Screening And Functional Study Of TFAP2B Mutations On Patent Ductus Arteriosus

Ductus arteriosus （DA） is a specific vascular structure between aorta and pulmonaryartery, which shunts the blood from the pulmonary artery into aorta for the development ofthe fetus and undergoes functional and anatomical closure soon after birth. The patentductus arteriosus （PDA） is defined as the condition that the continued patency in terminfants older than3months. The incidence of PDA in term infants is up to0.05%. If thePDA were not treated on time, it would result in the incidence of congestive heart failure,pulmonary artery hypertension and endocarditis. So, the pathogenesis of PDA alwaysabsorbs the scientists in the field of pediatric cardiology.The DA originates from the left dorsum part of the sixth pharyngeal arch.Histologically, the DA is derived from cardiac neural crest cells, so the division, growth,differention and migration of the neural crest cells have important effects on the normalclosure of the DA. TFAP2B is a transcription factor expressed in cardiac neural crest cells,so this gene should play an important role in the closure of the DA.It was reported that the recurrence increased up to5%in the siblings of patients withisolated PDA, so the genetic defect should be one of the factors of the PDA. Charsyndrome is the disease with PDA and deformity in the face and fingers. Masahiko Satodaidentified two missense point mutations in TFAP2B in the Char syndrome pedigrees andfound that the mutations have the dominant negative effect. And then other scientistsidentified base changes in the intron3and4, which have the haploinsufficiency effectresulted from the abnormal splicing in the mRNA.Most of the mutations on TFAP2B were identified from Char syndrome pedigrees. Inorder to study the effect of the TFAP2B mutations on isolated PDA and the geneticmechanism underlying the PDA, we were about to screen the TFAP2B of the patients with isolated PDA. And develop the plasmids contain these mutations based on the overlapPCR and then study the functions of these mutations to make analysis on the geneticmechanism of PDA caused by TFAP2B mutation.Part1Screen the TFAP2B gene of the patients suffered from PDAObjectiveThrough the gene screen on isolated PDA patients to identify novel mutations inTFAP2B and study the relation between the TFAP2B mutations and the PDA.Materials and MethodsThe patients were those with isolated PDA and the children without any congenitalheart disease were recruited for control. Peripheral blood samples were collected and theprimers were designed based on the gene sequence. All the exon sequences were amplifiedand sequenced. The sequence results were blasted in Genbank for analysis.ResultsThrouth the screen on the TFAP2B of patients with isolated PDA, two novelmutations c.435₄38delCCGG and c.601+5G>A were identified in two pedigrees, and thesamples from the control have none of the two mutations.ConclusionsTFAP2B c.435₄38delCCGG and c.601+5G>A is related to the pathogenesis of thePDA.Part2The development and of characterization of the TFAP2B mutationexpression vectorsObjectiveTo develop the TFAP2B c.435₄38delCCGG and c.601+5G>A mutation expressionvectors through the overlap PCR reactionMaterials and Methods Sequence analysis on mutations demonstrated that the c.435₄38delCCGG TFAP2Blose the CCGG at the3’ terminal of exon3and c.601+5G>A mutation cause the mRNAlosing the total exon3. We already have the TFAP2B wild-type expression plasmid, basedon which we would amplify the mutation DNA fragment through2turns of PCR by theproper primers. And then ligate the amplified DNA into the pcDNA3vectors through therestriction site in the primers.ResultsThe TFAP2B c.435₄38delCCGG and c.601+5G>A mutation expression vectorswere made successfully.ConclusionsThe overlap PCR reaction is suitable for the development of the expression plasmidscontaining the long deletion mutation.Part3Functional analysis on TFAP2B mutations in molecular and cellular levelObjectiveTo study the function of c.435₄38delCCGG and c.601+5G>A on TFAP2B gene andanalyze the genetic mechanism of the PDA caused by these two mutations.Materials and MethodsTransfect the U2-OS cells with the mutation expression plasmids andp3×AP2-luciferase and then detect the luciferase activity caused by the mutant TFAP2B;Transfect the U2-OS cells and study the expression and sub-cellular localization of themutant TFAP2B by Western blot and Immunofluorescence respectively; co-transfect thewild-type and mutant TFAP2B expression plasmids into U2-OS cells to study if themutant TFAP2B could inhibit the wild-type TFAP2B; develop the expression plasmidswhich fuse the Rluc1to c.435₄38delCCGG TFAP2B and Rluc2to TFAP2A respectively,and then determinate if the c.435₄38delCCGG TFAP2B could bind to the TFAP2Aaccording to the reconstituted renilla activity; finally, conduct the preliminary study of impact of the mutation on cell growth through the number difference of autophagosomesand lysosomes caused by the wild-type and mutant TFAP2B.ResultsBoth of the mutant TFAP2B lose the transactivation function and the Cited2was notable to enhance the function. Wild-tpye TFAP2B’s transactivation ability could beenhanced further by double dose of the Cited2, however the mutant TFAP2Bs did notrespond to the dose change made on the Cited2.Western blot and Immunofluorescence demonstrated that c.601+5G>A mutation wasnot able to expression any protein, yet the c.435₄38delCCGG mutation expressed thespecific protein and its localization inside the cell was very similar to the wild-typeTFAP2B.c.435₄38delCCGG mutant TFAP2B partly inhibited wild-type TFAP2B’stransactivation ability. Then the expression plasmids which fused the Rluc1toc.435₄38delCCGG TFAP2B and Rluc2to TFAP2A respectively were made successfully.The c.435₄38delCCGG TFAP2B can bind to the TFAP2A because the renilla activitywas reconstituted.The c.435₄38delCCGG TFAP2B and wild-type TFAP2B plasmids were transfectedinto U2-OS cells respectively; it demonstrated that the wild-type TFAP2B could increasethe number of autophagasomes and lysosomes, while the c.435₄38delCCGG TFAP2Bdid not.ConclusionsBoth of the c.435₄38delCCGG and c.601+5G>A mutation TFAP2B lose thetransactivation ability, and they might have haploinsufficiency or dominant negative effect.These two mutations down-regulate the function of the TFAP2B, the gene that has impactson the molecules that is related to the development of the ductus at different degree. Sothis should be the molecule mechanisms underlying PDA caused by those TFAP2Bmutation.