Relationship Between The Single Nucleotide Polymorphisms In The Upstream RegulatoryRegion Of The HLA-G And The Susceptibility To Sever Pre-eclampsia

Preeclampsia (preeclampsia, PE) is a serious pregnancy-specific complications, the incidence rate of which is 9.4% in our country. hypertension, proteinuria and edema are occurring after the 20th gestational week, physiological changes related to the various body systems, and usually with fetal growth restriction. The disease is one of the mainly reasons of morbidity and mortality for maternal and perinatal, which severely affected maternal and child health.The etiology and pathogenesis of the preeclampsia have not been well understood, and is the focus of research at home and abroad. Currently there are four commonly accepted theories as following:genetic susceptibility, immune maladaptation, placental ischemia, oxidative stress response. Epidemiological Investigates suggest that genetic susceptibility and immune maladaptation may interact, participate in the pathogenesis of preeclampsia. Around this central idea, The human leukocyte antigen (human leukocyte antigen, HLA) systems, which mainly cause rejection, is researched for screening of pre-eclampsia susceptibility loci and becoming an important idea of exploreing the pathogenesis of preeclampsia.What make the expression of HLA-G reduce may be related to HLA-G gene 5 'upstream regulatory region (5'-upstream regulatore region,5'-URR) single nucleotide polymorphisms (single nucleotide polymorphisms, SNP). SNP is single bases differences in normal genomic DNA in one (more) groups,and is the nature of human genetic polymorphism in the performance information.It has a number of more widely distributed, relatively stable features present in the upper chromosome. SNP may cause the protein production of phenotypic changes,so the SNP is the key influences on human body. people may be particularly vulnerable to or suffering from certain diseases. This study from the perspective SNP severe preeclampsia genetic background, suggesting that the upstream regulatory region of HLA-G gene may lead to onset of severe preeclampsia in a (few) SNP susceptibility locus, these changes result in base locus the HLA-G protein expression in the maternal-fetal interface changes, thus breaking the maternal-fetal immune tolerance, leading to the onset of severe preeclampsia.Taking into the important role of the HLA-G during pregnancy, and the clinical potential value probobly as a immune tolerance molecular, we research the factors that affect the expression of the HLA-G to further understand the biology characteristics of the HLA-G, and to provide valuable information for clinical practice.PurposeTherefore, we collected peripheral blood of severe preeclampsia and normal of pregnant women in Henan Han population. By sequencing the allele and genotype frequencies of HLA-G gene 5'-URR SNP are calculated to comparison of distribution between severe preeclampsia women and normal pregnancy women. From the perspective of immunogenetics we investigate whether the SNP of the upstream regulatory region of the HLA-G gene is associated with the severe preeclampsia.Materials and methods1 object39 Severe severe pre-eclampsia mothers are choosen from October 2008 to February 2009 in the N.O.3 Hospital of Zhengzhou University, whose mean age is (31.17±5.97) years; In the same period in normal late pregnant 43 women are choosen as the control group, who were due to social factors to caesarean section, with an average age (29.82±5.07) years The age of two groups are equilibrium-line test and the difference was not statistically significant (P> 0.05). The patients met the diagnostic criteria issued by Yue JIE on the 7rd edition of "Obstetric and Gynecology". Two groups of pregnant women had no blood pressure, heart, kidney, liver disease and diabetes mellitus, no the history of blood transfusion and immunotherapy.This study is approved by our hospital ethics committee and obtained by the informed consent of participants.2 Methods2.1 specimen collection We extracted with severe pre-eclampsia and normal pregnant women in late pregnancy peripheral blood 2ml before the cesarean, save these blood with 3.8% sodium citrate and set -20℃refrigerator spare. At the same time we collected personal information and medical history data to establish a database.2.2 Genomic DNA extraction-20℃frozen blood was gradually dissolved at room temperature.500μl the whole blood are taken into 1.5ml Eppendorf tube, Increased red blood cell lysate (triple-distilled water) 900μl, reversed mixing,4000 r/min, discard supernatant, repeat 2-3 times the above steps until the precipitation layer colorless. Then remove supernatant and add to white