Screening All The Exons Of Nkx2.5, ISLet1and MTHFR Gene In The Patients With Tetralogy Of Fallot

Background and ObjectsCongenital heart disease（CHD）is considerderd abnormalities of cardiovascularmorphology， structure， function and metabolism caused by abnormal embryonicdevelopment of cardiovascular system. Tetralogy of Fallot (TOF）accounted for6.8%ofall CHDs is the most common cyanotic CHD，it is seriously harmful to infant health. Heartdevelopment involves precise expression of multiple genes on different time and spaceand the regulation of various transcription factors.The variation of heart developmentgenes which control the development of heart is the common reason of CHD，abnormalexpression of them will lead to various CHD. With the application of the transgenic andgene knock-out technique，NK2transcription factor related locus5(Nkx2.5) and InsulinGene Enhancer Binding Protein1(ISLet1or ISLet1) were confirmed to be the mostimportant transcription factors regulating differentiation and development of myocardialprecursor cells’ in the first and second heart field. A great quantity of researches show thatthe variation of NKx2.5and ISLet1can lead to TOF.Methylenetetrahydrofolate Reductase (MTHFR) is the key enzyme of Methionine metabolism and folate metabolicpathways.Researches show that human MTHFR gene mutation may reduce the enzymaticactivity and Lead to the occurrence of CHD.The study is aimed to find the new variantgene site by screening of all exons of Nkx2.5，ISLet1and MTHFR gene in207ChineseHan people with TOF，as well as to investigate the mechanism of TOF caused by newvariant gene site.Methods207blood samples (including male118，female89，2.58±1.39years) were recruitedfrom the patients with TOF in the general Hospital of Shenyang military command fromJanuary2009to December2012.All cases were diagnosed as TOF by clinical symptomcombined with X-ray，electrocardiogram（ECG），ultrasonic cardiography（UCG）andcardiac angiography.Of all cases，hypertension，diabetes，tumor and serious diseases onliver and kidney were excluded by medical examination，laboratory examination，chest Xray，and ECG.213contrast blood samples，male136，female77，age2.87±1.67.All cases werediagnosed as non-CHD with X-ray，ECG and UCG，and hypertension，diabetes，tumorand serious diseases on liver and kidney were excluded.Genomic DNA in white blood cell was extracted from peripherally white blood cellswith reagent box. Screening all exons of Nkx2.5，ISLet1and MTHFR gene were determinedusing nested polymerase chain reaction-single strand conformation polymorphism (nestedPCR-SSCP）in207TOF patients. E137K variant gene site was first discovered by us usingDNA sequencing following by the proband’s family investigation. MTHFR genec.1333C>T was confirmed Single Nucleotide Polymorphism (SNP).We accessed thepotential association between the MTHFR genetic polymorphism and risk of CHDthrough PCR-Restriction Fragment Length Polymorphism(PCR-RFLP) and StatisticalMethods.Results1. Through detecting variant gene site in all the exons of NKx2.5and ISLet1geneby nested PCR-SSCP and DNA sequencing，we found the new variant gene site in ISLet1 gene，E137K，in Chinese patients with TOF.2. E137K in ISLet1gene exon3was confirmed by SSCP and repeated DNAsequencing. With sequencing analysis，the new alteration’s location was confirmed to be atG9557A in the whole sequence located in gene exon3and at G648A in cDNA，As a resultof base changes，Glutamic acid changed into Lysine acid，at the same time，the aminoacids’ hydrophilicity was increasing. With the high degree of homology and conservatism，the amino acids were found in the second LIM domain of ISLet1protein.3. The new variant gene site with G3456T locating in Nkx2.5gene’ exon2wasfound in the family suvey of ISLet1gene E317K.The new variant gene site was located in3′UTR.With the high degree of homology and conservatism，maybe the new gene site isthe target of hsa-miR-1225-3P microRNA.4. The rusults of family suvey of7relatives showed that new variant gene site ofNKx2.5and ISLet1gene appeared in genes of propand，his adoptive grand-father and hismother had.In genes of all the three ones，G/A heterozygote change was found at new genesite G648A in exon3cDNA of ISLet1gene(42.9%，3cases of7relatives）.While in genesof the other4relatives，GG wild-type change was found at this site(57.1%，4cases of7relatives）and no AA type was found. For G3456T site change in exon2of Nkx2.5gene，TT mutation was found in genes of proband（14.3%，1case of7relatives）and G/Theterozygote change in genes of his adoptive grand-father and his mother(28.6%，2casesof7relatives）.GG wild-type change was found at this site of genes of the other4relatives(57.1%，4cases of7relatives).5. A novel genetic polymorphism (c.1333C>T) of MTHFR gene was detected bythe PCR-RFLP and DNA sequencing methods. Results from the sequence analysessuggested that this genetic polymorphism was a non-synonymous mutation resulted from aC to T mutation in exon8of MTHFR gene.This substitution has lead to the arginine (Arg)to tryptophan (Trp) amino acid replacement (A445T).6. According c.1333C>T) of MTHFR gene，The PCR-amplified products weredigested with restriction enzyme and divided into three genotypes：CC，CTandTT.Thestudy summarizes the allelic and genotypic frequencies in the studied populations.The allelic frequencies of CHD patients(C，66.18%；T，33.82%)were significantly differentfrom those in non-CHD controls(C，73.00%；T，27.00%；χ2=4.6207，p=0.0316).Thegenotypic frequencies of CHD patients(CC，47.34%；CT，37.68%；TT，14.98%)were notconsistent with non-CHD controls (CC，52.11%；CT，41.79%；TT，6.10%；χ2=8.8129，p=0.0122).The study shows the potential association of c.1333C>T genetic polymorphismin MTHFR gene with the risk of CHD.Data indicated that this genetic variant wasstatistically associated with the increased risk of CHD in homozygote comparison(TTversus CC：OR=2.70，95%CI1.34–5.45，χ2=8.04，p=0.005).There was no significantassociation with the increased risk of CHD in heterozygote comparison (CT versus CC：OR=0.99，95%CI0.66–1.49，χ2<0.01，p=0.972).Conclusions1. The new variant gene sites of ISLet1gene E137K，Nkx2.5gene G3456T andMTFHR gene C1333T were all firstly found by us in Chinese patients with TOF.2. We concluded that gene distribution frequency of E137K variatnt site of ISLet1genes showed the most extensive GG type，less extensive AG type and the least AAtype.Family proneness was obvious on AG type and GG type.It was possible that changesof amino acids led to structural and functional changes of the second domain，whichprohibited it from binding with LIM homeodomain and coordinated regulation with otherproteins.It was uncertain if there was relationship between CHD and gene distributionfrequency of E137K variatnt site. It needed us to make further research on E137K variatntsite change，so as to detect why it influenced gene expression of ISLet1and heartdevelopment gene network related gene Mef2C，which increases the risk of CHD or TOF.3. We concluded that gene distribution frequency of G3456T variatnt site showedthe most extensive GG type，less extensive GT type and the least TT type.Familyproneness was obvious on GT type and TT type.We persumed that maybe the new genesite is the target of hsa-miR-1225-3P microRNA，where the translation of Nkx2.5genewas restrained.After base change of this site，hsa-miR-1225-3P microRNA can not bindwith it，leading to expression abnormality of Nkx2.5and heart development gene networkrelated gene.So，it would increase the risk of TOF and other CHD. 4. A novel genetic polymorphism (c.1333C>T) of MTHFR gene was statisticallyassociated with the increased risk of CHD in homozygote comparison.There was nosignificant association with the increased risk of CHD in heterozygote comparison.