1. Functional Analysis Of The HOXD13 Mutants Associated With Syndactyly Type V And Synpolydactyly 2. Mutation Analysis Of EDA Gene In Chinese Families With X-linked Hypohidrotic Ectodermal Dysplasia

Paper 1:Functional Analysis of the HOXD13 Mutants Associated with Syndactyly Typeâ…¤and SynpolydactylyIncidence of congenital limb malformation among the newborn population is 1‰-2‰.It can occur as an independent entity or as a part of a syndrome.Non-syndromic syndactyly is the most common type of human limb malformation and is inherited in an autosomal dominant pattern.Non-syndromic syndactyly is classified into five types according to Temtamy and McKusick.Polyalanine expansion(PAE) mutation in N terminal of HOXD13 can cause typeâ…¡syndactyly,which is also called synpolydactyly (SPD,MIM186000).The typical clinical manifestations of SPD are soft tissue syndactyly between the third and fourth fingers,and between the fourth and fifth toes,with a supernumerary digit in the syndactylous web.p.Q317R point mutation located in the homeodomain of HOXD13 can result in typeâ…¤syndactyly(MIM 186300),which is characterized by fusion of the fourth and fifth metacarpal.In this study,we focused on the functional analysis of HOXD 13 p.Q317R mutant and polyalanine expansion mutant.Partâ… :Functional Analysis of the HOXD13 p.Q317R Mutant Associated with Syndactyly Typeâ…¤Hox gene encodes a family of transcription factors of fundamental importance for body patterning during embryonic development.Humans,like most vertebrates,have 39 HOX genes organized into four clusters(HOXA-HOXD).HOX gene has 2 exons,the 2^nd exon encodes highly conserve homeodomain(HD),which serves as DNA binding domain of HOX protein.HD is composed of 60 amino acids,the 47^th,50^th and 51^st of which play vital role in DNA binding affinity and specificity of HD.5'Hoxa and Hoxd genes (paralogue groups 9-13) play essential roles during embryonic limb development.The first two limb malformations shown to be caused by mutations in the human HOX genes were synpolydactyly and hand-foot-genital syndrome,which result from mutations in HOXD13 and HOXA13,respectively.Several types of mutations have been identified in HOXD13 gene to be associated with distal limb malformation.HOXD13 polyalanine tract expansion mutation can cause typical sypolydactyly(SPD),whereas other point mutations and deletions in HOXD13 often result in an atypical form of SPD.HOXD13 p.I314L mutation which substituted the highly conserve isoleucine at amino acid position 47 of HD with leucine was identified in a family with brachydactyly type E and mild SPD.In the previous study,we identified a novel point mutation of HOXD13 in a Chinese family with syndactyly typeâ…¤.The novel mutation,p.Q317R,substituted the highly conserve glutamine at amino acid position 50 of HD with a basic arginine.This mutation is supposed to vary DNA binding affinity and specificity of HOXD13.Few of downstream target genes of HOXD13 have yet been identified.At present, EphA7 is the only well-studied direct target of Hoxd13.EphA7 is one of type A receptors of ephrin.Eph receptor-ephrin signaling pathway plays multiple roles in embryonic development process,such as development of cardiovascular and nerve system,axon guidance,tissue differentiation.Recently,several studies indicated that Eph-ephrin pathway regulates embryonic limb bud development.In order to investigate the consequences of the HOXD13 p.Q317R substitution in transactivating the promoter of EPHA7,we performed luciferase reporter assay.First,we constructed the expression vectors of wild type HOXD13 and two mutants(p.Q317R and p.I314L),cloned 660bp EPHA7 promoter region(-580～+80) into luciferase reporter (pGL3-Basic).Next,we transfected the HOXD13 expression vector,luciferase reporter driven by EPHA7 promoter and renilla luciferase reporter into mouse C3H10T1/2 cell. Thirty six hours after transfection,relative luciferase activity was tested by luminometer. The results showed that both mutations impaired HOXD13's capacity to transactivate EPHA7 promoter,but a remarkable difference in the decrease of transactivation was observed,p.Q317R more severely impaired HOXD13's transactivity,remaining only 13% of the reporter activity compared with the wild-type counterpart,whereas p.I314L showed merely moderate impairment,with 63%of reporter activity remaining.In order to test the EphA7 promoter binding ability of two HOXD13 homeodomain