Research On The Polymorphism Of The 14 Bp Deletion Polymorphism Of HLA-G In Severe Preeclampsia

Hypertensive disorder complicating pregnancy (HDCP) is occurring after the 20th gestational week.Its pathology, physiological changes related to the various body systems, including gestational hypertension,pre-eclampsia (Preeclampsia), eclampsia, chronic hypertension with pre-eclampsia as well as five types of chronic hypertension with pregnancy. In our country the incidence of 9.4%-10.4%,7%-12% abroad,which is seriously affect the safety of fetal and maternal morbidity and badly compromised the health of mothers and infants.Severe pre-eclampsia is a hypertensive disorder during pregnancy. Which is a common and important type of disease stage, Patients develop a transient high blood pressure, edema, proteinuria. Its characteristic pathological changes in uterine spiral arteries physiological recast obstacles and endothelial cell injury. With regard to the incidence of severe pre-eclampsia mechanisms are not yet entirely clear, has always been the focus of the study of obstetric. Domestic and foreign scholars have tried a lot of clinical observation and experimental study for its etiology and pathogenesis and put forward several theories as follows:mmunity doctrine,Genetics,abnormal trophoblast invasion of uterine myometriumv,ascular endothelial damage,nutritional deficiency and insulin resistance. In which the immune theory, genetics that are not mutually isolated, but interact with each other to promote and gradually formed the immunogenetics said it was the most active research areas in this part.The core of immune genetic compatibility is histocompatibility. Human leukocyte antigen (HLA) as an important etiology of immune genes, are triggered allograft rejection in a major antigen, The majority of scholars believe that the parent-derived genes fetus on the mother is a kind with the kinds of semi-allograft. Under normal circumstances, the mother and fetal immune tolerance existing between the Government can ensure that maternal-fetal peaceful co-existence. In pre-eclampsia state of the immune tolerance is broken, Which lead to acute graft rejection in a series of similar to the pathological changes. Once the fetus delivered, the symptoms can be reversed. We inferred that, pregnancy is a special physiological state,, and is the result of maternal-fetal both sides, regardless of normal or abnormal. Therefore, not only the disease-causing genes may come from the mother, but also may come from the fetus, or maternal-fetal interaction.Human leukocyte antigen-G (HLA-G) as a non-classical HLA-G I b antigen, mainly expressed in the maternal-fetal interface extravillous cytotrophoblasts (EVCT). This rigorous histological distribution and immune tolerance during pregnancy has played an important role in the particularly striking. As one molecule of immune tolerance, it can be through a variety of mechanisms involved in immune tolerance induction and maintenance.involved in the occurrence and development of pathological pregnancy, HLA-G is the most critical part to study the pathogenesis of the relationship of HLA and pre-eclampsia.Including my research group in the past, a large number of studies have shown that:in the placenta of pre-eclampsia women, the HLA-G mRNA and HIA-G protein expression is abnormal or lower than the normal pregnency women. When the HLA-G expression defects, maternal-fetal interface may lead to immune rejection enhanced protective response to weakened, impaired trophoblastic invasion of human capabilities, resulting in shallow placental implantation, placental insufficient for the flow and eventually lead to pre-eclampsia. In recent years, studies have found, HLA-G gene polymorphism may lead to the existence of the origin of this cascade of factors. Because a high degree of polymorphism of HLA genes lead to virious immune responses between individuals,and the capacity and differences in disease susceptibility genetic factors. Now, many of the HLA-related diseases need to genotyping technology to re-examine in order to locate susceptibility genes at the molecular level, in order to lay the foundation for gene therapyHLA-G gene polymorphism, including coding and non-coding region polymorphisms.The polymorphism in the coding region is limited, mostly meaningless mutation and deletion, the nucleotide sequence of the changes are not caused by amino acid changes, does not change the number and function of proteins, the most recent study found that HLA-G 3'-UTR Article 8 of the 14bp exon deletion/ insertion sites (+14bp/-14bp) polymorphisms affect the HLA-G transcription and expression, and