A common intronic variant in WDFY4 results in decreased allele-specific transcription through bindin

来源 :中华医学会2012年医学遗传学年会暨全国第十一次医学遗传学学术会议 | 被引量 : 0次 | 上传用户:PILIYADAN
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  Objective In two recent genome-wide association studies(GWAS)in east Asian population,three genetic variants,rs877819,rs7097397 and rs 1913517 in WDFY4/LRRC18 have been revealed to be associated with systemic lupus erythematosus(SLE).The aim of this study was to explore how WDFY4 and LRRC18 are risk factors of susceptibility to SLE.Method In order to confirm which gene is the causative one,the relative mRNA expression level of WDFY4 and LRRC18 was estimated in patients with SLE and healthy controls by RT-PCR and pyrosequencing.To investigate whether the intronic SNP rs877819 has effect on transcription activity,a dual-luciferase assay was performed.In addition,electrophoretic mobility shift assay(EMSA),supershift assays and CHIP assay was performed to analyse transcription factors binding to rs877819 and allelic difference in nuclear protein-binding affinity.To measure the effect of YY 1 on rs877819 activity,YY1 expression construct(CMV-YY1)or siRNA for YY1 were co-transfected HeLa cells with the pGL3Enh-G or A.Result The results showed WDFY4 was invovled in a down-regulated expression in patients,while no obvious difference oberved in LRRC18.Comparing of allelic expression of the other two SNPs,we found rs7097397 exhibited balanced allelic expression,while for rs877819,we observed a trend of decreased WDFY4 expression in individuals with the risk allele A.A dual-luciferase assay showed that the rs877819A allele had lower transcriptional activity than G allele.In EMSA and supershift assays,transcription factor Yinyangl (YY1)could bind to SNP rs877819 with a lower affinity to A allele,which led to a reduced transcriptional activity compared to G allele.This trend was further confirmed by siRNA,overexpression and ChIP experiments.Compared to the normal control,the relative luciferase activity of pGL3Enh-G Was significantly reduced in HeLa cells,as a result of YY1 knockdown mediated by siRNA.However,the risk allele was not significantly different with YY1 knockdown,suggesting that YY1 was a positive regulation factor of G allele,while almost no effect on A allele.Moreover,cells with CMV-YY1 constructs had high level of YY1 and gave rise to approximate 1.5-fold increase in the reiative luciferase activity of pGL3Enh-G.However,as for the A allele,the relative luciferase activity was not an obvious change in similar to the knockdown experiments.The relative activity of G allele increased in a concentration-dependent manner,with CMV-YY1 concentration from 20ng to 200ng,further supporting the role of YY1 as a positive regulation factor of G allele.Conclusions We performed functional analysis of WDFY4 and demonstrated that its downregulation may explain the increased risk of SLE.As well,an intronic SNP,rs877819,may be the functional variant,with the minor allele A associated with decreased expression of WDFY4.Our results might provide a fumctional link between a common intronic variant in WDFY4 gene and a causal variant for systemic lupus erythematosus.
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