| Systemic lupus erythematosus (SLE) is a prototypic autoimmune diseasecharacterized by the production of autoantibodies and deposition of immune complex,eventually leading to the damage of almost any organ systems by the autoimmunereactions. Though the exact etiology of SLE is unknown up to now, it is considered tobe a polygenic disease that involves both genetic and environment factors in the onsetand development of the disease.Great deals of data have shown that SLE represents strong familial aggregation,with a much higher frequency among first-degree relatives of patients. Theconcordance of the disease in identical twins is approximately 24%, and that indizygotic twins is about 0.4%. Genetic studies have mapped some susceptible loci ofchromosome, among them, 1q23,1q41,2q37,4p16,6p21,11p13,12q24,16q13loci have been reported to be associated with SLE at least in two independent studies.Both MHC (or HLA) and non-MHC regions included in the susceptibility to SLE.The most associated region in human HLA is HLA-DR2. HLA-Ⅲgene products suchas complement C2,C4 and tumor necrosis factor(TNF) have been confirmed to beassociated with SLE, moreover, C2 and C4 alone can result in the onset of SLE.Besides MHC genes, many non-MHC genes also take part in the development of SLE,such as complement C1q,FcγR,mannose-binding lectin (MBL),CTLA-4 andprogrammed cell death-1 (PDCD-1).Helper T cells (Th) play critical roles in the development of SLE. Th can beclassified into Th1 and Th2 subsets according to their different cytokine secretion.Th1 cells mainly secrete IL-2,IFN-γand TNF-α, involved in cell immunity toeliminate intracellular pathogens; Th2 secrete IL-4,IL-5,IL-10 and IL-13, taking partin humoral immunity to defense against extracellular pathogens. Keeping balancebetween Th1 and Th2 is very important for health, once the balance destroyed, manwill suffer from some diseases. Predominance of Th1 cytokines is associated with organ-specific autoimmune diseases, such as rheumatoid arthritis,type 1 diabetes andGRAVE disease, while, predominance of Th2 cytokines is associated with allergicdiseases and systemic autoimmune diseases like asthma,allergic rhinitis. Cytokinesproduced by Th1 and Th2 can cross-regulate partner subset's development andactivity. For instance, IFN-γ, a Th1-cytokine, can inhibit the production of a Th2cytokine, IL-4. And vice versa, IL-4 inhibits the secretion of IFN-γ.Animal experiments and clinical data confirmed that SLE is associated withTh1/Th2 bias, and it seems different stage and different manifestations of SLEresulted from different Th1/Th2 bias. Therefore, gene polymorphisms affectingTh1/Th2 balance might be associated with susceptibility to SLE.Forkhead-box jl(Foxj1) belongs to Forkhead-box (Fox) transcription factorfamily. All members in this family contain an about 100 amino-acid DNA bindingdomain. Up to now, more than 100 members included in this family, among them, atleast 43 members belong to human FOX gene family, these members classified into17 subfamilies, FoxA to FoxQ. Fox members take part in diverse biologicalprocesses, including development, metabolism, aging and cancer. Some members alsoparticipate in the regulation of immune responses, for example, Foxnl is associatedwith development of thymic epithelial cells; Foxo is important for immune toleranceof T and B lymphocytes; Foxp3 is closely related to regulatory T cells' function, andso on. Fox j1, also named HFH-4,FKHL-13, is mainly expressed in ciliated cells oflung, choroids plexus, testis and ovary, it's functions mainly linked to ciliated celldevelopment and left-right axis determination. Recently, Peng and his coworkersfound that Fox j1 expression was reduced in lupus-prone mice, and Fox j1-/- chimeramice represented lupus-like systemic autoimmune inflammation. In vitro studyrevealed that Th cells isolated from Fox j1-/- chimera mice produce more IL-2,IFN-γas well as Th1 specific transcription factor T-bet than wild type when stimulated withCD3 specific antibodies, indicating a Th1 bias state. The inhibition of T cell activity ispartially through the induction of IκBβwhich inhibited the nuclear translocation ofNFκB and reduced the production of IL-2 and IFN-γ, subsequently inhibited T cellactivation. Fox j1 still inhibit B cell activity through a similar mechanism. Furthermore, Fox j1 can also inhibit thymocyte egress. Therefore, Fox j1 plays criticalroles in preventing autoimmune diseases.Human FOXJ1 gene lies on chromosome 17q22-25, consisting of 2 exons and 1intron. 