Association of polymorphisms in non-classic MHC genes with susceptibility to autoimmune hepatitis

Hangzhou,China

Introduction

Autoimmune hepatitis (AIH) is a chronic,generally progressive in flammatory disorder of the liver.It is characterized by the presence of elevated serum levels of transaminase,immunoglobulins,and autoantibodies.[1]Frequently,it is associated with other autoimmune diseases.[2]Although the precise etiology of this disease is unknown,genetic factors have been implicated in its pathogenesis.[3,4]It is considered to be a consequence of autoantigen exposure,genetic predisposition,and impaired self-tolerance triggered by unidentified environmental agents (either infectious or noxious).[3,5]

Genetic factors are known to contribute to the susceptibility for most autoimmune diseases,such as Graves' disease,[6]systemic lupus erythematosus,[7]and insulin-dependent diabetes mellitus.[8]Therefore,AIH has a strong genetic predisposition which affects its occurrence,clinical phenotype and outcome.[3,4]

In patients with AIH,correlations are found with human leukocyte antigen (HLA) which resides in the major histocompatibility complex (MHC),located on the short arm of chromosome 6.Czaja[3]has summarized previous studies on susceptibility alleles for type 1 AIH,and considered that the HLA gene plays an important role in AIH,and shows genetic differences among ethnically different patients.For example,HLA DRB1*0301 and DRB1*0401 are associated with type 1 AIH in white north Americans and northern Europeans,[4]while HLA DRB1*0405 has susceptibility in Japanese.[9]

Although HLA plays an important role in the pathogenesis of AIH,HLA alone cannot explain the entire genetic predisposition,because 30%-50% of AIH patients lack those susceptibility alleles.[10]Current genome scanning studies suggest that genes outside the MHC may play a critical role in autoimmune disorders.This article focuses on our current understanding of these genes' polymorphisms and their association with susceptibility to AIH,as well as their significance in the therapy of AIH.

CTLA-4

CTLA-4,also known as CD152,is expressed on the surface of T cells,and induces peripheral tolerance through binding to B7-1 (CD80) and B7-2 (CD86)on antigen-presenting cells,in competition with costimulatory molecule CD28.Genetic associations of CTLA-4 polymorphisms with autoimmune diseases have been widely reported.[11]Most knowledge concerning the correlation between the CTLA-4 gene and AIH comes from genetic analysis at position 49 of exon 1 (+49 A/G).[10,12-15]Agarwal et al[12]first described this polymorphism (CTLA-4 G allele) as a second susceptibility allele (besides HLA-DRB1) in type 1 AIH in northern European Caucasoid patients,and speculated that there might be synergy between the HLA-DRB1*0301 and the GG genotypes in terms of disease risk.They analyzed the association between genotype distribution and clinical phenotype,and their statistical analysis revealed that patients with the GG genotype had a higher level of serum aspartate transaminase and a greater frequency of thyroid microsomal antibodies.[12]

However,some of the subsequent investigations which focused on this novel polymorphism showed different results.Several independent studies reported that this polymorphism did not confer susceptibility to type 1 AIH in either the Brazilian[10]or Japanese populations.[13]Another study from Djilali-Saiah showed the opposite result.It presented a genetic analysis of the CTLA-4/CD28 region in a panel of Caucasian children with AIH and their families from Canada and France.Compared with Agarwal's result,Djilali-Saiah's result emphasized that children carrying the A allele,not the G allele at the +49 position of CTLA-4 were predisposed to AIH type 1.[14]In China,Fan et al[15]found polymorphism of CTLA-4 gene exon 1 (+49 A/G) was not associated with AIH,but there was a strong correlation of this allelic variation in primary biliary cirrhosis patients,in whom the frequency of the G allele was significantly increased compared to controls.Furthermore,another novel polymorphism of CTLA-4 correlated with AIH was also reported by Fan and his colleagues.They found that the frequency of C alleles in promoter -318 was significantly increased in AIH patients,and considered that the -318 C allele might contribute to susceptibility to AIH in the Chinese population.[15]Unfortunately,there was no association found at the position of promoter -318 in the Japanese population.[13]