pathology of pregnancy are closely related. On this polymorphism and susceptibility to recurrent miscarriage has been confirmed, but the relationship between severe pre-eclampsia is not clear that further research needs to be done.More and more studies have shown that during pregnancy the mother's immune system to antigens of the immune tolerance of transplanted embryonic formation of the mother during pregnancy is not only a function of the immune system was suppressed, but the maternal-fetal immune regulation to achieve balance between the results. Many domestic and foreign studies have also confirmed that a single from his mother on the one hand consider the HLA-G gene polymorphism and the relationship between severe pre-eclampsia is not comprehensive, this particular state of pregnancy may be due to maternal-fetal gene interactions caused by the two sides. And this polymorphism in a result of geographic, racial and ethnic differences and still need to study.ObjectiveHLA-G Exon 8 genotyping was performed by polymerase chain reaction (PCR) in 42 Severe PE patients,45 normal pregnant women and their fetal,in which Chinese Han population in Henan Province. Respectively compare the distributions of the allele frequencies and genotype frequencies of the two groups, Combine maternal and fetal to analyze the differences of the genotype special bindings. in order to further explore the incidence of severe pre-eclampsia provide an important mechanism of clues, but also for clinical information between the use of maternal-fetal morbidity predict the risk of pre-eclampsia provide experimental evidence.Materials and methods1 MaterialsChoose from 42 Severe severe pre-eclampsia patients,45 normal pregnant women and their fetal, between October 2008 and February 2009 in the Third Affiliated Hospital of Zhengzhou University Hospital. The patients met the diagnostic criteria issued by the Sanitation Ministry of the People's Republic of China on the 6rd edition of "Obstetric and Gynecology",and the exclusion of endocrine disease, chronic hypertension, kidney disease, diabetes, cancer, inflammation etc.And all patients were followed up to 12 weeks postpartum. Average age of patients (30.60±5.76) years and the mean gestational age of (35.95±3.22) weeks. Another option the same period in normal late pregnant women and their newborns 45 pairs of the control group, were due to social factors to caesarean section, with an average age (30.09±4.80) years, with an average gestational age of (36.52±1.56) weeks. Two groups of pregnant women were residents of Henan Province, Han nationality, no history of mixed marriages, non-blood relations, and maternal age, gestational age, and maternal comparisons, there was no significant difference (P> 0.05).2 Methods2.1 Specimen CollectionWe extracted with severe pre-eclampsia and normal pregnant women in late pregnancy peripheral blood 2ml, at the same time in the two groups of pregnant women, cord blood taken during delivery sterile 2ml, the mother,and cord blood are used sodium citrate,-20℃refrigerator freezing for inspection.2.2 Genomic DNA extractionPut frozen blood into room temperature to lyze, then add leukocyte lysis to leukocyte marc which is got by centrifugation after the lyse of ereuthcyte, and at the same time shaking it to make it float. Throuhgout 30 minutes at 55℃water. Cooled to normal temperature, when it salts out, add the ethanol into the reaction system, then shake it and centrifuge. After finishing it, you'll see marc resembling cotton. Wash it by using 70% alcohol. When it dry, solute it in sterile triple-distilled water,assay the absorption by UV spectrophotometer and measured the content and purity. Adjust the dense of DNA to 100μg/ml, and store it at-80℃for use.2.3 HLA-G gene typing2.3.1 According to PCR primer design method, from Shanghai Invitrogne synthetic biotechnology company. Exon8 part of the fragment: Upstream:5'-GTGATGGGCTGTTTAAAGTGTCACC-3' Downstream:5'-GGAAGGAATGCAGTTCAGCATGA-3'β-actin: Upstream:5'-GTGGGGCGCCCCAGGCACCA-3' Downstream:5'-GTCCTTAATGTCACGCACGATTTC-3'2.3.2 PCR amplification reactionUsing 25μl reaction system, containing lOxTaqbuffer buffer 2.5μl,2mM dNTPMix 2.5μl,5μM HLA-G primer 2μl,5μM internal control primer 2μl, Taq DNA polymerase 0.5U,25 mM MgC12 1.5μl, genomic DNA 1μl,3 distilled water 12.8μl. 96-well PCR reaction plate stamped with paraffin oil.2.3.3 PCR reaction conditions94℃pre-denaturing for 5min,94℃30s,64℃lmin,72℃2min, a total of 35 cycles the final extension at 72℃10 min.2.4 PCR amplification products of the electrophoresis5μl PCR product obtained directly by adding that contains 0.5μg/ml 0.5 x 1000 Gelred