204 SLE patients and 418 controls were recruited from cheonbuk, cheonnamand chungbuk area in South Korea. Polymerase chain reaction (PCR) was performedto amplify FOXJ1 gene. Direct sequencing method was used to scan the wholeFOXJ1 gene including it's promoter(～2.2kb). Total of 7 single nucleotidepolymorphisms (SNPs) were found, among them, g.-460C＞T and g.-342G＞C lie onpromoter region; g. 1164G＞C, g. 1805G＞T, g. 1824C＞G and g. 1849G＞C lie on intronregion; and g.3375G＞C lies on 3'-UTR. Interestingly, g.-460C＞T lies on a bindingsite of a speculated transcription factor MZF1. Pairwise linkage disequilibriumbetween each pair of SNP loci was evaluated, the result indicate that g.-460C＞T and-342G＞C are in complete LD, g.1805G＞T,g.1824C＞G and g.1849G＞C are incomplete LD (D'=1,r2=1). According to LD and locations of SNPs, 3 of them werechosen to perform large sample genotyping, that is, g.-460C＞T in promoter region,g.1805G＞T in intron and g.3375G＞C in 3'-UTR. Genotyping was carried out bysingle base extension (SBE) method, a kind of minisequencing method. All genotypeswere recorded and were statistically analyzed.Hardy-Weinberg equilibrium test was performed by chi-square test, the resultindicates all genotypes were in Hardy-Weinberg equilibrium, indicating no samplebias.Chi-square test from 2×2 or 2×3 contingency table or Fisher's exact test wereperformed to compare the difference of the genotyping or allele frequency, the resultshowed that genotyping and allele frequency of g.-460C＞T and g.1805G＞Tpolymorphisms did not have significant difference between SLE and the controlgroup. But polymorphism g.3375G＞C in 3'-UTR showed significant differencebetween two groups (p=0.0072和0.0042). The genotype frequency of CC consistsof 1.7% in the control group, but only 0.5% in SLE group, indicating this genotypemay be a protective factor.Expectation maximization (EM) algorithm was used to construct haplotypes. Polymorphisms of g.-460C＞T,g. 1805G＞T和g. 3375G＞C in FOXJ1 constructed totalof 8 haplotypes. There are 3 main haplotypes, explaining 94% of distribution incontrols, and 2 major haplotypes in SLE accounting for 92.1% of distribution.Haplotype -460C-1805T-3375G consists of 5.1% in the control group, but only 1.5%in SLE group, represented significant difference(p=0.01), indicating this haplotype ispossibly a protective factor.ANOVA was used to evaluate the relationship between each genotype and ANAlevel in SLE patients, the result indicating no association between each genotype inthese 3 polymorphisms with ANA levels.Chi-square test from 2×3 contingency table was used to evaluate the associationbetween each genotype and anti-Sm, anti-Ro, anti-La, anti-RNP antibodiesrespectively in SLE patients, the results indicating no association between eachgenotype in these 3 polymorphisms with anti-Sm, anti-Ro, anti-La, anti-RNPantibodies.Chi-square test from 2×3 contingency table was used to evaluate the associationbetween each genotype and serositis, arthritis and lupus nephritis respectively, theresults indicating no association between each genotype in these 3 polymorphismswith serositis, arthritis and lupus nephritis.In a word, total of 7 SNPs were found in this study. Among them, g.-460C＞Tand g.-342G＞C lie on promoter region; g.1164G＞C,g.1805G＞T,g.1824C＞G andg.1849G＞C lie on intron; g.3375G＞C lies on 3'-UTR. g.-460C＞T lies on aspeculated transcription factor MZF1 binding site. Polymorphism g.3375G＞C mightbe associated with SLE susceptibility, and haplotype -460C-1805T-3375G might be aprotective factor on SLE. Polymorphisms of g.