Study On The Association Of The Gene Polymorphism Of Transcription Factors With Congenital Heart Disease

Objectives:(1)To evaluate the association between single nucleotide polymorphisms(SNPs)of NKX2.5,GATA4,TBX5 genes and congenital heart disease(CHD).(2)To explore the gene-gene interactions between NKX2.5,GATA4,TBX5 genes with CHD.(3)To explore the gene-environment interactions between NKX2.5,GATA4,TBX5 genes and maternal environmental factors with CHD.Methods:A hospital-based case-control study was conducted.The study participants were recruited in Hunan Children’s hospital from December2018 to June 2021.The CHD group was included the patients with non-chromosomal CHD and the control group was included the healthy individuals without any congenital defects or cardiac disease.Each individual was collected 3ml peripheral venous blood sample,and accomplished the investigation of maternal pregnancy information after permission.Target loci of NKX2.5,GATA4,TBX5 genes were selected by searching the common mutation variants in previous relevant studies and in the db SNP database of NCBI.Mass Array flight-time mass spectrometry was used to sequencing the polymorphism of target loci at NKX2.5,GATA4,TBX5 genes.Chi-squared tests and multivariate logistic regression analyses were used to evaluate the relationship between the polymorphism and the genetic model(dominant,additive and recessive)of target loci at NKX2.5,GATA4,TBX5 genes with CHD by SPSS26.0software.Linkage disequilibrium test and haplotype analyses were used to detect the relationship of loci and haplotype in single gene with CHD by Haploview4.2 software.Crossover analyses,logistic regression analyses and MB-MDR were used to explore the first-order to high-order of gene-gene and gene-environment interactions with CHD by R software(version3.6.3).Results:(1)The characteristics of the study paticipants:A total of 1185subjects were recruited in this study,including 585 in the CHD group and620 in the control group.All the study paticipants had completed the epidemiological investigation,blood sample collection and SNP sequencing,accounting for 108.5%of the designed sample size.(2)The environmental determinants of CHD:Multivariate logistic regression analysis showed that the mothers with history of adverse pregnancy outcomes(OR=1.50,95%CI:1.11-2.05),prepregnancy diabetes(OR=3.23,95%CI:1.69-6.18),consanguineous marriage history of family members(OR=13.88,95%CI:2.84-67.82),congenital malformation history of family members(OR=5.51,95%CI:1.64-18.50),gestational diabetes(OR=3.15,95%CI:1.61-6.15),gestational hypertension(OR=2.54,95%CI:1.19-5.42),took antibiotics 6 months before pregnancy(OR=2.84,95%CI:1.41-5.73),took ovulation medication 6 months before pregnancy(OR=4.38,95%CI:1.66-11.54),caught cold in early pregnancy(OR=1.57,95%CI:1.08-2.28),drunk alcohol in early pregnancy(OR=1.93,95%CI:1.16-3.23),had perm or dye hair during the peri-conception(OR=2.58,95%CI:1.50-4.44),used cosmetics frequently during the peri-conception(OR=1.69,95%CI:1.14-2.53)were associated with an increased risk of CHD in offspring.(3)The association between polymorphisms of target loci of NKX2.5,GATA4,TBX5 genes and CHD:After adjusting for the suspicious confounding factors,the analyses of polymorphisms at NKX2.5,GATA4,TBX5 genes and CHD showed that(1)the polymorphisms at rs118026695of NKX2.5 gene were significantly related to CHD(CC/TT:OR=5.28,95%CI:1.26-22.14;TC/TT:OR=1.58,95%CI:1.10-2.28;dominant model:OR=1.69,95%CI:1.18-2.41;additive model:OR=1.71,95%CI:1.23-2.37;recessive model:OR=4.72,95%CI:1.13-19.66).The polymorphisms at rs703752 of NKX2.5 gene were significantly related to CHD(CA/CC:OR=0.64,95%CI:0.41-0.99;dominant model:OR=0.64,95%CI:0.42-0.99).(2)The polymorphisms at rs4841588 of GATA4 gene were significantly related to CHD(GG/TT:OR=0.41,95%CI:0.23-0.73;additive model:OR=0.76,95%CI:0.60-0.96;recessive model:OR=0.42,95%CI:0.24-0.73).The polymorphisms at rs12458 of GATA4 gene were significantly related to CHD(AA/GG:OR=2.50,95%CI:1.64-3.83;AG/GG:OR=2.02,95%CI:1.41-2.89;dominant model:OR=2.16,95%CI:1.54-3.04;additive model:OR=1.59,95%CI:1.29-1.97;recessive model:OR=1.59,95%CI:1.12-2.26).The polymorphisms at rs904018 of GATA4gene were significantly related to CHD(AG/AA:OR=0.70,95%CI:0.51-0.97;dominant model:OR=0.70,95%CI:0.51-0.94;additive model:OR=0.78,95%CI:0.62-0.98).(3)The polymorphisms at rs11067101 of TBX5 gene were significantly related to CHD(GA/GG:OR=1.51,95%CI:1.08-2.11).The polymorphisms at rs6489956 of TBX5 gene were significantly related to CHD(TT/CC:OR=3.49,95%CI:1.10-11.08;CT/CC:OR=1.84,95%CI:1.29-2.62;dominant model:OR=1.93,95%CI:1.37-2.72;additive model:OR=1.85,95%CI:1.35-2.52;recessive model:OR=2.98,95%CI:1.94-4.58).(4)Linkage disequilibrium test and haplotype analyses:The linkage disequilibrium test of each gene had not found any strong correlation among loci.The haplotype analyses showed that the haplotype T-T(χ~2=22.228,P<0.001)and T-G(χ~2=25.824,P<0.001)formed of rs2645457-rs4841588 at GATA4 gene were significantly related to CHD.And the haplotype A-C-T(χ~2=9.397,P=0.002)formed of rs12426660-rs883079-rs10850326 at TBX5 gene were significantly related to CHD.