Analysis of patient-specific NF1 variants leads to functional insights for Ras signaling that can impact personalized medicine
Analysis of patient-specific NF1 variants leads to functional insights for Ras signaling that can impact personalized medicine
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DOI：
10.1002/humu.24290
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发表时间：
2021-11-15
期刊：
影响因子：
3.9
通讯作者：
Wallis, Deeann
中科院分区：
文献类型：
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作者：
Long, Ashlee;Liu, Hui;Wallis, Deeann
我们创建了一组 29 个代表错义变异的 NF1 变异互补 DNA (cDNA)，其中许多具有临床相关表型、框内缺失、剪接变异和无义变异。我们已经确定了这些变体的功能后果，评估了它们在 NF1 null (-/-) 细胞中产生成熟神经纤维蛋白和恢复 Ras 信号传导活性的能力。 cDNA 显示了神经纤维蛋白水平的变体特异性差异，表明某些变体导致 1 型神经纤维瘤病 (NF1) 基因或蛋白质不稳定或增强降解。当高水平表达时，一些变异蛋白仍然能够抑制 Ras 活性，表明 NF1 表型可能是由于蛋白丰度低所致。相比之下，其他变体蛋白无法抑制 Ras 活性，这表明某些变体蛋白不能在功能上与 Ras 结合并刺激 GTP 酶活性。我们观察到对蛋白质丰度和 Ras 活性的影响可能是相互排斥的。这些测定使我们能够根据功能效应对变异进行分类，可能有助于对未知意义的变异进行分类，并且可能对未来更有针对性的治疗产生影响。
We have created a panel of 29 NF1 variant complementary DNAs (cDNAs) representing missense variants, many with clinically relevant phenotypes, in-frame deletions, splice variants, and nonsense variants. We have determined the functional consequences of the variants, assessing their ability to produce mature neurofibromin and restore Ras signaling activity in NF1 null (-/-) cells. cDNAs demonstrate variant-specific differences in neurofibromin protein levels, suggesting that some variants lead to neurofibromatosis type 1 (NF1) gene or protein instability or enhanced degradation. When expressed at high levels, some variant proteins are still able to repress Ras activity, indicating that the NF1 phenotype may be due to low protein abundance. In contrast, other variant proteins are incapable of repressing Ras activity, indicating that some do not functionally engage Ras and stimulate GTPase activity. We observed that effects on protein abundance and Ras activity can be mutually exclusive. These assays allow us to categorize variants by functional effects, may help to classify variants of unknown significance, and may have future implications for more directed therapeutics.