Rare Variants Associated With Symptomatic Intracranial Atherosclerotic Stenosis Based On Target Region Sequencing

Objective:Symptomatic intracranial atherosclerotic stenosis(s ICAS)is a major cause of ischemic stroke(IS).The pathophysiological mechanisms of s ICAS remain unclear and mainly focus on oxidative stress,endothelial dysfunction,inflammatory response,hemodynamics and so on.s ICAS is a complex disease affected by many risk factors,and it’s the result of environmental and genetic factors.With the development of nextgeneration sequencing techniques,rare variant(minor allele frequency,MAF<0.01)association analysis has become an important approach to identify genetic susceptibility variants for complex diseases,and target region sequencing is the most cost-effective and suitable method for large sample studies.In this study,86 candidate genes associated with the pathophysiological mechanisms and risk factors of s ICAS were selected for target region sequencing.Rare variant association analyses were conducted and repeated validations were performed to confirm the associations.Furthermore,we carried out some molecular experiments to explore the biological functions of the rare variants of RARRES2 and its encoding protein Chemerin.Our study may provide additional information for the pathogenesis and new drug targets of s ICAS.Methods:This study was a two-stage case-control study.In the first stage,400 patients diagnosed with s ICAS from the Department of Neurology in Xiangya Hospital,Central South University from July 2015 to December2017 were enrolled,control individuals were obtained from Gene Sky inhouse database(1007 samples).According to literature reports and combing with Gene Cards,GWAS Catalog and other databases,we chose86 candidate genes related to the pathogenesis and risk factors of s ICAS.Targeted region sequencing of these candidate genes was performed on s ICAS subjects using the Agilent chip.Rare variants in the exon and splicing region of these genes were screened out.Gene-based association analyses were used to reveal the association between rare variants and s ICAS genetic susceptibility.In the second stage,repeated validation was performed in 400 s ICAS patients from the Department of Neurology in Xiangya Hospital from January 2018 to January 2020,and 1000 age-and sex-matched healthy controls were selected from the community health population.Fast Target target region sequencing technology was used to sequence the targeted region of RARRES2.Data from the two stages were combined for analysis.Fisher’s exact test or Chi-square test(dominant genetic model)was used to analyze the association between s ICAS group and the healthy control group in the level of single rare variant and whole gene.Point mutation plasmids of RARRES2 c.454G>C(p.G152R)and c.485G>A(p.R162H)were constructed.RT-PCR,ROS(reactive oxygen species),SOD(superoxide dismutants)and MDA(malondialdehyde)detection were performed to preliminarily explore the effects of Chemerin on inflammatory response,endothelial dysfunction and oxidative stress of human umbilical vein endothelial cells(HUVECs).Results:(1)A total of 917 rare variants were detected in 400 s ICAS patients,including 672 known variants and 245 unknown variants.Gene-based association analyses showed that rare variants in 42 genes including CMIP,RARRES2,SMARCA4,HTRA1,JMJD1 C and NAMPT were associated with s ICAS in at least one enrichment analysis.GO(gene ontology)analysis showed that these genes were associated with cholesterol homeostasis,post-translational protein phosphorylation,nitric oxide signal transduction,lipid and atherosclerosis,foam cell formation,and VEGFAVEGFR2 signaling pathways.(2)A total of 7 rare variants in RARRES2 were detected in 400 s ICAS patients and 1000 controls using Fast Target target region enrichment sequencing technique: c.472A>G(p.K158E),c.440G>A(p.S147N),c.397A>T(p.T133S),c.287G>A(p.R96Q),c.280-5C>A,c.182C>T(p.P61L),and c.133T>A(p.W45R).Combing the data of two stages together,c.175-15C>T、c.454G>C(p.G152R),and c.485G>A(p.R162H)were carried only in the s ICAS group,while the frequency of these variants in controls was 0.Based on the results of association analyses of single variant,the distribution of p.S147 N was significantly different between800 s ICAS patients and 1000 healthy controls(P=0.012).Chi-square test based on the whole gene showed that rare variants in RARRES2 were associated with the risk of s ICAS(P=0.031).(3)RARRES2 mutant plasmids(p.R162 H,p.G152R)was successfully constructed.The p.G152 R mutant plasmid could affect Chemerin expression level.The expression level of ICAM-1,VCAM-1 and Caspase-1 increased,ROS induced,MDA content increased,and SOD enzyme activity decreased in HUVEC treated with 100 ng/ml Chemerin,comparing to the blank control group.Conclusions:(1)Rare variants in CMIP,RARRES2,SMARCA4,HTRA1,JMJD1 C and NAMPT are possibly associated with the risk of s ICAS.(2)RARRES2 mutant plasmids p.G152 R may be involved in the pathogenesis of s ICAS by affecting endothelial function,inflammatory response and oxidative stress.