Screening Of Pathogenic Gene Mutations And Analysis Of Transcriptional Activation Activity Of Mutated Proteins In Polydactyly Families

ObjectivePolydactyly is an autosomal dominant limb malformation with clinical manifestations of distal limb abnormalities characterized by incomplete finger separation and the presence of excess fingers in the phalanges.As the most common hand and foot deformity in infants and young children,SPD has a high incidence.It can only recover part of its apparent function through surgery,but cannot fully achieve normal physiological function.Although SPD can be treated by surgery,and surgical techniques and minimally invasive techniques are constantly updated and improved,surgical treatment still fails to restore normal physiological functions to some extent.This not only has a great impact on the physical and mental health of the patients and their families,but also brings a great economic burden to the families and the society.Therefore,it is of special and far-reaching significance to further study the molecular biology and pathogenesis of SPD and provide a new therapeutic direction in clinic.In the molecular field of the pathogenesis of SPD,previous studies were limited to mutations in HOXD13 proteins located in and around the C '-terminal homologous box domain or in or around the N-' terminal polyalanine chain.The phenotype of the disease has been shown to be associated with mutations in the homologous box domain or polyalanine chain of the HOXD13 gene.But our team obtain a China 2 generation SPD padigree,screening out a mutations in HOXD13 the pathogenic mutations(A22S)domain is not in the same box structure or its nearby,which are not anywhere near of alanine polymer chain or its nearby,is the new position of the outside of the previously known structure,function and the location of the mutations are so far has not beenidentified in addition to outside HOXD13 homeobox structure and functional domains of alanine polymer chain.MethodsThe family members were diagnosed with polydactyly(toe)deformity in the affiliated central hospital of shenyang medical college,and the malformation phenotype and detection met the clinical characteristics of polydactyly(toe).The family members who could be traced back were examined in detail and questioned about their family history,based on clinical manifestations and family history,and an informed consent was signed.DNA was extracted from peripheral blood of family members,and HOXD13 gene was used as a candidate pathogenic gene.Two exons and the exon/intron junction of the gene were amplified by PCR polymerase chain reaction.UCSC software was used to analyze the conserved sequence analysis of the amino acid sites corresponding to the base changes among different species,and I-TASSER automatic threading method was used to predict and analyze the effect of the primary structural change of the protein on the spatial conformation of the tertiary structure of the protein,thus affecting the functional change of the mutant protein.The wild-type plasmid pc DNA3.1-HA-HOXD13 wt was used as a template to amplify full-length site-directed mutation of the HOXD13 gene containing the mutation site by PCR,and the mutant plasmid was constructed.Western Blotting experiments HOXD13A22 S mutant and wild-type protein expressed in cells that will build HOXD13MUT?HOXD13WT? pc DNA3.1-HA? HOXD13Q50 Rplasmid by transient transfection,cultivate 48 h after transfection in HEK293 cells,then extract the total proteins in cells,vertical plate after SDS-PAGE,transfer film,selecting specific antibody immune signatures.Dual luciferase reporter gene test HOXD13(A22S)mutations affect its transcriptional activity ability: we construct HOXD13 MUT ?HOXD13WT?pc DNA3.1-HA?HOXD13Q50R contains fireflies and sea renal luciferase report gene,plasmid transfection respectively by means of instantaneous transfection the cultivation HEK293 cells after 24 h,then the activity of dual luciferase report genedetection.ResultsThe family patient(II1)was a 5-year-old female with congenital right foot with five toes and multiple fingers,and no ossiosis or webbed skin.The father(I1)had 4?5digits of the left foot.No similar clinical symptoms and other related foot diseases were found in other members of the family,and they were clinically diagnosed as multiple digital(toe)malformations.The phenotype and genotype of the SPD family were analyzed,and the results showed that the family inheritance mode was autosomal dominant.PCR was performed to amplify two exons and the exon/intron junction of the HOXD13 gene,and the result was sequenced.It was found that there was a heterozygous mutation site in the HOXD13 gene of the patient.T-missense mutation occurred in the amino acid at position 64 of the first exon,which was transformed from non-polar alanine to polar serine(p.A22S).Analysis by UCSC software: the sequence of amino acid sites corresponding to the change of this base was highly conserved among different species;By predicting software I-TASSER automatic threading method forecast analysis: The mutation affected the shape of HOXD13 protein,22 alanine of wild type was in alpha helix formation,while Ser 22 after mutation was involved in formation of beta-turn.The sequencing results of mutant eukaryotic expression vector pc DNA3.1-HA-HOXD13A22 Swere correct.Western Blot results showed that proteins of the three plasmids,HOXD13WT? HOXD13 MUTand HOXD13Q50Rwere all expressed in vitro.The results of the experimental data of double luciferase reporter gene were analyzed by T test between HOXD13WT?HOXD13MUT and HOXD13Q50 R groups.The results showed that the significance level was P>0.05,no significant difference,no statistical significance.Conclusions1.This family is an autosomal dominant genetic disease: the family with multiple fingers(toes)(SPD1)has a heterozygous mutation on exon 1 of HOXD13 gene of chromosome 2(c.64 gb >t).2.Bioinformatics software analysis indicated that the amino acid site of the mutation was highly conserved among different species,and the mutation changed the primary structure of the protein,thus affecting the spatial conformation of the protein,which may lead to changes in the function of the protein.3.HOXD13A22 Smutant had no effect on the transcriptional activation ability of Eph A7 promoter.