The Folkhälsan Institute of Genetics, Haartmaninkatu 8, 00290 Helsinki, Finland.
Neuroscience Center, University of Helsinki, Viikinkaari 4, 00790 Helsinki, Finland.
Brain. 2017 May 1;140(5):1267-1279. doi: 10.1093/brain/awx040.
Progressive encephalopathy with oedema, hypsarrhythmia, and optic atrophy (PEHO) syndrome is an early childhood onset, severe autosomal recessive encephalopathy characterized by extreme cerebellar atrophy due to almost total granule neuron loss. By combining homozygosity mapping in Finnish families with Sanger sequencing of positional candidate genes and with exome sequencing a homozygous missense substitution of leucine for serine at codon 31 in ZNHIT3 was identified as the primary cause of PEHO syndrome. ZNHIT3 encodes a nuclear zinc finger protein previously implicated in transcriptional regulation and in small nucleolar ribonucleoprotein particle assembly and thus possibly to pre-ribosomal RNA processing. The identified mutation affects a highly conserved amino acid residue in the zinc finger domain of ZNHIT3. Both knockdown and genome editing of znhit3 in zebrafish embryos recapitulate the patients' cerebellar defects, microcephaly and oedema. These phenotypes are rescued by wild-type, but not mutant human ZNHIT3 mRNA, suggesting that the patient missense substitution causes disease through a loss-of-function mechanism. Transfection of cell lines with ZNHIT3 expression vectors showed that the PEHO syndrome mutant protein is unstable. Immunohistochemical analysis of mouse cerebellar tissue demonstrated ZNHIT3 to be expressed in proliferating granule cell precursors, in proliferating and post-mitotic granule cells, and in Purkinje cells. Knockdown of Znhit3 in cultured mouse granule neurons and ex vivo cerebellar slices indicate that ZNHIT3 is indispensable for granule neuron survival and migration, consistent with the zebrafish findings and patient neuropathology. These results suggest that loss-of-function of a nuclear regulator protein underlies PEHO syndrome and imply that establishment of its spatiotemporal interaction targets will be the basis for developing therapeutic approaches and for improved understanding of cerebellar development.
进行性脑水肿、高振幅失律和视神经萎缩（PEHO）综合征是一种早发性儿童发病、严重的常染色体隐性脑病，其特征是由于几乎完全颗粒神经元丧失而导致极度小脑萎缩。通过在芬兰家族中进行纯合子作图、对位置候选基因进行 Sanger 测序以及对外显子组进行测序，发现 ZNHIT3 中的亮氨酸到丝氨酸的同义替换杂合突变是 PEHO 综合征的主要原因。ZNHIT3 编码一种核锌指蛋白，先前涉及转录调控和小核仁核糖核蛋白颗粒组装，因此可能参与了前核糖体 RNA 加工。鉴定的突变影响 ZNHIT3 锌指结构域中高度保守的氨基酸残基。在斑马鱼胚胎中敲低和基因组编辑 znhit3 可重现患者的小脑缺陷、小头症和水肿。这些表型可以通过野生型而非突变型人 ZNHIT3 mRNA 挽救，表明患者的错义替换通过功能丧失机制导致疾病。用 ZNHIT3 表达载体转染细胞系表明，PEHO 综合征突变蛋白不稳定。对小鼠小脑组织的免疫组织化学分析表明，ZNHIT3 在增殖性颗粒细胞前体、增殖性和有丝分裂后颗粒细胞以及浦肯野细胞中表达。在培养的小鼠颗粒神经元和离体小脑切片中敲低 Znhit3 表明，ZNHIT3 对于颗粒神经元的存活和迁移是必不可少的，这与斑马鱼的发现和患者的神经病理学一致。这些结果表明，核调节蛋白的功能丧失是 PEHO 综合征的基础，并暗示建立其时空相互作用靶标将是开发治疗方法和更好地理解小脑发育的基础。
