Polymorphism Of HLA-G Gene 3'UTR Region And Its Relationship With HLA-G Expression In Dalian Han Population

Background: Human Leukocyte Antigen(HLA)is the expression product of human major histocompatibility complex(MHC),which plays a biological role in immunological recognition,antigen presentation,processing and regulation of immune response.Many studies have shown that HLA is an important genetic susceptibility factor for many diseases.HLA-G is a non-classical HLA class I molecule,and the3'untranslated region(3' UTR)of the gene is polymorphic.Studies on the gene polymorphism of 3'UTR of HLA-G gene in population in Jiangsu and Yunnan were reported in some domestic research.Some studies on gene polymorphism in Brazil and other countries were reported in some research abroad.There were differences of gene polymorphism of 3'utr of HLA-G gene among different countries and regions.shola-G transcribed from mRNA is an important molecule of HLA-G gene expression,which plays a systematic role in immunoregulation of the body.The polymorphisms at the 5'and 3' ends of HLA-G may affect the HLA-G expression,then it affects the function of HLA-G molecule.At present,there has been no research report on the gene polymorphism of 3'UTR of HLA-G gene and its correlation with the mRNA expression of HLA-G in Han population in Dalian Area.Purpose: To investigate the gene polymorphism of 3'UTR of HLA-G gene in Han population in Dalian Area and to discuss its correlation with mRNA expression of HLA-G.Method::Whole venous blood of 294 unrelated healthy blood donors in Dalian Area was collected.The blood donors were Han nationality,aged between 18 and 55,including 215 males and 79 females.The DNA of blood sample was extracted with automatic Nucleic Acid isolation machine.The polymerase chain reaction was used to amplify the 3'UTR of HLA-G gene of the sample DNA,and genes were sequenced.The direct counting method was used to count the number of genotypes of each siteof3'UTR of HLA-G gene,and calculate the genotypes and alleles frequencies.Thechi-square test in SPSS was used toperform hardy-weinberg equilibrium(HW)andcompare the allele frequency of polymorphic site of 3'UTR of HLA-G gene in different populations.Online SHEsis(http://analysis.bio-x.cn/)was applied to analyze linkage disequilibriumof polymorphic site of 3'UTR of HLA-G gene.Phase was applied to build haplotype to obtain haplotype frequency and possible double-type ofthe sample.The Poptree2 was adopted to calculate the genetic distance of 3'UTR of HLA-G gene in population from different countries and regions,and a genetic evolutionary tree.Peripheral blood mononuclear cells were isolated with lymphocyte separation medium and total RNA was extracted.After the total RNA was reverse-transcribed into c DNA with reverse transcription,Quantitative Real-time PCR was applied to test the mRNA expression of HLA-G with ?2M gene as reference gene so as to analyze the correlation between the double-type and mRNA expression of the sample.Results: After PCR amplification and gene sequencing,the sequence information of23 HLA-G polymorphisms of the 3'UTR of the HLA-G gene in 294 samples from Han population in Dalian were obtained.Among them,Twelve sites including +296114bp INDEL(rs37119462),+3003(rs1707)C/T,+3010(rs1710)C/G,+3027(rs17179101)A/C,+3035(rs17179108)C/T,+3092(rs180827037)A/C,+3120(rs760052251)C/G,+3121(rs138249160)A/G,+3142(rs1063320)C/G,+3183(rs187320344)C/T,+3187(rs9380142)A/G,+3196(rs1610696)C/G were polymorphic,which conform to hardy-weinberg equilibrium after testing and the remaining 11 sites were monomorphic without polymorphism.Allele frequencies of 8 representative HLA-G polymorphism sitesof3'-UTR of HLA-G of Han population in Dalian were compared with those of other 18 countries and regions.It was analyzed and found that the allele frequency distribution of8 representative polymorphism sitesof the Han population in Dalian Area was different from that in other 18 countries and regions.The allele frequencies of 8 polymorphism sitesof the 3'UTR of HLA-G gene in Han population in Dalian Area and population in18 other countries and regions were analyzed to calculate their genetic distance and buildthe genetic evolution tree,then it was found that the Han population in Dalian had a close genetic distance with the population in Jiangsu,China and Tokyo,Japan,deviating from the genetic background.It was found through analysis with SHEsis that8 sites(minimum frequency > 0.03)of the 3'UTR of HLA-Ggene in Han population in Dalian presented linkage disequilibrium.Phase was adopted to analyzeand obtain 8haplotypes,among which there were 5 haplotypes with the frequency more than 0.03,and haplotype UTR-1(DELACGGGCG,frequency=0.394558)was the most common.The correlation between the mRNA expression of HLA-G and different genotypes at 8 SNP sites of the 3'UTR including +2961 14 bp insertion/deletion(14bp INDEL),+3003 C/T,+3010 C/G,+3027 A/C,+3035 C/T,+3142C/G,+3187A/G and+3196 C/Gwere analyzed.It was found that there were differences in the mRNA expression of HLA-G different genotypes at +3010(rs1710)C/G,+3142(rs106330)C/G and +3187(rs9380142)A/G(P=0.0109,P=0.0105 and P= 0.0058),while no differences were found in the mRNA expression of HLA-G of different genotypes at other 5 sites.Based on multiple test by Dunn 's multiple comparisons,the results showed that at the site of + 3010(rs1710),the mRNA expression of genotype GG was lower than that of genotype CC(P < 0.01),and at the site of +3142(rs106330)the mRNA expression of genotype GG was higher than that of genotype CC(P < 0.01).And atthe site of+3187(rs9380142)the mRNA expression of genotype GG was lower than that of genotype AA(P < 0.01).Phase was applied to obtain the possible double-type of the3'UTR of HLA-G gene of the sample and analyze the relationship between the different double-type and the expression of HLA-GmRNA.The results showed that the differences of mRNA expression of 3'UTR of HLA-G gene of different double-type was statistically significant(P=0.0075).Among them,the mRNA expression of HLA-G of double-type UTR1-UTR1 was lower than that of double-type UTR1-UTR3(P=0.0268).Conclusion: In this study,the distribution data of genotypes,alleles and haplotype frequencies of polymorphisms of 3 '-UTR of HLA-G gene in the Han population in Dalian were obtained,which provided the basic data for the research on the correlation between polymorphisms of 3 '-UTR of HLA-G gene and diseases in population in Dalian.In this study,it was found that the distribution of allele frequency of HLA-G gene 3'-UTR in the Han population in Dalian was different from that in other domesticand foreign regions.It was obviously unique depending on regions,which may be related to differences of region and nationalities.In addition,it was still found that as for the Han population in Dalian,the gene polymorphism of 3'-UTR of HLA-G gene at3010(rs1710)C/G,+ 3142(rs106330)C/G and 3187(rs9380142)A/G was related to the mRNA expression of HLA-G,and HLA-G gene 3'UTRdiploid-to-haploid was related to HLA-G mRNA expression.