Analysis Of Environmental And Genetic Factors Of Clinical Characteristics Of Patients With Moyamoya Disease And Identification And Functional Study Of New Pathogenic Gene CAPN1

Objective:Moyamoya disease（MMD）is a chronic progressive intracranial vascular stenosis or occlusion disease,which can lead to stroke and other clinical symptoms.MMD has been reported all over the world,but there are obvious regional and ethnic differences in its incidence.In terms of global distribution,it is characterized by the"east-west gradient"of high incidence in East Asian countries,low incidence in Europe,America and other countries,and it also shows obvious regional clustering in China.At present,the etiology and pathogenesis of MMD are still unclear,and there is no effective radical treatment.Moreover,relevant research focuses on the relationship between gene polymorphisms and susceptibility to MMD from the perspective of genes,while ignoring or rarely involving specific environmental factors or the complex interaction between genes and the environment.The study of seasonal changes or temperature changes in the pathogenesis of cerebrovascular diseases is gradually attracting people’s attention,but there are few reports about the impact of geographical and seasonal environmental factors on the onset of MMD.The symptoms and course of MMD correlate with the age and type of initial onset,and its severity varies from transient ischemic attacks（TIA）to persistent neurological deficits.The onset age of MMD shows a bimodal distribution on children aged 5-10 and adults over 40.Many studies have found that MMD has obvious epidemiological characteristics such as race differences,gender differences and family aggregation,suggesting that the occurrence of the disease is closely related to genetic factors.The haplotype in the region of RNF213 p.R4810K was identified as unique to East Asians and was the main genetic factor for the high incidence of MMD in East Asians.Several studies confirmed its relationship with the clinical phenotype and prognosis of MMD,but the predictive effect of RNF213 p.R4810K on the clinical phenotype of Chinese patients with MMD and the impact of seasonal changes on the initial symptoms of patients have not been clarified.The carrying rate of RNF213 p.R4810K in Chinese patients with MMD is only about 20%,and has not been verified in its function.Based on the above analysis,we propose the hypothesis that RNF213 p.R4810K may indeed have a difference in the distribution of carrier rates in geographical space;The seasonal change may have some regularity with the incidence of initial symptoms of MMD.RNF213 p.R4810K may have different predictive effects on clinical characteristics of patients with MMD in China,Korea or Japan,and may have differences between children and adults;The carrier rate of RNF213 p.R4810K in Chinese patients with MMD is low,and there is genetic heterogeneity and the main pathogenic gene that has not been clarified.Methods:1、The Chinese map that indicated the geographical distribution of MMD patients and the distribution of RNF213 p.R4810K genotype were generated using Arc GIS 10.2software.One-way ANOVA was used to statistically analyze the incidence of the first symptoms（ischemic stroke and hemorrhage）of hospitalized patients in spring,summer,autumn and winter from January 2008 to December 2017.Based on the time series composed of the occurrence rate of the first symptoms of patients with MMD in the Department of Neurosurgery of the General Hospital of the People’s Liberation Army from2008 to 2017.Autoregressive Integrated Moving Average model（ARIMA）was built by using the Eviews 10.0 software to predict the monthly occurrence rate in 2018 in a short term,and the mean-absolute error（MAE）,mean-square error（MSE）and root mean squared error（RMSE）were calculated in combination with the actual monitoring value and prediction value to evaluate the prediction effect of the model.2.The blood samples of 2877 patients with sporadic MMD admitted by the Department of Neurosurgery of the General Hospital of the People’s Liberation Army from2008 to 2017 were collected,and the main clinical characteristic indicators were collected through clinical consultation,case report,imaging examination and other retrospective data.Quantitative Real-time PCR was performed to detect the genotype of RNF213p.R4810K using Taqman probe genotyping.To explore the predictive effect of RNF213p.R4810K on the clinical phenotype of Chinese patients with MMD,patients were divided into two subgroups,pediatric（<18 years old）and adult（≥18 years old）,based on the initial age,to explore whether mutations at this locus have different predictive effects for patients of different ages,respectively.3.A comprehensive and systematic search of published literature on RNF213p.R4810K genotype and clinical phenotype of MMD was conducted for quantitative meta-analysis.The retrieval time is from the establishment of the database to December 2022.Review Manage 5.3 and Stata 12.0 were applied to complete all statistical analysis.The statistical significance of the results was analyzed by Z-test.Subgroup analysis was conducted based on the countries of patients（China,Japan and Korea）to determine the difference in the predictive effect of RNF213 p.R4810K on clinical phenotype among patients in different countries.4.Whole-exome sequencing（WES）and systematic screening were performed using samples from 19 participants from six Chinese MMD families without RNF213 p.R4810K.The filtered variants were validated in an additional cohort of familial and sporadic subjects based on multiplex PCR sequencing.Statistically different loci were verified by Sanger sequencing after case-control analysis,combined with bioinformatics analysis to finalize newly identified variants,and case-control correlation analysis was performed in a cohort of 3054 independent