blood cell lysate 370μl (final concentration of proteinase K 750ug/ml, SDS of a final concentration of 1.0%), break precipitation with the oscillator,55℃water bath for 30 minutes. The next, when the mixture cooled to room temperature, add 100μl saturated NaCl, the full mix 15 seconds, 13,000 r/min for 6 minutes. After centrifugation, the supernatant was transferred to another sterile 1.5ml Eppendorf tube, then centrifuge 13,000 r/min for 3 minutes. After centrifugation, the supernatant then is transferred to another sterile 1.5ml Eppendorf tube, added ethanol 1ml, gently inverted several times, and centrifuged 13,000 r/min for 6 minutes.Tossed away the supernatant after centrifugal, and add 70% of ethanol 500μl, reversed mixing,13,000 r/min for 6 minutes. Taichung tossed away in the double decontaminated supernatant Eppendorf tube attached to the DNA and make it dry, plus three distilled water 50μl,then we detect the concentration and purity of DNA in UV spectrophotometer, and store it at -80℃for later use2.3 specific amplification of HLA-G 5'-URRNested polymerase chain reaction (nested polymerase chain reaction, nPCR) is adopted to specific amplification HLA-G 5'-URR. According to the National Center for Biotechnology Information (NCBI) GenBank database we get the upstream regulatory region of the HLA-G (L36549.1, the target gene) and human P actin (NC₀00006.11, internal reference gene) sequences, using Primer3 Input (version 0.4. 0) to design primers as follows.β-actin acts as the inner and outer nest PCR amplification of two experiments in the internal reference primers. Outer nest primers: upstream 5'-TGGGTGCTGTTTAAAGCCAAT-3', downstream 5'-CCCCGAGAGTA GCAGGAAGA-3', fragment of 1761bp; within the nest primers:upstream 5'-AGCA GGAGGTGAGGAAAAGG-3', downstream 5'-CCTTGGTA ACCCCTGAATGA-3' fragments of 593bp;βactin primers within the reference sequence:upstream 5'-TGCCAAGTGGAGCACCCAA-3', downstream 5'-GCATCTTGCTCTGTGCAG AT-3, fragments of 785bp. In our experiments, Reference gene within is separately PCR as a positive control and triple-distilled water as a template for amplification, as a negative control, and the two controls ensure the accuracy of experimental. PCR reaction components were added on ice.in the ultra-clean work Taichung2.3.1 the outside PCRthe following components are added in 0.2ml Eppendorf tube:5×PrimerStar Buffer 2μl,2.5mM dNTP 0.8μl, 10μM each of upstream and downstream primer 0.2μl,5×PrimerStar enzyme 0.1μl, genomic DNA 0.7μl, plus triple-distilled water to 10μl.Amplification parameter setting:98℃denaturation 10s,65℃annealing 8s,72℃extension of 2min,30 cycles.2.3.2 the within PCRthe following components are added in 0.2ml Eppendorf tube:5×PrimerStar Buffer 10μl,2.5mM dNTP 4μl, 10μM each of upstream and downstream primer 1μl, 5×PrimerStar enzyme 0.5μl, outside the nest after the products were diluted 10 times to take 3μl, triple-distilled water added to 50μl.Amplification parameter setting:98℃denaturation 10s,67℃annealing 8s,72℃extension 50s,5 cycles; 98℃denaturation 10s,65℃annealing 8s,72℃extension 50s,5 cycles; 98℃denaturation 10s,63℃annealing 8s,72℃extension 50s,5 cycles; 98℃denaturation 10s,61℃annealing 8s,72℃extension 50s,20 cycles. The total cycles is 35. (4) PCR amplification products of the 2.4 electrophoresis1μl product of PCR, 1μl 6×bromophenol sample buffer and 4μl 0.5×TBE electrophoresis buffer are mixed and adding into 1% agarose gel (which is used GOLD VIEW nucleic acid dye processing), 100bp ladder DNA ladder as maker,120V electrophoresis 25min, observation, photographs.The standard of a successful PCR amplification:Ladder is clear band; the background is clear; purpose fragments and reference fragments is clear, and the position is corresponds to the expected fragment sizes, without non-specific amplified band and the smear; no DNA fragments appears in negative control lane.In this study, the PCR of HLA-G 5'-URR amplified successfully (Figure 1). The purpose fragments is 593bp, the position is right farther than 600bp of the ladder, and the reference fragments is right farther than 800bp of the ladder. The negative controls showed no bands. Electrophoresis results show the success of our experiments.2.5 Purification of PCR productAfter the initial check of electrophoresis, the successful PCR amplification are judged. thick blocks are made, and the remaining PCR product re-electrophoresis (120V,25min), and under UV light gel DNA fragment are recoveried. The process of experiment can refer the illustration of DV805A produced by Takara Biotechnology Co., Ltd.Then the concentration and purity of the product are measured by UV spectrophotometer, and are saved in the ice box to send to the biotechnology company for sequencing.2.6 