The Association Of Mitochondrial Genetic Control Region Variation In Chronic HBV Infection

Objectives:The mutations data of mitochondrial DNA control region and the division of mitochondrial DNA haplogroups among chronic HBV infected crowd and healthy crowd were obtained by amplifing and sequencing mitochondrial DNA control region. Consequently the valuable variable sites and haplogroup related to HBV infection were selected by analysing the distribution of mitochondrial DNA haplogroup and mutations between the two groups.Lastly with combination of clinical data, the relationship were revealed between candidate mitochondrial DNA mutation and haplogroup with clinical manifestations of chronic HBV infection.MethodsrThe genomic DNA of peripheral blood was extracted from 117 patients with chronic HBV infected and 130 healthy donor controls from Yunnan province. Then mitochondrial DNA control region was amplified by PCR and the amplicon was sequenced. Consequently the revised Cambridge Reference Sequence (revised Cambridge Refrence Sequence; rCRS)was used as standard sequence for alignment to achieve all the mutations of mitochondrial DNA control region.Then referencing the mitochondrial DNA phylogenetic tree of the existed world population,classification was done based on haplogroup.Meanwhile the difference of mutation frequency of mitochondrial DNA control region and haplogroup frequency between chronic HBV infection group and control group was statisticated,and the significant difference of maternal genetic component was recognized. At last with the combination of clinical information, the relationship between the mutation of mtDNA control region and mtDNA haplogroup frequency and clinical manifestation was analysed.Results:1)The mtDNA D-loop regional sequence results:Totally there were 247 mtDNA control region sequences of peripheral blood from 117 chronic HBV infected people and 130 healthy people,with the length of 1124bp.Then all sequences was aligned with revised Cambridge Reference Sequence. The results indicated 152 polymorphic variation loci of case group in total at the variation rate of 13.52%,and of which 47 sites were high frequency of mutations(30.72%). Meanwhile,there were 163 polymorphic variation loci of control group at the variation rate of 14.50%,and of which 52 sites were high frequency of mutations (31.90%). Haplogroups were divided from 247 sequences into 21 categories, of which were 244 in total (case with 114, control with 130), respectively M、M*、M7、 M8、C、Z、M9、G、D、N9、Y、 A、R、R*、R9、F、B、B4、B5、K、L3*. With the combination of previous research, the data was analysed by principal component analysis and hierarchy. The analysis result proven that both HBV infected patient and control people were from Han of Yunnan province.The statistics results would be displayed at below.2) The distribution of the mutation sites in the case and the control group showed that there were significant differences in two polymorphic loci (m.523-524AC/del, m.16183A>C) between chronic HBV infected people and healthy people (P<0.05, 0<OR<1). The frequency of m.523-524AC/del mutation in patients with chronic HBV infection (27.35%) was significantly lower than that of the control group (43.85%). The frequency of m.16183A>C mutation in patients with chronic HBV infected (11.97%) was significantly lower than that of control group (26.15%).3)The level of ALT> AST in those who carried mtDNA mutation such as m.489T>C, m.16189T>C,m.16223C>T, m.16519T>C had significant differences between chronic HBV infected crowd and the healthy crowd. And the level of TP in those who carried mutation such as m.16223C>T or m.16519T>C also had significant differences between two groups.4)The mutation frequency(65.71%) of mtDNA 489 T>C in HBeAg positive infected patients was significantly higher than that of HBeAg negative patients(39.02%), P= 0.008 and OR=2.995,95%CI (1.31-6.85).5) The mutation frequency(11.43%) of m.523-524AC/del in HBeAg positive patients was significantly lower than that of m.523-524AC/del in HBeAg negative patients (31.15%), P= 0.012, OR=1.026,95%CI(0.23-2.35).6)There was no difference in the distribution of mitochondrial DNA haplotype betwe en chronic HBV infected people and healthy people. Meanwhile, there was no difference in the distribution of HBeAg positive and HBeAg negative chronic HBV infection.Conclusions:1) The frequency of mitochondrial DNA polymorphic site of m.l6183A>C and m.523-524AC/del in healthy population was significantly higher than those with chronic HBV infected group, which suggested that the two site mutations may be a potential protective factor for chronic HBV infection,or people who without those mutations was susceptibility to HBV.2) The frequency of HBeAg positive infected crowd carried m.489T>C mutation was significantly higher than that of HBeAg negative infected, which suggested that m.489T>C site may be a risk factor for HBeAg persistent positive in some patients.3) The frequency of HBeAg positive infected crowd who carried m.523-524AC/del mutation was lower than that of HBeAg negative infected, which suggested m.523-524AC/del may be associate with HBeAg disappearance or serological conversion.4)The finding of the susceptibility or resistance of mitochondrial DNA mutation sites and the distribution of mtDNA haplogroup type in the chronic HBV infected crowd need futher verify.