云南省间日疟患者G6PD基因编码区的突变分析

The mutation polymorphism of G6PD gene coding region in vivax malaria patients in Yunnan Province,China

目的分析云南省间日疟患者的葡萄糖-6-磷酸脱氢酶(G6PD)基因编码区突变多态及其与伯氨喹性溶血现象易发的规律。方法收集云南省2018年经"氯喹/伯氨喹八日疗法"治疗的间日疟病例血样。以服用伯氨喹期间患者出现"茶色"尿液体征、G6PD酶活性下降判断为伯氨喹诱发急性溶血。提取血样的人基因组DNA,PCR分别扩增含G6PD基因的12个外显子片段并测序。与G6PD基因非突变型序列比对,用DNAStar11.0、BioEdit 7.2.5等软件整理、拼接测序序列获得G6PD基因的cDNA序列。用MEGA 5.04、DnaSP 5.10软件分析cDNA序列的突变多态、选择效应等。计算突变多态与伯氨喹诱发性溶血发生的相关性系数(r)和比值比(OR)。结果共收集间日疟患者血样184份,获得44份血样的G6PD基因完整cDNA链(长度为1545 bp),其中1条来自溶血病例的基因组。44条c DNA链与非突变型序列比对的突变位点包括c.461 T>A、c.574 C>T、c.786 C>T、c.1059 C>T、c.1311 T>C和c.1376 G>T,频率分别为1.5/10万、1.5/10万、1.5/10万、1.5/10万、45.6/10万和1.5/10万。c.1311和c.1376的双位点突变连锁仅存在于溶血病例的基因组,且c.1376位点突变与伯氨喹性溶血的发生为正相关关系(r=1.000,P>0.05)。44条cDNA链定义为6种单倍型(Hap1~Hap6),单倍型期望杂合度(He)、核酸多样性指数(π)、Ka/Ks比值分别为0.493、0.001和0.062;c.1311单点突变的Hap2占比最多,为68.2%(30/44),高频率突变位点c.1311的等位组成亦最复杂,出现非突变半合(9/44,20.4%)、突变半合(16/44,36.4%)、非突变纯合(5/44,11.5%)、突变杂合(10/44,22.7%)、突变纯合(4/44,9.1%)等5种合子。c.1311位点突变与伯氨喹性溶血发生的OR值为0.6879(P>0.05),未显示关联性。Tjima’s中性检验D值为-1.414(P>0.05),且全段的D值均<1,表明研究序列检出的突变不属于定向选择压力下的非中性突变,Ka/Ks比值为0.062,说明研究序列非常保守、错义突变率低于同义突变率。结论云南省间日疟患者中,常见的G6PD基因编码区突变为c.1311位点的T>C碱基替换,但仅c.1376位点的G>T突变与氧化剂突变诱发性溶血的发生呈正相关性。

Objective To analyze the polymorphism of G6 PD(glucose-6-phosphate dehydrogenase)gene mutation in coding regions and its correlations with the proneness to primaquine hemolysis in vivax malaria patients in Yunnan Province,China.Methods Blood samples were collected from the vivax malaria patients receiving an treatment regime of"eight-day therapy of chloroquine/primaquine"in Yunnan Province in 2018.Patients with"teacolored"urine and reduced G6 PD enzyme activity during the primaquine therapy were defined as primaquine-induced acute hemolysis.Human genomic DNA was extracted from the blood samples,and of the G6 PD gene fragments containing 12 exon were amplified by PCR and sequenced.The sequences were compared with the nonmutation-type sequence,edited and spliced by DNAStar 11.0 and BioEdit 7.2.5 softwares to generate the cDNA sequence of G6 PD gene.MEGA 5.04 and DnaSP 5.10 softwares were used to analyze the mutation polymorphism the selection effect of the cDNAs.The correlation coefficient(r)and odds ratio(OR)for primaquine-induced hemolysis was estimated.Results A total of 184 blood samples were collected,of them 44 generated complete G6 PD cDNA strand sequence(length,1545 bp),including one sample from a hemolysis case.Alignment of the cDNA chain with the nonmutation-type sequence revealed the mutation sites including 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 a frequency of 1.5/100000,1.5/100000,1.5/100000,1.5/100000,45.6/100000 and 1.5/100000,respectively.The two-site mutation linkage between c.1311 and c.1376 was only present in the genome of the hemolysis cases,and the c.1376 site mutation was positively correlated with the primaquine-induced hemolysis(r=1.000,P>0.05).The 44 cDNA strands were defined into 6 haplotypes(Hap1-Hap6),with an expected heterozygosity,nucleic acid diversity indexπ,and Ka/Ks ratio were 0.493,0.001,and 0.062,respectively.The Hap2 type containing c.1311 single-site mutation accounted for the highest proportion of(68.2%,30/44).The site c.1311 with high frequency of mutation had the most complex allele composition,comprising 5 types of zygote including nonmutation hemizygote(9/44,20.4%),mutant hemizygote(16/44,36.4%),nonmutation homozygote(5/44,11.5%),mutant heterozygote(10/44,22.7%),and mutant homozygote(4/44,9.1%).The OR value of c.1311 mutation to the primaquine-induced hemolysis was 0.6879(P>0.05),showing no correlation in between.The D value of Tjima’s neutral test was-1.414(P>0.05),and the D value of the whole fragment was<1,indicating that the mutations detected in the sequence were not a non-neutral mutation under directional selection pressure;meanwhile the Ka/Ks ratio was 0.062,indicating that the sequence was highly conservative and the frequency of missense mutation was lower than that of synonymous mutation.Conclusion In malaria patients in Yunnan Province,the T>C base substitution at c.1311 is a common mutation in G6 PD coding region,but only the G>T mutation at c.1376 is positively correlated with the occurrence of oxidant induced hemolysis.