sequencingSequencing, according to Sanger dideoxy chain method, is being in introvigen biotechnology company by ABI 3730 sequencing instrument. Sequencing primers is the within nest downstream primers. Reverse sequencing means the sequence in map is just the reverse complementary sequence which is landing in GenBank(accession number L36549.1)2.7 DNA sequence analysisSequence obtained after sequencing are analysis by Chromas, DNASTAR software and manual to reduce error. The reverse complementary sequences are compaired with the sequence in the GeneBank to look for SNP sites.At last we find the amplified fragment in our experiment is similar with the 5'URR of the HLA-G and the existence of several SNP sites. THE SNPs of DNA sequence are shown in Figure 2 to 7In this study, high-fidelity DNA polymerase is used to keep the results reliable; and the DNA sequence is very clear and sequencing results are satisfied; discard the datas which is not sure.3 Statistical analysisSPSS 13.0 statistical analysis software package were used; goodness of fitχ2-test applied to determine whether the genotype distribution of Hardy-Weinberg equilibrium; The frequency of allele between two groups was analysised with direct counting, and the distribution differences used the column Table x2 test,χ2 test conditions that do not fit the data calculated with the Fisher exact test P value. Toα= 0.05 level as tested.Results1 Comparison of the clinical general informationAnalysis of severe preeclampsia group and the normal group of pregnant women in late pregnant clinical data, we can see that age difference between the two groups of pregnant women was not statistically significant (P> 0.05), Gestational age difference was statistically significant (P<0.05).2 the Hardy-Weinberg genetic equilibrium analysis of each SNP locusIn this study,39 pregnant women of severe preeclampsia and 43 normal pregnant cases in Henan, Chinese Han were detected the SNP of the 5'-URR fragments of the HLA-G gene through sequencing that is located between-1155bp to-716bp.The results show:8 SNPs were detected that is respectively,-1179A/G (rs1736935),-1155G/A (rs3823321),-1140A/T (rs1736934),-964G/A (rs1632947),-762C/T(rs1632946),-725C/G(rsl233334),-716T/G(rs2249863),-689A/G(rs2735022). The allele T and the genotype GG of the-725 was not detected that is different with the relevant reports.-1179A/G,-1155G/A,-689A/G is locate in the both ends of,DNA sequence which lead to the poor accuracy of their sequencing results,so we did not analysis. We analysis the Hardy-Weinberg genetic equilibrium of the data of the-1140A/T,-964G/A,-762C/T,-725C/G,-716T/G, and the results showed that the normal control group and patient group representative samples are good(χ2=0.01-2.11, P>0.05), suggesting its distribution in the two groups of pregnant women in the balance, from the same Mendelian population.Two SNPs in the fragment that were previously reported have not detected polymorphisms in this study, namely-1138bp (rs17875389),-1121bp (rs3115630).3 The frequencies comparision of genotype and allele of the HLA-G 5'-URR between the severe preeclampsia group and the normal pregnant groupComparison of -1140A/T,-964G/A,-762C/T,-725C/G,-716T/G genotype and allele in HLA-G 5'-URR between severe preeclampsia and normal pregnant group distribution shows:the frequencies of the -964 site of GG, GA, AA genotypes in severe preeclampsia were 15.4%,48.7%,35.9%,and the normal frequency of late pregnancy groups were 37.2%,39.5%,23.3%, The distribution between the two groups were significant statistically (P<0.05);-964 locus G, A allele in the severe preeclampsia group, the frequency was 39.7%,60.3%, and the frequencies of normal pregnant group were 57.0%,43.0% The distribution between the two groups were significant statistically (P<0.05).the frequencies of the -716 site of GG, GT, TT genotypes in severe preeclampsia were 38.5%,43.6%,17.9%,and the normal frequency of late pregnancy groups were 20.9%,37.2%,41.9%, The distribution between the two groups were significant statistically (P<0.05);-964 locus G, T allele in the severe preeclampsia group, the frequency was 60.3%,39.7%, and the frequencies of normal pregnant group were 39.5%,60.5% The distribution between the two groups were significant statistically (P<0.05).The differ of-1140A/T,-762C/T,-725C/G genotypes and alleles between severe preeclampsia and normal pregnant groups were no significant statistically (P>0.05).ConclusionThe differ between severe preeclampsia and normal pregnancy groups in the women of childbearing age of the Henan Han which happen in -716,-964 Genotype and allele frequencies suggesting that the 5'-URR SNP of HLA-G may be associated with the susceptibility of severe preeclampsia.