(5)The gene-gene interactions between NKX2.5,GATA4,TBX5genes with CHD:After adjusting for the suspicious confounding factors,the first-order gene-gene interactions of rs703752(NKX2.5)-rs6489956(TBX5)(OR=0.23,95%CI:0.08-0.71),rs4841588(GATA4)-rs6489956(TBX5)(OR=0.37,95%CI:0.18-0.75),rs904018(GATA4)-rs6489956(TBX5)(OR=0.26,95%CI:0.13-0.53)were significantly related to CHD.The second-order gene-gene interactions between NKX2.5-GATA4-TBX5gene of rs118026695-rs4841588-rs11067101(OR=2.54,95%CI:1.01-6.43),rs118026695-rs904018-rs6489956(OR=0.13,95%CI:0.02-0.83)and rs118026695-rs12458-rs6489956(OR=1.87,95%CI:1.06-3.28)were significantly related to CHD.MB-MDR analyses were used to identify the best model of first-order to fifth-order gene-gene interactions by a thousand permutation tests,and found that the best model of first-order was:rs12458(GATA4)-rs4841588(GATA4),the best model of second-order was:rs6489956(TBX5)-rs11067101(TBX5)-rs12458(GATA4),the best model of third-order was:rs6489956(TBX5)-rs11067101(TBX5)-rs12458(GATA4)-rs118026695(NKX2.5),the best model of fourth-order was:rs6489956(TBX5)-rs11067101(TBX5)-rs12458(GATA4)-rs703752(NKX2.5)-rs118026695(NKX2.5),the best model of fifth-order was:rs6489956(TBX5)-rs11067101(TBX5)-rs904018(GATA4)-rs4841588(GATA4)-rs703752(NKX2.5)-rs118026695(NKX2.5).(6)The gene-environment interactions between NKX2.5,GATA4,TBX5 genes with CHD:After adjusting for the suspicious confounding factors,the gene-environment interactions were found between NKX2.5gene with gestational diabetes and consanguineous marriage history of family members.The gene-environment interactions were found between GATA4 gene with consanguineous marriage history of family members,congenital malformation history of family members,prepregnancy diabetes,gestational diabetes,adverse pregnancy outcomes,took antibiotics 6 months before pregnancy,drunk alcohol in early pregnancy,had perm or dye hair during the peri-conception.The gene-environment interactions were found between TBX5 gene with consanguineous marriage history of family members,congenital malformation history of family members,prepregnancy diabetes,adverse pregnancy outcomes,caught cold in early pregnancy,drunk alcohol in early pregnancy.MB-MDR analyses were used to identify the best model of first-order to fifth-order gene-environment interactions by a thousand permutation tests,and found that the best model of first-order was:rs12458(GATA4)-prepregnancy diabetes,the best model of second-order was:rs12458(GATA4)-rs4841588(GATA4)-gestational diabetes,the best model of third-order was:rs6489956(TBX5)-rs12458(GATA4)-prepregnancy diabetes-took antibiotics 6 months before pregnancy,the best model of fourth-order was:rs6489956(TBX5)-rs12458(GATA4)-prepregnancy diabetes-took antibiotics 6 months before pregnancy-used cosmetics frequently during the peri-conception,the best model of fifth-order was:rs6489956(TBX5)-rs12458(GATA4)-prepregnancy diabetes-consanguineous marriage history of family members-took ovulation medication 6 months before pregnancy-used cosmetics frequently during the peri-conception.Conclusions:(1)The mothers with history of adverse pregnancy outcomes,prepregnancy diabetes,consanguineous marriage history of family members,congenital malformation history of family members,gestational diabetes,gestational hypertension,took antibiotics 6 months before pregnancy,took ovulation medication 6 months before pregnancy,caught cold in early pregnancy,drunk alcohol in early pregnancy,had perm or dye hair during the peri-conception,used cosmetics frequently during the peri-conception were independent factors of CHD in offspring.(2)Polymorphisms at rs118026695,rs703752 of NKX2.5 gene,rs4841588,rs12458,rs904018 of GATA4 gene,rs11067101,rs6489956of TBX5 gene were associated with CHD.The haplotype analyses showed that the haplotype T-T and T-G formed of rs2645457-rs4841588 at GATA4 gene and the haplotype A-C-T formed of rs12426660-rs883079-rs10850326 at TBX5 gene were significantly related to CHD.(3)The first-order gene-gene interactions of rs703752-rs6489956,rs4841588-rs6489956 and rs904018-rs6489956 were statistically related to CHD.The second-order gene-gene interactions of rs118026695-rs4841588-rs11067101,rs118026695-rs904018-rs6489956and rs118026695-rs12458-rs6489956 were statistically related to CHD.The best model of first-order to fifth-order gene-gene interactions among loci at NKX2.5,GATA4 and TBX5 genes were related to CHD.(4)Gene-environment interactions were found between NKX2.5,GATA4,TBX5 genes with adverse pregnancy outcomes,prepregnancy diabetes,consanguineous marriage history of family members,congenital malformation history of family members,gestational diabetes,ook antibiotics 6 months before pregnancy,took ovulation medication 6months before pregnancy,caught cold in early pregnancy,drunk alcohol in early pregnancy,had perm or dye hair during the peri-conception and used cosmetics frequently during the peri-conception.