Molecular Genetic Studies Of Three Congenital Limb Malformations

Molecular Genetic Studies of Three Congenital Limb MalformationsCongenital limb malformations occur in 1 in 500 to 1 in 1000 human live births, including the alterations of the number,length and anatomic morphology of the digits.The major causes are the abnormal genetic programming and the infaust factors during the development,such as the poor uterine environment.The identification of the responsible gene mutations is important for genetic counseling and the understanding of the mechanisms controlling limb development.In this paper,we performed the molecular genetic studies in three pedigrees with different kinds of congenital limb malformations including brachydactyly type B1(BDB1) combinds syndactyly typel(SD1),synpolydactyly (SPD) and split-hand/split-foot malformation(SHFM).PartⅠ:Detection and Functional Analysis of a Pathogenic MutationAssociated with a Novel complex malformation with BDB1 and SD1Brachydactyly(BD) refers to shortening of the fingers or toes due to hypoplasia or aplasia of metacarpals(metatarsals) or phalanges,which can occur as an isolated trait or in association with other malformations.Isolated BD has been categorized to five types, among which the brachydactyly type B(BDB) can be further divided into two subtypes, which are BDB1(MIM 113000) and BDB2(MIM 611377).The prominent features of BDB1 are hypoplasia or absence of the distal phalanges and nails,hypoplastic middle phalanges and symphalangism.Nonsense and frameshift mutations,either proximal or distal with respect to the tyrosine kinase domain in ROR2,have been identified to be the main cause of this disease.Syndactyly refers to the fusion of soft tissues of fingers and/or toes with or without the fusion of bones.It can be divided into five types,and the most common one is syndactyly typeⅠ(SD1,MIM 185900).SD1 has been linked to chromosome 2q34-36,manifested as complete or partial webbing between the 3^rd and 4^th fingers associated with the 2^nd and 3^rd toes.It can occur on both sides of the limb,and can also appear asymmetry.Till now,no genes have been reported to be associated with it. We identified a three-generation Chinese Han family with complex phenotypes of BDB1 and SD1.Two-point linkage analysis was performed and a maximal LOD score of 2.71 was obtained for the markers D9S1815 and D9S1841,showing the great possibility of linkage between the positions and this disease.We then directly sequenced the 8^th and 9^th exons of ROR2 gene and found a 1-bp deletion,c.2243delC,in exon 9,which leads to a frame shift mutation at Trp749 and predicts a truncated protein with 24 novel amino acids before the first stop codon.This mutation was further confirmed by restriction analysis among all the family members.During the functional analysis of this novel mutation,we constructed the ROR2^WT/ ROR2^W749fsX24-GFP-fusion expression vector to observe the subcellular location of these two proteins.With the use of HeLa cells as targeting transfected cells,we noticed that ROR2^WT was located in the cytoplasma membrane while ROR2^W749fsX24 was scattered throughout the cytoplasm.When we use U2OS cells,we found that the filopodia of ROR2 ^W749fsX24 were much more than those of ROR2^W749X.To investigate the interaction between ROR2^WT/ROR2^W749fsX24and 14-3-3β,we performed both yeast two-hybrid assay and mammalian two-hybrid assay by using the cytoplasmic part of ROR2^WT/ ROR2 ^W749fsX24and the full length of 14-3-3β.Immunoprecipitation was also used as a test in vitro.No obvious clues of interactions between the two proteins were found.In conclusion,we report a new limb malformation of BDB1 associated with SDL The disease gene in this family was mapped to 9q22 by two-point linkage analysis and a novel mutation,c.2243delC,was identified by directly sequencing of ROR2 gene,which is the first report of this mutation internationally.Further functional analysis reveals that there's obvious differences in the subcellular location between ROR2^WT and ROR2^W749fsX24,and filopodia formation between ROR2^W749X and ROR2^W749fsX24,which give us the inspiration that ROR2^WT,ROR2^W749fsX and ROR2^W749fsX24may play different roles in cells or even during the limb development.Further study should be done to confirm this. PartⅡ:Mutation Identifications of HOXD13 gene in Two ChineseFamilies with SynpolydactylyThe HOX genes encode a highly conservative family of transcriptional factors,which play a fundamental role in embryonic morphogenesis.In human,as in most vertebrates, there are 39 HOX genes organized into four clusters named HOXA through HOXD,which are believed to have arisen from a single ancestral cluster by duplication and divergence. The four clusters are distributed on different chromosomes including 7p15,17p21,12q13 and 2q31.HOXD 13 gene is located at the 5' end of HOXD cluster,containing two coding exons and encoding 335 amino acids.There is an imperfect GCN(N=A or C or G of T) triplet repeats in exon 1,encoding a 15-residue polyalanine tract.The expansion of this repeat,resulting in an additional 7-14 alanine residues,would lead to synpolydactyly(SPD, MIM 186000).SPD is a rare,dominantly- inherited limb malformation,which belongs to Syndactyly typeⅡ.Typically,patients have 3^th/4^th fingers and 4^th/5^th toes syndactyly,with partial or complete digit duplications of the digits in the syndactylous web.All patients, with or without polydactyly,will have syndactyly.The subject of this study is two SPD families with incomplete penetrance and variable expressivity as the common traits.One affected individual in family 1 has the rare phenotype of pre-axial polydactyly in hands and post-axial polydactyly in feet.This is the first report of SPD associated with pre-axial polydactyly in the world so far.The affected individuals in family 2 also have the phenotype of BDD and BDE.This is also a rare case of SPD.We found a 7-alanin expansion in the patients in family 1 and a 9-alanin expansion in family 2 using T-A cloning and sequencing of HOXD13 gene.Meanwhile,the exact insertion site and the specific constitution of the bases in additional alanine residues were revealed,which may provide strong theoretical knowledge to the understanding of the mechanism of SPD. PartⅢ:Analysis of Genettic Loci and Pathogenic MutationsAssociated with EctrodactylyEctrodactyly,also known as split-hand/split-foot malformation(SHFM),is a congenital autopod malformation characterized by cleft of the hands and/or feet due to the absence of the central rays.Typical cases may be the lobster-claw variety(absence of central rays) or monodactyly type(deficiency of radial rays with no cleft).It may occur as an isolated entity or as a part of a syndrome.SHFM is a disease of great clinical heterogeneity.Till now,six genetic loci have been identified,including SHFM1(MIM 183600),SHFM2(MIM 313350),SHFM3(MIM 600095),SHFM4(MIM 605289), SHFM5(MIM 606708) and a new locus reported in 2006,which are on human chromosome regions of 7q21,Xq26,10q24,3q27 2q31 and 8q21.11-q22.3,respectively. Among them,only two pathogenic mutations have been identified,which are large-scale DNA duplications in SHFM3 locus and point mutations in TP63 gene in SHFM4 locus.According to the prior study,we chose DLX5,DLX6 and the adjacent region of chromosome 7q21 as the major screening region.The range of the sequencing started from the 5' end of DLX5 to about 20kb upstream of DLX6.hi total,20 variations have been identified,14 of which are known SNPs and the others are changes unreported in introns. We also selected some highly conserved elements in this region to perform Real-time PCR considering the possibility of copy number variations in this region.No obvious pathogenic mutations have been found.Associated with the distribution of SNPs identified in sequencing and Real-time PCR,we deduce that if deletions are the main reason for this disease,they would be present in chr7:96,469,328-96,474,996,where we have not detected any SNPs.Array CGH or TAR(transformation-associated recombination) cloning should be performed to confirm this conclusion in the future.