Study Of Mutation Identification And Pathogenic Mechanism In Limb Malformations

IntroductionCongenital limb malformation(LM) is one of the most common birth defects in general population,it may affect the appearance and functional movement,even makes the individuals loss of the ability to work and selfcare.For the past few years,along with the completion of the human genome project(HGP),The identification of causatibe gene mutation about the human single gene inheritance diseases is continuously developed, and various pathopoiesis genes of LM had been identified gradually,the study data of human congenital LM not only could replenish the deficiency of animal and cell experiments,but also help to understand the process of embryonic development, Moreover,it could provide solid therotical basis for the study of human tissue engineering and medicine of gene therapy.So it is important to study the mutation identification and pathogenic mechanism in limb malformationsThe etiology can be subdivided into two general categories including environmental and genetic factors.The genetic factors refer to gene mutations and chromosomal aberrations.These LMs may present as an isolated trait or as part of a genetic syndrome.Many gene mutations and chromosomal aberrations may lead to abnormal morphogenesis thereby causing various developmental defects,such as brachydactyly(BD),ectrodactyly(ED),polydactyly(PD) and syndactyly(SD).Proximal symphalangism(SYM1,MIM 185800),which is characterized by brachydactyly and fusions of autopod skeletal elements and sometimes regarded as part of BDC(Brachydactyly type C,MIM 113100).SYM1 would be transmitted in antosomal dominant pattern and be caused by mutation of GDF5(growth/ differentiation factor 5,MIM 601146) or NOG(MIM 602991 ).Ectrodactyly,also known as split-hand/split-foot malformation(SHFM),is a congenital autopod malformation characterized by cleft of the hand and/or foot due to the absence of the central rays.It showed variable limb phenotype ranging from typical lobster-claw malformations to monodactyly.Up to now,five loci of SHFM have been identified,including SHFM1(MIM 183600),SHFM2(MIM 313350),SHFM3(MIM 600095),SHFM4(MIM 605289) and SHFM5(MIM 606708),at human chromosome regions 7q21 Xq26,10q24,.3q27 and 2q31,respectively.An about 0.5Mb large-scale DNA duplication at the SHFM3 locus and point mutations in TP63(MIM 603273) at the SHFM4 locus have been identified.Hand-foot-genital syndrome(HFGS,MIM 140000) is a malformation that affected the distal limbs and genitourinary.In the limbs,the abnormality is brachydactyly,delayed ossification,fusion,and shortening of the carpals and tarsals. Urinary abnormalities include ectopic ureteric orifices,vesicoureteric reflux and pelviureteric junction obstruction and can lead to chronic pyelonephritis,renal insufficiency,and renal transplant.Genital abnormalities include hypospadias in males and Mullerian duct fusion defects in females,the latter defects range from isolated longitudinal vaginal septum to double uterus with double cervix.HFGS would be transmitted in antosomal dominant pattern and be caused by mutation of Hox gene HOXA13(Homeobox A13,MIM 142959) at chromosome 7p15.In the present study,we recruited three Chinese families with different LMs (SYM1,SHFM and HFGS) for research.First of all,Haplotyping was performed to locate the chromosome position of pathopoiesis genes,then,we undertook DNA sequencing or quantitative PCR to identify the mutations,further,western blot, dual-luciferase reporter assay and chromatin immunoprecipitation assay were undertaken to analyze the preliminary functional characterization of the mutant genes Materials and methods1,SubjectFamily investigations were performed in the three LM Chinese families, including family medical history and pedigree,then medical examinations or X-ray examinations were performed and peripheral venous blood samples from all available family members were collected after informed consent.2,Haplotype analysis was performed to locate the causative genesUsing the UCSC Genome Browser,20 perfect microsatellite markers covering five SHFM loci were selected for haplotype analysis.The PCR products of the microsatellite markers were separated by electrophoresis on denaturing polyacrylamide gel and allele fragments were detected with routine silver staining,then on the basis of individual's genotype and kinship,the haplotypes were deduced according to the Mendel's law of inheritance.3,DNA sequencing of the candidate gene coding sequenceAfter determining the candidate gene by haplotyping,PCR was performed to amplify the exons of the gene,and then the amplicons were sequenced.4,Quantitative PCR was performed to detect genomic copy number variationReal time fluorescence quantitative PCR was performed to compare the affected individual's genomic copy number to that of control.5,Long range PCR coupled with DNA sequencing was performed to determine the break point of the duplicationAfter minimizing the extent of the breakpoint to about 2-4kb by quantitative PCR, the proximal and distal breakpoint positions were alnplified by long range PCR by telomeric forward primer R2-2F and centromeric reverse primer L10-5R,then the amplicons were sequenced. 