Correlation of CYP2D6 allelic polymorphism to outcome of acute coronary syndrome in mid-Euphrates Iraqi patients on metoprolol therapy

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.

Section: Discussioncontrasting

confidence: 93%

Section: Discussionmentioning

confidence: 99%

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

Duan²,

et al. 2020

Preprint

“…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: 91%

“…The primers of CYP2D6 for polymerase chain reaction (PCR) ampli cation were designed by using GenBank (https://www.ncbi.nlm.nih.gov/gene/), reference sequence (ID NC_000022.11) was used as template, following the reaction condition in previous studies [23,31]. The primers for rst-round nested PCR covering the coding region of CYP2D6 gene exons1~4 were: 5'-CCAGTGACAGATAAGGGTGC-3' and 5'-GACGTGGATAGGAGGTACAGAG-3'; the primers for second-round nested PCR were: 5'-GGTGACTTCTCCGACCAGG-3' and 5'-TTCCCAAACCCATCTATGC-3'.…”

Section: Pcr Ampli Cation Of Cyp2d6 Gene Fragmentmentioning

confidence: 99%

“…The heterogeneity of CYP2D6 monooxygenase activity varies from complete dysfunction to ultra-rapid enzyme metabolism, depending on the various genotypes of CYP2D6 [14]. Among the 150 identi ed alleles, multiple mutations such as CYP2D6*3, CYP2D6*4, CYP2D6*5 and CYP2D6*6 are considered to represent null CYP2D6 enzyme activity [31,34].…”

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

confidence: 99%

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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

The association of CYP2D6 gene polymorphisms in the full-length coding region with higher recurrence rate of vivax malaria in Yunnan Province, China

Duan

et al. 2021

Malar J

Background Accumulating evidence suggest that compromised CYP2D6 enzyme activity caused by gene mutation could contribute to primaquine failure for the radical cure of vivax malaria. The current study aims to preliminarily reveal the association between the recurrence of vivax malaria in Yunnan Province and CYP2D6 gene mutation by analysing polymorphisms in the entire coding region of human CYP2D6 gene. Methods Blood samples were collected from patients with vivax malaria, who received "chloroquine and 8-day course of primaquine therapy" in Yunnan Province. The suspected relapsed cases were determined by epidemiological approaches and gene sequence alignment. PCR was conducted to amplify the CYP2D6 gene in the human genome, and the amplified products were then sequenced to compare with the non-mutation “reference” sequence, so as to ensure correct sequencing results and to determine 9 exon regions. Subsequently, the DNA sequences of 9 exons were spliced into the coding DNA sequence (CDS), which, by default, is known as maternal CDS. The paternal CDS was obtained by adjusting the bases according to the sequencing peaks. The mutation loci, haplotypes (star alleles), genotypes and odds ratios (OR) of all the CDSs were analysed. Results Of the119 maternal CDS chains in total with 1491 bp in length, 12 mutation sites in the 238 maternal and paternal CDS chains were detected. The c.408G > C mutation was most frequently detected in both suspected relapsed group (SR) and non-relapsed group (NR), reaching 85.2% (75/88) and 76.0% (114/150), respectively. The c.886C > T mutation was most closely related to the recurrence of vivax malaria (OR = 2.167, 95% CI 1.104–4.252, P < 0.05). Among the 23 haplotypes (Hap_1 ~ Hap_23), Hap_3 was non-mutant, and the sequence structure of Hap_9 was the most complicated one. Five star alleles, including *1, *2, *4, *10 and *39, were confirmed by comparison, and CYP2D6*10 allele accounted for the largest percentage (45.4%, 108/238). The frequency of CYP2D6*2 allele in the SR group was significantly higher than that in the NR group (Χ2 = 16.177, P < 0.05). Of the defined 24 genotypes, 8 genotypes, including *4/*4, *4/*o, *2/*39, *39/*m, *39/*x, *1/*r, *1/*n, and *v/*10, were detected only in the SR group. Conclusion Mutation of CYP2D6*10 allele accounts for the highest proportion of vivax malaria cases in Yunnan Province. The mutations of c. 886C > T and CYP2D6*2 allele, which correspond to impaired PQ metabolizer phenotype, are most closely related to the relapse of vivax malaria. In addition, the genotype *4/*4 with null CYP2D6 enzyme function was only detected in the SR group. These results reveal the risk of defected CYP2D6 enzyme activity that diminishes the therapeutic effect of primaquine on vivax malaria.