Mutations in PROSC Disrupt Cellular Pyridoxal Phosphate Homeostasis and Cause Vitamin-B6-Dependent Epilepsy

Abstract

Pyridoxal 5'-phosphate (PLP), the active form of vitamin B₆, functions as a cofactor in humans for more than 140 enzymes, many of which are involved in neurotransmitter synthesis and degradation. A deficiency of PLP can present, therefore, as seizures and other symptoms that are treatable with PLP and/or pyridoxine. Deficiency of PLP in the brain can be caused by inborn errors affecting B₆ vitamer metabolism or by inactivation of PLP, which can occur when compounds accumulate as a result of inborn errors of other pathways or when small molecules are ingested. Whole-exome sequencing of two children from a consanguineous family with pyridoxine-dependent epilepsy revealed a homozygous nonsense mutation in proline synthetase co-transcribed homolog (bacterial), PROSC, which encodes a PLP-binding protein of hitherto unknown function. Subsequent sequencing of 29 unrelated indivduals with pyridoxine-responsive epilepsy identified four additional children with biallelic PROSC mutations. Pre-treatment cerebrospinal fluid samples showed low PLP concentrations and evidence of reduced activity of PLP-dependent enzymes. However, cultured fibroblasts showed excessive PLP accumulation. An E.coli mutant lacking the PROSC homolog (ΔYggS) is pyridoxine sensitive; complementation with human PROSC restored growth whereas hPROSC encoding p.Leu175Pro, p.Arg241Gln, and p.Ser78Ter did not. PLP, a highly reactive aldehyde, poses a problem for cells, which is how to supply enough PLP for apoenzymes while maintaining free PLP concentrations low enough to avoid unwanted reactions with other important cellular nucleophiles. Although the mechanism involved is not fully understood, our studies suggest that PROSC is involved in intracellular homeostatic regulation of PLP, supplying this cofactor to apoenzymes while minimizing any toxic side reactions.

Keywords: PROSC; YggS; developmental delay; epilepsy; homeostasis; microcephaly; pyridoxal 5’-phosphate; pyridoxine; treatment; vitamin B6.

Figure 1

Enzymes and Transporters Involved in Mammalian CNS PLP Synthesis and Homeostasis and Known Human Genetic Vitamin-B₆-Dependent Epilepsies Pyridoxal 5′-phosphate (PLP); pyridoxamine 5′-phosphate (PMP); pyridoxal (PL); pyridoxine (PN); pyridoxine 5′-phosphate (PNP); pyridoxine-5′-β-D-glucoside (PNG); intestinal phosphatases (IP); transporter (identity unknown; T1); pyridoxal kinase (PK); pyridox(am)ine 5′-phosphate oxidase (PNPO); tissue non-specific alkaline phosphatase (TNSALP); pyridoxal-phosphatase (PLPase); aldehyde oxidase (Mo cofactor)/β-NAD dehydrogenase (AOX/DH). (1) PNPO is controlled by feedback inhibition from PLP. (2) PLP functions as a co-factor, forming Schiff bases with the ε-amino group of lysine residues of proteins. (3) PLP can be formed by recycling the cofactor from degraded enzymes (salvage pathway). (4) PLP levels are maintained, in part, by circadian-clock-controlled transcription factors with PAR bZip transcription factors (DBP, HLF, and TEF) targeting PK. (5) PNPO mutations cause a B₆-dependent epilepsy disorder. (6) Disorders resulting in accumulation of L-Δ¹-pyrroline-5-carboxylic acid (P5C) and Δ¹-piperideine-6-carboxylic acid (P6C) (hyperprolinaemia type II and pyridoxine-dependent epilepsy due to mutations in ALDH4A1 and ALDH7A1, respectively) cause decreases in bioavailable PLP as do reactions with exogenous small molecules.

