Identification Of Functional Variant And The Mechanisms Of WDFY4Gene In The Pathogenesis Of Systemic Lupus Erythematosus

Systemic lupus erythematosus (SLE) is a prototype autoimmune disease, characterized by autoantibody production and immune complex (IC) deposition leading to severe inflammatory reaction multi-organ damage. Epidemiological studies suggest strong contribution of genetic factors in the etiology of SLE. SLE has high tendency of familial aggregation and a higher concordance rates between monozygotic twins (25-56%).Since2005, Genome-wide association studies (GWASs) have greatly drived the genetics study of SLE. By now, GWASs have confirmed genetic associations of over40genes/loci with SLE during the past several years. In2009, two GWASs in east Asian populations have discovered WDFY4-LRRC18region was a genetic locus for SLE, with3susceptibility SNPs rs877819, rs7097397and rs1913517. Wang C et al have confirmed the association of rs1913517, which is located both in WDFY4and LRRC18, with Caucasian populations.As the fourth member in WDFY family, WDFY4encodes multiple transcripts and the longest transcript encodes a huge protein with3184amino acids residues. Its closest paralog is WD repeat and FYVE domain containing3(WDFY3), and both proteins contain five WD40domains and a BEACH (Beige and chediak-kigashi) domain. However, the best characterized FYVE zinc finger domain in the family is truncated for WDFY4. WD40domain is found in a number of eukaryotic proteins which are mainly involved in a wide variety of functions including adaptor modules in signal transduction, pre-mRNA processing and cytoskeleton assembly. Most of BEACH domain containing proteins (BDCPs) act as scaffolding proteins that facilitate membrane events, including both fission and fusion, and recent studies found that mutations in individual BDCPs cause several human diseases and cell dysfunction, such as abnormal lysosome size, apoptosis and autophagy. In a variety of mammals, the sequence of WDFY4protein is highly conserved. So far, very little is known about the potential function of this well conserved protein. An interesting phenomenon is that WDFY4is predominantly expressed in immune tissues such as lymph node, spleen, thymus and tonsil, which implies the important role on immunity of organism.Although GWASs have shown that the association of WDFY4-LRRC18with SLE in Chinese Han population, given the genetic heterogeneity between different populations, the restriction of previous studies to only Central and Southern Chinese Han populations limited the generalizability of this association to the whole Chinese Han ethnicity. Then, whether WDFY4-LRRC18is the susceptibility region of SLE in Northern Chinese Han population? Whether there is the functional variation in the three SNPs in WDFY4-LRRC18gene or not? And how to involved in SLE pathogenesis? What is the mechanisms of susceptibility gene contributing to the pathogenesis of SLE? To answer these questions, we carried out the following study.Part IReplicated association of WDFY4-LRRC18with Systemic Lupus Erythematosus in a Northern Chinese Han Population To explore the possible association of WDFY4-LRRC18variants with SLE in a Northern Chinese Han population, the3SNPs rs877819, rs7097397and rs1913517were genotyped by a case-control association study.400patients with SLE were recruited from the Department of Rheumatology and Immunology of Qilu Hospital and Provincial Hospital affiliated to Shandong University, and1000unrelated, random-sampled healthy controls from the two hospitals were recruited during the same time. All healthy individuals were matched to cases by age, ethnicity and geographic region.The allelic and genotypic frequency distributions of the three SNPs were in agreement with Hardy-Weinberg Equilibrium(HWE)(P>0.05) for both patients and controls, with the MAFs consistent with the International HapMap Project data for CHB. The SNPs rs877819and rs7097397presented strong associations with SLE (P=0.015, OR1.36,95%CI1.06-1.75; P=0.0004, OR1.365,95%CI1.148-1.622). Whereas rs1913517was not observed association with SLE (P=0.244, OR1.131,95%CI0.919-1.392).Our replication study revealed that two SNPs, rs877819and rs7097397in WDFY4gene, were significantly associated with SLE in a Northern Chinese Han population.Part IIIdentifying susceptibility gene and the functional variant in WDFY4-LRRC18region associated with SLEAlthough WDFY4-LRRC18is a susceptibility region of SLE, but the functional variant is still unclear. Usually, the SNPs rs877819, rs7097397and rs1913517, which were found to be related to SLE by GWASs, are not functional, and they might be just in strong linkage disequilibrium (LD) with the causal variation.The susceptibility SNP rs1913517is located in an intron in LRRC18, which itself is located in an intron of WDFY4, thus the exact susceptibility gene with SLE is unclear. To identify the susceptibility gene, we examined the mRNA levels of the two genes in peripheral blood mononuclear cell (PBMC) samples from58SLE patients and58healthy controls. Relative WDFY4mRNA expression was lower in patients with SLE than in controls (P<0.05), with no significant difference in LRRC18expression.Because the novel variant in WDFY4-LRRC18might have a functional effect on SLE susceptibility, we sequenced all61coding exons of WDFY4in70normal individuals, however, no novel variant was found in the coding sequences.To identify the functional variant among3SNPs, we conducted an independent contribution test and allele specific expression (ASE) assay. We found rs1913517and rs877819were in strong linkage disequilibrium (LD) with r2=0.93. Conditional logistic regression revealed rs877819had an independent contribution to SLE with P=2.5×10-4, when controlling the effect of rs1913517. However, the P value for independent rs1913517was just0.7758, with no significant assiociation with SLE. These results suggest the assiociation of rs1913517with SLE was dependent on rs877819.To detect whether WDFY4expression was associated with rs877819or rs7097397, we performed allele specific expression (ASE) assay. For rs7097397located at the transcript sequence, pyrosequencing was performed in31healthy subjects heterozygous for rs7097397, and no significant difference of ASE was identified on