Abstract: AbstractObjective: To detect ZFPM2 mutations in conotruncal abnormality (CTA) patients by Sanger sequencing and study its pathogenicity and related molecular mechanism in CTA.Methods: Sanger sequencing was performed to detect ZFPM2 mutations in myocardial tissues of non-syndromic CTA patients in Children's Hospital of Fudan University. Plasmids were constructed, including expression constructs encoding GATA4, ZFPM2 wild type(WT) or variant and a reporter construct containing ANF promoter. The co-immunoprecipitation (Co-IP) and luciferase reporter gene assays were performed to study the change of interaction between ZFPM2 and GATA4 or the effect on ANF promoter activity in ZFPM2 WT or mutant. Injection of mRNA was performed to determine the effect of hZFPM2 mutant on cardiac phenotypes of zebrafish.Results: 107 non-syndromic CTA patients including 64 males and 43 females, aged from 1 to 168 months (median 8 months), were sequenced. Among them, there were 39 tetralogy of Fallot (TOF), 29 TOF with patent foramen ovale (PFO), 6 TOF with atrial septal defect (ASD), 8 TOF with PFO and patent ductus arteriosus (PDA), and other 25 multiple phenotypes, such as atrioventricular septal defects (AVSD) and left superior vena cava (LSVC). A heterozygous missense variant c.397A>G, p.M133V (rs77117583) was found in a CTA patient with TOF and PFO, which was located in the exon 4 and N-terminal transcriptional repression domain with 0.3‰ mutation frequency in Asian population. The Co-IP and luciferase reporter assays in HEK293T and HL-1 cells revealed that the M133V variant protein enhanced significantly the interaction between ZFPM2 with GATA4. And M133V mutant displayed a higher repression for GATA4-mediated transcriptional activation of ANF. Injection of hZFPM2 mRNA in zebrafish embryos demonstrated that the rate of cardiac abnormalities in M133V group(27.5%) was higher than control group without injection(11.3%) and WT group(15.0%), P＜0.05. The cardiac malformations were observed, including abnormal looping and the ventricle and atrium displayed the complete mirror image of normal.Conclusion: A low frequency variant of ZFPM2 c.397A>G, p.M133V was detected in a CTA patient and it was involved in the pathogenesis of CTA by affecting the combination with GATA4, and GATA4 transcriptional regulation.