Polymorphism in coding region of human CYP2D6 gene contributes to failed radical cure of vivax malaria using primaquine

Background: So far, it has been confirmed that primaquine is the only medicine could effectively kill the hypnozoites of P. vivax. Therefore, World Health Organization (WHO) recommends primaquine as the only anti-relapsing drug for the treatment of vivax malaria patient. However, due to the active component of primaquine for killing P. vivax hypnozoite is the 5-hydroxy-primaquine, which has to be catalyzed by the Cytochrome P450, family 2, subfamily D, polypeptide 6 (CYP2D6) enzyme in human hepatocytes, the decrease of CYP2D6 enzyme activity caused by CYP2D6 gene mutation has been considered as an risk factor for the use of primaquine. Based on the analysis of CYP2D6 gene polymorphisms in relapsed vivax malaria patients, this study preliminarily revealed the genetic association between human CYP2D6 genotype and the curative effect of primaquine 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. Then, the relapsed cases were determined by identifying the homogeneity of P. vivax circumsporozoite protein（pvcsp）gene sequences between the Plasmodiums isolated from patients’ first and second infected blood samples. Human genomic DNA was extracted from blood samples, and 9 exons of CYP2D6 gene were amplified by polymerase chain reaction (PCR) and then sequenced. The coding DNA sequence (CDS) of CYP2D6 gene was obtained by splicing after alignment with the wild-type reference sequence. The mutation types of CDS and their association with vivax malaria relapse were analyzed, and the activity phenotype of CYP2D6 isozyme was predicted by the allelic form of CYP2D6 gene. Results: One hundred and fifty-six blood samples from 75 relapsed cases of vivax malaria and 75 non-relapsed cases were collected for nested-PCR amplification. Two kinds of nested-PCR products，the CYP2D6 gene fragments containing exon1-4 and exon5-9 (2411bp and 2388bp)，were obtained from every sample. After splicing and combining these two amplification products, the complete CDS of CYP2D6 (1491 bp) of each sample could be gotten. Clustered CYP2D6 full-CDS of 150 patients, 24 haplotypes (Hap_1~Hap_24) were defined, including 17 haplotypes in relapsed patients and 15 haplotypes in non-relapsed patients. The Hap_6 showed G>C, C>T and G>C base substitutions at c.408, c.886 and c.1457, resulting in V136V invariance and the variations of R296C and S486T at 136th, 296th and 486th amino acid sites. The odds ratio of Hap_6 sequence type was 5.615 (P˂0.05) as compared with the relapse of vivax malaria. For the relapsed cases, the mutation at c.408 of CYP2D6 gene is 100% homozygous allele (C/C, 10/10), while at c.886 is 80% homozygous allele (T/T, 8/10) and 20% heterozygous allele (C/T, 2/2). But the non-relapsed cases only showed mutation heterozygote allele (C/T, 2/2) at c.886 locus. Moreover, most of mutant allele types in relapsed patients were CYP2D6 *2, and the CYP2D6 enzyme activity of them was predicted as normal metabolizer (NM) by scoring the diploid allele of Hap_6 sequence. Conclusion: Among the numerous mutations of CYP2D6 gene, the triple mutations at three loci (c.408, c.886, c.1457) are most closely related to the decreased CYP2D6 enzyme activity, while whether the c.886 locus mutation plays a critical role needs to be further verified by expanded sample size.