BACKGROUND Kinesin motor proteins transport intracellular cargo, including mRNA, proteins, and organelles. Pathogenic variants in kinesin-related genes have been implicated in neurodevelopmental disorders and skeletal dysplasias. We identified de novo, heterozygous variants in KIF5B, encoding a kinesin-1 subunit, in four individuals with osteogenesis imperfecta. The variants cluster within the highly conserved kinesin motor domain and are predicted to interfere with nucleotide binding, although the mechanistic consequences on cell signaling and function are unknown. METHODS To understand the in vivo genetic mechanism of KIF5B variants, we modeled the p.Thr87Ile variant that was found in two patients in the C. elegans ortholog, unc-116, at the corresponding position (Thr90Ile) by CRISPR/Cas9 editing and performed functional analysis. Next, we studied the cellular and molecular consequences of the recurrent p.Thr87Ile variant by microscopy, RNA and protein analysis in NIH3T3 cells, primary human fibroblasts and bone biopsy. RESULTS C. elegans heterozygous for the unc-116 Thr90Ile variant displayed abnormal body length and motility phenotypes that were suppressed by additional copies of the wild type allele, consistent with a dominant negative mechanism. Time-lapse imaging of GFP-tagged mitochondria showed defective mitochondria transport in unc-116 Thr90Ile neurons providing strong evidence for disrupted kinesin motor function. Microscopy studies in human cells showed dilated endoplasmic reticulum, multiple intracellular vacuoles, and abnormal distribution of the Golgi complex, supporting an intracellular trafficking defect. RNA sequencing, proteomic analysis, and bone immunohistochemistry demonstrated down regulation of the mTOR signaling pathway that was partially rescued with leucine supplementation in patient cells. CONCLUSION We report dominant negative variants in the KIF5B kinesin motor domain in individuals with osteogenesis imperfecta. This study expands the spectrum of kinesin-related disorders and identifies dysregulated signaling targets for KIF5B in skeletal development.

中文翻译：

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KIF5B 的显性阴性变异通过下调 mTOR 信号传导导致成骨不全。

背景技术驱动蛋白马达蛋白运输细胞内货物，包括mRNA、蛋白质和细胞器。驱动蛋白相关基因的致病性变异与神经发育障碍和骨骼发育不良有关。我们在四名成骨不全症患者中发现了编码驱动蛋白 1 亚基的 KIF5B 的从头杂合变异。这些变体聚集在高度保守的驱动蛋白运动结构域内，预计会干扰核苷酸结合，尽管对细胞信号传导和功能的机制影响尚不清楚。方法 为了了解 KIF5B 变体的体内遗传机制，我们通过 CRISPR/Cas9 编辑对在两名患者中发现的线虫直系同源物 unc-116 相应位置 (Thr90Ile) 的 p.Thr87Ile 变体进行建模，并进行泛函分析。接下来，我们通过显微镜、NIH3T3 细胞、原代人成纤维细胞和骨活检中的 RNA 和蛋白质分析，研究了复发性 p.Thr87Ile 变异的细胞和分子后果。结果 unc-116 Thr90Ile 变体杂合的线虫表现出异常的体长和运动表型，这些表型被野生型等位基因的额外拷贝所抑制，与显性失活机制一致。GFP 标记的线粒体的延时成像显示 unc-116 Thr90Ile 神经元中的线粒体运输有缺陷，为驱动蛋白运动功能受损提供了有力的证据。人类细胞的显微镜研究显示扩张的内质网、多个细胞内空泡和高尔基复合体的异常分布，支持细胞内运输缺陷。RNA 测序、蛋白质组分析和骨免疫组织化学证明 mTOR 信号通路下调，而患者细胞中补充亮氨酸可以部分挽救这一信号通路。结论 我们报告了成骨不全患者 KIF5B 驱动蛋白运动结构域的显性失活变异。这项研究扩大了驱动蛋白相关疾病的范围，并确定了骨骼发育中 KIF5B 失调的信号靶标。