Pathogenic variants in TMEM184B cause a neurodevelopmental syndrome associated with alteration of metabolic signaling

Transmembrane protein 184B (TMEM184B) is an endosomal 7-pass transmembrane protein with evolutionarily conserved roles in synaptic structure and axon degeneration. We report six pediatric cases who have de novo heterozygous variants in TMEM184B. All individuals harbor rare missense or mRNA splicing changes and have neurodevelopmental deficits including developmental delay, corpus callosum hypoplasia, seizures, and/or microcephaly. TMEM184B is predicted to contain a pore domain, wherein many human disease-associated variants cluster. Structural modeling suggests that all missense variants alter TMEM184B protein stability. To understand the contribution of TMEM184B to neural development in vivo, we suppressed the TMEM184B ortholog in zebrafish and observed microcephaly and reduced anterior commissural neurons, aligning with symptoms of affected individuals. Ectopic TMEM184B expression resulted in dominant effects for Lys184Glu and Gly162Arg. However, in vivo complementation studies demonstrate that all other variants tested result in diminished protein function and indicate a haploinsufficiency basis for disease. Human induced pluripotent stem cells (iPSC) with monoallelic expression of Lys184Glu show mRNA disruptions in key metabolic pathways and molecules including those controlling mechanistic target of rapamycin (mTOR) activity. Expression of Lys184Glu and other variants increased apoptosis in cell lines and altered nuclear localization of transcription factor EB (TFEB), a master regulator of lysosomal biogenesis, further supporting disruptions to nutrient signaling pathways. Together, our data indicate that TMEM184B variants cause cellular metabolic disruption likely through divergent molecular effects that all result in abnormal neural development. Overall design: RNAseq profiling of induced pluripotent stem cells (iPSCs) to determine mRNA levels of TMEM184b and expression of transcripts related to metabolism and cellular pH. The assessed cell lines belonged to one of three conditions, each containing four biological replicates: wild type (WT), Lys184Glu/+, or Lys184Glu/Lys184Glu. Non-WT samples exhibited either a heterozygous or homozygous mutation to TMEM184b that mimic mutations observed in human patients.

跨膜蛋白184B（TMEM184B）是一种定位于内体的7次跨膜蛋白，在突触结构与轴突变性过程中发挥进化保守的功能。本研究报道6例携带TMEM184B基因新发杂合变异的儿科病例。所有受试者均携带罕见错义变异或mRNA剪接改变，且存在神经发育缺陷，包括发育迟缓、胼胝体发育不全、癫痫及/或小头畸形。预测TMEM184B含有一个孔结构域，该区域聚集了大量与人类疾病相关的变异位点。结构建模结果显示，所有错义变异均会改变TMEM184B蛋白的稳定性。为阐明TMEM184B在体内对神经发育的调控作用，本研究敲低了斑马鱼中的TMEM184B同源基因，并观察到小头畸形以及前连合神经元数量减少的表型，这与患者的临床症状相符。异位表达TMEM184B的实验显示，Lys184Glu与Gly162Arg变异存在显性效应。但体内互补实验结果表明，其余所有被检测的变异均会导致蛋白功能受损，提示该病的发病机制为单倍体剂量不足。携带单等位基因Lys184Glu变异的人类诱导多能干细胞（human induced pluripotent stem cells，iPSC）在关键代谢通路及分子（包括调控雷帕霉素靶蛋白（mechanistic target of rapamycin，mTOR）活性的相关分子）中出现mRNA表达紊乱。表达Lys184Glu及其他变异的细胞系中细胞凋亡水平升高，同时溶酶体生物合成的主调控因子转录因子EB（transcription factor EB，TFEB）的核定位发生改变，进一步证实营养信号通路存在紊乱。综上，本研究数据表明，TMEM184B变异可能通过多种不同的分子机制引发细胞代谢紊乱，最终均导致神经发育异常。整体实验设计：对诱导多能干细胞（iPSC）进行RNA测序（RNAseq）分析，以检测TMEM184B的mRNA水平以及与代谢、细胞pH相关的转录本表达情况。所评估的细胞系分为3组，每组均包含4个生物学重复：野生型（wild type，WT）组、Lys184Glu/+杂合突变组以及Lys184Glu/Lys184Glu纯合突变组。非野生型样本均携带模拟人类患者的TMEM184B杂合或纯合突变。