Study On The Association Between Gene Polymorphisms Of IL-2 And IL-12 And Susceptibility Of HBV Intrauterine Infection

Hepatitis B virus (HBV) infection is still a worldwide public health problem. It has high prevalence in China and 30%⁵0% HBV carriers result from mother to infant transmission. Among patients due to HBV intrauterine infection, around 90% of them become chronic HBV infection. The characters of chronic HB patients attributed to HBV intrauterine infection are worse response to antiviral therapy and susceptible to developing cirrhosis and hepatocellular carcinoma. So the research for interruption vertical transmission is very important for our country. The major reason of HBV intrauterine infection is because of the lower ability of host to clean up HBV during early phase of HBV infection. T helper 1 type cytokines play an important role in cleaning up HBV and interleukin 2 (IL-2) and interleukin 12 (IL-12) are the presents of T helper 1 type cytokines. And we have found in our prophase study that the level of IL-2 and IL-12 was decreased in HBV intrauterine infection group compared with without HBV intrauterine infection group. It suggests that the two cytokines maybe have association with HBV intrauterine infection. IL-12 is a heterodimeric cytokine composed of two disulfide-linked subunits designated p35 and p40. The p35 mRNA (IL-12A) is composing expression while p40 (IL-12B) mRNA is modulatory expression, so the polymorphisms of IL-12B have more important effects at the expression of IL-12 than those of IL-12A. As further researches for genome medicine, the effect of genetic background to the occurence and development of non-inherited diseases of human are got more and more attention. Although there are many SNPs in one gene, the research for the whole of several important SNPs as well as haplotype maybe benefit to find the relationship between gene and diseases.The objective of this research is to study the relationship between IL-2 gene -330(A/C) site and IL-12B gene four polymorphisms including 3'UTR +-1188(A/C) site, G deletion site SNP, promoter region 4bp insert polymorphism (IL-12Bpro, C7CTAA/CG) and ATT_8/9 STR polymorphism, and the susceptibility to HBVintrauterine infection. To constitute and analyze the haplotype of IL-12B gene polymorphism sites, to explore the genetic susceptible factors of high-risk children developing intrauterine HBV infection. To explore the cooperating function of IL-2 and IL-12's effect on intrauterine HBV infection of our country.Subjects and MethodsThe subjects were selected from outpatients of the hepatitis B vaccine follow-up clinics of our hospital. To collect peripheral blood of all subjects who have been accepted initiative or initiative combined passive inmmunication. Intrauterine HBV infection was defined as peripheral blood HBsAg and/or HBV DNA positive at birth and at least 6 months(Group I). Normal immune children were defined as peripheral blood negative for HBV marker since birth and afterwards HBsAb titers level were of >10 mIU/L (Group II). Mothers of subjects in group I and II were HBV carriers and parts of them were injected hepatitis B immunoglobulin (HBIG) during the end course of pregnancy. There were some normal and health children as control group. To extract genome DNA of all subjects, then a polymerase chain reaction for aimed segment and sequency or performance capillary electrophoresis to test gene polymorphisms of IL-2 and IL-12B gene were performed in three groups. To contribute and analyze the haplotype of IL-12B gene polyphorphisms by PHASE software and compare the difference of frequency of every genotype, allele, haplotype of IL-12B and genotype combination of IL-2 and IL-12B between group I , group II and control group.ResultsFrequency of AA, AC and CC genotype of IL-2 gene -330 sits were 32.4%, 56.8% and 10.8% in Group I , and 51.3%, 38.2% and 10.5% in Group II, in control group the frequency was 54.8%, 38.1%, 7.1%, respectively. A significant difference was found in frequency distribution of IL-2 -330 site genotype between Group I and Group II (x²- 6.237, p=0.044); And there are significant difference of AA genotype and non-AA genotype between Group I and Group II (p=0.019), 0R_AA = 0.455 (95%CI, 0.234-0.883); OR_AC=2.132 (95%CI, 1.107-4.645). Frequency distribution of IL-2 -330 allele in three groups was A allele 64.3%, 72.3% and 73.8%, C allele 35.7%, 27.7% and 26.2% respectively. A significant difference was found in allelefrequency