-460C＞T,, g. 1805G＞T and g. 3375G＞Cin FOXJ1 gene seems no association with ANA levels, anti-Sm,anti-RNP,anti-Roand anti-La antibodies as well as serositis, arthritis and lupus nephritis. This studymight be helpful in uncovering the biological function of FOXJ1 gene further, andwill provide new evidence.Asthma is a Th2 predominant disease characterized by airwayhyper-responsiveness, eosinophil counting increase and IgE elevation. There are about 150 million of Asthma patients all over the world, and 20 million of that in China. Itsmorbidity and mortality are increasing year by year. Therefore, Asthma becomes acommon social problem that attracting more and more attention. It is considered thatAsthma is a kind of polygenic disease influenced by both genetic and environmentfactors. Searching and localizing the susceptible genes is a hotspot. Up to now,5q31-33,6p,,11q,12q are considered to be main susceptible loci of Asthma, andIL-4,TNF-α,IL-4R,IL-10,β2-Adrenoceptor are confirmed to be susceptible genes.But, for Asthma is a complex polygenic disease associated with many minor effectedgenes, there are still a lot of works to be done.IL-27 is a newly identified heterodimeric cytokine of IL-6/IL-12 family. It iscomposed by Epstein-Barr virus induce gene 3 (EBI3, IL-12p40 homology) and p28(IL-12p35 homology). IL-27 is mainly secreted by activated APCs likemacrophages and dendritic cells). Its signal is conducted through IL-27 receptor(IL-27R), a heterodimeric chain of IL-27ra (WSX-1, TCCR) and gpl30. IL-27R ismainly expressed on the surface of naive T cells and natural killer (NK) cells, it'sligation with IL-27 resulted in the activation of JAK/STAT pathways. Recently, it isreported that IL-27 also can activate p38 MAPK and ICAM-1/LFA-1 pathways.IL-27 is associated with IL-12 and IL-23 in many ways, and IL-12 is a keyplayer in Th1 differentiation of naive T cells, whereas, IL-23 is associated with Th1maintenance in memory T cells. Therefore, early researches on IL-27 are emphasizedon Th1 differentiation. In vitro studies identified that IL-27 can induce T-bet, a Th1specific transcription factor, through STAT1 pathway on naive T cells. Subsequently,T-bet induce the expression of IL-12Rβ2, a subunit of IL-12 receptor which is notexpressed on na(?)ve T cells, in this way, na(?)ve T cells can response to IL-12 stimulation,resulting Th1 differentiation. So, IL-27 is a critical player in the early stage of Th1differentiation. On the other hand, IL-27 can inhibit the expression of a Th2 specifictranscription factor-GATA3, resulted the decrease of IL-4 and IL-13. These in vitrostudies indicate that IL-27 might make the Th1/Th2 balance skewing to Th1 state.Whereas, quite confused results were obtained from in vivo studies. It isfounded that IL-27 is required for Th1 differentiation only when IL-4 exists in vivo, but is not required when IL-4 is absent. When IL-4 exists, the role of IL-27 is toantagonize IL-4 through inhibiting the expression of GATA3 on na(?)ve T cells. GATA3is a Th2 specific transcription factor, closely related to the expression levels of IL-4and IL-5. Otherwise, GATA3 can inhibit the expression of IL-12Rβ2 and STAT4,blocking Th1 differentiation and decreasing IL-12,IFN-γsecretion. Therefore, theinfluence of IL-27 in vivo seems more important in inhibiting Th2 differentiation,making the Th1/Th2 balance skewing to Th1.IL-27 also plays an important role in Th1 negative regulation in vivo. IL-27ra-/-mice infected with T. gondii could produce efficient Th1 responses in the early stageto control infection. But these animals will develop tissue damage even lethalinflammation as a result of sustained T cell activation. T. cruzi infected and ConAinjected IL-27ra-/- mice develop more serious liver damage than wild type, this kind ofdamage is induced by Th1 responses. These studies indicated that IL-27 could inhibitoveractivated Th1 responses. The immunosuppressive activity may be resulted byIL-2 and IL-6 suppression.Taken together, IL-27 drive naive T cells to Th1 differentiation at the early phase,whereas in activated Th1 cells, IL-27 inhibits Th1 cells' proliferation, limit Th1 cells'strength and duration to prevent organ damage by overactivated immune responses.Therefore, abnormal expression of IL-27 might be associated with autoimmune and/or allergic diseases. Many studies have confirmed the relationship between IL-27 andimmune diseases. It is reported that neutrolizing IL-27p28 subunit could suppressongoing of adjuvant-induced arthritis. In a experimental autoimmuneencephalomyelitis (EAE) model, the expression levels of IL-27p28,EBI3 and IL-27raare highly elevated. Otherwise, IL-27/IL-27ra signaling is also related to theexacerbation of intestinal inflammation in a colitis model resembling humaninflammatory bowel disease. Besides Th1 predominant diseases, IL-27 is alsoassociated with the development of Th2 predominant diseases. In an experimentalasthma model, IL-27ra deficient mice were induced more serious