Although those previous studies present con flicting results on the correlations between CTLA-4 polymorphisms and AIH,the role of CTLA-4 in the pathogenic mechanisms of autoimmune diseases has been increasingly recognized.[16,17]A number of autoimmune diseases have been associated with CTLA-4 gene exon 1 (+49 A/G),including type 1 diabetes[18]and multiple sclerosis.[11]There are four domains in full-length CTLA-4,termed the leader peptide,ligand-binding domain,transmembrane domain and cytoplasmic tail.Each domain is encoded by exon 1 to exon 4.[16]The substitution of dimorphism (A/G) at position 49 of exon 1 leads to Thr/Ala substitution at codon 17 in the leader peptide,but the true function of this substitution is elusive.Some investigators suggested that this substitution might have a subtle effect on posttranslational processing or localization of the CTLA-4 protein.[12]

According to current opinion,autoimmune diseases are a result of the failure of the immune system to develop tolerance to self-proteins.Impaired peripheral immune tolerance is also involved in the pathogenesis of AIH.Over the past few years,the CTLA-4/B7 pathway has been considered a major coordinator in immune regulation,by inducing peripheral tolerance.In contrast to CD28,CTLA-4 negatively regulates T cell function in several ways,[19]including altering T cell activation through suppressing TCRζ chain phosphorylation and ERK activation,regulating signal transduction in regulatory T cells,and in fluencing the balance of Th1/Th2.Many studies have focused on the potential value of the CTLA-4 pathway in therapeutic application in order to translate knowledge into clinical applications.[20-24]Using CTLA-4 to block interaction between CD28 and the B7 costimulatory ligands is considered to be a potential approach to inhibit pathologic autoimmune responses.A recombinant CTLA-4-Ig fusion protein,called CTLA-4-Ig,was used to block the normal costimulatory function of CD28,and it was considered a novel therapeutic agent to inhibit the abnormal immune response.Previous studies have shown that CTLA-4-Ig prevented T-cell expansion by disturbing CD28 engagement[20]and inducing immunosuppression in vivo.[21]More recently,the humanized form of CTLA-4-Ig,named Abatacept,was licensed by the FDA in the United States for the treatment of rheumatoid arthritis in the case of inadequate response to anti-TNF-α therapy.[22]However,the functional significance of CTLA-4-Ig in AIH is still not well established.Thus,more studies on the therapeutic effect of CTLA-4-Ig in AIH should be investigated.Furthermore,there is a long way to go before we can transfer basic research into clinical use.

Fas gene

The Fas receptor is encoded by the Fas gene,which is located on chromosome 10 in humans.It belongs to the tumor necrosis factor receptor superfamily and induces receptor-mediated programmed cell death(apoptosis) through engagement with its ligand.The Fas cell death pathway has been described as a critical mechanism to maintain peripheral tolerance by removal of autoreactive T cells that escaped central tolerance.[17]A genetic link of Fas polymorphisms to the development of AIH was first reported by Hiraide et al,[25]while many studies on Fas gene susceptibility in other autoimmune disorders have been reported.[26]The result of Hiraide et al revealed significant differences between AIH patients and controls in the allele frequencies of Fas -670 A/G(in the enhancer region),Fas IVS (intervening sequence) 2nt176,Fas IVS3nt46,and Fas IVS5nt82 polymorphisms.[25]Despite the lack of evidence to support the assumption that the Fas gene in fluences susceptibility to AIH,Agarwal et al found that the polymorphism at position -670 may affect the early development of hepatocirrhosis.[27]Current evidence suggests that the -670 A allele may increase the binding capacity of STAT1[28]and Fas expression on the activated T-cell surface.[27]Based on these results,we postulate that receptor-mediated programmed cell death may also play a role in the progression of AIH.

