Discovery And Functional Study Of Susceptibility Loci In Psoriasis And Leprosy/Diabetes

The incidence of most infectious diseases has declined,but the incidence immune/inflammatory and metabolic diseases has been steadily rising.Complex genetic diseases arise from the interaction of multiple genetic and environmental risk factors.Since 2005,genome-wide association studies(GWAS)have greatly increased knowledge of the genetics of complex diseases.Today,>3,000 GWAS are published.Interestingly,one clear finding emerging from the published GWAS is that some shared susceptibility loci might have a similar or discordant role across different diseases.These studies show that there are intrinsic genetic relationships between these complex diseases:for some susceptibility loci play opposite or discordant effects on risk in immune/inflammatory and infectious diseases;some susceptibility loci play concordant effects on risk in immune/inflammatory and metabolic diseases.This study intends to search for the same Single Nucleotide Polymorphisms(SNP)that play an antagonistic role in immune/inflammatory diseases such as psoriasis and infectious diseases such as leprosy,and for the shared loci that show a similar pathogenic role between immune/inflammatory skin diseases such as psoriasis and metabolic diseases such as diabetes mellitus.This study will be helpful for the elucidation of the link in the pathogenesis of the psoriasis and leprosy/diabetes.Psoriasis is a chronic inflammatory,immune-mediated skin disorder,characterized by altered keratinocyte differentiation,angiogenesis and infiltration of immune cells into the dermis and the epidermis.Leprosy,an ancient infectious dermatosis,is caused by the obligate intracellular pathogen Mycobacterium leprae(M.leprae)in susceptible individuals,which mainly affects the skin and peripheral nerves.Diabetes is due to either inability of the pancreas to produce enough insulin or the cells of the body not responding properly to the insulin produced,with irreversible hyperglycemia and related complications.Psoriasis is an inflammatory skin disease,leprosy is a chronic granulomatous infection of skin.Psoriasis and leprosy are almost mutually exclusive.Leprosy has now been eliminated in developed countries(especially in European countries),but in contrast,psoriasis has increased in these areas.It is worth noting that there is an increased prevalence of psoriasis in areas where epidemics of leprosy occurred in the past in some regions.However the prevalence of psoriasis among leprosy patients was astonishingly reported to be as low as 0.014%in a questionnaire survey of global physicians,which is much lower than that in the world population as a whole.It has been hypothesized that psoriasis protects against the development of leprosy,and that leprosy has been contained because of an increase in the prevalence of psoriasis.Consequently,the genotype conferring susceptibility to psoriasis has expanded in certain populations because of selective pressure exerted by leprosy epidemics,favoring survival of an individual during a leprosy epidemic.The 'leprosy-resisting'genes may have conferred an evolutionary advantage associated with more vigorous immunity against infectious agents.The HLA-Cw*06 allele has been associated with>50%of psoriasis cases,and is also associated with a statistically-significant negative association with leprosy in the Indian population.In addition,HLA-DR B1*04 has been associated with a protective effect against the development of leprosy,particularly of lepromatous leprosy.It is also associated with susceptibility to psoriasis and psoriatic arthritis.Some studies have suggested that genetic factors such as HLA-Cw*06,HLA-DR B1*04,IL23R and IL12B may play a role in protecting psoriatic patients from leprosy.The mechanism(s)of protection remain unclear,it is likely that multiple complex immune-mediated mechanisms are responsible for the protective effect of psoriasis against leprosy.Psoriasis is characterized by a state of enhanced innate and adaptive immunity of antibacterial defense mechanisms in the skin of patients.This implies that the immune activation induced in psoriasis may prevent the onset of leprosy.Thus,various epidemic,genetic and immunological studies support the hypothesis that psoriasis and leprosy are almost mutually exclusive.This inverse phenomenon needs genetic and paleoepidemiological confirmation.However,to date,there has been no systematic investigation of whether previously-reported psoriasis susceptibility genes are shared