tissues. Data were generated by the GTEx Consortium. Expression in tibial nerve is highlighted with a dark gray arrow and is among the tissues with the highest PDXK expression. TPM = Transcripts Per Kilobase Million. (B) Expression of PDXK in multiple cell types of the mouse central nervous system (CNS) and peripheral nervous system (PNS) generated using single cell RNA‐seq. PDXK gene expression across single cells isolated from the mouse central and peripheral nervous systems and displayed using a heatmap demonstrates highest expression of this gene in neurons of the mouse hindbrain,38 with expression in the peripheral neurons including sensory neurons. CB = cerebellum; GC = glial cells; HC = hippocampus; MSN = medium spiny neurons; NBL = neuroblasts; OB = olphactory bulb; Symp = sympathetic. (C) Top‐down plot of the black module genes in the tibial nerve tissue. Only the most connected genes are shown. PDXK gene is highlighted in yellow. Genes known to be associated with the Gene Ontology term GO:0055114, oxidation–reduction process, are highlighted in red. The size of gene nodes reflects their connectivity with the rest of the genes in the module. PDXK is among the top 60 most connected genes. Proximity of genes in the plot reflects their similarity in terms of shared connections with other genes. Interestingly, within the PDXK regulon from the tibial nerve, we found DHTKD1 already linked to Mendelian disorders and associated with primary peripheral axonal neuropathy.59, 60 (D) Conservation of p. Ala228Thr and p.Arg220Gln in PDXK across species. (E) Crystal structure of human pyridoxal kinase with bound adenosine triphosphate (ATP; PDB accession number 3KEU). PDXK is a dimeric enzyme with 1 active site per monomer65 (monomers A and B are depicted in green and yellow, respectively). In the PDXK structure, the backbone‐carbonyl oxygen of alanine 228 establishes a hydrogen bond with the adenine NH₂ group of ATP. The active site of each monomer binds 1 ATP molecule, two Mg²⁺ ions, and 1 Na²⁺ ion. The ATP‐binding site is composed of a β‐loop‐β structure, often referred as a flap, which provides numerous hydrogen‐bond interactions to the ATP β‐ and γ‐phosphates, and sequesters the ATP for catalysis.39 Arginine 220 is located in the β9, in the vicinity of the ATP‐binding site. PDB = Protein Data Bank.