Cytokines

The role of cytokines in the pathogenesis of AIH has been widely investigated in the past decade,including polymorphisms of the IL-1B,IL-1RN,TNF-A,and IL-10 promoter genes.[29-33]The only significant outcome came from TNF-A,encoded tumor necrosis factor alpha,at position -308 in the promoter region.[29-31,33]The TNFA*2 (-308A allele) polymorphism was considered to confer susceptibility to AIH in white patients.[29,30]Although it was associated with poorer treatment outcome,the differences in these outcomes were not significant.[30]This polymorphism might be another susceptibility allele on chromosome 6p21,affecting both disease expression and behavior,[29,30]besides the HLA DRB1*0301 haplotype.However,the linkage between TNFA*2 and HLA DRB1*0301 is still not well established.[30]Furthermore,con flicting conclusions come from different studies.TNFA*2 has been associated with susceptibility to type 1 AIH in European and North-American Caucasian patients,[29,30]while such an association has not been found in Brazil.[31]Subsequently,two independent investigations in China have shown different results.One considered TNF -308A might be involved in the pathogenesis of AIH,[32]but the other found no genetic link of this polymorphism with AIH and primary biliary cirrhosis in Chinese.[33]In Japanese,there was no relationship between TNF-α gene polymorphisms and AIH after stratification analysis for the effect of HLA-DBR1*0405.[34]Although the relevance of TNF to both in flammatory and autoimmune diseases[35]has been demonstrated,and anti-TNF antibodies (In fliximab) have been used in treatment of disorders such as Crohn's disease,[36]there is no clearly established functional significance in the promoter region of TNF-A.It has been described as regulation of TNF translation,[35]and other investigators have reported a functional binding site for NF-κB in the transcriptional start site of the TNF gene.[37]Thus the functional association between TNF and AIH still requires investigation in detail.

TGF-β1 is a multifunctional peptide that controls cell growth,tumor development and immune suppression.[38]The anti-in flammatory effect of TGF-β1 plays an important role in maintaining immune homeostasis and preventing autoimmune diseases.Recently,a report from Paladino and Flores showed that TGF-β1 codon 10 (T/C) and codon 25 (G/C)polymorphisms might be useful in the prediction of AIH severity.[39]The TGF-β1 T allele at codon 10 produces lower levels of TGF-β1,while the homozygous GG genotype at codon 25 has been associated with increased levels of TGF-β1.The associations of these two functional polymorphisms between pediatric (PAH)and adult (AAH) forms of type I AIH were reported in their study.According to the histological findings of hepatitis severity,they divided these AIH patients into subgroups.They found the frequency of codon 10 CC("high producer") and 25 CC ("low producer") genotypes increased in AAH patients with low in flammation andfibrosis,while in PAH patients the increase of 25 GG("high producer") genotype was only detected in children with the highest in flammatory reaction and the 10 CC genotype increased in non-cirrhotic PAH.Furthermore,the analysis of codon 25 GG and 10 CC haplotypes revealed that the combined presence of the two position-haplotypes seems to neutralize anti-cirrhotic effect observed in AAH patients with the 10 CC haplotype.[39]These results may indicate the mechanisms underlying the pathogenesis of AIH,and explain the different clinical outcomes of AAH and PAH.More studies are needed in ethnically different countries.

TBX21

T-bet is a key regulator in initiating Th1 lineage development from naive Th cells.It is encoded by the TBX21 gene and controls the expression of the hallmark Th1 cytokine,interferon-gamma.[40]The expression level of T-bet is associated with autoimmune,infectious,and allergic diseases.[41]A Th1-like immune response is implicated in the pathogenesis of AIH.[42]Chen and colleagues[42]detected two polymorphisms (T-1993C and T-1514C) in the TBX21 gene promoter and considered that the T-1993C polymorphism might confer susceptibility to type 1 AIH in a Chinese population.They also found that the -1993C allele has a high affinity for the Yin Yang 1 (YY1) transcription factor,a suppressor of the TBX21 promoter.Thus,cells with the -1993C allele express less TBX21 and interferon-gamma but more IL-4 than those with the T allele.This study demonstrates that the TBX21 T-1993C polymorphism represses gene expression and Th1 cytokine production through interaction with YY1,resulting in an imbalance between Th1 and Th2 responses in autoimmune diseases.[43]

