Polymorphism in coding region of human CYP2D6 gene contributes to failed radical cure of vivax malaria using primaquine
Authors: Huang Herong , Dong Ying , Xu Yanchun , Deng Yan , Zhang Canglin , Liu Shuping , Chen Mengni , Liu Yan 
Published: March 2020

Abstract
Background: Recent studies found that damaged enzyme activity of cytochrome P450 isoenzyme 2D6 (CYP2D6) can lead to compromised efficacy of primaquine in killing Plasmodium during liver dormancy and consequently relapsed malaria. Therefore, WHO has listed the decrease enzyme activity of CYP2D6 as one of the four scenarios that are not suitable for primaquine therapy. Based on analysis of CYP2D6 gene polymorphisms in relapsed vivax malaria patients, this study preliminarily revealed the genetic association between human CYP2D6 genotype and the declined efficacy of primaquine treatment in vivax malaria. Methods: Blood samples of vivax malaria cases treated with "chloroquine/primaquine eight-day therapy" from 2014 to 2018 in Yunnan Province were collected, and the vivax malaria relapsed cases after clinical curing were determined by identifying the infected Plasmodium isolates through gene traceability analysis. Human genomic DNA was extracted from blood samples, and 9 exons of CYP2D6 gene were amplified by PCR and then sequenced. The CDS of CYP2D6 gene was obtained by alignment and splicing with the wild-type reference sequence. The mutation types of coding DNA sequences and their association with vivax malaria relapse were analyzed, and the activity phenotype of CYP2D6 isozyme was predicted by its allelic form. Results: One hundred and fifty-six blood samples from 75relapsed cases of vivax malaria and the same number of 75 non-relapsed vivax malaria cases were collected for PCR amplification. Two amplification products (2411bp and 2388bp) containing exon1-4 and exon5-9 of CYP2D6 gene were obtained from every sample. The 1491-bp-length CDS chains of CYP2D6 gene were obtained from those 150 samples, and were defined as 24 haplotypes (Hap_1~Hap_24).17 haplotypes were determined from the sequences of vivax malaria relapsed patients and 15 haplotypes were from those of non-relapsed patients.Hap_6 sequencing showed G>C, C>T and G>C base substitutions at three loci of c.408, c.886 and c.1457, resulting in V136V invariance and R296C and S486T variation at 136th, 296th and 486th amino acid, and the odds ratio of Hap_6 to vivax malaria relapse was 5.615 (P˂0.05).The diploids of CYP2D6 gene in relapsed cases were 100% mutation homozygous (C/C, 10/10) at c.408, 80% (8/10) at c.1457 and 20% (2/10) at c.886, respectively. However, the coding DNA sequences from non-relapsed cases only showed mutation heterozygote (C/T, 2/2) at c.886 locus. The allelic form of relapsed patients was CYP2D6 *2, and diploid score was used to indirectly predict the CYP2D6 enzyme activity of Hap_6, and the relapsed patients belong to normal metabolizer (NM).Conclusion: Among the numerous mutations of CYP2D6 gene, the joint mutations at three loci (c.408, c.886, c.1457) are most closely related to the damagedCYP2D6 enzyme activity. Whether c.886 locus mutation plays a critical role contributing to such damage warrants further verification with expanded sample size.