Association between CBL gene polymorphism and susceptibility of microscopic polyangiitis in a Chinese population: A case-control analysis.

OBJECTIVE: To assess whether Casitas B-lineage lymphoma (CBL) gene polymorphism influences the risk of microscopic polyangiitis (MPA) in Chinese populations. METHODS: In total, 266 MPA patients and 297 healthy controls were recruited for a case-control study. Five CBL SNPs were genotyped using multiplex polymerase chain reaction and high-throughput sequencing. The relationship between SNPs and the risk of MPA under different genetic models was evaluated by SNPstats. SNP-SNP interaction was analyzed by generalized multifactor dimensionality reduction (GMDR). Finally, the association between CBL SNPs and treatment effects were assessed. RESULTS: The results showed that CBL rs2276083 was associated with decreasing MPA risk under dominant (OR: 0.53; p = 0.014) and recessive models (OR: 0.52; p = 0.0034). Stratification analysis indicated that rs2276083 and rs2509671 in age < 60 years, rs2276083 in female or in Han population were protective factors for MPA. The CBL haplotype (A-A-G-C-T) was associated with an increased risk of MPA. GMDR suggested that CBL rs2276083, phosphatidylinositol-4, 5-bisphosphate 3-kinase catalytic subunit alpha (PI3KCA) rs1607237, and autophagy-related gene 7 (ATG7) rs7549008 might interact with each other in MPA development (p = 0.0107). CBL rs1047417 with AG genotype and rs11217234 with AG genotype had better clinical treatment effects than other two genotypes (p = 0.048 and p = 0.025, respectively). CONCLUSION: The genetic polymorphism of CBL had a potential association with the risk of MPA and clinical treatment effects in Guangxi population in China.

Association between the AKT1 single nucleotide polymorphism (rs2498786, rs2494752 and rs5811155) and microscopic polyangiitis risk in a Chinese population.

Microscopic polyangiitis (MPA) is an autoimmune disease, characterized by ANCA in blood and necrotizing inflammation of small and medium-sized vessels, one of the three clinical phenotypes of ANCA-associated vasculitis (AAV). Autophagy has been confirmed to be involved in the pathogenesis of AAV. AKT1 is one of the autophagy-regulated proteins. Its single nucleotide polymorphisms (SNPs) are associated with multiple immune-related diseases, but there are rarely studies in AAV. The incidence rate of AAV has a notable geographic difference, and MPA is predominant in China. The aim of this study was to investigate the association between AKT1 SNP and MPA risk. Genotypes of 8 loci in AKT1 were evaluated by multiplex polymerase chain reaction (PCR) and high-throughput sequencing in 416 people, including 208 MPA patients and 208 healthy volunteers from Guangxi in China. Additionally, data of 387 healthy volunteers from China were obtained from the 1000Genomes Project on public database. Differences were observed between the loci (rs2498786, rs2494752, and rs5811155) genotypes in AKT1 and MPA risk (P = 7.0 × 10-4, P = 3.0 × 10-4, and P = 5.9 × 10-5, respectively). A negative association was detected in the Dominant model (P = 1.2 × 10-3, P = 2.0 × 10-4 and P = 3.6 × 10-5, respectively). A haplotype (G-G-T) was associated with MPA risk negatively (P = 7.0 × 10-4). This study suggests that alleles (rs2498786 G, rs2494752 G and rs5811155 insT) are protective factors for MPA and alleles (rs2494752 G and rs5811155 insT) for MPO-ANCA in patients with MPA. There is a haplotype (G-G-T), which is a protective factor for MPA. It suggests that the role of AKT1 in MPA/AAV needs further study to provide more intervention targets for MPA/AAV.

Microscopic polyangiitis plasma-derived exosomal miR-1287-5p induces endothelial inflammatory injury and neutrophil adhesion by targeting CBL.

Background: An inflammatory environment around the vessel wall caused by leukocyte infiltration is one of the characteristic histopathological features of microscopic polyangiitis (MPA); however, the pathogenic mechanisms are not fully understood. Studies have found that circulating microRNA (miRNA) can be used as potential biomarkers for the diagnosis and classification of anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitides (AAV), and the E3 ubiquitin ligase casitas B-lineage lymphoma (CBL) seems to be associated with inflammation. In addition, evidence indicates that miRNA can be tracked into exosomes and transferred into recipient cells to mediate the process of vascular endothelial injury. Herein, we aimed to identify the profiles of exosomal miRNA, and determine the effect of exosomal miR-1287-5p and its target gene CBL on vascular endothelial cells in MPA. Method: We isolated plasma exosomes from patients with MPA (MPA-exo) and healthy controls (HC-exo) by ultracentrifugation and conducted exosome small-RNA sequencing to screen differential miRNA expression in MPA-exo (n = 3) compared to HC-exo (n = 3). We measured the expression levels of miR-1303, miR-1287-5p, and miR-129-1-3p using quantitative reverse transcription-polymerase chain reaction (qRT-PCR, n = 6) and performed dual luciferase reporter gene assays to confirm the downstream target gene of miR-1287-5p. In addition, we treated human umbilical vein endothelial cell (HUVEC) with MPA-exo, or transfected them with miR-1287-5p mimic/inhibitor or with CBL-siRNA/CBL-siRNA+ miR-1287-5p inhibitor. After cell culture, we evaluated the effects on vascular endothelial cells by examining the mRNA levels of IL-6, IL-8, MCP-1, ICAM-1 and E-selectin using qRT-PCR and performed neutrophil adhesion assay with haematoxylin staining. Result: Transmission electron microscopy, Western blot and nanoparticle tracking analysis showed that we successfully purified exosomes and MPA-exo could be absorbed into HUVEC. We screened a total of 1,077 miRNA by sequencing and observed a high abundance of miR-1287-5p in the exosomes obtained from MPA plasma. The dual luciferase reporter assay identified CBL as a downstream target gene of miR-1287-5p, and the results revealed that MPA-exo decreased CBL protein expression in HUVEC. In addition, treatment with MPA-exo, up-regulating miR-1287-5p or silencing of CBL in HUVEC significantly increased the mRNA expression of inflammatory factors (including IL-6, IL-8, and MCP-1) and adhesion molecules (including ICAM-1 and E-selection) and promoted the adhesion of neutrophils to HUVEC. However, down-regulating miR-1287-5p had the opposite effect. Conclusion: Our study revealed that MPA-exo was involved in the intercellular transfer of miR-1287-5p and subsequently promote the development of acute endothelial injury in MPA. MiR-1287-5p and CBL agonists may be promising therapeutic approach for MPA-induced vascular inflammatory injury.

