Abstract: Objective · To investigate the pathogenesis of total anomalous pulmonary venous connection (TAPVC), and to identify ARHGEF16 gene through full exon sequencing screening and analyze its mutation function. Methods · The blood, clinical data and auxiliary examination results of 78 children with TAPVC and 100 healthy controls were collected, and the genomic DNA was extracted for ARHGEF16 mutation screening. ARHGEF16 wild-type and mutant plasmids were constructed and transfected into 293T cells. mRNA and protein levels were detectedquantitative real-time PCR (RT-qPCR) and Western blotting, respectively. Protein-protein interaction exploration was performedCytoscape software. Results · Two novel variants c.C236>T (A79V) and c.G619>C (G207R) were found in TAPVC patients and were not found in healthy controls. Compared with the wild type, the mutants ARHGEF16 were up-regulated in both mRNA and protein levels. Protein interaction analysis showed that ARHGEF16 and RAC1 were directly associated; RAC1 was up-regulated in HEK293 cells with ARHGEF16 over through RT-qPCR. Conclusion · The missense mutations of ARHGEF16 affect the mRNA and protein levels of ARHGEF16. Over of ARHGEF16 up-regulates the of RAC1, suggesting that it may participate in the development and formation of TAPVCregulating RAC1.

Key words: total anomalous pulmonary venous connection, ARHGEF16, missense mutations, RAC1, development