Effect of potential pathogenic gene PDX1 variants of total anomalous pulmonary venous connection on its gene function

Abstract

Objective·To explore the possible pathogenic gene of total anomalous pulmonary venous connection (TAPVC) by whole exon sequencing and verify its function.Methods·One hundred TAPVC children (case group) and one hundred and twenty healthy children (control group) in Xinhua Hospital and Shanghai Children's Medical Center, Shanghai Jiao Tong University School of Medicine from 2014 to 2019 were included. The blood samples from the two groups of children were collected, and whole blood genomic DNA was extracted for exon sequencing to screen out the potential pathogenic genes of TAPVC. Harmful mutation sites of pathogenic genes were screened through Mutation Taster, SIFT and PolyPhen-2 websites, and then conducted by Sanger sequencing. The wild-type (wild-type group) and mutant (mutant group) plasmids of PDX1 were transfected into HUVEC cells. Quantitative real-time PCR (qPCR) and Western blotting were used to detect the effects of mutations on mRNA and protein levels of PDX1, respectively. The STRING database was used to analyze the interaction between proteins, and qPCR was used to determine the expressions of downstream genes regulated by PDX1.Results·Pathogenic PDX1 was found in TAPVC children, and Sanger sequencing revealed two novel variants in the gene: c.C237A (P33T) and c. C237G (P33A). Compared with the wild-type group, there was no significant difference in PDX1 mRNA levels in the two mutant groups, but there was a significant increase in relative protein expression of the CA group and CG group, which was 2.9 and 3.4 times higher than the wild-type group, respectively (P=0.000, P=0.001). Protein interaction analysis demonstrated that PDX1 was associated with SOX17. qPCR results showed that overexpression of PDX1 could downregulate the expression of SOX17 in HUVEC.Conclusion·The two novel PDX1 missense mutations can affect the process of PDX1 post-transcriptional translation, indicating that PDX1 may participate in the occurrence and development of TAPVC by regulating SOX17.

Keywords

• total anomalous pulmonary venous connection (tapvc)
• pdx1
• missense mutation
• sox17
• human umbilical vein endothelial cell (huvec)