6,Clone of the wild and mutant type eukaryotic expression vectorThe wild-type GDF5 or HOXA13 coding sequences were amplified by PCR and then were ligated with pLXSN vector or p3xFLAG-CMV vector,respectively,and transformed the competent cell.then the positive clones were verified by sequencing. The mutant expression vectors were cloned by site-directed mutagenesis using the wild-type vectors as templates.7,Western blot was performed to detect the dimerization and secretion of GDF5^1.373RCOS7 cells that stable expression wild type and mutant GDF5 were obtained by G418 selection,nonreducing SDS-PAGE and immunodetection was applied to detect dimerization and secretion of GDF5(1.373R) in the transgene cell lysate and medium supernate.8,Using dual luciferase reporter assay to investigate the consequences of the HOXA13^V375F in transactivating the promoter of EphA7We transfected the wild-type and mutant expression vector,luciferase reporter driven by EphA7 promoter and renilla luciferase reporter into mouse C3H10T1/2 cell, relative luciferase activity was tested by luminometer.9,ChIp was undertook to test the binding ability of HOXA13^V375F to downstream target gene promoterChromatin immunoprecipitation(ChIP) assay was performed using anti-Flag monoclonal antibody after transfection of the expression vectors of wild type or mutant HOXA13 into nlouse C3H10 T1/2 cell.Then quantitative PCR were perfomed to test the enrichment of downstream target gene promoter region containing HOXA13 binding site. Results1,SubjectWe performed clinical classification for the limb malformations families that had been recruited,and regarded them as SYM1.SHFM and HFGS,respectively.2,Haplotype analysisMicrosatellite markers,D20S787,D20S601,D20S909 and D20S914 at chromosome 20q11 were cosegregation with malformations of SYM1 individuals;The potential haplotype shared by all affected individuals of SHFM was detected in the microsatellite markers of LBX1T1,D10S1239,DactylinTAAT,Dactylin2 and Dactylin3 at chromosome 10q24.3,DNA sequence of the candidate gene coding sequenceOne heterozygosis missence mutation c.1118T＞G(p.L373R) in GDF5 in SYM1 family,and another heterozygosis missence mutation c.1123G＞T(p.V375F) in HOXA13 in HFGS family were identified,both mutations caused restriction fragment length polymorphism(RFLP),and were confirmed by restriction analysis to cosegregate with the LM phenotypes in all affected individuals,but not detected in all unaffected individuals of the families or 50 unrelated control individuals.4,Quantitative PCR was performed to detect genomic copy number variationResults of quantitative PCR suggested that all affected individuals had an extra copy in the rearrangement region including LBX1,BTRC,POLL,DPCD and FBXW4 at chromosome 10q24 locus.5,Long range PCR coupled with DNA sequence was performed to determine the break point of the duplicationThe proximal and distal breakpoint positions were refined by quantitative PCR and were amplified by long range PCR using forward primer R2-2F and reverse primer L10-5R with a 2000bp production in 3 affected individuals,but no production in the normal family members.Sequence and blat analysis of the chimeric sequence localized the telomeric and the centromeric breakpoint to position 103453895bp and 102965035bp,respectively.The duplication were tandem head-to-tail in orientation region,spanning 488,859bp in length,and encompassed the entire LBXI,BTRC,POLL, DPCD and FBXW4 genes,We concluded that the rearrangement may be NHEJ (nonhomologous end joining) or a simplified FoSTeS event(replication fork stalling and template switching,FoSTeS).6,Clone of the wild-type and mutant eukaryotic expression vectorThe wild-type and the mutant GDF5 or HOXA13 coding sequences were cloned into the pLXSN vector or p3xFLAG-CMV vector to drive expression of the fusion proteins tagged with myc or flag,respectively.The sequence and reading frame of clone vectors were verified by sequencing.7,Western blot was performed to detect the dimerization and secretion of GDF5^L373RSpecific band was detectable in the cell lysate and medium supernate of cells transfected with the mutant GDF5 as well as the wild-type GDF5 by western blot, indicating that the mutant proteins could be dimerizated and secreted out of cytoplasm as the wild-type one.8,Using dual luciferase reporter assay to investigate the consequences of the HOXA13^V3755F in transactivating the promoter of EphA7The dual luciferase reporter assay results showed that the mutant HOXA13^V375F impaired HOXA13's capacity to transactivate EphA 7 promoter,remaining 66.72%of the reporter activity compared to the wild-type counterpart.9,ChIP was undertook to test the binding ability of HOXA13^V375F to downstream target gene promoterChromatin immunoprecipitation(ChIP) assay results indicated that mutant HOXA13^V375F impaired the DNA binding ability of HOXA13 at EphA7,EphA6 and Bmp2 promoters or Sostdc13'UTR,remaining 65.98%.72.95%,42.89%and 68.03%of the binding ability compared to the wild-type counterpart,respectively.No significant change was observed in theBmp 7 and Sostdcl promoters.Conclusion(1) The pathogenic mutation for SYM1 patients was a novel missence mutation in GDF5,c.1118T＞G(p.L373R).The mutant protein GDF5^L373R could be dimerizated and secreted out of cytoplasm as the wild-type one.(2) The pathogenic mutation for SHFM patients was a tandem duplication spanning 488859 bp in length,and encompassed the entire LBX1,BTRC,POLL,DPCD and FBXW4 genes.(3) The pathogenic mutation for HFGS patients was a novel missence mutation in HOXA13,c.1123G＞T(p.V375F).The mutant protein HOXA13^V375F impaired HOXA13's capacity to transactivate EphA7 promoter,and damaged the DNA binding ability of HOXA13 at EphA 7,EphA6 and Bmp2 promoters or Sostdc13'UTR.