nucleic acid stain 3.5% agarose gel electrophoresis tank,100V electrophoresis 45min, in the gel image analysis system, observation, photographs. according to the different 14bp deletion polymorphism,There are three types of HLA-G gene Exon 8 PCR amplification products in electrophoresis, namely:amplification products of a belt, a length of 210bp, can be interpreted as a homozygous deletion (-14bp/-14bp); amplified products as a belt, a length of 224bp, can be interpreted as the insertion homozygous (+14bp/+14bp); amplified products as two bands, respectively, the length of 210bp and 224bp, can be interpreted as a lack of heterozygotes (-14bp/+14 bp); length is restricted reference materials for the 548bp fragment-action.2.5 224bp and the 210bp PCR products were sequencedSelect a zone electrophoresis PCR products were separate, deletion homozygote (-14bp/-14bp) and insertion homozygous (+14bp/+14bp) of the PCR reaction stock solution, sent them to the Invitrogen company to be purified and sequenced. Results and GenBank Blast sequence comparison with the HLA-G Exon8 fragment match (GenBank accession number J52705).224bp product of a period of more than 210bp product of a length 14bp gene sequences:ATTTGTTCTTGCCT.3 Statistical analysisUse SPSS13.0 software package for Statistical analysis., goodness of fitχ2-test applied to determine whether the genotype distribution of Hardy-Weinberg equilibrium; measurement data between the two groups compared with the single-factor analysis of variance; direct calculation method to calculate allele of each group frequency; allele frequency distribution differences between fourfold table method usingχ2-test,χ2-test conditions that do not fit the data with the Fisher exact probability method to calculate P values. When the two groups between the allele or genotype frequencies of compatibility with a significant difference between genotypes (P<0.05), to further calculate the odds ratio associated with intensity (OR value) and 95% confidence interval (CI). In order to test the level of a= 0.05.Results1 Comparison of clinical general informationAnalysis of severe pre-eclampsia group and normal group of pregnant women in late pregnancy clinical data, we can see that comparing the two groups of pregnant women in age, gestational age differences were not statistically significant, (P> 0.05)2 HLA-G gene polymorphism 14bp deletionSevere pre-eclampsia group and the normal late-pregnant group HLA-G gene 14bp deletion polymorphism analysis:The genotype frequency distribution in two groups of mother and their infents, were consistent with Hardy-Weinberg equilibrium(P> 0.05). The population genetic equilibrium genetic line.2.1 HLA-G 14bp deletion polymorphism allele and genotype frequency comparison in two groups of pregnant womenHLA-G 14bp deletion polymorphism in the allele frequencies between the two groups of pregnant women were no significant difference (P>0.05); The genotype comparison in two pregnant women:Severe preeclampsia group (-14bp/-14bp), (-14bp/+14 bp), (+14 bp/+14 bp) genotype frequencies were 42.9%,45.2%,11.9%, while the normal pregnant group, the frequency were51.1%,44.4%,4.4%, compared the genotype frequency distribution, the differences were not statistically significant (P> 0.05);2.2 HLA-G 14bp deletion polymorphism allele and genotype frequency comparison in two groups of newbornsNewborns with severe pre-eclampsia group of 14bp deletion polymorphism allele frequency was+14bp 44.4%,-14 bp 56.0%, normal group was+14 bp30.0%,-14 bp70.0%, There is no significant difference between the two groups(χ2=3.678 P=0.055); The genotype comparison in two newborns:Severe preeclampsia group (-14bp/-14bp), (-14bp/+14 bp), (+14 bp/+14 bp) genotype frequencies were 28.5%, 54.8%,16.7%, while the normal pregnant group, the frequency were 48.9%,45.2%, 8.9%, compared the genotype frequency distribution, the differences were not statistically significant(P> 0.05);2.3 Two groups of maternal/fetal HLA-G 14bp deletion polymorphism genotype compared CompatibilitySevere preeclampsia mother/child are homozygous deletion (-14bp/-14bp) compatibility genotype frequency was 14.3%, significantly lower than the normal late pregnant women 33.3%, respectively, statistically significant difference[χ2= 4.304, P = 0.038; OR=3.000 (95% CI 1.036 8.690)]. Athers genotype binding between the two groups showed no significant difference (P> 0.05).Conclusion1 It suggested that HLA-G 14 bp deletion polymorphism was relative to the susceptibility of Severe PE in Chinese Han population in Henan Province;2 Maternal/fetus (-14bp/-14bp) genotype binding may release the risk of the severe pre-eclampsia.