by leprosy or vice versa.Psoriasis and diabetes are both chronic immune-and inflammation-related diseases.The biological connections between psoriasis and diabetes have been.suggested by epidemiological,immunological and genetic studies.Several genetic studies of psoriasis or diabetes have suggested that many genes are associated with psoriasis and also with diabetes.For example,four susceptibility genes of psoriasis(CDKAL1,PSORS2,PSORS3 and PSORS4)have been found to be associated with type 2 diabetes,while PTPN22 has been identified as a susceptibility gene for type 1 diabetes,psoriatic arthritis and early-onset psoriasis.The discovery of shared genetic susceptibility will help to reveal the common biological pathway(s)between the two diseases.GWAS have identified many genes which are involved in different diseases,but the studies all focused on a single disease,rather than on the same single nucleotide polymorphisms(SNPs),and there was strong ethnic genetic heterogeneity in these studies.Thus there is a lack of systematic research involving two diseases in a cohort which could provide strong evidence of genetics to reveal the relationship between psoriasis and leprosy/diabetes.Next we explored whether previously-reported psoriasis susceptibility genes are shared with leprosy,whether previously-reported leprosy susceptibility genes are also psoriasis susceptibility genes,and whether previously-reported diabetes susceptibility genes are shared with psoriasis.We also studied whether or not there is functional variation,and investigated how the mechanisms of genetic susceptibility contribute to the pathogenesis of diseases.To answer these questions,we carried out the following study in the Chinese Han ethnic population.Chapter 1Association study of shared genetic susceptibility loci betweenpsoriasis and leprosySection 1Identification of shared genetic susceptibility loci between psoriasisand leprosyIn this section,we studied the genetic association between psoriasis and leprosy by testing 23 leprosy-associated SNPs and 25 psoriasis-associated SNPs in 3,312 psoriasis cases,3,312 leprosy cases,and 3,312 controls from a Chinese Han population.We aimed to identify the genes which confer resistance to leprosy but susceptibility to psoriasis or play opposed pleiotropic roles,and in this way possibly contribute to better understanding of the correlation of the two diseases.We found that the T allele at rsl465788 was associated with increased risk of psoriasis(P = 2.39 × 10-3,OR= 1.13).Interestingly,rs1465788_T(A)demonstrated opposite effects in psoriasis and leprosy.Psoriasis patients were further classified as early-onset(disease onset occurring before 16 years of age)or later-onset(disease onset occurring after 45 years of age).Patients and controls were matched for gender,age and geographic region.We found that rsl465788 was particularly associated with early-onset psoriasis(P = 7.84 × 10-5,OR = 1.21).For rs1465788,'T' or 'A' has opposite effects on psoriasis and leprosy(as shown by opposing odds ratios),providing a protective effect against leprosy,but increasing the risk of psoriasis in Chinese Han population.This study reinforces the evidence that the opposite association of psoriasis and leprosy.Further functional investigations will likely help to understand the impact of the shared loci on psoriasis and leprosy.Section 2The effect of the variant rs1465788 on the transcriptional activity ofthe gene ZFP36L1As mentioned in Section 1,rs1465788_T(A)has been shown to increase the risk of psoriasis but protect against leprosy.This result may be interpreted to indicate that rsl465788_T(A)in the ZFP36L1 regions have opposing biological effects in psoriasis and leprosy.SNP rsl465788 locates 629 bp upstream of gene ZFP36L1.Since gene ZFP36L1 is reverse encoded,rs 1465788 is in the upstream sequence of ZFP36L1 in the transcription orientation and possibly locates in the promoter region of ZFP36L1.Consequently,we next explored the effects of rs1465788 on the transcriptional activity of the ZFP36L1 promoter region.We analyzed the mRNA expression of ZFP36L1 in samples from healthy individuals with different genotypes of rs 1465788 using quantitative polymerase chain reaction(PCR)analysis.Genotyping for rsl465788,performed by the Taqman method,was implemented in 60 healthy samples.The results showed no difference in the effects of GG homozygotes(N = 30)or AG heterozygotes(N = 30)on ZFP36L1 expression(P>0.05).This confirmed that rsl465788 is located in the region upstream of ZFP36L1(transcription