PLPHP deficiency: clinical, genetic, biochemical, and mechanistic insights

Abstract

Biallelic pathogenic variants in PLPBP (formerly called PROSC) have recently been shown to cause a novel form of vitamin B6-dependent epilepsy, the pathophysiological basis of which is poorly understood. When left untreated, the disease can progress to status epilepticus and death in infancy. Here we present 12 previously undescribed patients and six novel pathogenic variants in PLPBP. Suspected clinical diagnoses prior to identification of PLPBP variants included mitochondrial encephalopathy (two patients), folinic acid-responsive epilepsy (one patient) and a movement disorder compatible with AADC deficiency (one patient). The encoded protein, PLPHP is believed to be crucial for B6 homeostasis. We modelled the pathogenicity of the variants and developed a clinical severity scoring system. The most severe phenotypes were associated with variants leading to loss of function of PLPBP or significantly affecting protein stability/PLP-binding. To explore the pathophysiology of this disease further, we developed the first zebrafish model of PLPHP deficiency using CRISPR/Cas9. Our model recapitulates the disease, with plpbp-/- larvae showing behavioural, biochemical, and electrophysiological signs of seizure activity by 10 days post-fertilization and early death by 16 days post-fertilization. Treatment with pyridoxine significantly improved the epileptic phenotype and extended lifespan in plpbp-/- animals. Larvae had disruptions in amino acid metabolism as well as GABA and catecholamine biosynthesis, indicating impairment of PLP-dependent enzymatic activities. Using mass spectrometry, we observed significant B6 vitamer level changes in plpbp-/- zebrafish, patient fibroblasts and PLPHP-deficient HEK293 cells. Additional studies in human cells and yeast provide the first empirical evidence that PLPHP is localized in mitochondria and may play a role in mitochondrial metabolism. These models provide new insights into disease mechanisms and can serve as a platform for drug discovery.

Keywords: PLPBP; PROSC; epilepsy; pyridoxine; vitamin B6-responsive epilepsy.

Figure 1

Axial T₂ (first three columns) and sagittal (last column) T₁-weighted images of Patients 1, 3 and 4. At age 3 years, the MRI of Patient 1 is normal. Patients 3 and 4 show a simplified gyral pattern, cyst-like structures connected to the anterior horns and a T₂-hyperintense signal of the hilus of the dentate nuclei. White matter signal is T₂ hyperintense and appears swollen. These abnormalities are more severe in Patient 4 (who additionally has intraventricular blood) than in Patient 3. The corpus callosum lacks the most posterior part.

Figure 2

Pathogenic variants reported so far and their genetic location, predicted secondary structure and 3D structure in the PLPHP protein in the context of PLP-binding. (A) Human PLPBP gene structure, protein coding exons shown in dark blue and 5′ and 3′ UTR shown in light blue. Position of the variants reported previously by Darin et al. (2016) and Plecko et al. (2017) are shown in black, seven novel variants identified by this study are shown in red and a splicing variant reported previously but also observed in our cohort is shown in green. (B) 2D graphical representation of the PLPHP protein based on secondary structure prediction and the tridimensional model (shown in D). Blue cylinders represent the outer α-helices and pink arrows represent the inner β-strands that comprise the (β/α)8-TIM barrel structure. Residues observed mutated in PLPHP-deficiency are shown in circles, black for variants reported previously or red for novel variants reported here. Residues located within 6Å of the modelled PLP position are shown in grey. (C) 3D structure of the human PLPHP model showing the PLP molecule in green, the lysine 47 residue in blue and the positions of the residues found mutated in PLPHP deficiency in black or red according to A. (D) Predicted PLP-binding pocket showing the key lysine 47 (K47) as a PLP-Lys adduct (blue and green), PLP-pocket residues (<6Å radius) and the residues found mutated in PLPHP deficiency in black or red according to A. Non-covalent contacts as calculated by Arpeggio are shown; black dashed lines indicate hydrophobic interactions, orange and red dashed lines represent weak and strong hydrogen bonds, grey dashed line represents carbon-pi interaction and a yellow dashed line indicates a methyl-sulphur-pi interaction. Note that the variant p.His275Asp was co-inherited homozygously with p.Thr116Ile in Patient 1, we report this as a variant of unknown significance.

