Children's Hospital of Eastern Ontario Research Institute, Ottawa, ON, Canada.
Department of Biology, University of Ottawa, Ottawa, ON, Canada.
Brain. 2019 Mar 1;142(3):542-559. doi: 10.1093/brain/awy346.
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.
PLPBP（以前称为 PROSC）中的双等位致病性变异最近被证实可导致一种新的维生素 B6 依赖性癫痫形式，其病理生理基础尚不清楚。如果不治疗，疾病会在婴儿期进展为癫痫持续状态和死亡。在这里，我们介绍了 12 名以前未描述的患者和 PLPBP 中的 6 种新的致病性变异。在鉴定 PLPBP 变异之前，疑似临床诊断包括线粒体脑病（2 名患者）、叶酸反应性癫痫（1 名患者）和运动障碍与 AADC 缺乏症一致（1 名患者）。编码蛋白 PLPHP 被认为对 B6 稳态至关重要。我们对变异的致病性进行了建模，并开发了一种临床严重程度评分系统。最严重的表型与导致 PLPBP 功能丧失或显著影响蛋白稳定性/PLP 结合的变异有关。为了进一步探索这种疾病的病理生理学，我们使用 CRISPR/Cas9 开发了第一个 PLPHP 缺乏症的斑马鱼模型。我们的模型重现了疾病，plpbp-/- 幼虫在受精后 10 天出现癫痫活动的行为、生化和电生理迹象，并在受精后 16 天早期死亡。用吡哆醇治疗可显著改善 plpbp-/- 动物的癫痫表型并延长其寿命。幼虫的氨基酸代谢以及 GABA 和儿茶酚胺生物合成受到干扰，表明 PLP 依赖性酶活性受损。使用质谱，我们观察到 plpbp-/- 斑马鱼、患者成纤维细胞和 PLPHP 缺陷型 HEK293 细胞中的 B6 维生素水平发生显著变化。人类细胞和酵母的进一步研究提供了第一个经验证据，表明 PLPHP 定位于线粒体，可能在线粒体代谢中发挥作用。这些模型为疾病机制提供了新的见解，并可以作为药物发现的平台。
