关键词: 云南省, 伯氨喹, 溶血, 葡萄糖-6-磷酸脱氢酶, 突变, 空间结构

Abstract:

Objective Glucose-6-phosphate dehydrogenase (G6PD) deficiency is related to primaquine-induced hemolysis. This study aims to determine the mutations of G6PD gene isolated from a patient with primaquine-induced hemolysis and the possible structure change related to the pathology. Methods Blood sample was collected from a primaquine-induced hemolysis patient infected with Plasmodium vivax. The G6PD enzyme activity was measured to reduce up to 75% in this patient. The genomic DNA was extracted from the blood sample, and G6PD gene fragments containing exon2, exon3-7, exon8-9 and exon10-13 were amplified by PCR using primers designed based on the G6PD coding sequence. The amplified PCR products were sequenced to obtain the full-length coding sequence of G6PD and then aligned with the wildtype (GenBank: NC_000023.11) and mutant (GenBank: X55448.1) G6PD sequences to identify the mutations using MEGA5.04 software. The 3D structure of G6PD was predicted based on the deduced amino acid sequences by SWSS-MODEL(www.swissmodel.expasy.org/interactive) and modified by PyMOL 2.2.0 software. Results The DNA fragments containing G6PD exon2, exon3-7, exon8-9, exon10-13 were amplified from genomic DNA isolated from the blood sample of a P. vivax infected patient with primaquine-induced hemolysis, with the length of 336 bp, 2 277 bp, 976 bp and 1 421 bp, respectively. The full-length DNA of 1 545 bp coding for G6PD of the patient was obtained by aligning with reference wild-type and mutant G6PDs deposited in GenBank. Alignment of patient’s G6PD sequence with wild-type and mutant reference sequences identified two mutations with c.1 311T > C and c.1 376G > T. The first mutation(c.1 311T > C) was synonymous without changing the coding amino acid(Y), the second mutation(c.1 376G > T) leads to the coding amino acid changed from Arg(R) to Leu(L) at position 459. However, the mutation at position 459 was far away from the binding site of G6PD with NADP⁺ and glycolic acid ligands. The structure of patient G6PD could be the dimer form or tetramer form with the dimer more stable according to the GMQE and QMEAN modeling systems. Compared with the wild-type model of the dimer G6PD (6e07.1.A), the patient G6PD mutant amino acid at 459 was on the surface of the structure, and the binding region with NADP⁺ ligands contained 16 residues including 238 and 357 positon amino acids, similar to the wild-type form. However, the 3D structure of patient’s G6PD tetramer form was not as stable as wild-type G6PD. The ligand binding regions of glycolic acid contained only 6 residues including 47 position amino acid which is less than 11 residues of wild-type model. Conclusion The sequence of G6PD isolated from a P. vivax infected patient with primaquine-induced hemolysis contained a synonymous mutation of c.1 311T > C and a nonsynonymous mutation of c.1 376G > T compared to the wild-type G6PD. The nonsynonymous mutation at c.1 376G > T leads to the coding amino acid change from Arg(R) to Leu(L) at postion 459 which may not change the dimer structure, but interfere with the formation of tetramer structure of G6PD.

Key words: Yunnan Province, Primaquine, Hemolysis, Glucose-6-phosphate dehydrogenase, Mutation, Spatial structure