mutants(p.Q317R and p.I314L),we conducted chromatin immunoprecipitation(ChIP) assay after transfection of the expression vectors of wild type HOXD13 or two mutants into mouse NIH3T3 cell.PCR were perfomed to test the enrichment of EphA7 promoter region containing HOXD13 binding site.The results indicated that both mutations impaired the DNA binding ability of HOXD13 at EPHA7 promoter.However,no significant difference was observed between the two mutants.Our results indicated that one possible pathogenic mechanism of p.Q317R mutation is the impairment of DNA binding ability of HOXD13 at EPHA7 promoter,which resulted in decreased transactivating capacity of HOXD13.Moreover,the difference in the impairment of transactivaing EPHA7 promoter may provide an explanation for the different phenotype caused by two HOXD13 homeodomain mutations.In order to further investigate the pathologic role of HOXD13 p.Q317R mutation during embryonic development and elucidate the molecular mechanism of typeâ…¤syndactyly,we constructed mouse Hoxd13 Q50R replacement gene targeting vector,which will contribute to future generation of mouse model carrying the corresponding mutation. Partâ…¡:Functional Analysis of the HOXD13 Polyalanine Tract Expansion Mutant Associated with SynpolydactylyPolyalanine tract expansion(PAE) is a novel type of mutation identified recently in 9 different types of human congenital malformations.The 1^st exon of HOXD13 encodes N-terminal region of the protein,which contains a 15-residue polyalanine in normal individuals.Expansion of this polyalanine tract to 22-29 alanine can cause synpolydactyly (SPD),a distal limb malformation characterized by syndactyly and polydactyly. Phenotype-genotype analysis showed that both the penetrance and severity of phenotype correlate with expansion size.The function of polyalanine and the molecular mechanism of polyalanine expansion mutation are currently unknown.Studies of 5'Hoxd gene (Hoxd11-13) knockout mouse and spdh(synpolydactyly homologue) mouse indicated that Hoxd13 polyalanine expansion mutant may have a dominant-negative effect on wild type Hoxd13 as well as other 5'Hoxd protein.Hox proteins bind DNA through homeodomain.During embryonic development, Hox proteins can interact with different cofactors and regulate downstream gene expression in a tissue specific manner.Recently,numerous studies showed that many Hox proteins can interact with Smad proteins which serve as intracellular signaling transducers for TGF-βpathway.Smad protein is composed of MH1 domain,MH2 domain and link region.MH1 domain is DNA binding domain,both MH1 and MH2 can interact with various cofactors.Previous study showed that Smad1,2,5 can interact with Hoxd13.BMPs belong to TGF-βligand superfamily,involve in genesis and development of many organs and tissues,including developing limb bud.In order to test whether polyalanine expansion mutation may impair HOXD13-SMAD1 interaction,we cloned HOXD13 polyalanine contraction and expansion mutants into BD vector(pGBKT7),which result in a fusion protein of HOXD13 and GAL4 DNA binding domain.We also cloned the coding region of MH2 domain from SMAD1 into AD vector(pGADT7),which gave a fusion protein of MH2 domain and GAL4 transactivation domain.We next tested the interactions between HOXD13 and MH2 using yeast-two hybridization system.The results showed that the polyalanine tract contraction mutants(-13A,-11A,-7A) can interact with MH2 domain just like the wild-type. However,polyalanine expansion mutants +9A and +14A failed to interact with MH2 domain in yeast whereas +7A seems to present a weaker interaction strength compared with wild-type.The results ofα-galactosidase quantitative assay showed that polyalanine expansion mutation did impair HOXD13-MH2 interaction,and also indicated the interaction strength correlate with expansion size.In order to narrowed down the HOXD13 peptide sequence necessary for HOXD13-SMAD1 interaction,we cloned 6 types of truncation mutants,the peptide length of which were 1-71,72-264,72-167,168-264,136-200,266-335,respectively.We subcloned these truncation mutants into BD vector and checked the interactions between these mutants and MH2 domain of SMAD1 using yeast-two hybridization.The results showed that the minimal peptide sequence necessary for HOXD13-SMAD1 interaction ranged from amino acid position 168 to 200,which did not contain polyalanine region. These results indicated that