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Published on: October 12, 2017
GEN1 as a risk factor for human congenital anomalies of the kidney and urinary tract
Xuanjin Du1, Chunyan Wang1, Jialu Liu1
1Department of Nephrology, Shanghai Kidney Development and Pediatric Kidney Disease Research Center, Children's Hospital of Fudan University, 201102, Shanghai, China.
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.
Conclusion:
Overall, our findings indicated GEN1 as a risk factor for human CAKUT.
Insights
GEN1 variants are linked to congenital anomalies of the kidney and urinary tract (CAKUT). This study confirms GEN1 as a risk factor in humans, impacting protein stability and function in CAKUT patients.
Area of Science:
- Genetics
- Developmental Biology
- Urology
Background:
- Congenital anomalies of the kidney and urinary tract (CAKUT) are common birth defects with incompletely understood genetic causes.
- Previous research identified GEN1 as a CAKUT-associated gene in mice.
- This study investigates the role of GEN1 in human CAKUT.
Purpose of the Study:
- To determine the correlation between GEN1 variants and human CAKUT.
- To functionally characterize identified GEN1 variants in vitro.
- To establish mouse models for GEN1-related CAKUT.
Main Methods:
- Collected DNA from 910 individuals with CAKUT and identified GEN1 rare variants.
- Performed in vitro experiments to assess mutant protein stability, enzymatic hydrolysis, and DNA-binding ability for selected variants.
- Utilized mini-gene splicing assays to evaluate splice variants.
- Generated mouse models for key GEN1 variants.
Main Results:
- Identified 26 GEN1 rare variants in individuals with CAKUT.
- Demonstrated impaired protein stability, enzymatic hydrolysis, and DNA-binding ability in several CAKUT-associated GEN1 variants.
- Confirmed significant splicing defects caused by a specific GEN1 variant (c.1071+3(IVS10) A>G).
- Mouse models carrying specific GEN1 variants replicated CAKUT phenotypes.
Conclusions:
- GEN1 is a significant risk factor for human CAKUT.
- Functional impairments in GEN1 variants contribute to the development of CAKUT.
- Further research into GEN1 is warranted for understanding and potentially treating CAKUT.
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