Zhonghua yi xue yi chuan xue za zhi = Zhonghua yixue yichuanxue zazhi = Chinese journal of medical genetics · 2021
[Analysis of PKD2 gene variant and protein localization in a pedigree affected with polycystic kidney disease].
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Cheng Jianping, Li Ping, Li Yujun, Zhou Yong'an, Ren Ruirui, Han Yaxin, Li Xingxing, Li Zhe, Bai Yuan
Аннотация
OBJECTIVE: To detect the mutation site in a pedigree affected with autosomal dominant polycystic kidney disease (ADPKD) and verify its impact on the protein function.
METHODS: Peripheral blood samples were collected from the proband and his pedigree members for the extraction of genomic DNA. Mutational analysis was performed on the proband through whole-exome sequencing. Suspected variant was verified by Sanger sequencing. A series of molecular methods including PCR amplification, restriction enzyme digestion, ligation and transformation were also used to construct wild-type and mutant eukaryotic expression vectors of the PKD2 gene, which were transfected into HEK293T and HeLa cells for the observation of protein expression and cell localization.
RESULTS: The proband was found to harbor a c.2051dupA (p. Tyr684Ter) frame shift mutation of the PKD2 gene, which caused repeat of the 2051st nucleotide of its cDNA sequence and a truncated protein. Immunofluorescence experiment showed that the localization of the mutant protein within the cell was altered compared with the wild-type, which may be due to deletion of the C-terminus of the PKD2 gene.
CONCLUSION: The c.2051dupA (p. Tyr684Ter) mutation of the PKD2 gene probably underlay the pathogenesis of ADPKD in this pedigree.
METHODS: Peripheral blood samples were collected from the proband and his pedigree members for the extraction of genomic DNA. Mutational analysis was performed on the proband through whole-exome sequencing. Suspected variant was verified by Sanger sequencing. A series of molecular methods including PCR amplification, restriction enzyme digestion, ligation and transformation were also used to construct wild-type and mutant eukaryotic expression vectors of the PKD2 gene, which were transfected into HEK293T and HeLa cells for the observation of protein expression and cell localization.
RESULTS: The proband was found to harbor a c.2051dupA (p. Tyr684Ter) frame shift mutation of the PKD2 gene, which caused repeat of the 2051st nucleotide of its cDNA sequence and a truncated protein. Immunofluorescence experiment showed that the localization of the mutant protein within the cell was altered compared with the wild-type, which may be due to deletion of the C-terminus of the PKD2 gene.
CONCLUSION: The c.2051dupA (p. Tyr684Ter) mutation of the PKD2 gene probably underlay the pathogenesis of ADPKD in this pedigree.