Parent-derived HLA-A Gene Associated With Preeclampsia

Pre-eclampsia belongs to hypertensive disorders in pregnancy(HIP). It is a unique disease to pregnancy, the incidence rate was 9.4%～10.4% in our country. This disease has the following characteristics:the blood pressure≥140/90 mm Hg, urine protein≥300mg/24h after 20 weeks of pregnancy. This disease is bad for heart, brain, liver, kidney and some other important organs, resulting in multiple organ failure, which badly compromised the health of mothers and infants. The pathogenesis of preeclampsia has not yet been clarified yet. Its research is one of the hotspots and difficulties in the field of Obstetrics and Gynecology. 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:l:placental trophoblast cells in hypoxic-ischemic; 2:vascular endothelial cell injury; 3:immunity doctrine; 4:Genetics; 5:doctrine of insulin resistance. Immunogenetics of these theories has been widely recognized by scholars and has become the research hotspot in recent years.Pregnancy can be regarded as a successful semi-allogeneic transplantation, some studies found that:pre-eclampsia can occur in patients with acute organ transplant rejection is similar to the acute vascular inflammation pathological changes. Allograft rejection is against the immune response to allograft tissue antigens.The most important tissue antigens is a product encoded by major histocompatibility complex (major histocompatibility compiex, MHC) gene. The human MHC is called human leukocyte differentiation antigen (human leucocyte antigen, HLA), its coding gene on the six human chromosome P21.31, full-length 3600kb, accounting for the entire human genome 1/3000, is by far the most polymorphic of human genes found in complex. The complex not only contains multiple gene loci (locus), but also the same HLA locus with multiple alleles (allele), and the allele frequencies has racial, ethnic, geographic differences. HLA gene products can be divided into three categories: classical HLA genes, including the classic HLA-Ⅰgenes (HLA-A,-B,-C) and the classic HLA-Ⅱgenes (HLA-DR,-DP,-DQ); immune function-related genes, including serum complement components (C2, C4, B factor) encoding genes, antigen processing of commission-related genes, non-classical HLA-Ⅰgenes (such as HLA-E,-F,-G, etc.) and inflammation-related genes (tumor necrosis factor family, etc.); immune related genes, including 21-hydroxylase (CYP21) genes.HLA complex with cell type (hyplotype) genetic, but people are diploid animals, any one individual carried two alleles of HLA genes must come from the father and mother. Embryonic with allogeneic antigen components of the father is a graft to the the mother. Mother,s immune system identify it and produce the immune response. Under normal circumstances, the mother and the fetus develop an immune tolerance. But in patients with preeclampsia, the balance between maternal-fetal disorders, carrying half of the father's genes embryonic antigen were excluded. The results suggest that the parent-derived genes may be play an important role in preeclampsia.HLA-A belongs to the classical HLA-Ⅰgenes, widely distributed in the surface of nucleated cells. It is a key molecule involved in antigen presentation, and a major antigen leading to the same kinds of transplant rejection. It was endogenous peptide gave toxic CD8 positive T cells, and specificity relevant with the particularly immune response.ObjectiveThe topics collected samples of preeclampsia and normal pregnant women and their spouses and their spouses in Zhengzhou Han people,using PCR-SSP technology for HLA-A alleles typing, for exploration parent-derived HLA-A gene associated with the pre-eclampsia. From the HLA polymorphism perspective to discuss the genetic susceptibility or resistance of preeclampsia and clarify the father's immune genetic background with preeclampsia, and to provide theoretical and experimental basis predicting the risk of preeclampsia and early intervention to the high-risk populations. 1 MaterialThe studied population included 53 Chinese patients in our hospital at Zhengzhou of Henan province with pre-eclampsia clinically diagnosed between October 2008 and March 2009 and their spouses. 