| Objective:The current study aims to preliminarily reveal the association between the recurrence of vivax malaria in Yunnan Province and CYP2D6 gene mutation by analysing genetic polymorphisms in the entire coding region of human CYP2D6(Cytochrome P450,family 2,subfamily D,polypeptide 6)gene.Methods:Collect blood samples of vivax malaria cases treated with"chloroquine/primaquine eight-day therapy"in Yunnan Province.Confirm the suspected relapsed cases and Non-relapsed cases of vivax malaria through epidemiology and genetic sequence comparison.Polymerase chain reaction(PCR)amplifies the CYP2D6gene in the human genome,and sequence the amplified products.The sequence of the CYP2D6 gene obtained by sequencing was compared with the non-mutation reference sequence to determine the correctness of the sequencing and 9 exons region.Manually intercept the DNA sequence of 9 exons and splice them into the DNA coding region sequence(CDS).This chain defaults to the maternal CDS chain,and the paternal CDS chain is obtained by adjusting the base information according to the sequencing peak map.The mutation polymorphism and haplotype of CDS chain were identified by Dna SP6.11.01 software.The haplotype(allele)of CYP2D6 gene was named according to the database of Human cytochrome P450 allele Nomenclature Committee,and the CYP2D6 activity was predicted by diploid genotype.The mutation loci,alleles and the ratio of genotype to vivax malaria recurrence(oddsratio,OR)of all CDS chains were analyzed by IBM?SPSS?21 software.Results:A total of 326 maternal CDS chains with a length of 1491bp were obtained.A total of 12 mutation loci were detected in all 652 maternal and paternal CDS chains.The c.1457 G>C locus mutation was the most detected in the SR and NR groups,81.2%(112/138)and 68.5%(352/514);c.271、c.281、c.294、c.505、c.408、c.1457mutation loci was most closely related to the recurrence of vivax malaria.Among the 23haplotypes(Hap＿1～Hap＿23),Hap＿3 is non-mutant,and the sequence structure of Hap＿9 is the most complex;11 are known suballeles,which can be combined into*1,*2,*4,*10 and*39 5 kinds of star alleles,and the most detected allele form is*10alleles(45.1%,294/652);The detection rate of*1 allele in non-relapsed group was significantly higher than that in suspected relapsed group(x~2=10.484,P<0.05),while the detection rate of*39 allele in suspected relapsed group was significantly higher than that in non-relapsed group(x~2=19.188,P<0.05),and*4 allele was only detected in suspected relapsed group(P<0.05).A total of 10 genotypes that can be accurately named were defined in 326 patients with vivax malaria.Except for the null enzyme activity genotype*4/*4,which was only found in the SR group,the other 9 genotypes were detected in both groups.The most common genotypes in both groups were 27.6%(19/69)and 26.5%(68/257),respectively.But the difference was not statistically significant(x~2=0.032,P>0.05).The detection rate of genotype*1/*1 with normal enzyme activity in NR group was significantly higher than that in SR group(x~2=8.096,P<0.05),while the detection rate of genotype*10/*10 in SR group was higher than that in NR group(x~2=8.851,P<0.05).Compared with other genotypes,the risk of recurrence of vivax malaria was increased by 8.063 times.The results of Tajima’s neutral test showed that the Tajima’s D neutral test value was 0.42819(P>0.10),and the D value fluctuated greatly,indicating that the mutation detected in the coding region of the whole CYP2D6 gene dose not belonged to the non-neutral mutation under the pressure of directional selection,and the Ka/Ks ratio was more than 2.9.it indicated that the research sequence was very active,the missense mutation rate was higher than the synonymous mutation rate,and the effect of positive multiple selection was strong.Conclusions:In the population exposed to primaquine in Yunnan Province,c.1457(rs1135840),CYP2D6*10 and*10/*10 were the most common mutation loci,alleles and genotypes of CYP2D6 gene,respectively.The mutation loci of c.271,c.281,c.294,c.505,c.408,c.1457 and CYP2D6*4 allele,which correspond to impaired primaquine 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. |
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