mesp2 variants contribute to conotruncal heart defects by inhibiting cardiac neural crest cell proliferation

Conotruncal heart defects (ctds) are closely related to defective outflow tract (oft) development, in which cardiac neural crest cells (cnccs) play an indispensable role. however, the genetic etiology of ctds remains unclear. mesoderm posterior 2 (mesp2) is an important transcription factor regulating early cardiogenesis. nevertheless, mesp2 variants have not been reported in congenital heart defect (chd) patients. we first identified four mesp2 variants in 601 sporadic nonsyndromic ctd patients that were not detected in 400 healthy controls using targeted sequencing. reverse transcription-quantitative pcr (rt-qpcr), immunohistochemistry, and immunofluorescence assays revealed mesp2 expression in the oft of carnegie stage (cs) 11, cs13, and cs15 human embryos and embryonic day (e) 8.5, e10, and e11.5 mouse embryos. functional analyses in hek 293t cells, hl-1 cells, joma1 cells, and primary mouse cnccs revealed that mesp2 directly regulates the transcriptional activities of downstream ctd-related genes and promotes cncc proliferation by regulating cell cycle factors. three mesp2 variants, c.346g>c (p.g116r), c.921c>g (p.y307x), and c.59a>t (p.q20l), altered the transcriptional activities of myocd, gata4, nkx2.5, and cfc1 and inhibited cncc proliferation by upregulating p21cip1 or downregulating cdk4. based on our findings, mesp2 variants disrupted mesp2 function by interfering with cncc proliferation during oft development, which contribute to ctds.