2. Academic Validation
  3. Integrative identification of the pathogenic role of a novel G6PD missense mutation c.697G>C

Integrative identification of the pathogenic role of a novel G6PD missense mutation c.697G>C

  • Ann Transl Med. 2021 Feb;9(3):194. doi: 10.21037/atm-20-3941.
Hongyang Zhang 1 2,  Danyi Peng 1 2 3,  Yi Shu 1 2,  Dan Zhu 1 2,  Weiwei Hu 4,  Chaowen Yu 1 2,  Juan Zhang 1 2,  Shan Liu 1 2,  Kexing Wan 1 2,  Zhaojian Yuan 1 2,  Hao Liu 1 2,  Dongjuan Wang 1 2,  Tingting Jiang 1 2,  Jie Yu 1 5,  Penghui Zhang 1 6,  Lin Zou 1 2
Affiliations

Affiliations

  • 1 Center of Clinical Molecular Medicine & Newborn Screening Center, Ministry of Education Key Laboratory of Child Development and Disorders; National Clinical Research Center for Child Health and Disorders (Chongqing), China International Science and Technology Cooperation base of Child development and Critical Disorders, Children's Hospital of Chongqing Medical University, Chongqing, China.
  • 2 Chongqing Engineering Research Center of Stem Cell Therapy, Chongqing, China.
  • 3 Department of Respiratory Medicine, Children's Hospital of Chongqing Medical University, Chongqing, China.
  • 4 Department of respiratory and critical care medicine, First Affiliated Hospital of Chongqing Medical University, Chongqing, China.
  • 5 Department of Hematology, The Children's Hospital of Chongqing Medical University, Chongqing, China.
  • 6 Center of Clinical Laboratory, The Children's Hospital of Chongqing Medical University, Chongqing, China.
PMID: 33708821 DOI: 10.21037/atm-20-3941
Abstract

Background: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a hereditary disease caused by pathogenic mutations of G6PD. While most of the pathogenic variants of G6PD have been annotated, hemolysis of unknown etiology but analogous to that in G6PD deficiency persists, implying the existence of undocumented pathogenic variants. In our previous study, we reported four novel G6PD variants in China, for which the pathogenicity remains to be verified.

Methods: The variants were verified by exogenous expression in HEK-293 cells, and their functions were predicted by PolyPhen-2 and SIFT. The CRISPR/Cas9 system was exploited to edit the G6PD c.697G>C variant in HEK-293 cells and K562 cells. The expression of G6PD was detected by quantitative PCR (qPCR) and western blotting. The cell growth capacity was detected by the CCK-8 assay and crystal violet staining. The G6PD Enzyme activity was reflected by the G6P/6PG ratio test. The Apoptosis of cells was detected by Annexin V-APC/7-AAD staining. The secondary and crystallographic structures were denoted according to the literature and PyMOL software. The G6PD protein was purified from lysis of transformed Escherichia coli (E. coli) cell with Ni-charged Resin Column. The enzymatic activity was detected at different temperatures.

Results: The G6PD activity of exogenous G6PD c.697G>C in HEK-293 cells was significantly lower than that of wild type (WT) G6PD, a finding that was consistent with the observation in clinical samples. The functional predictions conducted by different algorithms indicated the damage role of the G6PD c.697G>C variant in its enzymatic activity. We recapitulated the G6PD c.697G>C variant both in HEK-293 cells and K562 cells by adapting the CRISPR/Cas9 strategy. Using distinct cell lines expressing the G6PD c.697G>C variant endogenously, we confirmed the deteriorative role of the G6PD c.697G>C variant in its enzymatic activity. Regarding the secondary and crystallographic structure, we found a mutated amino acid approaching the structural NADP+ binding site. Finally, we demonstrated the c.697G>C variant compromised the thermal stability of G6PD protein.

Conclusions: Our data delineated the pathogenic role of G6PD c.697G>C variant for G6PD deficiency, implying the wide usage of CRISPR/Cas9 for genetic Disease Research.

Keywords

CRISPR/Cas9; Glucose-6-phosphate dehydrogenase deficiency (G6PD deficiency); genome editing; novel missense mutation; pathogenic variant.

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