polyalanine expansion mutation may impair HOXD13-SMAD1 interaction by changing the conformation of HOXD13 protein.We next performed mammalian two-hybrid assay to verify the results obtained from yeast-two hybridization.We cloned full-length SMAD1 coding region into BD vector (pBIND) of the mammalian two-hybrid assay,which gave a fusion protein of SMAD1 and GAL4 DNA binding domain.We also cloned wild type and polyalanine expansion mutants into AD vector(pACT),which resulted in a fusion protein of HOXD13 and VP16 transactivation domain.After transfection of BD,AD and luciferase reporter vector (pG51uc) into HeLa cell,we examined the relative luciferase activity.The results showed similar tendency as yeast-two hybrid assay.To investigate whether the decreased interaction strength between HOXD13 polyalanine expansion mutants and SMAD proteins could impair the transcriptional activity of Smad downstream genes,we transfected polyalanine expansion mutation or wild type HOXD13 expression vectors into HepG2 together with pGli3ti-(SBE)₄,which is Smad3/4 responsive luciferase reporter vector.After TGF-β1 induction,we tested relative luciferase activity.The results showed that wild-type HOXD13 antagonized TGF-β1-inducible Smad3/4 transactivating capacity,whereas polyalanine expansion mutants showed weaker inhibition ability correlated with polyalanine size.Moreover,we also showed that the inhibition ability of wild type HOXD13 was not affected by polyalanine expansion mutants.In general,polyalanine expansion mutation disrupted the interaction between HOXD13 and SMAD,possibly due to the change in local protein conformation.The impaired interactions decrease the co-factor ability of HOXD13 toward SMAD proteins, which maybe result in uncontrolled expression of downstream genes.Our results provided molecular basis for the pathogenesis of the synpolydactyly caused by HOXD13 polyalanine expansion mutation. Paper 2:Mutation analysis of EDA gene in Chinese families with X-linked hypohidrotic ectodermal dysplasiaX-linked hypohidrotic ectodermal dysplasia(XLHED,MIM305100) is a recessive disease characterized by severe hypohidrosis,hypotrichosis and hypodontia.XLHED is the most common type of anhidrotic/hypohidrotic ectodermal dysplasia(EDA) and accounts for 95%of clinical EDA cases.It is mainly caused by mutations in the EDA(ectodysplasin A) gene.This gene contains 12 exons and undergoes extensive alternative splicing which produce various isoforms of signaling molecule of the tumor necrosis factor(TNF)-related ligand family.Of these variants,the longest EDA-A1 isoform encodes a protein of 391 amino acids.As a new member of the TNF ligand superfamily,EDA-A may play a role in the early epithelial-mesenchymal interaction that regulates ectodermal appendage formation.To date,more than 100 pathogenic mutations in the EDA gene have been described to be associated with XLHED,including single base-pair substitutions,deletions and insertions.At present,there is no effective treatment besides symptomatic therapy for XLHED.Molecular prenatal diagnosis is the only way to identify disease status of fetus in high risk pregnancy.In the present study,using PCR amplification and automatic sequencing of EDA gene coding region,we report five mutations identified in 6 Chinese families with XLHED, including three novel ones:p.M1T,p.L62P and p.G195E.The exon3 deletion mutation identified in a Urgur Chinese family with XLHED was further tested for deletion range using PCR amplification of the genomic fragment located in 5kb,7.5kb,10kb upstream and 10kb,20kb,25kb,30kb downstream of EDA exon 3.The results showed that deletion was ranging from 7.5kb upstream of exon3 to 30kb downstream of exon3.Using Gap-PCR, we the deletion size was determined to 36kb.Sequence alignment of Gap-PCR product and wild type sequence indicated that the deletion was caused by unequal homologous recombination between two LINE-1 elements located upstream and downstream of EDA exon3.Based on the results of mutation analysis,we performed prenatal diagnosis for 3 female carriers.The results showed that 2 fetuses were male and inherited normal allele of EDA gene,1 fetus was affected male inherited mutant allele.In general,our findings enrich our knowledge on the spectrum of mutation in the EDA gene.Furthermore,we first provide evidence for an unequal homologous recombination between two LINE-1 elements in EDA gene as pathogenic mechanism of XLHED.