distribution of IL-2 -330 between Group I and Group II (p=0.010), and OR_A=0.65 (95%CI, 0.404-1.054); ORc=1.53 (95%CI, 0.948-2.476). Frequency of AA, AC and CC genotype at IL-12B gene +1188 site were 25.7%, 44.3% and 30.0% in Group I , and 36.6%, 47.9% and 15.5% in Group II, in control group the frequency was 48.8%, 39.0%, 12.2%, respectively. A significant difference was found in frequency distribution of the CC genotype and non-CC genotype between Group I and Group II (x² =17.78, p＜0.001), OR_CC = 2.338 (95%CI, 1.028-5.035); Frequency distribution of IL-12B +1188 allele in three group was A allele 47.8%, 60.7% and 67.8%, C allele 52.2%, 39.3% and 32.2% respectively. There are significant difference was found in allele frequency distribution between Group I and Group II (x²= 4.586, p=0.032), OR_A=0.597 (95%CI, 0.372-0.959), OR_c= 1.673 (95%CI, 1.043-2.684).IL-12B gene promoter 4bp deletion polymorphismdeletion homozygous (IL-12Bpro2.2, CG/CG) play an advantage in Group I (49.6%), there were only 29.3% and 27.9% in Group II and control group respectively. To compare distribution of three genotypes among three groups, a significant difference was found between Group I and Group II (x² -7.223, p=0.027). The difference of IL-12Bpro2.2 (CG/CG) genotye and non-IL-12Bpro2.2 genotye between Group I and Group II was more significant (x²=6.286, p=0.012), OR_pr02.2=2.346 (95%CI, 1.198-4.595). No significant difference was found in distribution of two alleles (CTCTAA/CG) X² =3.519, p=0.061.No significant difference was found in distribution of alleles and genotypes of IL-12B G/—and ATT_(8/9) polymorphisms between Group I and Group II.The PJCICTAA haplotype's distribution of IL-12B +1188 site and IL-12Bpro polymorphism has significant difference between Group I (19.29%)and Group II (31.43%), p=0.028, OR_a/ctcAA=0.521(95%CI, 0.300-0.904). No significant difference was found in distribution of haplotype "—/A/CTCTAA/ATTg" combined by IL-12B gene four polymorphisms between Group I and Group II (p=0.065), But the frequency in Group I (15.11%) was obviously lower than in Group II (24.24%). Other haplotypes' distribution was no significant difference between two groups. Then haplotype C/G and A/- combined by G/— and +1188 of IL-12B gene play an advantage in two groups(>90%), but the frequency of haplotype A/G å’Œ C/-was very low (<10%) in two groups. Their distribution was no significant difference between two groups.The AA/AA, AA/(-/-), AA/(CTCTAA/CTCTAA) genotype combination which were made ofbyIL-2 gene-330 (A/C) site with +1188, G/- and IL-12Bpro of IL-12B respectively played an advantage in Group II (Group I vs Group II = 4.92%:20.63%, 4.69%: 17.65%, 1.56%: 13.24%), and their distribution between Group I and GroupII had significant difference (p = 0.015, 0.026, 0.018). But the AC/CC, AC/GG, AC/(CG/CG) genotype combination of them played an advantage in Group I (GroupI vs II - 21.31% : 4.72%, 18.75% : 2.94%, 26.56% : 11.76%), and there are significant difference in distribution between two groups (p = 0.007, 0.004, 0.030). Other haplotypes' distribution has no significant difference between two groups.ConclusionThere is a relationship between IL-2 gene -330 site SNP and susceptibility to intrauterine HBV infection. IL-2 gene -330 site A allele and AA genotype play an advantage in group II. It suggests that IL-2 -330 site A allele and AA genotype maybe protect high-risk infants to develop intrauterine HBV infection. But high-risk children carring with AC genotype are susceptible to develop to intrauterine HBV infection.There is an relationship between IL-12B +1188 site, IL-12Bpro polymorphisms and susceptibility to intrauterine HBV infection: high-risk children carring C allele and CC genotype at +1188 site maybe susceptible to develop HBV intrauterine infection, contrarily A allele maybe protect high-risk infants to develop intrauterine HBV infection; IL-12Bpro2.2 (CG/CG) is the susceptible genotype to high-risk infants to develop intrauterine HBV infection. No association of IL-12B gene G/— and ATT_(8/9) polymorphisms and HBV intrauterine infection could be demonstrated. The haplotype of IL-12B gene +1188 site SNP and IL-12Bpro polymorphism has association with susceptibility to intrauterine HBV infection. A/CTCTAA haplotype maybe protect high-risk infants to develop intrauterine HBV infection; G/- and + 1188 (A/C) sits SNPs are of linkage disequilibrium. And IL-2 and IL-12 have the cooperated effect in intrauterine HBV infection in our country.