Identifying A Pathogenic Variant Of Neuropsychiatric Systemic Lupus Erythematosus By Whole-exome Sequencing And Searching For The Pathogenesis

BackgroudNeuropsychiatric systemic lupus erythematosus(NPSLE)is one of the major factors leading to disability and mortality in systemic lupus erythematosus(SLE),with complicated pathogenesis which can lead to injuries of various parts of the nervous system through the destruction of brain barrier,generation of pathogenic autoantibodies,inflammation mediated by cells and cytokines,etc.Therefore,NPSLE is of high clinical heterogeneity and great difficulty in diagnosis and treatment.Study of the pathogenesis and optimization of treatment for the disease is still challenging.For many complex diseases,rare variants have higher genetic potency than common variants,and play important roles in the exploration of pathogenesis,diagnosis,and treatment strategies.Whole-exome sequencing(WES)is currently one of the most effective tools for identifying rare variants of complex diseases.Trio-based WES can reduce the difficulty of data analysis and increase the level of evidence for classifying the variants.Many risk allele loci of SLE are enriched around the nuclear factor-?B(NF-?B)pathway,suggesting that the pathogenesis is closely related to this pathway.Activated NF-?B plays a role in the pathogenesis of SLE by causing defective clearance of autoreactive T and B cells in bone marrow,spontaneous maturation and activation of dendritic cells,release of pro-inflammatory factors,and activation of peripheral lymphocytes.Meanwhile,NF-?B pathway is one of the important inflammatory pathways in vascular endothelial cells,astrocytes,and microglia.Tumor necrosis factor alpha-induced protein 3(TNFAIP3),also known as A20 protein,regulates ubiquitination of several key molecules in NF-?B pathway,and is an important negative regulator of NF-?B signaling cascade system.Several single nucleotide polymorphisms in TNFAIP3 are associated with SLE.Recent studies have shown that loss of function germline mutations in TNFAIP3 can lead to a range of autoimmune and autoinflammatory diseases,known as haploinsufficiency of A20.Objectives1.To identify a pathogenic variant of NPSLE based on trio-based WES.2.To explore the impact of TNFAIP3(c.1806delG)on the pathogenic mechanismof NPSLE.Methods1.Peripheral blood samples from a trio with a rare phenotype of NPSLE were collected.DNA was extracted for WES.Pathogenic variants were screened and confirmed by Sanger sequencing.2.Peripheral blood of the proband and controls were collected.Western Blot was used to detect expression of A20 and molecules related to NF-?B signaling pathway in peripheral blood mononulear cells(PBMCs)before and after tumor necrosis factor-?(TNF-?)stimulation.Real-time quantitative PCR(RT-PCR)was used to detect relative expression of downstream cytokines of NF-?B before and after TNF-?stimulation.3.Mutant TNFAIP3 expression vector was constructed according to the mutant site of the proband.Human embryonic kidney 293T cells(HEK293T)were transfected with empty vectors,wild-type TNFAIP3 vectors and mutant TNFAIP3 vectors,respectively.Co-immunoprecipitation was used to detect deubiquitination function of the mutant protein.Human umbilical vein endothelial cells(HUVEC),normal human astrocytes(NHA),and human microglial cells(HM06)were transfected with the above vectors.Western Blot and RT-PCR were used to explore the pathogenic effects of the mutation on nervous system.Results1.Identification of a pathogenic variant in TNFAIP3 in a trio with NPSLEThe proband was a 36-year-old female with a rare phenotype and recurrent course of NPSLE.Trio-based WES was performed.After initial filtering of harmful variants,compound heterozygous model in recessive inheritance and de novo mutation model in autosomal dominant inheritance were used to screen pathogenic variants combining with analysis of gene biologic function,pathogenic effect of the variation,and criteria for classification of sequence variation recommended by the American College of Medical Genetics and Genomics in 2015.A likely pathogenic variant TNFAIP3(c.1806delG)was identified.This de novo frameshift mutation located in the important functional region of ZnF4 motif,leading to premature presence of the stop codon and formation of a truncated A20(p.T602fs*95).Co-segregation was confirmed by Sanger sequencing.2.Research on the impact of TNFAIP3(c.1806delG)on the pathogenic mechanism of NPSLE.First,in order to figure out the expression of the mutant protein in the proband,we collected PBMCs of the proband and 2 normal controls.Western Blot showed that expression of wild-type A20 in peripheral T cells and monocytes of the proband was lower than that of normal controls.At the same time,wild-type A20 expression of the proband was lower than mutant A20.Secondly,HEK293T were co-transfected with the constructed TNFAIP3(c.1806delG)expression vectors and K63-linked ubiquitin vectors.Co-immunoprecipitation assay showed that deubiquitination function of mutant A20 was impaired.Then,in order to explore the impact of TNFAIP3(c.1806delG)on peripheral immune cells,Western Blot was performed and showed that expression of phosphorylated proteins of NF-?B pathway in T cells and monocytes of the proband was higher than that of normal controls.NF-?B pathway of the proband was more easily activated by TNF-? stimulation.RT-PCR results showed that the relative expression of NF-?B downstream inflammatory cytokines in PBMCs was significantly higher than that in SLE patients without neuropsychiatric symptoms and normal controls,and was further increased after TNF-? stimulation.These results indicated that TNFAIP3(c.1806delG)activated NF-?B pathway in peripheral immune cells and increased the secretion of pro-inflammatory factors.Finally,the pathogenic impact of TNFAIP3(c.1806de1G)on neuroimmunity was explored.Western Blot results showed that after TNF-? stimulation,NF-?B pathway of HUVEC transfected with mutant TNFAIP3 vectors was significantly activated,and expression of proteins that formed tight junctions and adherent junctions decreased.RT-PCR results showed that relative expression of pro-inflammatory factors was significantly increased in NHA transfected with mutant TNFAIP3 vectors after TNF-?stimulation.In resting HMO6 transfected with mutant TNFAIP3 vectors,RT-PCR showed that relative expression of pro-inflammatory factors was significantly increased.These results suggest that TNFAIP3(c.1806delG)has a pathogenic impact on nervous system by destruction of blood-brain barrier and activation of innate immune cells of central nervous system.SignificanceA pathogenic variant TNFAIP3(c.1806delG)was identified in a trio with extreme phenotype of NPSLE,whose impact was explored in systemic immune and neuroimmune system.This mutation could lead to occurrence of NPSLE by means of activation of immune cells in peripheral blood and central nervous system,and destruction of blood-brain barrier.This study provides a theoretical basis and research direction for individualized therapy and pathogenesis of NPSLE in the future.