Variants within the Neurotrophic Tyrosine Kinase Receptor Type 2 (NTRK2) gene have been discovered to play a role in developmental and epileptic encephalopathies, a group of debilitating conditions for which little is known about cause or treatment. Here, we determine the functional consequences of two variants: p.Tyr434Cys (Y434C) (located in the transmembrane domain) and p.Thr720Ile (T720I) (located in the catalytic domain). Wild-type and variant cDNAs were constructed and transfected into HEK293 cells. In cell culture, variant Y434C exhibited ligand-independent activation of tropomyosin-related kinase B (TRKB) signaling with an associated abnormal response to brain-derived neurotrophic factor (BDNF) stimulation and increased levels of phosphorylated extracellular signal-regulated kinase (ERK) and ETS like-1 protein (ELK1) activity. Expression of variant T720I resulted in decreased TRKB signaling with reduced mTor activity as determined by decreased levels of phosphorylated S6. With the deleterious mechanisms characterized, we utilized mediKanren (a novel artificial intelligence tool) to identify therapeutics to compensate for the pathological effects. Downregulation of TRKB through inhibition with mediKanren-predicted compound 1NM-PP1 led to decreased MEK activity. Upregulation of TRKB signaling by mediKanren-predicted valproic acid led to subsequent increase of mTor activity. Overall, our results provide further characterization of the pathogenicity of these two variants in the NTRK2 gene. Indeed, Y434C is the first patient-specific NTRK2 variant with demonstrated hypermorphic activity. Furthermore, we observed that variants Y434C and T720I result in distinct functional consequences that require distinct therapeutic strategies. These data suggest the possibility that unique mutations within different regions of the NTRK2 gene results in separate clinical presentations, representing distinct genetic disorders requiring unique therapeutics.

中文翻译：

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引起发育性脑病和癫痫性脑病的 NTRK2 基因变异的功能特征和潜在治疗途径


神经营养性酪氨酸激酶受体 2 型( NTRK2)基因内的变异体被发现在发育性脑病和癫痫性脑病中发挥作用，这是一组令人衰弱的疾病，其原因或治疗方法知之甚少。在这里，我们确定了两个变体的功能后果：p.Tyr434Cys (Y434C)（位于跨膜结构域）和 p.Thr720Ile (T720I)（位于催化结构域）。构建野生型和变体 cDNA 并转染至 HEK293 细胞中。在细胞培养中，变体 Y434C 表现出原肌球蛋白相关激酶 B (TRKB) 信号传导的配体独立激活，并伴有对脑源性神经营养因子 (BDNF) 刺激的异常反应以及磷酸化细胞外信号调节激酶 (ERK) 和ETS like-1 蛋白 (ELK1) 活性。变体 T720I 的表达导致 TRKB 信号传导减少，同时磷酸化 S6 水平降低确定 mTor 活性降低。根据有害机制的特征，我们利用 mediKanren（一种新型人工智能工具）来确定治疗方法以补偿病理效应。通过 mediKanren 预测的化合物 1NM-PP1 抑制来下调 TRKB 导致 MEK 活性降低。 mediKanren 预测的丙戊酸上调 TRKB 信号传导导致 mTor 活性随后增加。总体而言，我们的结果进一步表征了NTRK2基因中这两种变异的致病性。事实上，Y434C 是第一个具有超形态活性的患者特异性NTRK2变体。 此外，我们观察到变体 Y434C 和 T720I 会导致不同的功能后果，需要不同的治疗策略。这些数据表明， NTRK2基因不同区域内的独特突变可能会导致不同的临床表现，代表需要独特治疗的不同遗传性疾病。