Glutamatergic synapse dysfunction is believed to underlie the development of Autism Spectrum Disorder (ASD) and Intellectual Disability (ID) in many individuals. However, identification of genetic markers that contribute to synaptic dysfunction in these individuals is notoriously difficult. Based on genomic analysis, structural modeling, and functional data, we recently established the involvement of the TRIO-RAC1 pathway in ASD and ID. Furthermore, we identified a pathological de novo missense mutation hotspot in TRIO’s GEF1 domain. ASD/ID-related missense mutations within this domain compromise glutamatergic synapse function and likely contribute to the development of ASD/ID. The number of ASD/ID cases with mutations identified within TRIO’s GEF1 domain is increasing. However, tools for accurately predicting whether such mutations are detrimental to protein function are lacking. Here we deployed advanced protein structural modeling techniques to predict potential de novo pathogenic and benign mutations within TRIO’s GEF1 domain. Mutant TRIO-9 constructs were generated and expressed in CA1 pyramidal neurons of organotypic cultured hippocampal slices. AMPA receptor-mediated postsynaptic currents were examined in these neurons using dual whole-cell patch clamp electrophysiology. We also validated these findings using orthogonal co-immunoprecipitation and fluorescence lifetime imaging (FLIM-FRET) experiments to assay TRIO mutant overexpression effects on TRIO-RAC1 binding and on RAC1 activity in HEK293/T cells. Missense mutations in TRIO’s GEF1 domain that were predicted to disrupt TRIO-RAC1 binding or stability were tested experimentally and found to greatly impair TRIO-9’s influence on glutamatergic synapse function. In contrast, missense mutations in TRIO’s GEF1 domain that were predicted to have minimal effect on TRIO-RAC1 binding or stability did not impair TRIO-9’s influence on glutamatergic synapse function in our experimental assays. In orthogonal assays, we find most of the mutations predicted to disrupt binding display loss of function but mutants predicted to disrupt stability do not reflect our results from neuronal electrophysiological data. We present a method to predict missense mutations in TRIO’s GEF1 domain that may compromise TRIO function and test for effects in a limited number of assays. Possible limitations arising from the model systems employed here can be addressed in future studies. Our method does not provide evidence for whether these mutations confer ASD/ID risk or the likelihood that such mutations will result in the development of ASD/ID. Here we show that a combination of structure-based computational predictions and experimental validation can be employed to reliably predict whether missense mutations in the human TRIO gene impede TRIO protein function and compromise TRIO’s role in glutamatergic synapse regulation. With the growing accessibility of genome sequencing, the use of such tools in the accurate identification of pathological mutations will be instrumental in diagnostics of ASD/ID.

中文翻译：

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通过基于结构的新型方法检测自闭症谱系障碍相关的致病性三重变异

谷氨酸能突触功能障碍被认为是许多人患上自闭症谱系障碍 (ASD) 和智力障碍 (ID) 的基础。然而，识别导致这些个体突触功能障碍的遗传标记非常困难。基于基因组分析、结构建模和功能数据，我们最近确定了 TRIO-RAC1 通路在 ASD 和 ID 中的参与。此外，我们在 TRIO 的 GEF1 结构域中发现了一个病理性从头错义突变热点。该域内与 ASD/ID 相关的错义突变会损害谷氨酸能突触功能，并可能导致 ASD/ID 的发生。 TRIO 的 GEF1 结构域内发现突变的 ASD/ID 病例数量正在增加。然而，缺乏准确预测此类突变是否损害蛋白质功能的工具。在这里，我们部署了先进的蛋白质结构建模技术来预测 TRIO 的 GEF1 结构域内潜在的从头致病性和良性突变。突变体 TRIO-9 构建体在器官型培养海马切片的 CA1 锥体神经元中生成并表达。使用双全细胞膜片钳电生理学检查这些神经元中 AMPA 受体介导的突触后电流。我们还使用正交免疫共沉淀和荧光寿命成像 (FLIM-FRET) 实验验证了这些发现，以测定 HEK293/T 细胞中 TRIO 突变体过表达对 TRIO-RAC1 结合和 RAC1 活性的影响。 TRIO 的 GEF1 结构域中的错义突变预计会破坏 TRIO-RAC1 结合或稳定性，经过实验测试，发现会极大地损害 TRIO-9 对谷氨酸能突触功能的影响。相比之下，在我们的实验测定中，预计对 TRIO-RAC1 结合或稳定性影响最小的 TRIO GEF1 结构域中的错义突变并没有削弱 TRIO-9 对谷氨酸突触功能的影响。在正交测定中，我们发现大多数预测会破坏结合的突变表现出功能丧失，但预测会破坏稳定性的突变体并不能反映我们从神经元电生理学数据中得到的结果。我们提出了一种方法来预测 TRIO 的 GEF1 结构域中可能损害 TRIO 功能的错义突变，并在有限数量的测定中测试效果。这里使用的模型系统可能产生的局限性可以在未来的研究中解决。我们的方法没有提供证据证明这些突变是否会带来 ASD/ID 风险，或者此类突变是否会导致 ASD/ID 的发展。在这里，我们表明，基于结构的计算预测和实验验证的结合可以用于可靠地预测人类 TRIO 基因中的错义突变是否会阻碍 TRIO 蛋白功能并损害 TRIO 在谷氨酸能突触调节中的作用。随着基因组测序的日益普及，使用此类工具准确识别病理突变将有助于 ASD/ID 的诊断。