| Polydactyly of thumb is one of the most common congenital deformities of human body,which is characterized by more fingers than normal.Thumb polydactyly is one of the most common polydactyly.According to the statistics of China's birth defect prevention and control report in 2012,about 30000 children will be born every year.The proportion of familial polydactyly is 4.8%,while the proportion of sporadic polydactyly is 95.2%.The proportion of axial(thumb side)and axial posterior polydactyly is 74.7% and 25.3%respectively.Thumb plays an important role in hand function,so thumb multi finger deformity will have a huge impact on hand function.According to the severity of the deformity,thumb polydactyly can be divided into different types.The light one will affect the appearance of the hand,while the heavy one will also affect the grasping function of the hand.The loss or reduction of hand function will have a huge impact on the individual's social life,and the appearance of hand deformity will also bring greater psychological burden to patients and their families.Thumb polydactyly can occur in the fetal stage,because the conventional prenatal diagnosis is mainly based on ultrasound,and the fetus is often clenched in the womb,which makes the diagnosis of polydactyly difficult.Most of the children's deformities are found after birth.In the early stage of childhood,patients with multiple fingers deformity often remove redundant fingers by surgical treatment.If they grasp the opportunity of operation,they can get better function of thumb reconstruction.At present,with the development of society,the treatment of thumb polydactyly is not only limited to the requirements of hand function,but also focused on reshaping the beautiful appearance of the hand,which also brings great challenges to surgeons.According to whether congenital polydactyly is accompanied by symptoms of other organs,it can be roughly divided into syndrome type and non syndrome type.The former is oftenaccompanied by symptoms of other organs of the body,the more common ones are: Greg cephalopolysyndactyly syndrome,Pallister hall syndrome and Ellis van Creveld syndrome.According to the anatomical structure of the multi fingers,it can be roughly divided into axial multi fingers,axial multi fingers and complex multi fingers.It has been proved that polydactyly is related to multiple gene mutations,including fbln1,HOXD13,lmbr1,FGFR2,GLI3 and shh.It was found by consulting the literature that the previous studies on polydactyly were mostly focused on genetic pedigree studies,and few cases of sporadic type were reported.At present,sporadic type of thumb polydactyly with non family inheritance is the most common in clinical work.Therefore,in order to further explore the mutation type,genetic characteristics and molecular formation of non hereditary sporadic type of thumb polydactyly(axial polydactyly)patients Mechanism,in this study,we mainly carried out the following aspects of work.Research content and resultsIn the first part,the clinical information of 20 Chinese thumb polydactyly patients was collected through the hand and foot surgery of the first hospital of Jilin University.All of the20 patients were non syndromic thumb polydactyly.Type of disease: sporadic,no family genetic history.Gender: 13 males and 7 females.Age distribution: June to 6 years old.Affected limbs: 10 patients with polydactyly of the left thumb and 10 patients with polydactyly of the right thumb.Wassel typing: type II 1,type III 2,type IV 8,type V 3,type VI 3,type VII 3.Team McKusick: ppd1 17,ppd2 3.Peripheral blood samples were collected from all patients,and DNA samples of patients were collected for whole exome sequencing(WES)analysis.The results showed that 668088 total heterotopia points were screened by whole exome sequencing,8312 predicted harmful mutation points were obtained by conservative(harmful)annotation for gene function and pathway annotation,and the genes of mutation points were queried and annotated in the related disease database,so as to obtain the information related to the mutation point and the disease through database comparison and literature search We were asked to determine the gene mutations of S4,S7 and S20 inthree patients: among them,S4 carries the heterozygous mutations of kiaa0586(nm_),p.s68t(dbSNP ID: rs147119902)and p.p772l(rs139493302).Patient S7 carries the heterozygous mutation p.m948i(nm_)in GLI3 gene.S20 of patients carries 2-BP deletion in EVC gene,which will lead to early termination of the translation of p.q470 fs and EVC protein.In order to evaluate the effect of missense mutation on protein level,protein conservation analysis was carried out in 7 species.The data showed that missense mutations in kiaa0586 and GLI3 affected highly conserved amino acids,indicating that mutation sites were crucial to protein function.After that,Sanger sequencing of these mutant genes confirmed that the mutation really existed in patients.The proteins encoded by these genes play an important role in the cilia and Shh(Sonic hedgehog)protein signaling pathway.Previous studies have found that if Shh protein signaling pathway appears abnormal or gene mutation during embryonic development,it will cause abnormal embryonic development,which plays a key role in the formation of polydactyly.In the second part,through literature review,it was found that in vertebrates,the Hh signal was transmitted by GLI transcription factors(GLI1,GLI2,GLI3),in which GLI3 played a leading role in limb development.GLI3 protein is a transcription factor of sonic hedgehog gene signaling pathway.GLI3 is located at the top of primary cilia in a hedgehog dependent manner.The interaction of Shh and GLI3 in the whole process of limb bud development ultimately determines the formation of five fingers and their corresponding morphological characteristics.It has been reported that the formation of thumb requires HOXA13 to directly regulate the transcription of GLI3.Shh / GLI3 signal system and Hoxd transcription factor interact with each other continuously during the development of limb to make thumb formation.GLI3 and its related factors play an important role in thumb formation.In the previous experiment,we found a new heterozygous mutation p.m948i(nm_)in GLI3 gene by exome sequencing.Through bioinformatics analysis,we confirmed that the mutation of the gene has not been reported in literature.Through literature review,we thought that the mutation may be a mutation gene that forms thumb polydactyly.In orderto further clarify the biological significance of the new mutation of GLI3 gene,we constructed The p.m948 i point mutation vector of GLI3 was constructed and transfected into mouse embryonic fibroblasts to further study the effect and potential mechanism of the mutant gene on mouse embryonic fibroblasts(MEFs).First,the primary mouse embryonic fibroblasts were isolated and cultured,and the cell purity was confirmed by vimentin and ?-SMA immunofluorescence staining.Then,the GLI3 mutant plasmid was constructed by molecular cloning technology and transfected into mouse embryonic fibroblasts.The RNA of GLI3 mutant cells was extracted and analyzed by transcriptome sequencing.The sequence was analyzed by RNA sequencing and bioinformatics.The results showed that there were2452 mouse embryonic fibroblasts transfected with GLI3 mutant plasmid compared with wild-type mouse embryonic fibroblasts The results of differential expression analysis showed that cbx3 gene was significantly up-regulated.The third part,the function of cbx3 in mouse embryonic fibroblasts.Construction of cbx3 overexpression plasmid pcdna-cbx3,design of siRNA for gene silencing,respectively transfection of mouse embryonic fibroblasts,CCK-8 reagent to detect the proliferation of mouse embryonic fibroblasts,Transwell to detect the change of cell invasion ability.The results showed that after overexpression of cbx3 gene,the value-added rate of the cells was significantly higher than that of the silence group and the control group,and the difference was extremely significant.However,after the silence of cbx3,the proliferation ability of the cells was lower than that of the control group,and there was significant difference.After overexpression of cbx3 gene,the number of cell invasion was significantly more than that of the control group and the silence group.According to the above results,cbx3 gene can promote the proliferation and invasion of mouse embryonic fibroblast.Conclusion:Through the research of this project,we found a new mutation gene which may cause the thumb polydactyly.The function of GLI3 gene after mutation was verified,which suggested that it might be related to up regulating cbx3 and then affecting the downstreampathway.This study further clarified the potential function of GLI3 gene in limb development,established a new relationship between gene mutation and polydactyly,and preliminarily clarified the possible signal pathway,which laid a foundation for further study on the etiology of polydactyly. |
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