Genetic Polymorphism Of PfKelch13 And Its Correlation With Resistance To Artemisinin In Plasmodium Falciparum Cases Reported From Yunnan Province

【Objective】To understand the genetic polymorphism of PfKelch13 gene and its molecular epidemiological characteristics in P.falciparum isolates from Yunnan province;To evaluate the correlation between PfKelch13 gene and resistance to artemisinin,provide a theoretical basis for the molecular monitoring of artemisinin resistance of Plasmodium falciparum in Yunnan Province【Methods】(1)202 P.falciparum cases were collected from 2012 to 2015 in Yunnan province and 384 P.falciparum infected cases were collected from 2009 to 2011 in Myanmar;P.falciparum cases from Myanmar、age 2~60 years、in 2 weeks without antibiotic medication or / and antimalarial drug history,at the same time accept the artesunate in vivo testing of drug resistance.(2)Refer to the QIAamp Mini Kit DNA kit for the extraction of Plasmodium falciparum genomic DNA,then use nested PCR to amplify the PfKelch13 gene kelch domain,sequence of the PCR positive products;Analysis the haplotype and expected heterozygosity(He)、gene polymorphism(H)and Fxiation index(Fst);Analyze the genetic background of Plasmodium falciparum isolates from Yunnan and Mynamar.(3)Analysis the correlation between mutations of PfKelch13 gene and resistance to artemisinin of Plasmodium falciparum isolates.(4)Refer to the QIAamp Mini Kit DNA kit for the extraction of Plasmodium falciparum genomic DNA,then use PCR to amplify the exon 2 region of the Pfhrp2 gene,sequence of the PCR positive products;Analysis the haplotype and expected heterozygosity(He)、gene polymorphism(H)and Fxiation index(Fst);Analyze the genetic background of Plasmodium falciparum isolates from Yunnan and Mynamar.【Results】(1)DNA sequences of kelech domain in PfKelch13 gene from Yunnan bloods samples had 13 haplotypes,the expected heterozygosity(He)was 0.0449 and the nonsynonymous mutations sequences accounted for 34.7%(66/190),at 446、676 and16 loci showed only single mutation and the F446 I mutation rate was the highest with25.3%(48/190);DNA sequences from Myanmar bloods samples had 19 haplotypes,the expected heterozygosity(He)was 0.0449 and the nonsynonymous mutations sequences accounted for 58.8%(114/194),in these sequences,at 446、76 and 492 loci showed only single mutation,F446 I mutation rate was the highest with 43.3%(84/194).(2)The index of genetic differentiation of Yunnan and Myanmar populations in PfKelch13 gene kelch domain of Plasmodium falciparum was 0.0410(Fst=0.0410,P<0.05),variation within the population and amomg the populations accounted for95.90% and 4.10% respectively,the haplotypes media network diagram showed“Satellite” layout in this two populations.(3)The index of genetic differentiation of Yunnan and Myanmar populations in Pfhrp2 gene exon2 region was 0.0047(Fst = 0.0047,P < 0.05),the differentiation degree was low,there was a similar genetic background;Yunnan reported cases and Myanmar infected cases of Plasmodium falciparum in the haplotype network showed no significant geographic domain clustering branch.(4)In the report of Myanmar,the OR value between artemisinin resistance and PfKelch13 gene kelech domain at 446 site mutation was 1.640(95%CI:1.284~2.095),the OR value of the 12 site mutations of PfKelch13 gene kelech domain was 1.840(95%CI:1.412~2.398).【Conclusion】(1)The PfKelch13 gene kelech domainof Plasmodium falciparum cases from Yunnan and Myanmar present the polymorphism.(2)The differentiation degree of Plasmodium falciparum was low in the PfKelch13 gene kelech domain from Yunnan and Myanmar,there was a similar genetic background.(3)In the report of Myanmar,the OR value between artemisinin resistance and PfKelch13 gene kelech domain at 446 site mutation was 1.640(95%CI:1.284~2.095),the OR value of the 12 site mutations was 1.840(95%CI:1.412~2.398);The genetic association between the PfKelch13 gene mutation and artemisinin resistance in Myanmar is suitable for the report of Plasmodium falciparum population in Yunnan.(4)The differentiation degree of Plasmodium falciparum was low in the Pfhrp2 gene exon2 region from Yunnan and Myanmar,there was a similar genetic background.