| ObjectiveTotal exon sequencing was performed in peripheral blood of URSA patients using WES technology to screen out gene variations that may be related to URSA.Based on bioinformatics analysis,the correlation between NCF1-339 variation and unexplained recurrent abortion(URSA)was investigated.And the mechanism of ROS/ NF-κB signaling on immune regulation.To explore the potential risk factors of URSA from the perspective of genetics,and provide scientific information for determining clinical prevention and targeted strategies.Methods53 URSA patients with peripheral blood all exons sequencing,selection of exon region non-synonymous mutations,four prediction software to find related immune function through the literature and auto-immune ovarian disease gene,using RNA-seq,bioinformatics analysis and study the function of the gene enrichment,screening may be associated with URSA gene mutation;NCF1 gene c.269G>A,p.R90 H was detected by nested PCR combined with Tag Man fluorescence probe in 152 URSA patients and 72 healthy controls.A,P.r90h(NCF1-339)typing,chi-square test,Logistic regression analysis was used to calculate OR value and 95% confidence interval,and the correlation between NCF1-339 variation and URSA was investigated.According to NCF1 genotyping,URSA patients were divided into variant group(GA)and non-variant group(GG),and all healthy controls were GG.RNA-seq and GSEA enrichment analysis were performed in the mutant group and non-mutant group.The levels of MYD88,NOX2,TLR4,IFN-γ,ROS,TNF-α,IL-6,IL-8 and other cytokines in three groups were detected by ELASA.The expression of NCF1 m RNA,RELA m RNA and NFKBIA m RNA were detected by q RT-PCR.ResultsEleven high frequency mutations of immune-related genes(ABCD1,AIRE,ATM,DOCK8,FOXN1,STIM1,TBX1,JAK3,PTPN22,PTPRC,NCF1)were screened by analyzing the whole exon sequencing results of 53 URSA patients.NCF1-339 genotyping validation of 152 URSA patients and 72 healthy controls showed that NCF1-339 variant(GA)was significantly associated with the risk of URSA(variable-vs non-variable-group: OR =3.257,95%CI: 1.494-7.102).The NCF1-339 A allele was significantly associated with the risk of unexplained recurrent abortion(A vs G: OR =3.313,95%CI: 1.588-6.911).By RNA-SEQ and GSEA enrichment analysis,it was found that the biological process of enrichment was the differentiation and activation of B cells in the down-regulated genotypes in the mutant group.ROS levels in the non-variant group were significantly higher than those in the non-variant group(73.05±35.82 vs 101.20±15.72),and the difference was statistically significant(P>0.05);The expression of RELA in the mutant group was significantly higher than that in the non-mutant group(5.89±5.47 vs 1.48±1.20),and the difference was statistically significant(P<0.05),and the variation group was higher than the non-variation group(5.89±5.47 vs 3.64±2.63,P>0.05);There were no significant differences in TNF-α,IL-6 and IL-8 levels of downstream cytokines of NF-κB signaling pathway(P>0.05).Conclusions1.The results of total exon sequencing in peripheral blood of some URSA patients revealed the variation of genes related to immune function regulation;2.NCF1 mutation leads to decreased ROS,which may affect the occurrence of abortion through the ROS/ NF-κB signaling pathway. |
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