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GAPVD1 and ANKFY1 Mutations Implicate RAB5 Regulation in Nephrotic Syndrome
Journal of the American Society of Nephrology ( IF 10.3 ) Pub Date : 2018-08-01 , DOI: 10.1681/asn.2017121312
Tobias Hermle 1, 2 , Ronen Schneider 1 , David Schapiro 1 , Daniela A Braun 1 , Amelie T van der Ven 1 , Jillian K Warejko 1 , Ankana Daga 1 , Eugen Widmeier 1 , Makiko Nakayama 1 , Tilman Jobst-Schwan 1 , Amar J Majmundar 1 , Shazia Ashraf 1 , Jia Rao 1 , Laura S Finn 3 , Velibor Tasic 4 , Joel D Hernandez 5 , Arvind Bagga 6 , Sawsan M Jalalah 7 , Sherif El Desoky 8 , Jameela A Kari 8 , Kristen M Laricchia 9 , Monkol Lek 9 , Heidi L Rehm 9 , Daniel G MacArthur 9 , Shrikant Mane 10 , Richard P Lifton 10, 11 , Shirlee Shril 1 , Friedhelm Hildebrandt 12
Affiliation  

Background Steroid-resistant nephrotic syndrome (SRNS) is a frequent cause of CKD. The discovery of monogenic causes of SRNS has revealed specific pathogenetic pathways, but these monogenic causes do not explain all cases of SRNS.

Methods To identify novel monogenic causes of SRNS, we screened 665 patients by whole-exome sequencing. We then evaluated the in vitro functional significance of two genes and the mutations therein that we discovered through this sequencing and conducted complementary studies in podocyte-like Drosophila nephrocytes.

Results We identified conserved, homozygous missense mutations of GAPVD1 in two families with early-onset NS and a homozygous missense mutation of ANKFY1 in two siblings with SRNS. GAPVD1 and ANKFY1 interact with the endosomal regulator RAB5. Coimmunoprecipitation assays indicated interaction between GAPVD1 and ANKFY1 proteins, which also colocalized when expressed in HEK293T cells. Silencing either protein diminished the podocyte migration rate. Compared with wild-type GAPVD1 and ANKFY1, the mutated proteins produced upon ectopic expression of GAPVD1 or ANKFY1 bearing the patient-derived mutations exhibited altered binding affinity for active RAB5 and reduced ability to rescue the knockout-induced defect in podocyte migration. Coimmunoprecipitation assays further demonstrated a physical interaction between nephrin and GAPVD1, and immunofluorescence revealed partial colocalization of these proteins in rat glomeruli. The patient-derived GAPVD1 mutations reduced nephrin-GAPVD1 binding affinity. In Drosophila, silencing Gapvd1 impaired endocytosis and caused mistrafficking of the nephrin ortholog.

Conclusions Mutations in GAPVD1 and probably in ANKFY1 are novel monogenic causes of NS. The discovery of these genes implicates RAB5 regulation in the pathogenesis of human NS.



中文翻译:


GAPVD1 和 ANKFY1 突变暗示肾病综合征中 RAB5 的调节



背景类固醇抵抗性肾病综合征 (SRNS) 是 CKD 的常见原因。 SRNS 单基因病因的发现揭示了特定的发病途径,但这些单基因病因并不能解释所有 SRNS 病例。


方法为了确定 SRNS 的新单基因病因,我们通过全外显子组测序筛选了 665 名患者。然后,我们评估了通过测序发现的两个基因及其突变的体外功能意义,并在足细胞样果蝇肾细胞中进行了补充研究。


结果我们在两个早发性 NS 家族中发现了保守的GAPVD1纯合错义突变,在两个患有 SRNS 的兄弟姐妹中发现了ANKFY1纯合错义突变。 GAPVD1 和 ANKFY1 与内体调节因子 RAB5 相互作用。免疫共沉淀测定表明 GAPVD1 和 ANKFY1 蛋白之间存在相互作用,当在 HEK293T 细胞中表达时,它们也共定位。沉默任一蛋白质都会降低足细胞迁移率。与野生型 GAPVD1 和 ANKFY1 相比,携带患者衍生突变的GAPVD1ANKFY1异位表达时产生的突变蛋白表现出与活性 RAB5 的结合亲和力改变,并降低了挽救敲除诱导的足细胞迁移缺陷的能力。免疫共沉淀测定进一步证明了去氧肾上腺素和 GAPVD1 之间的物理相互作用,免疫荧光揭示了这些蛋白质在大鼠肾小球中的部分共定位。患者来源的GPVD1突变降低了去氧肾上腺素-GAPVD1 结合亲和力。在果蝇中,沉默Gapvd1会损害内吞作用并导致去氧肾上腺素同源物的错误运输。


结论GAPVD1突变(可能还有ANKFY1突变)是 NS 的新单基因原因。这些基因的发现表明 RAB5 在人类 NS 发病机制中的调节。

更新日期:2018-08-01
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