orientation)and predicted that rs 1465788 might bind to the three transcriptional factors CTCF,CREBBP and SIN3A by HaploReg v4.1 and Regulome DB.To investigate the activity of the different alleles of rs 1465788 and whether the promoter activity was due to the effect of the different alleles of rs1465788 on the transcription factor binding,we synthesized a 2.0 kb long rs1465788 flanking reverse complementary sequence containing rs1465788_A or G and cloned it into the upstream region of the firefly luciferase gene in vector GV238.We transfected the luciferase vector containing rs1465788_A,G or empty vector into 293T cells,then cotransfected the expression vector of CTCF or SIN3A with reporter vectors to determine whether the effect of the allele of rs1465788 on the binding capacity of the transcription factor effected promoter activity.Our finding revealed that the allele A of rs1465788 exhibited significantly higher activity than the allele G in 293T cells(P<0.05)and transcription activity of the ZFP36L1 promoter region containing either of the alleles was notably increased in cells with the addition of CTCF.We did not find any regulatory role of SIN3 A on the allele.Compared to the risk transcription allele A(forward,T),the protective transcription allele G(forward,C)significantly reduced the transcriptional activity of this promoter region.Further,we found that the transcriptional activity of the alleles A and G on rs1465788 is changed by the positive regulation of CTCF in the luciferase reporter activity assay,but unfortunately not dependent on CTCF.According to published results from studies in mouse macrophages,the inefficient expression of Zfp3611 may lead to increased mRNA stability of mitogen-activated protein kinase phosphatases-1(Mkp-1),which would inhibit p38 mitogen-activated protein kinase(MAPK)activation and tumor necrosis factor(Tnf)-a production upon lipopolysaccharide stimulation.So we speculated that down-regulation of ZFP36L1 caused by the leprosy risk allele rs1465788_G reduces the expression of TNF-a via the MKP-1-p38MAPK-dependent pathway,which plays a critical role in leprosy;in contrast,the psoriasis risk allele rs1465788_A increases the expression of TNF-a to regulate inflammatory responses in psoriasis.The activity test result was consistent with the observation described in Section 1.Thus the discovery of ZFP36L1 provided new clues and could help us understand the mechanism involved in leprosy and psoriasis more comprehensively.Chapter 2Association studies of diabetes susceptibility genes in psoriasisTo identify additional shared susceptibility loci and investigate shared pathogenesis between these two diseases,we genotyped reported diabetes susceptibility loci in 4,456 psoriasis cases and 6,027 controls from the Chinese population using the MassARRAY system from Sequenom.Cases and controls were matched for gender,age and geographic region.We reviewed the reported findings from all diabetes(including both Type 1 diabetes and Type 2 diabetes)GWASs in the Catalog of Published GWASs and calculated their linkage disequilibrium scores(LDs)using the ASN 1000G Phase 1 dataset and the SNAP Version 2.2 and HaploReg v4.0.A total of 131 SNPs were selected for the association study.Of these,105 were successfully designed and after quality control,89 SNPs were retained for further association analysis.We identified three significant associations at rs6679677 on 1p13.2(P = 6.15 ×10-5,OR = 5.07),rs16861329 on 3q27.3(P = 2.02 × 0-4,OR = 0.87),and rs849135 on 7p15.1(P = 6.59 × 10-9,OR = 1.78),which suggested that PTPN22,ST6GAL1 and JAZF1 may be novel susceptibility genes for psoriasis in the Chinese Han population.These three significant SNPs as well as the SNPs that exhibited a high LD(r2>0.9 and D'>0.9)with the three significant SNPs were also investigated with expression quantitative trait locus(eQTL)analysis.A significant eQTL effect at rs6679677 near the PTPN22 gene was found to be significantly associated with expression of PTPN22 in whole blood.A significant eQTL effect at rs849135 was found to be associated with expression of JAZF1 in muscle skeletal.We did not find any evidence for an eQTL effect at rs 16861329.The results in Chapter 2 demonstrated that there are shared genes between psoriasis and diabetes.Our findings,particularly the association of PTPN22 with psoriasis,implicated the involvement of the T-cell receptor signaling pathway in the pathogenesis of both psoriasis and diabetes and further confirmed the shared genetic susceptibility between psoriasis and diabetes.