Objective:In this study,we aim to screen MESP1 variants in congenital heart disease(CHD),and explore the biological functions of these variants.Methods:Targeted sequencing was used to screen MESP1 variants in a cohort...Objective:In this study,we aim to screen MESP1 variants in congenital heart disease(CHD),and explore the biological functions of these variants.Methods:Targeted sequencing was used to screen MESP1 variants in a cohort of patients with CHD and healthy controls from Shandong Province,China,and confirm the variants with Sanger sequencing.Dual-luciferase reporter assay was used to assess the effects of MESP1 variants on the expression of downstream target genes in HEK293T and H9C2 cells.Binding affinity with NKX2-5 between variants and wild-type MESP1 was detected in HEK293T cells using an electrophoretic gel mobility shift assay(EMSA).The teratogenic effects of the MESP1 variants compared to the wild-type were explored with zebrafish embryo microinjection.Results:We identified two rare mutations,namely c.359T>C(p.L120P)and c.526A>T(p.T176S),in MESP1.The impact of these mutations was investigated using dual-luciferase reporter assay.Our findings demonstrate that compared to the wildtype,the mutation c.359T>C(p.L120P)significantly inhibits the expression of downstream target genes,whereas the mutation c.526A>T(p.T176S)does not exhibit altered activity.Further insights from EMSA revealed that the mutation c.359T>C(p.L120P)displays a reduced binding affinity with NKX2-5.Notably,our investigation involving zebrafish embryo microinjection unveiled a significant increase in the rate of malformation associated with the mutation c.359T>C(p.L120P).Conclusions:Two rare mutations in MESP1 were identified in the CHD cohort.Our in vitro and in vivo studies showed that MESP1 c.359T>C(p.L120P)is a loss-of-function mutation that may increase the risk of CHD.展开更多
基金National Natural Science Foundation of China(82171845)Gansu Provincial Natural Science Foundation of China(23JRRA1046)。
文摘Objective:In this study,we aim to screen MESP1 variants in congenital heart disease(CHD),and explore the biological functions of these variants.Methods:Targeted sequencing was used to screen MESP1 variants in a cohort of patients with CHD and healthy controls from Shandong Province,China,and confirm the variants with Sanger sequencing.Dual-luciferase reporter assay was used to assess the effects of MESP1 variants on the expression of downstream target genes in HEK293T and H9C2 cells.Binding affinity with NKX2-5 between variants and wild-type MESP1 was detected in HEK293T cells using an electrophoretic gel mobility shift assay(EMSA).The teratogenic effects of the MESP1 variants compared to the wild-type were explored with zebrafish embryo microinjection.Results:We identified two rare mutations,namely c.359T>C(p.L120P)and c.526A>T(p.T176S),in MESP1.The impact of these mutations was investigated using dual-luciferase reporter assay.Our findings demonstrate that compared to the wildtype,the mutation c.359T>C(p.L120P)significantly inhibits the expression of downstream target genes,whereas the mutation c.526A>T(p.T176S)does not exhibit altered activity.Further insights from EMSA revealed that the mutation c.359T>C(p.L120P)displays a reduced binding affinity with NKX2-5.Notably,our investigation involving zebrafish embryo microinjection unveiled a significant increase in the rate of malformation associated with the mutation c.359T>C(p.L120P).Conclusions:Two rare mutations in MESP1 were identified in the CHD cohort.Our in vitro and in vivo studies showed that MESP1 c.359T>C(p.L120P)is a loss-of-function mutation that may increase the risk of CHD.
