| Backgroud:G6PD deficiency is the most common with X-linked incomplete dominance of one of the genetic diseases.There are about 400 million people around the world.Patients with G6 PD deficiency can cause acute hemolysis under the inducement of eating broad beans,taking oxidative drugs,bacterial and viral infections,which may be accompanied by hypovolemic shock or acute renal failure,which can lead to death in severe cases.In my country,G6 PD deficiency presents a characteristic of "high in the south and low in the north".Yunnan Province is also an area with a high incidence of G6 PD deficiency.The main reasons may be related to the high incidence of malaria,the majority of ethnic minorities,and the border with Southeast Asian countries.Objective:To investigate the epidemiology and clinical characteristics of G6 PD deficiency among Dai people in Dehong and Xishuangbanna Prefecture,Yunnan Province.The LD block and haplotype construction of Dai people in Dehong and Xishuangbanna Prefecture are carried out to provide a basis for elucidating the origin of G6 PD gene mutation types.Method:1.The NGS detects the G6 PD genotype of 1596 samples from Dai people in Dehong and Xishuangbanna Prefecture,Yunnan Province.Extract genomic DNA from 75 samples of G6 PD gene mutation.Using PCR technology to amplify 7 G6 PD gene fragments.The Gene Sequencing Technology was used to detect 14 G6 PD gene mutations in 75 samples.2.Fluorescence spot method was used to quantitatively detect G6 PD enzyme activity in1596 samples from Dai people in Dehong and Xishuangbanna Prefecture(1009 samples from Dehong Prefecture and 587 samples from Xishuangbanna Prefecture).The ROC curve was used to analyze the cut-off value of G6 PD enzyme activity.3.SPSS 20.0 was used to analyze the genotype and clinical phenotype of G6 PD gene mutation samples(Dehong Prefecture 508 and Xishuangbanna Prefecture 260)of Dai people in Dehong and Xishuangbanna Prefecture.4.Using Haploview 4.2,SNP linkage inequality analysis and LD block construction were performed on the Dai people in Dehong and Xishuangbanna Prefecture.Result:1.Using NGS to carry out genetic screening for G6 PD deficiency among the Dai people in Dehong and Xishuangbanna Prefecture.The G6 PD gene mutation type gene carrying rate in the two regions was 48.12%(male 40.89%,female 53.73%).The carrying rate in Dehong Prefecture was 50.35%(men 45.37% and women 54.25%).The carrier rate in Xishuangbanna Prefecture was 44.29%(men 33.58% and women 53.11%).A total of 44 types of G6 PD gene mutations(12 single-base mutations and 32 compound mutations)were found in the Dai people of Dehong and Xishuangbanna Prefecture.The common pathogenic G6 PD gene mutations carrying rate in Dehong Prefecture: c.487 G>A(9.12%),c.1388 G>A(5.35%),c.392 G>T(3.47%),c.1376 G>T(2.77%).The common pathogenic G6 PD gene mutations carrying rate in Xishuangbanna Prefecture: c.1388 G>A(3.92%),c.1376 G>T(2.90%),c.392G>T(1.70%),c.487 G> A(1.36%).2.Fluorescence spot method was used to detect the G6 PD enzyme activity in Dai people in Dehong and Xishuangbanna Prefecture,and the results showed that the positive rate was14.53%;Dehong Prefecture 14.97%(male 21.99%,female 9.71%);Xishuangbanna Prefecture 13.80%(male 15.85%),female 12.11%).When the cut-off value of G6 PD enzyme activity was 2.25 U/g Hb,the specificity was 97% and the sensitivity was 27%;If five SNPs were eliminated(c.1311 C>T,rs1050757 A>G,IVS11-nt93 T>C,rs2472393 A>G,rs2472394A>G),the enzyme activity cut-off value was 2.25 U/g Hb,The specificity was 94% and the sensitivity was 67%;Analysis of the cut-off value of G6 PD enzyme activity in men of Dai people in Dehong and Xishuangbanna Prefecture.When the cut-off value of enzyme activity was 2.25 U/g Hb,the specificity was 94% and the sensitivity was 41%;If the genes at five SNPs(c.1311 C>T,rs1050757 A>G,IVS11-nt93 T>C,rs2472393 A>G,rs2472394 A>G)G6PD enzyme activity cut-off value analysis,the ROC curve area was 0.921.When the enzyme activity cut-off value was 2.25 U/g Hb,the specificity was 92% and the sensitivity was 84%.Analysis of the cut-off value of G6 PD enzyme activity of in women of Dai people in Dehong and Xishuangbanna Prefecture.When the cut-off value of enzyme activity was2.25 U/g Hb,the specificity was 97% and the sensitivity was 17%.If the genes at five SNPs(c.1311 C>T,rs1050757 A>G,IVS11-nt93 T>C,rs2472393 A>G,rs2472394 A>G)enzyme activity cut-off value analysis,the enzyme activity cut-off value was 2.25 U/g Hb,the specificity was 94% and the sensitivity was 30%.3.In Dehong and Xishuangbanna Prefecture c.1376 G>T mutation sites,male enzyme activity decreased most significantly;in Dehong female patients with G6 PD deficiency,c.392G>T mutation site enzyme activity changed significantly.The 4 types of G6 PD gene mutations in Dehong Prefecture(c.487 G>A,c.1388 G>A,c.392 G>T,c.1376 G>T)male G6 PD enzyme activity decreased significantly than females,statistically Significance(P<0.001).In Xishuangbanna Prefecture,c.1376 G>T and c.143 T>C two types of G6 PD gene mutations,the enzyme activity of males decreased significantly than that of females,which was statistically significant(P<0.05).There was no significant difference in enzyme activity between males and females of the three gene mutation types c.1388 G>A,c.392 G>T,and c.487 G>A(P>0.05).4.Dehong and Xishuangbanna Prefecture carried out LD block and haplotype construction of about 11 Kb in length at 14 SNPs,suggesting that the two regions c.1311 C>T,rs1050757 A>G,IVS11-nt93 T>C,rs2472393 A >G and rs2472394 A>G,these 5 mutations have strong linkage.Conclusion:1.The second-generation genetic testing technology was used to test the G6 PD deficiency gene in the Dai people of Dehong Prefecture and Xishuangbanna Prefecture in Yunnan Province.The G6 PD gene carrier rate in the two regions is 48.12%,which is the highest reported domestic and international gene carrier rate.The main pathogenic G6 PD gene mutation types in Dehong Prefecture was c.487 G>A(9.12%).The main pathogenic G6 PD gene mutation types in Xishuangbanna Prefecture was c.1388 G>A(3.92%).2.Fluorescence spot method was less sensitive to G6 PD enzyme activity screening,and limited significance in the diagnosis of G6 PD deficiency.3.Different gene mutation types have different changes in enzyme activity.In the c.1376 G>T mutation sites in Dehong and Xishuangbanna,males have the most obvious decline in enzyme activity.4.The results of constructing the LD block of Dai people in Dehong and Xishuangbanna Prefecture suggest that: c.1311 C>T,rs1050757 A>G,IVS11-nt93 T>C,rs2472393 A>G and rs2472394 A>G have five SNPs Strong linkage. |
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