Spectrum Of Congenital Heart Disease In Yangbi County Of Yunnan Province And Identification Of Candidate Atrial Septal Defect Predisposing Gene Variants In Sporadic Atrial Septal Defect Using Whole Exome Sequencing

[Background and Objective]Congenital heart disease （CHD） is one of the most common congenital anomalies. Previous studies showed that the prevalence of CHD in different regions and nationalities have difference. Epidemiological study of CHD in Yunnan province had been carried out by Yan’an Affiliated Hospital of Kunming Medical University since 2001 and over 200,000 participants had been involved in our study. The CHD investigation of Yangbi County which is in the section one belongs to a part of the whole systemic CHD investigation in Yunana province. In this section, we aimed to investigate the spectrum of CHD between 3 to 16 aged populations in Yangbi Yi Autonomous County and to analyze the related factors, which could understand the region status and distribution characteristics of CHD in Yangbi.Studies in section one and other epidemiological data showed that atrial septal defect （ASD） was one of the most common subtypes of CHD and the morbidity rate was higher than ventricular septal defect （VSD） and patent ductus arteriosus （PDA）. However, the etiology of ASD remains largely unknown. To explore the whole exome genetic backgrounds in ASD, we employed whole exome sequencing （WES） combined with individualized bioinformatics analysis methods to identify candidate ASD predisposing gene variants in section two.[Methods]Section one:Cross-sectional studies were carried out among 9945 children between 3 to 16 aged populations in January 2015. To diagnose suspicious CHD, the methods that combined questionnaire survey and conventional cardiac physical examination were used, and then Echocardiography were employed to verify the subtypes of CHD.Section two:Cardiac tissues near the defects were isolated during ASD repair operation and peripheral blood were collected as a self-control in the cardiovascular department of Yan’an Affiliated Hospital of Kunming Medical University. DNA in tissues and blood were extracted. By employing HiSeq2500 equipment, the whole exome sequence （WES） derived from five cardiac tissues have been captured.Using ANNOVAR software, we transformed raw date and single nucleotide variants （SNV） in exome regions have been obtained. Candidate atrial septal defect predisposing gene variants were gained by filtering 1000Gemo, dbSNP and NHLBI database and using individualized bioinformatics analysis methods. False positive variants was excluded by using Sanger sequencing. Peripheral blood derived from the five WES ASD patients were as a self-control and positive variants were detected by using Mass-Array equipment. According to compared results, we employed Mass-Array to verify the other positive variants in 421 ASD cases and 507 healthy controls. Lastly, H9C2 cells were transfected with HYDIN siRNA. The expression of HYDIN was detected by qRT-PCR. After transfection, cell migrating ability was measured with wound healing assay, and cell cycle and apoptosis were assessed by flow cytometry instrument.[Results]Section one:505 suspicious CHD cases have been found through conventional cardiac physical examination and 79 CHD have been lastly verified. The total CHD prevalence in Yangbi is 7.94‰. （Male 7.17%o, Female:8.69‰） The CHD prevalence of Yi minority is 8.25%o which was higher than the average level in Yangbi. According to statistics in different aged group, the CHD morbidity rate was 4.63%o between 3 to 6 years,6.45%o in 7-12 and 11.11%o in 13-16 years. There were statistically significant differences in different groups （P<0.05）. The CHD prevalence varied among different towns and the CHD prevalence in each town was positively correlated with their GDP. The most common subtypes of CHD was ASD.Section two:20.7 GB raw data were gained by WES in five ASD cases and 181762 SNV were obtained transformed from raw data by using ANNOVAR software. Filtering 1000Gemo, dbSNP and NHLBI database 713 rare variants were obtained. From this 713 variants, we chose 33 variants in 25 ASD candidate genes by using personal bioinformatics analysis methods. One false positive variants was excluded by using Sanger sequencing. Compared with the DNA of tissues and peripheral blood, we did not detect any somatic mutations in other 32 variants. Moreover, Mass-Array equipment were used to check the other 32 variants, and we found that TTN> HYDIN、ZFPM1、XPO1 and FOXL2 were new ASD candidate predisposing gene. The rare variants 23862952 in MYH6 gene and 61708404 in XPO1 gene may be associated with ASD. The 138665410 variant （NM₀23067_c.C155G:p.A52G） which was a high mutation frequency in the ASD. group （P<0.05） may be highly associated with the occurrence of ASD. Lastly, transfection siRNA could effectively inhibit H9C2 cell expression of HYDIN. Compared with the blank control and empty vector group, HYDIN-silencing cells had higher apoptosis rate as well as lower migration rate and proliferation.[Conclusion]1. The CHD morbidity is significantly higher than the average of Yunnan, and the prevalence of Yi is higher than the other nationality in Yangbi.2. The CHD prevalence in female is higher than male.3. The CHD prevalence varied among different towns in Yangbi and there is a negative correlationship between the incidence of CHD and the regional economic level.4. The incidence of CHD has declined in past 15 years in Yangbi 5. ASD is the most common subtypes of CHD in Yangbi.6. The method that WES combined with personal bioinformatics analysis is an efficient tool to identify candidate predisposing gene variants in sporadic ASD.7. Large-scaled verification in ASD clinical trial is necessary because the complex background of ASD.8. Somatic mutation do not represent an important an etiology pathway in ASD.9. TTN、HYDIN、ZFPM1、XPO1 and FOXL2 are newly found ASD candidate predisposing gene.10. The rare variants 23862952 in MYH6 gene and 61708404 in XPO1 gene may be associated with ASD.11. The 138665410 variant could be highly associated with the occurrence of ASD, and the mechanism of leading ASD need further study.