| Objective: To analyze the polymorphism of the coding region of glucose-6-phosphate dehydrogenase(G6PD)gene in patients with Plasmodium vivax in Yunnan province and its correlation with primaquine-induced hemolysis;analyze the mutation sites in the G6 PD gene coding region that often occur in people with reduced G6 PD enzyme activity,It lays the foundation for further screening for genetic markers with reduced G6 PD enzyme activity in Yunnan Province.Methods:(1)Collect 184 blood samples of P.vivax in Yunnan Province treated with "chloroquine / primaquine eight-day therapy" from January to December 2018.The samples used in this experiment include the G6 PD gene spliced into the complete c DNA strand of the vivax malaria report case sample and the exon 10～13 coding segment of the vivax malaria report case sample,Samples of vivax malaria report include dizziness,fatigue,and abdominal discomfort during the initial period of taking primaquine.Jaundice,urine-like "brown"-like changes,continued decrease in hemoglobin indications,and any decrease in G6 PD enzyme activity measured by the NADPH / NADP + ratio method were determined to be acute hemolysis induced by primaquine.(2)To extract human genomic DNA from filter paper blood samples,use the QIAamp DNA Mini Kit kit,refer to the DNA extraction kit instructions(QIAgen DNA Mini Kit),extract human genomic DNA,and nested PCR amplify G6 PD containing exon 2～13 fragments Genes are sent for sequencing.The sequencing results were collated with software such as DNAStar11.0 and Bio Edit7.2.5.The obtained DNA sequence was compared with the wild-type reference sequence and mutant reference sequence of the G6 PD gene.The start and end points of exon13 exon region,and the DNA sequences of these 12 exons are spliced into the c DNA sequence of G6 PD gene along the order of exon2 to exon13.(3)The MEGA5.04 software was used to adjust the file format and amino acid conversion of the c DNA strand and the exon10～13 coding segments,and compared with the wild-type sequence to confirm the missense mutations and synonymous mutation sites.Expected heterozygosity(He),nucleic acid diversity index(π),synonymous substitution rate(Ks),and heterosynthetic substitution rate(Ka)ratio of the haplotype of the G6 PD gene sequenced gene fragment using Dna SP 5.10 software Ka / Ks ratio,analysis and calculation analysis.(4)Correlation coefficients(Relevance,R)and odds ratios(OR)of G6 PD gene coding site mutations with Primaquine-induced hemolysis were calculated using the "cross-listing" program of IBM SPSS 21 software in statistics.Perform analytical calculations.Results:(1)In this study,184 blood samples of G6 PD gene of reported cases of P.vivax in Yunnan Province were collected,and 44 samples of complete c DNA strands of G6 PD gene(1545 bp)in blood samples of reported cases of P.vivax were obtained.One of them was from hemolysis.G6 PD genome of the case;91 samples of exon 10～13(495 bp in length)of the G6 PD gene partial blood sample of the reported vivax malaria case were obtained.The mutation sites of 44 c DNA strands aligned with the wild-type sequence include c.461 T> A,c.574 C> T,c.786 C> T,c.1059 C> T,c.1311 T> C,and c.1376 G> T,with frequencies of 1.5 / 100,000,1.5 / 100,000,1.5 / 100,000,1.5 / 100,000,47.1 / 100,000 and 1.5 / 100,000.The mutation site of 91 exon10～13 part of the G6 PD gene alignment with the wild type sequence was c.1311 T> C,no mutation site positively related to primaquine-induced hemolysis occurred c.1376 G> T,the mutation site is c.1311T> C,the frequency is 168.72 / 100,000.(2)The two-site mutation linkage of c.1311 and c.1376 exists only in the genome of one hemolytic case,and the c.1376 site mutation is positively correlated with the occurrence of Primaquine-induced hemolysis(R = 1.000,P ? 0.05).(3)44 c DNA strands are defined as 6 haplotypes(Hap＿1～Hap＿6),and He,π,Ka / Ks are 0.493,0.001,and 0.062,respectively;c.1311 single-point mutation Hap＿2 haplotypes account for the most,It is 68.2%(30/44),and the allele composition is also the most complicated.Wild hemizygotes(6/44,13.6%),mutant hemizygotes(17/44,38.6%),and wild homozygotes(5/44,11.5%),Mutant homozygotes(11/44,25%),mutant heterozygotes(5/44,11.4%)and other 5 types of zygotes.The T value of Tjima’s neutral test was-1.414(P> 0.05),and the D value of the entire segment was <1,indicating that the mutations detected in the study sequence were not non-neutral mutations under directional selection pressure,Indicating that the study sequence is very conservative,and the missense mutation rate is lower than the synonymous mutation rate.(4)91 c DNA strands are defined as 2 haplotypes(Hap＿1～Hap＿2),He,π,0.2784,0.001 and 0.001 respectively;c.1311 single point mutation Hap＿2 haplotype accounted for the most,83.5%(76/91),with the most complicated allelic composition,including wild hemizygous(12/91,13.2%),mutant hemizygous(56/91,61.5%),wild homozygous(3/91,3.3%),There are 5 types of homozygotes,including mutant homozygotes(18/91,19.8%)and mutant heterozygotes(2/91,2.2%).Tjima ’s neutral test has a D value of 0.48503(P> 0.10),but its D value <1,indicating that the mutations detected in the studied sequence are not non-neutral mutations under balanced selection pressure.G6 PD gene exon nucleotide variation is not affected by selection pressure,or is not greatly affected by selection pressure,which is consistent with the neutral mutation hypothesis.Conclusions:1.In this experimental sample,6 mutation sites were detected from 44 G6 PD whole genes,3 of which were synonymous mutations,and the other 3 caused amino acid mutations,of which c.461,c.574 and c.786 were new Three types of base substitution mutations found.2.By analyzing the G6 PD gene mutation in a case of P.vivax malaria suspected of primaquine-induced hemolysis in Yunnan,the double site mutation linkage of c.1311 and c.1376 only exists in the genome of the hemolytic case.The study found that c.The 1376 mutation is related to primaquine-induced hemolysis.Only the G>T mutation at position c.1376 was positively correlated with the occurrence of oxidant-induced hemolysis(R=1.000,P?0.05).3.Of the 44 whole-gene samples in this study,32 cases had mutations at c.1311,and 91 cases of G6 PD gene exon10-13 had 76 mutations at c.1311.It shows that in the malaria population in Yunnan province,c.1311 is a common mutation site of the exon11 coding region of G6 PD gene exon11,and the exon11 of G6 PD gene is a high-risk region of genes susceptible to mutation in exon 2～13 coding region.4.A total of 8 mutant genotypes were identified in 44 samples,18 hemizygous 3 mutant genotypes(461,1311,1376),6 heterozygous 2 mutant genotypes(786,1311),and 13 pure mutations Three mutant genotypes of zygote(574,1059,1311).5.In this study,the primers and PCR technology of optimized design were used to amplify the entire gene encoding sequence of G6 PD gene exon with a total length of 1545 bp,which laid the foundation for the analysis of G6 PD gene exon polymorphism. |
PDF Full Text Request