The Impact Of TGFBR1^A230T Variants In Loeys-Dietz Syndrome On Lineage-Specific Smooth Muscle Cell

Background:Loeys-Dietz Syndrome（LDS）is an inherited disorder predisposing individuals to thoracic aortic aneurysm and dissection（TAAD）.Currently,there are no medical treatments except surgical resection.The genetic basis of LDS is autosomal dominant mutation in TGF-βsignaling pathway.However,the molecular mechanisms underlying the disease remain elusive impeding the development of a therapeutic strategy.In addition,aortic smooth muscle cells（SMC）have heterogenous embryonic origins depending on their spatial location,and lineage-specific effects of pathogenic variants on SMC function,likely causing regionally constrained LDS manifestations,have been unexplored.Objectives:Through human induced pluripotent stem cell（hiPSC）modelling the LDS,explore the lineage specific impact of TGFBR1^A230Tmutation in SMC from two lineages to reveal the underlying pathological mechanisms and investigate potential therapeutic target.Methods:1.Based on the LDS family carrying novel TGFBR1^A230T mutation,generating hiPSC from LDS peripheral blood monocyte from LDS patient and healthy control,Inducing and correcting TGFBR1^A230T mutation in hiPSC by CRISPR-Cas9 gene editing2.In vitro differentiation from hiPSC to neural crest stem cell derived smooth muscle cell（NCSC-SMC）and cardiovascular progenitor cell derived smooth muscle cell（CPC-SMC）3.Exploring lineage specific impact of TGFBR1^A230T mutation on the contractile genes expression and contractile function of NCSC-SMC and CPC-SMC by traditional molecule biology methods and single cell RNA-seq（scRNA-seq）4.Exploring how correcting TGFBR1^A230T mutation affect CPC-SMC from LDS patient by traditional molecule biology methods and scRNA-seq5.Exploring lineage specific impact of TGFBR1^A230T mutation on TGF-βsignaling of NCSC-SMC and CPC-SMC by traditional molecule biology methods and scRNA-seq6.Testing whether Rapamycin could alleviate the impact of TGFBR1^A230Tmutation on CPC-SMCResults:1.Generating hiPSC from healthy control and LDS patient carrying novel TGFBR1^A230T mutation,inducing the heterozygote and homozygote mutation in hiPSC from healthy control and correcting the mutation in hiPSC from LDS patient2.hiPSC induced to NCSC-SMC and CPC-SMC3.TGFBR1^A230T mutation impairing the contractile genes expression and contractile function of CPC-SMC but not in NCSC-SMC4.Correcting TGFBR1^A230T mutation from LDS patient promoting the contractile genes expression and contractile function of CPC-SMC5.TGFBR1^A230T mutation impairing the TGFBR1/SMAD3 and TGFBR1/AKT signaling of CPC-SMC but not in NCSC-SMC6.Rapamycin treatment alleviating the inhibition of TGFBR1^A230T in CPC-SMCConclusion:Based on the clinical finding of novel LDS family which carried TGFBR1^A230T mutation,we find the mutation impairs contractile genes expression and contractile function of CPC-SMC by inhibiting TGFBR1/SMAD3 and TGFBR1/AKT signaling instead of NCSC-SMC.d Rapamycin recue the inhibition of the mutation in CPC-SMC.Our results implicate that the lineage specific impact of LDS mutation might leads to aortic root aneurysm and activation of TGFBR1/SMAD3 and TGFBR1/AKT signaling could be potential therapeutic target to prevent the aortic root dilation in LDS patients.