| BackgroundMoyamoya disease?MMD?is a progressive occlusive cerebrovascular disorder.It is characterized by progressive narrowing or occlusion of the intracranial portion of the internal carotid artery and its proximal branches,which followed by the abnormal collateral vessels angiogenesis.As one of the main causes of stroke in children and youth,the researches on MMD have made some progresses,but its etiology and pathogenesis are still unknown.Our team has conducted a 2-stage GWAS study on moyamoya disease in Chinese han population.And the tissue enrichment analysis showed that the genes of related loci were highly enriched in the immune system.It suggested that MMD may be associated with immunity in Chinese han population.It is well known that HLA plays an important role in the immune system,but there are few studies on the correlation between HLA and MMD in Chinese han population.In addition,Our previous study has also found a significantly correlated Single Nucleotide Polymorphism?SNP?,rs2107595,in the gene of HDAC9.It has reported that HDAC9is associated with large-vessel diseases.Combined with previous studies,we hypothesize that MMD in Chinese han population may be related to immunity and have some common pathogenesis with large-vessel diseases.ObjectiveTo verify our hypothesis,Study 1:Genetic analysis method was used to study the HLA region of MMD patients and healthy control in Chinese han population;Study 2:By verifing the large-vessel diseases risk gene HDAC9 in MMD patients and healthy control to identify the possible risk loci;Study 3:By using homocysteine?Hcy?,a risk factor of moyamoya disease,to treat the primary cultured rat cerebral vascular smooth muscle cells?VSMCs?,to investigate the role of HDAC9 in cerebral VSMCs and explore the possible role of HDAC9 in MMD.MethodsStudy 1:?1?The Illumina Human Omni Zhong Hua-8 chip was used to genotype on 755patients and 2031 controls,and the genotyping data in the HLA region was sorted out.?2?After quality control,we performed HLA imputation by using the SNP2HLA software.?3?Applying stepwise regression logic analysis and conduct relevant analysis of the imputation results,to find independent signal,classical allele or amino acid polymorphism in the HLA region;Study 2:?1?The Illumina Human Omni Zhong Hua-8 chip was used to genotype on 755patients and 2031 controls,and the genotyping data of HDAC9 region was sorted out.?2?By using Plink1.07 software,a trendwise chi-square test was performed to compare the distribution of allele frequency between the group of case and control;Study 3:?1?The CCK-8 experiment was used to observe the proliferation of cerebral VSMCs after treated with different concentrations of Hcy for 24 h or 48 h.?2?The cell scratch test was used to observe the migration of cerebral VSMCs after treated with Hcy.?3?RT-PCR and Western-blot were used to detected the expression of HDAC9 in the cerebral VSMCs after treated with Hcy.?4?HDAC9 shRNA was constructed to interfere cerebral VSMCs,and The efficiency of HDAC9 shRNA was detected by RT-PCR and Western-blot.?5?Cerebral VSMCs were treated with HDAC9 shRNA,then CCK-8 and cell scratch assay were used to observe the proliferation and migration of cerebral VSMCs after treated with 500?M Hcy.ResultsStudy 1:After quality control,we finaly gained 24862 SNPs,178 classical HLA alleles and 688 amino acid polymorphisms.The most significant SNP was rs3129731(P=3.69×10-16,OR=1.79),The most significant classical allele was HLA-B*46(P=4.12×10-11,OR=0.49),and the most significant amino acid was the 24th alanine residue of HLA-B(Ala,A,P=3.48×10-14,OR=1.58).Through stepwise regression analysis,we found two independent signals:rs3129731(P=3.69×10-16,OR=1.79)and rs1071817(P=1.26×10-11,OR=0.62).The result of eQTL analysis showed that rs3129731 was related to HLA-DQA2 gene expression;Study 2:A total of 321 SNPs were obtained by extracting the HDAC9 region within7p21 from the genotyping data.There were 114 SNPs which were significantly differences between the moyamoya disease cases and controls,including rs2107595(P=1.40×10-14,OR=1.69)and rs2389995?P=0.03,OR=1.16?;Study 3:The proliferation of primary cultured cerebral VSMCs was enhanced with the increase of Hcy treatment concentration.In addition,we found that after treated with500?M Hcy for 48 h,the proliferation of cerebral VSMCs was most increased significantly.The migration of cerebral VSMCs was significantly accelerated,After treated with 500?M Hcy for 48 h.And the expression of HDAC9 protein in cerebral VSMCs was also significantly increased.Then the HDAC9 shRNA was constructed to interfere with the expression of HDAC9 in cerebral VSMCs.It was found that HDAC9shRNA could significantly reduce the expression of HDAC9 mRNA and HDAC9protein in cerebral VSMCs.After 48 h of 500?M Hcy treatment,the proliferation and migration of cerebral VSMCs in the HDAC9 shRNA treatment group were significantly reduced compared with the control.ConclusionIn this study,we found two independent signals of HLA regions in Chinese han population,which proved that the HLA regions plays an important role in the pathogenesis of MMD,and indicated that MMD is an immune-related disease.Then,The sites which previously reported related to large-vessel stroke were found in the HDAC9 gene region,suggesting that MMD and large-vessel stroke may have some common pathogenesis,where HDAC9 may involved.In addition,we treated primary cultured cerebral VSMCs with the risk factor of moyamoya disease,Hcy,to study HDAC9's role in cerebral VSMCs.The results showed that it plays an important role in maintaining the function of cerebral VSMCs.The above researches illustrate MMD in Chinese han population is an immune-related disease,and may possibly exist some common pathogenic mechanisms with the large-vessel stroke,where HDAC9 may participate.Our study illustrates part of the pathogenesis of MMD,and provides a new direction and some theoretical basis for further study on MMD. |
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