Figure 3

PDXK mutations lead to reduced pyridoxal (PL) kinase enzymatic activity and low PL 5′‐phosphate (PLP). (A) Circular dichroism analyses of recombinant PDXK wild‐type (WT) and p.Ala228Thr mutant proteins. The left and right panels show the normalized far‐ultraviolet (UV) and near‐UV spectra of the 2 proteins, respectively. A clear difference in secondary structure content between the 2 proteins is observed from the far‐UV experiment. CD = circular dichroism. (B) Analysis of the interaction of nonhydrolyzable analogue adenosine 5′‐(3‐thiotriphosphate) tetralithium salt (ATPγS) with PDXK WT and p.Ala228Thr mutant proteins by isothermal titration calorimetry. The left panel shows the titration of ATPγS (250μM) into a PDXK WT solution (25μM). The thermogram shows that the interaction was entropically and enthalpically favored, with ΔH = −3.27 ± 0.42kcal/mol, TΔS = −4.42 ± 0.48kcal/mol, K_D = 2.33 ± 0.25μM, and ΔG = −7.69 ± 0.06kcal/mol. The stoichiometry was 0.80 ± 0.02μM, indicating that each molecule of PDXK binds to 1 molecule of ATPγS. The right panel reports the titration of ATPγS (250μM) into a PDXK p.Ala228Thr solution (25μM). The experiment showed no interaction under the experimental conditions tested, suggesting that the mutation affected the ability of the kinase to bind the analogue substrate ATPγS. (C) Western blot analysis shows normal expression of the PDXK protein in cases compared to controls. (D) Activity of recombinant WT and p.Ala228Thr PL kinase protein measured as PLP formation. Conditions: 0–100μmol/l PL, 300μmol/l MgATP, 20mmol/l potassium phosphate, pH 7.0, 37°C, 10‐minute incubation with 100ng recombinant protein. Points displayed are a mean of 3 repeats. V_max: WT = 2.17μmol/l/h, p.Ala228Thr = 2.52μmol/l/h. K_m: WT = 14.53μmol/l, p.Ala228Thr = 31.93μmol/l. MUT = mutant. (E) Kinetics of recombinant WT and p.Ala228Thr PL kinase protein upon variation of PL concentration. PL kinase activity of recombinant human WT and p.Ala228Thr PDXK protein is measured as PLP formed after incubation with the substrate PL. Incubations were performed in the presence of variable concentrations of MgATP (0–500μmol/l) and 50μmol/l PL. Kinetics were sigmoidal, and parameters established were as follows. WT: k_0.5 = 53.4μmol/l, V_max = 16.8pmol/h; p.Ala228Thr: k_0.5 = 174.4μmol/l, V_max = 6.3pmol/h. Results indicate a dramatic reduction in the catalytic efficiency of the p.Ala228Thr PDXK protein. n = 3 at each data point. (F) Erythrocyte PDXK activity in dried blood spots (DBSs) from cases homozygous for the p.Ala228Thr and p.Arg220Gln versus controls (age = 15–92 years). Patients homozygous for p.Ala228Thr and p.Arg220Gln have lower activity than all controls. Activity measured as PLP formed after incubation of a 3mm DBS punch with PL. Each sample was analyzed in duplicate, and the mean is shown. There was no correlation of PDXK activity with age. (G, H) Comparison of plasma PLP concentrations (retention time = 2.78/2.84 minutes) in control (red) and cases carrying the PDXK mutation (blue) p.Arg220Gln (G) and p.Ala228Thr (H) show a significant reduction of PLP in the case samples (7.8 and 9nmol/l, respectively) versus control (control range = 25–75nmol/l).

Figure 4

Pyridoxal (PL) 5′‐phosphate (PLP) supplementation in patients with PDXK mutations can rescue the biochemical phenotype. (A) Concentrations of plasma B₆ vitamers in affected homozygous (hom) cases for p.Ala228Thr (F1‐II‐5), p.Arg220Gln (F2‐II‐2), and a heterozygous (het; F1‐III‐1) PDXK mutation carrier. The levels prior to supplementation were compared to the published range of B₆ vitamers in adult controls (n = 523)66 not receiving PLP. All units are nmol/l, except for PNP, which is given in concentration units. nd = not detected; PA = 4‐pyridoxic acid; PM = pyridoxamine; PN = pyridoxine; RI = reference interval. (B) The effect of PLP supplementation on plasma PLP concentrations in a case with PDXK mutations. The red bar represents the PLP levels in a group of adult controls with no B₆ supplementation. There is a significant difference in the plasma PLP concentration of F1‐II‐5 before supplementation (blue bar) and on PLP replacement (magenta bar; ***p < 0.05). The difference between groups was tested with the use of a 1‐way analysis of variance test followed by the Tukey–Kramer test. The horizontal lines on the bars indicate mean value ±1 standard deviation. (C) Neurofilament light chain (NFL) concentrations in plasma from cases with homozygous p.Arg220Gln and p.Ala228Thr PDXK mutations and a heterozygous carrier (F1‐III‐1). The blue bars show that NFL levels prior to PLP supplementation are high and consistent with values published in other inherited peripheral neuropathies (solid line),44 indicating ongoing axonal damage. The orange, gray, and yellow bars show the NFL levels in the cases from Family 1 at 4, 12, and 24 months on PLP supplementation, respectively. The levels have reduced to that of normal controls (dashed line) and continued to improve with longitudinal follow‐up, suggesting an amelioration of the axonal breakdown.