Study On The Discovery Of A New Pathogenic Gene BMP9 In Idiopathic Pulmonary Hypertension By Whole-exome Sequencing

Background Idiopathic pulmonary arterial hypertension（IPAH）is a rare but fatal disease with high heritability.Although about 10 predisposing genes have been linked to IP AH and account for more than 80%of familial pulmonary hypertension（FPAH）,only 20-30%of IP AH patients could detect rare deleterious mutant on known genes.the genetic etiology remains unknown for a large number of IP AH cases.Objective The aim of our study was to discover germline genes associalated with IPAH,especially in Chinese patients.Methods This is a two-stage,case-control study.In the first phase,we enrolled a total of 251 IPAH patients from two pulmonary hypertension centers as a discovery cohort,identified their genetic background by next-generation sequencing.The exonm informations of 1884 normal populations from Novo-Zhonghua project were used as control group.We conducted an exome-wide gene-based burden analysis to screen the susceptible genes.In the second phase,a validation cohort containing 80 patients with IPAH was recruited,which completely independent from discovery cohort,and genotyped by whole exome sequencing.The exomenomic information from the public database GnomeAD East Asian population（8624 cases）was used as a control.The susceptibility genes were screened with the same procedure as first-phase,to verified the results of discovery cohort.The study included a total of 331 IPAH cases and 10508 controls.The clinical impacts of the new susceptibility genes were identified.And functional assessments of the mutation sites were conducted to analyze the effects of genetic mutations on protein biosynthesis and function.Results（1）The gene encoding human bone morphogenetic protein-9（BMP9）demonstrated strong association with IPAH in the discovery cohort,reaching the exome level significance(odds ratio,18.8;P=1.9 × 10^-11),that was identified as a novel genetic locus with IPAH.This correlation was authenticated in the independent verification cohort(P=1.0 x 10^-5).（2）In the combined cohort of two phase,6.7%of patients carried BMP9 rare deleterious mutations,significantly higher than the normal population control（0.30%）,odds ratio=21.1,95%confidence interval（11.7-37.6）,raw P value was 2.7×10^-19,and Bonferr P was 3.74×10^-15.BMP9 mutation was considered to be strongly associated with IPAH,ranking second to BMPR2.（3）In IPAH patients carrying BMP9 mutation,the average plasma BMP9 level was significantly lower than those in IP AH patients without BMP9 mutation（median 12.1 pg/ml,quartile interval 8.6-21.8 pg/ml vs.median 19.8 pg/ml,quartile interval 15.2 pg/mL-43.4 pg/mL,P=0.002）.Both of IPAH patients had lower BMP9 level than the normal population.（4）In vitro expression experiments demonstrated that most BMP9 rare,deleterious,missense mutations lead to a decrease in BMP9 secretion in pulmonary artery endothelial cells,especially at mature BMP.A few nonsense mutations could reduce the expression of BMP9.Intriguingly,some special missense mutations may lead to barriers of processing of BMP.（5）The ability of BMP9 to activate ACVRL1 channel is damaged in variant protein.Furthermore variant proteins were usually unable to protected pulmonary artery endothelial cells from apoptosis induced by TNFa,displaying the impaired anti-apoptosis ability.Conclusions The rare deleterious mutations of BMP9 had strongly association with IPAH.The circulating active BMP9 is decreased in IPAH patients carring BMP9 mutation.BMP9 mutation could negatively affect BMP9 function,including synthesis,secretion,processing,and/or activation of ACVRL1 signaling.In summary,We identify BMP9 as an IPAH culprit gene.