Genetic Polymorphism of human CYP2D6 gene coding region results in failed radical cure of vivax malaria using primaquine

Background: Primaquine is the traditional medicine could effectively eliminate the hypnozoites of P. vivax, and therefore has been recommended by World Health Organization (WHO) as the anti-relapsing drug for the treatment of vivax malaria patient. However, 5-hydroxy-primaquine, the active component in primaquine to kill P. vivax hypnozoite has to be catalyzed by Cytochrome P450, family 2, subfamily D, polypeptide 6 (CYP2D6) enzyme in human hepatocytes. Nevertheless, suppressed CYP2D6 enzyme activity caused by CYP2D6 gene mutation has been considered as a risk factor for the efficacy of primaquine. Based on the analysis of CYP2D6 gene polymorphism in relapsed vivax malaria patients, the current study aims to preliminarily reveal the genetic association between human CYP2D6 genotype and the curative effect of primaquine in vivax malaria. Methods: Blood samples of vivax malaria cases received "chloroquine/primaquine eight-day therapy" from 2014 to 2018 in Yunnan Province were collected. The relapsed cases were determined by identifying the homogeneity of P. vivax circumsporozoite protein (pvcsp) gene sequences between the Plasmodiums isolated from patients’ infected blood samples. Human genomic DNA was extracted from the blood samples, and 9 exons of CYP2D6 gene were amplified by polymerase chain reaction (PCR) and were then sequenced. The coding DNA sequence (CDS) of CYP2D6 gene was obtained by splicing and aligning 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: A total of 156 blood samples from 75 relapsed cases of vivax malaria and 75 non-relapsed cases were collected for nested-PCR amplification. Two nested-PCR products, the CYP2D6 gene fragments containing exon1-4 and exon5-9 (2411bp and 2388bp), were obtained from the sample. After splicing and combining these two amplification products, we found that the CDS of CYP2D6 gene in each sample was 1491bp in length. Moreover, we identified 24 haplotypes (Hap_1~Hap_24) in the Clustered CYP2D6 full-CDS of 150 patients, including 17 haplotypes in relapsed patients and 15 haplotypes in non-relapsed patients (8 haplotypes co-existed in two groups of patients). 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 variations of R296C and S486T at 136th, 296th and 486th amino acid sites, respectively. The risk of relapsing vivax malaria in Hap_6 was about 5.615-fold higher than in other haplotypes. In the relapsed cases, c.408 locus mutation of CYP2D6 gene was 100% homozygous allele (C/C, 10/10), whereas c.886 locus mutation was 80% homozygous allele (T/T, 8/10) and 20% heterozygous allele (C/T, 2/2). However, 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 was predicted as normal metabolizer (NM) according to the scoring of diploid allele of Hap_6 sequence.Conclusion: Among the numerous mutations of CYP2D6 gene, triple mutations at three loci (c.408, c.886, c.1457) are most closely related to impaired CYP2D6 enzyme activity, and greater sample size should be applied to ascertain whether c.886 locus mutation plays a critical role.