MTOR gene polymorphism may be associated with microscopic polyangiitis susceptibility in a Guangxi population of China.

OBJECTIVE: Microscopic polyangiitis (MPA) onset is affected by genetic predisposition. Autophagy plays a certain role in antineutrophil cytoplasmic antibody-associated vasculitis developing. A key factor in autophagy regulating, the genetic polymorphism of MTOR gene is essential. The objective was to explore the associations between MTOR gene polymorphism and MPA susceptibility in a Guangxi population of China. METHODS: A sum of 208 MPA cases and 209 healthy volunteers from Guangxi in this case-control study, four important single nucleotide polymorphism (SNP) loci of MTOR gene including rs3806317, rs1064261, rs1883965 and rs2295080 were examined. Multiplex polymerase chain reaction combined with high-throughput sequencing was performed. Subgroup analysis was evaluated by gender and ethnicity. Linkage disequilibrium and haplotype analysis were tested. Multi-SNPs interaction among mTOR signaling pathway was assessed. RESULTS: For rs2295080, homozygous mutant GG genotype was associated with a decreased susceptibility of MPA in recessive model (OR = 0.38, 95%CI: 0.14-1.00, p = 0.040), particularly in the subgroup of female (OR = 0.16, 95%CI: 0.03-0.74, p = 0.006) and Han population (OR = 0.32, 95%CI: 0.10-1.00, p = 0.034). Individual carrying G allele was linked with decreasing MPA susceptibility in Han population of Guangxi (OR = 0.65, 95%CI: 0.44-0.97, p = 0.036). In haplotype analysis, the haplotype AAT was correlated with increasing susceptibility of MPA (OR = 1.347, 95%CI: 1.004-1.807, p = 0.046). Moreover, in the multi-SNPs interaction analysis, the six-locus model was identified as the best interaction model (p < 0.05). CONCLUSION: These findings suggest that rs2295080 polymorphism of MTOR gene may be associated with MPA susceptibility in a Guangxi population of China and G allele might be an important protective factor.

Association of ATG7 gene polymorphisms with microscopic polyangiitis in Chinese individuals.

Microscopic polyangiitis (MPA) is a type of antineutrophil cytoplasmic antibody (ANCA)-related vasculitis. Autophagy-related gene 7 (ATG7) protects against complicated disorder states in model organisms, but the way ATG7 dysfunction contributes to MPA remains elusive. This investigation assessed the impacts of ATG7 single-nucleotide polymorphisms (SNPs) on microscopic polyangiitis (MPA) in China. A total of 211 controls and 214 MPA patients were recruited and analyzed. Polymerase chain reaction (PCR) and high-throughput sequencing were adopted to detect the ATG7 SNPs (rs75492008, rs2594966, rs6442260 and rs8154), and stratification analysis, different genetic models and differences in allele and genotype frequencies were evaluated. Haplotype evaluation was performed after linkage disequilibrium (LD) analyses, and interactions between alleles were assessed. Generalized multifactor dimensionality reduction (GMDR) was adopted to analyze SNP-SNP interactions among the four ATG7 SNPs and phosphatidylinositol-4, 5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) and unc-nc-like autophagy activating kinase 1 (ULK1) SNPs previously studied by our team. Relationships between ATG7 polymorphisms, disease activity biomarkers and therapeutic effects in MPA were analyzed. Sex stratification analysis of the rs2594966 GG genotype with codominant and recessive models showed OR=3.42, 95% CI [1.19-9.80], P=0.041 and OR=3.31, 95% CI [1.23-8.90], P=0.012, respectively. Haplotype G-G-C-T was related to an increased MPA risk (OR=1.5, 95% CI [0.999-2.266], P=0.029). Permutation testing of GMDR suggested that ATG7 rs6442260 and rs8154, PIK3CA rs1607237, and ULK1 rs4964879 might interact with each other in MPA development (P<0.05). Among 214 MPA patients, 79 available complete follow-up clinical datasets were gathered from September 2009 to October 2020, showing that rs75492008 and rs4964879 affect the correlation between C-reactive protein (CRP) and the erythrocyte sedimentation rate (ESR) in MPA activity. Patients with rs8154 TT and rs1607237 CC genotypes had better clinical treatment effects (P<0.05). Gene polymorphisms may be related to MPA in China, exhibiting correlation with MPA activity indicators, treatment and prognosis.