Comprehensive Identification Of Potential Idiopathic Pulmonary Arterial Hypertension Causal Genetic Factors By Whole Exome Sequencing

Research BackgroundWhole exome sequencing(WES)was used in the research of familial pulmonary arterial hypertension(FPAH).CAV1 and KCNK3 were found as two novel candidate genes of FPAH.However,few pathogenic genes were identified in idiopathic pulmonary arterial hypertension(IPAH).Research PurposesDue to the lack of suitable research technology,the research of IPAH is progressing slowly.Therefore,we tried to explore the genetic factor related to IPAH by WES and look forward to find out several useful information to provide clues for future research.Research MethodsWe conducted WES in 20 unrelated IPAH patients that did not carry the known PAH-pathogenic variants among BMPR2,CAV1,KCNK3,SMAD9,ALK1 and ENG and conducted a comprehensive bioinformatics analysis.We also conducted validation studies in five mutant genes related to PLC-activating GPCR signaling pathway potentially involved in intracellular calcium regulation through Sanger sequencing for mutation accuracy,q RT-PCR for m RNA stability,immunofluorescence for subcellular localization,western blot for protein level,fura-2 imaging for intracellular calcium,CCK8 and cell cycle for the ability of cell proliferation and apoptosis assay and Caspase-3 activity for the apoptosis rate of cell.Research ResultWe found a total of 4950 variants in 3534 genes including 4444 SNPs and 506In Dels.Through the comprehensive and multi-level analysis,we disclosed several novel signaling cascades significantly connected to IPAH,including variants related to cadherin signaling pathway,dilated cardiomyopathy,glucose metabolism,immune response,mucin-type O-glycosylation,PLC-activating GPCR signaling pathway,vascular contraction and generation,and voltage-dependent Ca²⁺channels.The validation experiments showed that those variants in CCR5 and C3AR1 significantly increased the rise of intracellular calcium and the variant in CCR5 profoundly enhanced proliferative capacity of human pulmonary artery smooth muscle cells.ConclusionOur study suggests that multiple genetically-affected signaling pathways take effect together to cause the formation of IPAH and the development of right heart failure,the mutation of CCR5 and C3AR1 may play an important role in the IPAH,and may further provide new therapy targets or putative clues for the current treatments such as limited therapeutic effectiveness of Ca²⁺channel blockers.