Figure 2

Pedigree of Index Family and Position of PROSC Variants (A) Pedigree of index family (subjects 1–3) and segregation analysis of c.233C>G (p.Ser78Ter) for this family. Affected individuals are homozygous for GG. Analysis of extended family DNA demonstrated that both parents are heterozygous carriers of the identified variant, and all other individuals were found to either be wild-type (CC) or heterozygous (GC) for this variant. Squares represent males, circles represent females, and a double line represents a consanguineous union. Black shapes represent affected individuals, shown subsequently to be homozygous for c.233C>G. The diagonal line through the square indicates that this individual is deceased. Genotypes: II.2, II.4 = GC; III.1, III.2, III.3, III.4, III.5 = GC; III.6 = CC; IV.1, IV.2, IV.6 = GG; IV.3, IV.4 = GC; IV.5 = CC. (B) Predicted features of PROSC and position of mutations. This gene spans 17.17 kb and consists of eight exons. The translated protein is 275 amino acids in length. Blue arrows indicate positions of mutations detected. The green line represents the PLP-binding barrel domain (amino acids 21–250), and the blue line represents the alanine racemase N-terminal domain (amino acids 17–251). K47 is proposed to have an N6-pyridoxal-phosphate modification (by similarity). A cAMP- and cGMP-dependent protein kinase phosphorylation site, RKGS (amino acids 132–135), and a putative N-linked glycosylation site, NTGS (amino acids 146–149), are also predicted. aa, amino acids.

Figure 3

CT and MRI Brain Features of Individuals with Mutations in PROSC (A) Computed tomography (CT) head scan (axial) of subject 1 at 2 months of age showing underdevelopment of brain with broad gyri and shallow sulci, as well as a cyst adjacent to the left frontal horn (white arrow). (B and C) MRI scan (axial T2 weighted) of subject 4 at 2 months of age showing underdevelopment of brain with broad gyri and shallow sulci, as well as subcortical and deep white matter edema and white matter petechial hemorrhages (black arrow). (D) MRI scan (axial, T2 flair) from subject 5 at 16 days of age showing global underdevelopment of brain with coarse gyral pattern and bilateral large cysts adjacent to the frontal horns. (E) MRI scan (axial, T2 flair) from subject 5 at 1 year and 6 months of age showing global underdevelopment of the brain with a coarse gyral pattern, more severe at the frontal poles, and underdevelopment of white matter.

Figure 4

Loss of PROSC Expression Affects Intracellular PLP Homeostasis (A) qRT-PCR showing PROSC expression in affected individuals and wild-type. Reference genes: β-Actin and GAPDH. Data are presented as means ± SD. Statistical analysis was performed with Student’s two tailed t test; ^∗∗∗p < 0.001. Controls, n = 8. Samples from affected individuals and heterozygotes, n = 3. (B) Western blot analysis of control fibroblasts, fibroblasts from PROSC-deficient individuals and a Ser78Ter PROSC heterozygote. β-Actin was used as a control. Full-length wild-type PROSC is 30 kDa, faint band (subject 5) is 26 kDa. (C) PROSC cDNA products from wild-type and subject 5 fibroblasts. (D) Total fibroblast PLP of controls, PROSC-deficient individuals, and Ser78Ter heterozygote. PLP was quantified relative to D2-PLP internal standard and normalized (mg cell protein). Data presented as means ± SD. Statistical analysis performed with Student’s two tailed t test; comparison of controls with PROSC-deficient samples; ^∗∗∗p < 0.0001. QC, quality control sample. QC, n = 5. Controls, n = 18. PROSC-deficient subjects and heterozygote, n > 3.

Figure 5

Complementation of E. coli ΔyggS with Human Wild-Type and Mutant PROSC E. coli ΔyggS were transformed with an empty vector (pBAD33) as a negative control, human wild-type PROSC, and the mutant versions encoding p.Ser78Ter, p.Leu175Pro, p.Pro87Leu, or p.Arg241Gln and grown in the presence of discs impregnated with (1) 20 μL 0.1 mg/mL pyridoxine or (2) 20 μL 1 mg/mL pyridoxine. Pyridoxine produces a ring or rings of growth inhibition around the disc. This is prevented by transfection with wild-type PROSC or PROSC encoding the p.Pro87Leu variant. It is not prevented by transfection with PROSC encoding p.Ser78Ter, p.Leu175Pro, or p.Arg241Gln. The latter exacerbates growth inhibition, producing a filled circle rather than a ring.