cases and in public databases.5.RT-PCR,Western-blot and immunofluorescence methods were used to detect the changes of gene/protein expression in human aortic smooth muscle cells（HASMCs）after gene silencing,overexpression and mutation plasmid transfection.CCK-8,Transwell and scratch test were used to observe the proliferation,migration and invasion of HASMCs under the above different treatments.Results:1.The number of patients with MMD and the RNF213 p.R4810K carrier rate showed obvious regional aggregation in China.The analysis indicated that more than half of the cases come from the Central Plains and its surrounding areas,such as Henan Province,Shandong Province and Hebei Province.The higher carrying rate of RNF213 p.R4810K is distributed in eastern provinces of China such as Shandong and Jiangsu,and northeastern provinces such as Heilongjiang and Liaoning.2.Seasonal factors affect the incidence of first ischemic symptoms in patients with MMD.The incidence of patients whose initial symptom was cerebral ischemia was significantly higher in summer than in spring（P=0.014）and autumn（P=0.036）.However,there was no significant statistical difference in the incidence of cerebral hemorrhage in the four seasons.Therefore,the time series of monthly incidence of cerebral ischemia were handled after a series of operations such as smoothing（first-order difference,first-order seasonal difference）,model identification,screening and testing,the ARIMA（1,1,2）（0,1,1）₁₂ model was finally constructed.3.RNF213 p.R4810K homozygous AA and heterozygous GA have different predictive effects on the clinical phenotype of Chinese pediatric and adults with MMD,and also have different predictive effects on patients with MMD in China,Korea and Japan.Analysis showed that the proportion of patients with the following characteristics was significantly higher in the GA group than in the GG group（P<0.017）:female,age at onset<18 years,infarction after TIA,family history of MMD,posterior cerebral artery（PCA）,and angiographic manifestation of Suzuki intermediate stage at the first consultation.For pediatric patients,GA showed more cerebral hemorrhage（CH）at the first consultation（odds ratios（ORs）[95%confidence intervals（CIs）]=3.99（1.61-9.88）,P=0.003）,more patients were in the Suzuki intermediate stage（P=0.001）and fewer patients were in the advanced Suzuki stage（P=0.001）,while for the adult group,GA indicated more female onset（OR[95%CIs]=1.43[1.15-1.79],P=0.001）,fewer patients with diabetes（0.58[0.38-0.86],P=0.007）and early Suzuki stage（P=0.01）significantly less than the GG genotype.Meta-analysis demonstrated the inclusion of a total of 10 published domestic and international studies of RNF213 p.R4810K and clinical phenotype prediction,with a total of 4259patients with MMD enrolled,to determine the effect of heterozygous or homozygous RNF213 p.R4810K variants on 18 clinical features.The result showed that patients under15 years old were more common in GA and AA groups（AA vs GA:P=0.004;AA vs GG:P=0.0005;GA vs GG:P<0.00001）.The number of patients with MMD before 4 years old was higher in the AA group（AA vs GA:P<0.00001;AA vs GG:P<0.00001）.The frequency of brain infarction in AA was significantly higher（AA vs GA:P=0.004;AA vs GG:P=0.0005）,and the frequency of TIA in GA was significantly higher（GA vs GG:P<0.0001）.However,the incidence of CH in patients with GA genotype was lower than that in patients with GG genotype（P<0.00001）.The frequency of patients with AA and GA genotypes having a family history of MMD is higher（AA vs GG:P<0.00001;GA vs GG:P=0.002）.Patients with AA and GA were more commonly involved in the PCA（AA vs GG:P=0.008;GA vs GG:P<0.00001）.The results of subgroup analysis based on country suggested that the prediction effect of homozygous and heterozygous RNF213p.R4810K on patients with MMD in China,Korea and Japan was different.4.CAPN1 was identified as a new pathogenic gene of moyamoya disease in China by whole-exome sequencing.After WES sequencing and conditional filtering,12 of the 30 newly identified variants were identified to reappear in 113 patients with sporadic and 59 with familial MMD.Significant evidence of an association between five candidate variants（NLRP14rs11041150,AHI1 rs1186817196,CAPN1 rs139570056,BLM rs55880859,and VPS13C rs77673743）and the risk of MMD was identified.Based on follow-up analyses,including Sanger sequencing and bioinformatics prediction of the remaining candidate variants,CAPN1 p.R616C was finally validated as the only variant that increases the risk of MMD development.5.In vitro experiments verify that CAPN1 can cause characteristic pathological changes of MMD.In vitro assays have shown that CAPN1 knockdown leads to failure of Filamin A（FLNA）cleavage,which results in its excessive accumulation and ultimately promotes phenotypic transformation of smooth muscle cells and significantly enhances their proliferation and migratory functions.Conclusion:1.The distribution of RNF213 p.R4810K carrier rate in patients with MMD in China shows a geographical regional difference decreasing from East to West and from North to South.2.Patients with MMD are more prone to cerebral ischemia in summer.3.RNF213 p.R4810K has different predictive effects on the clinical phenotypes of pediatric and adults with MMD in China,and there are also significant differences in the predictive effects on the clinical phenotypes of patients with MMD in China,Japan and Korea.4.WES identified CAPN1 p.R616C as a new rare mutation site in Chinese patients with MMD.5.The silence of CAPN1 can lead to the failure of FLNA cleavage,resulting in the phenotypic transformation of aortic smooth muscle cells from"contractile"to"synthetic",which can promote the proliferation,migration and invasion of cells,accelerate vascular remodeling and angiogenesis,and lead to the occurrence of characteristic vascular manifestations of MMD.