airwayhyper-reactivity,increased eosinophil counting and elevated IgE level, indicating thatIL-27 can down-regulate airway hyper-reactivity and pulmonary inflammation. Human IL-27p28 lies on chromosome 16. To detect whether IL-27 genepolymorphisms associated with asthma susceptibility, IL-27p28 promoter region,exons and it's boundary regions were scanned for searching single nucleotidepolymorphisms(SNPs), and the relationship of these polymorphisms with asthmasuscetptibility was evaluated by a case-control study.288 asthma patients and 444 normal controls were included in this study. Allmembers were recruited from Cheonbuk area in South Korea. Polymerase chainreaction (PCR) was used to amplify IL-27p28 gene. Then, direct sequencing methodwas used to scan the IL-27p28 gene including it's promoter(～1.9kb),5 exons andtheir boundary regions. Total of 4 SNPs were found, among them, g.-964A＞G lies onpromoter region; g.2905T＞G in the exon 2, g.4603G＞A in intron 3, and g.4730T＞Cin exon 4. Interestingly, a single nucleotide change T to G at g.2905T＞G in exon 2resulted in a amino acid change of p.Ser59Ala and a single nucleotide change T to Cat g.4730T＞C in exon 4 resulted in a amino acid change of p.Leu119Pro. Pairwiselinkage disequilibrium between each pair of SNP loci was evaluated, the resultindicate that g. g.4603G＞A and g.4730T＞C are in complete LD, g.1805G＞T,g.1824C＞G and g.1849G＞C are in complete LD (D'=1,r2=1). According to LD andlocations of SNPs, 3 of them were chosen to perform large sample genotyping, that is,g.-964A＞G in promoter region, g.2905T＞G in exon 2 and g.4730T＞C in exon 4.Genotyping was carried out by single base extension (SBE) method, a kind ofminisequencing method. All genotypes were recorded and were statistically analyzed.Hardy-Weinberg equilibrium test was performed by chi-square test, the resultindicates all genotypes were in Hardy-Weinberg equilibrium, indicating no samplebias.Chi-square test from 2×2 or 2×3 contingency table or Fisher's exact test wereperformed to compare the difference of the genotyping or allele frequency, the resultshowed that genotyping and allele frequency of g.2905T＞G and g.4730T＞Cpolymorphisms did not have significant difference between the asthma and the controlgroup. But polymorphism g.-964A＞G in promoter region showed a significant difference between two groups (p=0.006和0.003), indicating this polymorphismmay be associated with susceptibility to asthma.Expectation maximization (EM) algorithm was used to construct haplotypes.Polymorphisms of g.-964A＞G,g.2905T＞G and g.4730T＞C in IL-27p28 constructedtotal of 8 haplotypes. There are 4 main haplotypes in each group, explaining 95.3% ofdistribution in controls, and 94.7% of distribution in asthma. Haplotype964A-2905T-4730T consists of 65.8% in the control group, but 71.2% in asthma group,represented significant difference (p=0.035), indicating this haplotype is possibly asusceptible gene polymorphism; Haplotype -964G-2905G-4730T consists of 8.4% inthe control group, but 5.4% in asthma group, represented significant difference(p=0.035), indicating this haplotype is possibly a protective polymorphism.ANOVA was used to evaluate the relationship between each genotype and serumtotal IgE level in asthma patients, the result indicating no association between eachgenotype in these 3 polymorphisms with IgE level.To summary, total of 4 SNPs were found in this study. Among them, g.-964A＞Glies on promoter region; g.2905T＞G in exon 2, g.4603G＞A in intron 3, andg.4730T＞C in exon 4. Interestingly, a single nucleotide change T to G at g.2905T＞Gin exon 2 resulted in a amino acid change of p.Ser59Ala and a single nucleotidechange T to C at g.4730T＞C in exon 4 resulted in a amino acid change ofp.Leu119Pro. Polymorphism g.-964A＞G might be associated with asthmasusceptibility, and haplotype -964A-2905T-4730T might be associated withsusceptbility to asthma, whereas, haplotype -964G-2905G-4730T in IL-27p28 mightbe a protective factor. And IL-27p28 gene polymorphisms of g.-964C＞T,g.2905G＞Tand g.4730G＞C seems no association with serum total IgE levels, anti-Sm,anti-RNP,anti-Ro and anti-La antibodies as well as serositis. This study might behelpful in uncovering the biological function of IL-27 gene further, and will providenew evidence for gene diagnosis and prevention of asthma.
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