VDR

As 1,25-dihydroxyvitamin D3 (vitamin D) is regarded as an immunomodulator,the vitamin D receptor(VDR) has been implicated in the pathogenesis of many autoimmune diseases.Recently 1,25-dihydroxyvitamin D3 has been implicated in the function of inhibiting the T cell production of in flammatory cytokines and promoting the development of regulatory T cells expressing CTLA-4 and FoxP3 combined with interleukin-2.[44]Polymorphisms of the VDR have been linked to several autoimmune disorders such as multiple sclerosis,[45]type 1 diabetes mellitus,[46]Graves'disease[47]and especially AIH[48,49]and primary biliary cirrhosis.[48-50]Vogel et al[48]reported a significant association of the exon 2 initiation codon polymorphism,detected by a Fok restriction site,in AIH patients in comparison to controls.Also,the same result was found in a Chinese population,but the distribution of gene types might differ between Chinese and Caucasians.[49]

Table.Association between genetic polymorphisms of non-classic MHC and AIH

Conclusion and perspectives

It is a fact that genetic and immunologic factors are involved in its pathogenesis of AIH,although the pathogenic mechanism is unclear.Many investigators focus on this issue to better understand the etiology of this disease,and hope tofind potential for treatment of AIH.Although the association between the HLA gene and AIH has been well-established as supported by a great deal of evidence from independent investigators in the past two decades,[2]few studies about its therapeutic potential have been reported.Thus,the non-classic MHC might be considered to have an important role not only in the pathogenesis but also in the treatment of this disease.

Previous case-control studies on genetic polymorphisms (Table) revealed that at least six genes are involved in AIH besides HLA.[10,12-15,25,27,29-33,39,42,48,49]Data analyses of these studies showed that there has been no agreement about gene susceptibility.Although the actual clinical significance of these genes is still controversial,it still implies that ethnicity may affect the association of gene polymorphisms with susceptibility to AIH.Compared with the polymorphism studies of other diseases,the subjects involved in these studies are few.The studies only consist of about 150 subjects,which might contribute to the confusing results.A larger population is essential in future studies,though the collection of patients is difficult.More recently,Miyake and his group[51]did a meta-analysis to identify the associations between CTLA-4 gene polymorphisms at position +49 and the susceptibility to type 1 AIH and primary biliary cirrhosis.The data fromfive independent studies confirmed the association of this polymorphism with the susceptibility to type 1 AIH and primary biliary cirrhosis,which implied that the A/A genotype might be protective against both of these diseases.The in fluence of the CTLA-4 gene +49 A/G polymorphism might be significant in both the susceptibility and clinical phenotype of type 1 AIH.[51]Similarly,analogous systematic reviews should be undertaken on the other gene polymorphisms.In addition,a genome-wide association study can also be useful in detecting new genes that predict risk of the disease.

Some genes should also be included in later investigations,such as programmed death 1 (PD-1)and its ligands.So far,the association between polymorphisms of the PD-1 gene and AIH has not been reported.Similar to CTLA-4,PD-1 and its ligands,PD-L1 and PD-L2,also deliver inhibitory signals that regulate the activation of T cell,induction and maintenance immune tolerance.[52]The hypothesis suggests that CTLA-4 plays an important role in early tolerance induction while PD-1 plays the dominant role during the maintenance of tolerance.[52]Therefore,the PD-1 pathway might also have effects on the pathogenesis of AIH,and manipulation of the PD-1 signaling pathway may be applied as a new therapeutic strategy for AIH.

Despite the heterogeneity among clinical manifestations,the major therapies for AIH use a nonspecific anti-in flammatory or immunosuppressive regimen,or both.Liver transplantation has been provided to patients who have no response to medical management.Now,inducing immunologic tolerance might be an effective approach to cure autoimmune diseases.New specific immunosuppressive agents are urgently needed.Studies on gene polymorphisms outside MHC and knowledge of genetic predispositions for AIH may not only elucidate pathogenic mechanisms,but also provide new targets for therapy.

Contributors:ZSS designed and proposed this review.TJ and ZC searched for information and wrote the draft.TJ and ZC contributed equally to this work.ZZJ discussed and gave suggestions for the draft.ZSS is the guarantor.

Funding:This study was supported by grants from the National Basic Research Program of China (973 Program,2009CB522403)and Key Program of the National Natural Science Foundation of China (30731160620).

Ethical approval:Not needed.

Competing interest:No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

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