rs7097397.To determine whether intronic variant rs877819was associated with WDFY4and LRRC18expression, we analyzed the expression of subjects with different genotypes on the SNP. Quantitative PCR analysis showed significantly lower WDFY4expression was seen in AG heterozygotes than in GG homozygotes (P<0.05), but no difference on LRRC18expression. Thus, risk allele A of rs877819was significantly associated with a lower WDFY4expression.The result in this part suggest WDFY4is directly associated with SLE. The functional variant might be the intronic SNP rs877819, with the risk allele A associated with the lower expression of WDFY4, which might be consistent with the result that the expression of WDFY4is decreased in SLE.Part ⅢThe mechanisms of the functional variant rs877819involved in the pathogenesis of SLESince the risk allele A of rs877819is associated with the decreased expression of WDFY4, the intronic SNP might affect gene expression through transcriptional regulation. We examed the transcriptional activity of the allele G or A on rs877819by the luciferase reporter activity assay. As expected, the activity was significantly lower for the A allele than G allele in HeLa and HEK-293T cells (P<0.001), which was consistent with our observation of the rs877819risk allele A associated with decreased expression of WDFY4.Gene transcription might be modulated by transcription factor binding sites in intronic regions. Electrophoretic mobility shift assay (EMSA) was used to investigate whether the allelic difference in transcription activity on rs877819might be explained by their different binding capacities for nuclear factors. We found both alleles on rs877819could form DNA-protein complexes with the nuclear extracts, with the higher affinity of G allele than A.Three transcriptional factors Freac2, ZBTB7and Yinyangl (YY1), predicted by Matlnspector, might bind with rs877819, so we synthesized conserved motifs for the three factors as competition probes. The detected complex was completely abrogated only by the conserved YY1binding motif, thus YY1might be the transcription factor binding to the rs877819site. Supershift assay suggested YY1 antibody could bind with the DNA-protein complex, while the mutant YY1conserved motifs could not. Chromatin immunoprecipitation assay (ChIP) also indicated that YY1could bind to the rs877819site in vivo.These DNA-protein binding analyses indicated that endogenous YY1protein specifically interacted with intronic variant rs877819within WDFY4, and the risk allele A had lower affinity than the G allele with transcription factor YY1, which might explain that allele A was associated with reduced transcription of WDFY4.To investigate the effect of transcription factor YY1on different alleles of rs877819, we transfected HeLa cells with the luciferase vector containing rs877819-G or-A and siYYl or the expression vector of YY1(CMV-YY1). The result showed that YY1had a positive regulation on rs877819G, with little effect on the allele A.In this part, our findings suggest transcription factor YY1could bind to rs877819, with lower affinity to the A allele, which might lead to the reduced transcriptional activity of WDFY4.Part IVThe effects of WDFY4gene on the autophagy pathwayThe closest paralog of WDFY4is WDFY3in the same gene family and both contains WD40domains and a BEACH domain, however, the best characterized FYVE zinc finger domain in the family is truncated for WDFY4. WDFY3protein, involved in selective autophagy, is also called autophagy-linked FYVE protein (ALFY). There has been a growing studies for the possibility that autophagy may be involved in autoimmune disease. In addition, the elevated autophagy has been detected in lupus mice. Since the protein structure of WDFY4is so similar with ALFY, whether WDFY4is also involved in autophagy? Whether the susceptibility of WDFY4to SLE is related to autophagy process?With these questions, we first examined the expression patterns of Wdfy4and autophagy in spleen from lupus mice. We found WDFY4gene was predominantly expressed in immune tissues in both human and mouse, such as spleen, thymus and bone marrow. In addition, compared with the wild-type mice, the expression of Wdjy4decreased in lupus mice spleen, however, the level of autophagy increased.What’s more, the expression level of WDFY4in Raji (B lymphoma cells) was higher than Jurkat, NK92and THP-1. In order to investigate the effect of WDFY4on autophagy, we transfected cells with the shRNA of WDFY4and expression vector of WDFY4containing WD40-BEACH domain with starvation induced and chloroquine (CQ) treatment, and autophagy was detected by Western Blot. Our results showed that decreased expression of WDFY4led to a higher autophagy flux, and expression of BEACH-WD40domain inhibited autophagy flux.To study the mechinsms of WDFY4involved in autophagy, the expression of several key proteins in autophagy pathyway were detected in cells transfected with shWDFY4and BEACH-WD40domain expression vector. We found the expression of Bc12decreased in cell with shWDFY4, while increased in cell with BEACH-WD40domain expression vector. Bc12is an apoptosis-related protein, and its high expression might inhibit cell apoptosis. In addition, Bc12is also involved in the autophagic process, and it can be combined with the autophagy protein Beclinl, which cause Beclinl involved in autophagy reduced. As we all known, autophagy can lead to autophagic cell death. Therefore, Bc12can directly inhibit cell apoptosis, or inhibit autophagic cell death.Next, the effects of WDFY4gene on cell apoptosis was analysed by flow cytometry. Consistent with our expectation, the result indicated that decreased expression of WDFY4promoted cell apoptosis, and the expression of BEACH-WD40domain inhbited cell apoptosis to a certain extent.In this part, the expression of WDFY4gene is higher in B cells than other immunocyte. Knockdown of WDFY4increased cell autophagy and downregulated the expression of Bcl-2, which might contribute to the elevated cell apoptosis. Consistently, the expression vector containing BEACH-WD40domain caused cell autophagy decreased and the expression level of Bcl2increased evidently, protecting cells from apoptosis. Therefore, WDFY4may contribute to the pathogenesis of SLE through the autophagy pathway.