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". Their average ages were (30.49±5.03) years and average pregnant weeks were (35.98±3.04) week. At the same term randomly selected 51 pairs healthy pregnant women and their spouses as control groups. The average age of pregnant women (29.97±4.80) years and the mean gestational age of (37.45±1.51) weeks. Two groups of patients were excluded from medical complications and gestational diabetes. Two groups of pregnant women are Han Chinese, no blood relationship, no history of mixed marriages and no significant difference in their ages, pregnancy weeks and delivery times.Every one participated in this study signed an informed consent.2 Methods2.1 Sample collectionExtract 2ml peripheral blood of two groups of pre-eclampsia patients and normal pregnant women and 2ml peripheral blood of their spouses under the sterile operating, EDTA anticoagulant,-20℃freeze for test of allele genes.2.2 Gene group extractThis process is done by salting out. Put 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 thirty 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'11 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 100ng/μl, and store it at-20℃for later use. 2.3 HLA-A genotypingIt was performed by polymerase chain reaction-sequence-specific primer (PCR-SSP). According to the nucleotide sequence of HLA-Ⅰtype released in 1995, 24 pairs of HLA-A special primers were designed. Each reaction system includes an control primers which upstream primers is 5-TGC CAA GTG GAG CAC CCA A-3 and downstream is 5-GCA TCT TGC TCT GTG CAG AT-3. All primers were produced by Shanghai Yingjun Biologic Engineering. were produced by Shanghai Biologic Engineering Corporation. PCR was performed in the total 25μl volume containing genomic DNA 1μl,10×buffer 2.5μl, Mg2+1.5μl,2mM dNTP2.5μl,5μMHLA-A primer 2μl,5μM control oligonucleotide primer 2μl, Taq DNA polymerase 0.7U, dist-water 12.8μl. PCR amplification conditions are as follows:preparing denature at 96℃for 5 min and followed by 5 cycles of 96℃for 30s,64℃for50s,72℃for50s,5 cycles of 96℃for 30s,62℃for 50s,72℃for 50s, 10 cycles of 96℃for 30s,60℃for 50s,72℃for 50s,15 cycles of 96℃for 30s,55℃for 50s,72℃for 50s and final extension at 72℃for10min.6μl of PCR products was added directly into 2% agarose gel by 140V electrophoresis for 20min, and observed by UV gel imaging analysis system.3 Statistical analysisUse SPSS16.0 software package for Statistical analysis.Allelic frequencies were directly counted,χ2 or Fisher's exact test were used to compare the difference of allelic distribution between the two groups. Calculate the odds ratio for calculating the disease (oddsratio, OR) and 95% confidence interval (confidenceinterval, Cl)when a significant difference between the two groups. Analyse some specific maternal/father genic bindings,α=0.05 was regarded as testing level.Because each patient's HLA-A locus has two alleles, the gene frequency calculation of the total gene number (the number of patients or controls X 2) as the denominator, the allele number as the molecules.Results1 Comparison of clinical general information Analysis of pre-cclampsia group and normal group of pregnant women in late pregnancy clinical date, we can see that comparing the two groups of pregnant women in age,gestational age differences were not statistically significant(P>0.05).2 Analysis of HLA-A allelic frequencies in normal pregnant women and in pre-eclamptic patients14 kinds of HLA-A alleles were obtained in all samples. The allelic frequencies of HLA-A2(19.61%),-A24(15.69%) and-A11(14.71%) were higher in control group, while the allelic frequencies of HLA-A2(26.42%),-Al 1(16.04%) and-A24(10.38%) were higher in pre-eclampsia group. Statistical differences were not observed in all allelic frequencies.3 Analysis of HLA-A allelic frequencies in spouses of normal pregnant women and pre-eclamptic patients13 kinds of HLA-A alleles were obtained in all samples. The allelic frequencies of HLA-A24(18.63%),-A2(16.67%) and-Al 1(13.73%) were higher in control group, while the allelic frequencies of HLA-A2(20.75%),-A11(15.09%) and-A24(5.66%) were higher in pre-eclampsia group. Statistical analysis showed that the frequency HLA-A24 was lower in spouses of pre-eclamptic patients than in normal pregnant women's (P<0.05),OR=3.050,95%CI(1.236-7.690).Statistical differences were not observed in other HLA-A allelic frequencies.4 Analysis of HLA-A father/mather special bindings in normal pregnant women and pre-eclamptic patientsWe analysized some special allelic bindings (HLA-A2,-A11,-A24) (higher frequencie and P<0.05 in analysis of allelic frequencies).The results showed:the frequency of mother with HLA-A11 positive and father with HLA-A11 positive was higher in pre-eclamptic patients than in normal pregnant women(P<0.05); the frequency of father with HLA-A24 positive and mother with HLA-A24 negative was lower in pre-eclamptic patients than in normal pregnant women while mother negative and father negative higher in pre-eclamptic patients than in normal pregnant women(P<0.05). Conclusions1 Paternal factors play an important role in preeclampsia.2 Parent-derived HLA-A24 may be a Protective factor of the preeclampsia.3 Analysing Some HLA-A mather/father special bindings associated with thepre-eclampsia are more meaningful than simply analysing paternal or maternal.