Genetic Polymorphism of human CYP2D6 gene coding region from vivax malaria cases in Yunnan Province, China

Background: Primaquine is one of two medications able to 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 patients. However, 5-hydroxy-primaquine, the presumed active component, must be generated by CYP2D6 enzyme in human hepatocytes and reduced CYP2D6 enzyme activity caused by gene mutations has been considered cause of primaquine failure. Based on the analysis of CYP2D6 gene polymorphisms in suspected relapsed vivax malaria patients, the current study aims to determine the association between human CYP2D6 genotype and the efficacy of primaquine. Methods: Blood samples from vivax patients in Yunnan Province who received "chloroquine/primaquine eight-day therapy" and experienced suspected relapses were collected from 2014 to 2018. Human genomic DNA was extracted, and 9 exons of CYP2D6 gene were amplified by polymerase chain reaction (PCR) and were then sequenced. The mutation types of CDS and their association with CYP2D6 enzyme activity changes were analyzed. Results: A total of 156 blood samples from 75 relapsed cases of vivax malaria and 75 non-relapsed cases were collected and 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). In addition, we identified 33 diplotypes (A_1~A_33) in 150 patients. The A_10 and A_12 diplotypes showed higher frequency in suspected relapsed group.Conclusion: Among the numerous mutations of CYP2D6 gene， Hap_2 accounted for the largest proportion in 24 haplotypes. And the combined homozygous mutations at c. 408, c. 1457, especially the two loci combined mutations with c. 886, may be related to the poor efficacy of primaquine in the radical cure P. vivax in Yunnan, however, greater sample size is required confirm these findings.