Figure 3

Evidence of mitochondrial enrichment of PLPHP in HeLa cells and growth defects in yeast null for the PLPHP ortholog in several energy sources requiring active mitochondrial metabolism. (A) Western blot of wild-type HeLa cells and HeLa cells with HA-tagged mitochondria (HeLa HA-MITO) that were immunoprecipitated for mitochondrial purification, showing PLPHP enrichment in the mitochondrial fraction, other antibodies show minimal contamination from the cytosol or other organelles. (B) Growth curves of wild-type yeast cells and mutant strains on rich oleate medium. The strains shown are: wild-type (WT, BY4742, blue), fox1Δ (green) and ybl036cΔ (purple). (C) Growth of wild-type and mutant cells after 18 h on 20 g/l glucose and non-fermentable carbon sources: rich oleate, 2% ethanol and 2% glycerol medium. Values are expressed as % growth relative to wild-type. The strains shown are: wild-type (BY4742) (blue), fox1Δ (green) and ybl036cΔ (purple). (D) Growth of wild-type cells and mutant cells on 2% ethanol medium. The strains shown are: wild-type (BY4742) (blue), ybl036cΔ + pPROSC1a (human PLPHP under catalase promoter) (orange) or pPROSC2a (human PLPHP under Tef promoter) (red) and ybl036cΔ + empty vector (purple).

Figure 4

Development of plpbp^−/− zebrafish model by CRISPR/Cas9 and epileptic phenotypic analysis. (A) Chromatograms of zebrafish larvae showing wild-type and the genotypes for homozygous mutants plpbp^ot101/ot101 and plpbp^ot102/ot102. Compound heterozygous mutant larvae (plpbp^ot101/ot102) (not shown) were used for most experiments with the same phenotype as the homozygotes. (B) Cropped western blot (for clarity) showing that no Plphp protein was detected in mutant larvae. Total protein (stain free blot) is shown underneath for standardization. Full blot available in the Supplementary material. (C) Survival curves showing reduced survival of mutant larvae compared to wild-type and the two heterozygous parental types (n = 20 larvae per group). (D and E) Mutant larvae moved a greater total distance during fast speed (>20 mm/s) movements and spent more time in fast movements, respectively (n = 16 larvae per group). (F) Relative mRNA expression showing increased expression of c-fos in mutant larvae compared to wild-type larvae, pentylenetetrazol (PTZ) treatment was used as a positive control. (G) Example electrophysiology recordings of mutant (top) and wild-type (bottom) larvae showing increased number of ictal-like events. Insets are magnified examples (4 s) of ictal-like, interictal and wild-type recordings. Significance: **P < 0.01, *P < 0.05.

Figure 5

Vitamin B6-responsive epilepsy in plpbp^−/− zebrafish larvae. Survival in mutants was moderately improved using PLP (A) but showed a better response that was clearly dose-dependent with pyridoxine (B). (C) Five-minute trace recordings of 11 dpf zebrafish larvae showing increased hyperactivity in the mutants which was alleviated with 10 mM pyridoxine treatment, as measured by (D) time spent in fast movements and (E) distance moved in fast movements. (F) Highest seizure-like behaviour category identified by blinded observers. Only untreated mutant larvae showed evidence of S2 or S3 seizure-like activity. (G) Electrographic activity in mutant larvae was normalized by treatment with 5 mM pyridoxine. (H) Example electrophysiology recordings of untreated and treated mutant larvae. Significance: **P < 0.01, *P < 0.05. PN = pyridoxine; WT = wild-type.

Figure 6

Targeted mass spectrometry studies of plpbp^−/− zebrafish larvae indicates changes in B6 vitamer, amino acid, and neurotransmitter profiles. (A) B6 vitamer profile of mutant and wild-type 10 dpf larvae. (B) Amino acid and neurotransmitter profile of whole larval mutant, heterozygous and wild-type 11 dpf larvae after 24 h fasting. (C) Metabolic pathways for the synthesis and degradation of PLP. (D) Biosynthetic pathways of catecholamines and trace amines, highlighting (in blue) the role of AADC. (E) The serotonin biosynthesis pathway, highlighting the role of AADC. 3-MT = 3-methoxytyramine; 5-HIAA = 5-hydroxyindoleacetic acid; 5-HTTP = 5-hydroxytrytpophan; AADC = aromatic-l-amino acid decarboxylase; AO = aldehyde oxidase I; DH = β-NAD dehydrogenase; PA = 4-pyridoxic acid; PK = pyridoxal kinase; PL = pyridoxal; PM = pyridoxamine; PMP = pyridoxamine 5′-phosphate; PN = pyridoxine; PNPO = PNP oxidase. Significance: ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05.