PDF(Pubmed)
Abstract：
BACKGROUND: Congenital anomalies of the kidney and urinary tract (CAKUT) are prevalent birth defects. Although pathogenic CAKUT genes are known, they are insufficient to reveal the causes for all patients. Our previous studies indicated GEN1 as a pathogenic gene of CAKUT in mice, and this study further investigated the correlation between GEN1 and human CAKUT.
METHODS: In this study, DNA from 910 individuals with CAKUT was collected; 26 GEN1 rare variants were identified, and two GEN1 (missense) variants in a non-CAKUT group were found. Mainly due to the stability results of the predicted mutant on the website, in vitro, 10 variants (eight CAKUT, two non-CAKUT) were selected to verify mutant protein stability. In addition, mainly based on the division of the mutation site located in the functional region of the GEN1 protein, 8 variants (six CAKUT, two non-CAKUT) were selected to verify enzymatic hydrolysis, and the splice variant GEN1 (c.1071 + 3(IVS10) A > G) was selected to verify shear ability. Based on the results of in vitro experiments and higher frequency, three sites with the most significant functional change were selected to build mouse models.
RESULTS: Protein stability changed in six variants in the CAKUT group. Based on electrophoretic mobility shift assay of eight variants (six CAKUT, two non-CAKUT), the enzymatic hydrolysis and DNA-binding abilities of mutant proteins were impaired in the CAKUT group. The most serious functional damage was observed in the Gen1 variant that produced a truncated protein. A mini-gene splicing assay showed that the variant GEN1 (c.1071 + 3(IVS10) A > G) in the CAKUT group significantly affected splicing function. An abnormal exon10 was detected in the mini-gene splicing assay. Point-mutant mouse strains were constructed (Gen1: c.1068 + 3 A > G, p.R400X, and p.T105R) based on the variant frequency in the CAKUT group and functional impairment in vitro study and CAKUT phenotypes were replicated in each.
CONCLUSIONS: Overall, our findings indicated GEN1 as a risk factor for human CAKUT.
METHODS: In this study, DNA from 910 individuals with CAKUT was collected; 26 GEN1 rare variants were identified, and two GEN1 (missense) variants in a non-CAKUT group were found. Mainly due to the stability results of the predicted mutant on the website, in vitro, 10 variants (eight CAKUT, two non-CAKUT) were selected to verify mutant protein stability. In addition, mainly based on the division of the mutation site located in the functional region of the GEN1 protein, 8 variants (six CAKUT, two non-CAKUT) were selected to verify enzymatic hydrolysis, and the splice variant GEN1 (c.1071 + 3(IVS10) A > G) was selected to verify shear ability. Based on the results of in vitro experiments and higher frequency, three sites with the most significant functional change were selected to build mouse models.
RESULTS: Protein stability changed in six variants in the CAKUT group. Based on electrophoretic mobility shift assay of eight variants (six CAKUT, two non-CAKUT), the enzymatic hydrolysis and DNA-binding abilities of mutant proteins were impaired in the CAKUT group. The most serious functional damage was observed in the Gen1 variant that produced a truncated protein. A mini-gene splicing assay showed that the variant GEN1 (c.1071 + 3(IVS10) A > G) in the CAKUT group significantly affected splicing function. An abnormal exon10 was detected in the mini-gene splicing assay. Point-mutant mouse strains were constructed (Gen1: c.1068 + 3 A > G, p.R400X, and p.T105R) based on the variant frequency in the CAKUT group and functional impairment in vitro study and CAKUT phenotypes were replicated in each.
CONCLUSIONS: Overall, our findings indicated GEN1 as a risk factor for human CAKUT.
摘要：
背景：先天性肾脏和泌尿道异常（CAKUT）是普遍存在的出生缺陷。虽然致病性CAKUT基因是已知的，它们不足以揭示所有患者的病因。我们以前的研究表明GEN1是小鼠CAKUT的致病基因，本研究进一步探讨了GEN1与人CAKUT的相关性。
方法：在本研究中，收集了910名CAKUT患者的DNA；鉴定出26个GEN1罕见变异，在非CAKUT组中发现了两个GEN1（错义）变体。主要是由于网站上预测的突变体的稳定性结果，在体外，10个变体（八个CAKUT，选择两个非CAKUT）来验证突变蛋白的稳定性。此外,主要基于位于GEN1蛋白功能区的突变位点的划分，8个变体（六个CAKUT，选择两个非CAKUT)来验证酶促水解，选择剪接变体GEN1（c.10713（IVS10）A>G）以验证剪切能力。根据体外实验的结果和更高的频率，选择功能变化最显著的三个位点构建小鼠模型.
结果：CAKUT组中6种变体的蛋白质稳定性发生了变化。基于八种变体(六种CAKUT，两个非CAKUT)，CAKUT组突变蛋白的酶促水解和DNA结合能力受损。在产生截短蛋白的Gen1变体中观察到最严重的功能损伤。微型基因剪接实验显示CAKUT组中的变体GEN1(c.1071+3(IVS10)A>G)显著影响剪接功能。在微型基因剪接测定中检测到异常外显子10。构建点突变小鼠品系(Gen1:c.1068+3A>G，p.R400X,和p.T105R）基于CAKUT组的变异频率和功能损害的体外研究，并且在每个中复制了CAKUT表型。
结论：总体而言，我们的研究结果表明GEN1是人类CAKUT的危险因素.
方法：在本研究中，收集了910名CAKUT患者的DNA；鉴定出26个GEN1罕见变异，在非CAKUT组中发现了两个GEN1（错义）变体。主要是由于网站上预测的突变体的稳定性结果，在体外，10个变体（八个CAKUT，选择两个非CAKUT）来验证突变蛋白的稳定性。此外,主要基于位于GEN1蛋白功能区的突变位点的划分，8个变体（六个CAKUT，选择两个非CAKUT)来验证酶促水解，选择剪接变体GEN1（c.10713（IVS10）A>G）以验证剪切能力。根据体外实验的结果和更高的频率，选择功能变化最显著的三个位点构建小鼠模型.
结果：CAKUT组中6种变体的蛋白质稳定性发生了变化。基于八种变体(六种CAKUT，两个非CAKUT)，CAKUT组突变蛋白的酶促水解和DNA结合能力受损。在产生截短蛋白的Gen1变体中观察到最严重的功能损伤。微型基因剪接实验显示CAKUT组中的变体GEN1(c.1071+3(IVS10)A>G)显著影响剪接功能。在微型基因剪接测定中检测到异常外显子10。构建点突变小鼠品系(Gen1:c.1068+3A>G，p.R400X,和p.T105R）基于CAKUT组的变异频率和功能损害的体外研究，并且在每个中复制了CAKUT表型。
结论：总体而言，我们的研究结果表明GEN1是人类CAKUT的危险因素.
