Functional and structural characterisation of common cytochrome P450 2D6 allelic variants—roles of Pro34 and Thr107 in catalysis and inhibition

Duan²,

et al. 2020

Preprint

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.

Section: Discussioncontrasting

confidence: 86%

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

Duan²,

et al. 2020

Preprint

“…Among the 24 haplotypes de ned, Hap_2 makes up the largest proportion of the sequences, reaching as high as 36.6% (55/150). This haplotype shows multiple mutations at four loci, including c.100C > T, c.336C > T, c.408G > C, c.1457G > C. These mutations are different from previous studies which reported that CYP2D6*10 allele is frequently detected in Asian populations [32,34]. CYP2D6*10 allele has multiple mutations at c. 100(C > T), c. 408(G > C) and c. 1457(G > C), yet is devoid of c. 336C > T mutation as opposed to Hap_2.…”

Section: Discussioncontrasting

confidence: 83%

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

Duan²,

et al. 2020

Preprint

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.

“…Among the 23 alleles de ned from 23 haplotypes in all the CDSs chains, the proportion of CYP2D6*10 reached as high as 45.4% (108/238). The composition of CYP2D6 gene alleles is the same as that found by Qian et al [32] in Chinese Han population, also consistent with previous studies which reported that CYP2D6*10 allele is most frequently detected in Asian populations [31,[35][36]. Although the frequencies of CYP2D6*10 allele de ned from SR group and NR group did show signi cant different, but the frequency of CYP2D6*10 allele was the highest in both groups, and CYP2D6*10 has been an allele leading to the decrease of CYP2D6 enzyme activity.…”

Section: Sample Information and Pcr Ampli Cation Of Human Cyp2d6 Genesupporting

confidence: 90%

Genetic Polymorphism of Cyp2d6 Gene Coding Region and Association Between Them With Vivax Malaria Relapse in Yunnan Province, China

Duan

et al. 2020

Preprint

BackgroundPrimaquine 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 vivax malaria patients. However, the reduced CYP2D6 enzyme activity caused by gene mutations has been considered to result in the failure of primaquine relapse vivax malaria. Based on the analysis of CYP2D6 gene polymorphisms in vivax malaria cases the current study aims to determine the association between human CYP2D6 polymorphism and the relapse of vivax malaria. MethodsBlood samples of vivax malaria cases who received "chloroquine/primaquine eight-day therapy" from 2014 to 2018 in Yunnan Province were collected. The suspected relapsed cases were determined by epidemiology and gene sequence alignment. Human genomic DNA was extracted from the blood samples, nine exons of CYP2D6 gene were amplified by polymerase chain reaction (PCR) and PCR products were then sequenced. The coding DNA sequence (CDS) of CYP2D6 gene was obtained by splicing and aligning with the non-mutation reference sequence. The mutation loci, haplotypes (star alleles) and genotypes of CYP2D6 gene were inferred and their association with vivax malaria relapse were analyzed. ResultsA total of 120 blood samples from 45 suspected relapsed cases of vivax malaria and 75 non-relapsed cases were collected and two nested-PCR products (2411bp and 2388bp) containing exon1-4 and exon5-9, were obtained from 119 samples. 119 CDS chains (1491bp length) were formed by splicing and combining sequencing sequences, A total of 12 mutation loci were identified in 238 CDS chains (including 119 chains from direct sequencing and another 119 chains identified from sister chromosome). Among them, the mutation locus at c.886 has the closest relationship with the relapse of vivax malaria (OR=2.167, 95%CI: 1.104~4.252, P<0.05). There were 23 haplotypes (Hap_1~Hap_23) to be identified and be defined as 23 alleles of CYP2D6 gene, Among them, the 5 star alleles (*1, *2, *4, *10 and *39) were confirmed by comparison, and the CYP2D6*10 allele accounted for the most (45.4%, 108/238). the frequency of CYP2D6*2 allele in the SR group was significantly higher than that in the NR group (P<0.05). However, among the defined 24 genotypes, there were 8 genotypes (*4/*4, *4/*o, *2/*39, *39/*m, *39/*x, *1/*r, *1/*n, and *v/*10) appeared only in the SR group.ConclusionAmong the numerous mutations of CYP2D6 gene，CYP2D6*10 allele accounts for the highest proportion of vivax malaria cases in Yunnan Province. The mutation at c. 886 locus is most closely related to the relapse of vivax malaria. In addition, there was the genotype *4/*4 with null CYP2D6 enzyme function. These results suggest that Yunnan Province should pay attention to the risk of reduced CYP2D6 enzyme activity on the radical cure therapeutic effect of primaquine