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doi: 10.1242/10.1242/dev.00396

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An I47L substitution in the HOXD13 homeodomain causes a novel human limb malformation by producing a selective loss of function

Giuliana Caronia1,*, Frances R. Goodman2,*, Carole M. E. McKeown3, Peter J. Scambler2 and Vincenzo Zappavigna1,4,{dagger}

1 Department of Molecular Biology and Functional Genomics, DIBIT-H San Raffaele, Via Olgettina 58, 20132 Milano, Italy
2 Molecular Medicine Unit, Institute of Child Health, 30 Guilford Street, London WC1N 1EH, UK
3 Clinical Genetics Unit, Birmingham Women's Hospital, Edgbaston, Birmingham B15 2TG, UK
4 Department of Animal Biology, University of Modena and Reggio Emilia, Via Campi 213/d, Modena 41100, Italy



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  		Fig. 1. Pedigree drawing of the family with brachydactyly-polydactyly. Black symbols represent affected individuals. The arrow indicates the proband. A horizontal line above a pedigree symbol indicates that the individual was examined clinically.

 


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  		Fig. 5. HOXD13(I47L) displays impaired DNA-binding ability in EMSAs. Probe sequences are shown below the gels, with potential Hox core binding motifs (5'-TAAT-3' or 5'-TTAT-3') on the forward strand and reverse strands over- and underlined respectively. Arrows indicate bands corresponding to HOX protein:DNA complexes. Asterisks indicate nonspecific bands produced by the reticulocyte lysate. (A) Binding of in vitro translated HOXD13 (lanes 3-6) and HOXD13(I47L) (lanes 7-10) to 32P-labelled HCR{alpha} (lane 1). (B) Binding of in vitro translated HOXD13 (lane 3) and HOXD13(I47L) (lane 4) to 32P-labelled HCR I (lane 1). In A and B, to identify HOX protein:DNA complexes, 200 ng of anti-HA antibody (Santa Cruz Biotechnology) was added to the binding reactions (A, lanes 6 and 10; B, lanes 5 and 6). In B, to assess binding specificity, a 300-fold molar excess of cold competitor HCR I was added to the binding reactions (lanes 7 and 8). (C) Binding of bacterially expressed, purified GST-HOXD13HD (lanes 2-6), GST-HOXD13HD(I47L) (lanes 7-11) and GST-HOXD13HD(IQN) (lanes 12-16) to 32P-labelled SelD13 (lane 1).

 


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  		Fig. 6. HOXD13HD(I47L) binds a subset of the sites recognised by HOXD13HD. The sequences of 100 oligonucleotides selected by each homeodomain in site selection experiments were aligned using the program CLUSTAL W. Those selected by HOXD13HD fell into two equal-sized groups, one with a TTAT core (site 1) and the other with a T(T/A)AC core (site 2), whereas those selected by HOXD13HD(I47L) formed a single group with a T(T/A)AC core. (A) Numbers of oligonucleotides containing sites 1 and 2 selected by each homeodomain. (B) Results displayed as consensograms (Wilson et al., 1993Go). (C) Binding of GST-HOXD13HD and GST-HOXD13HD(I47L) to site 1 (TTATTGG, lanes 1-7) and the two variants of site 2 (TTACGAG, lanes 8-14, and TAACGAG, lanes 15-21). The arrow indicates bands corresponding to HOX protein:DNA complexes.

 


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  		Fig. 2. The brachydactyly-polydactyly phenotype. (A,B) Hands of IV.8, showing small fifth distal phalanges and very short third to fifth metacarpals. (C,D) Hands of V.2, showing absent fifth distal phalanges and nails, small second and fourth distal phalanges, short third to fifth metacarpals, and hypoplasia of the styloid process of the ulna. (E,F) Hands of VI.2, showing very small fourth and fifth distal phalanges, absent or hypoplastic fourth and fifth nails, and short third to fifth metacarpals. (G-J) Hands of VI.7, showing partial fourth finger duplication (abnormally wide middle phalanges, with a bracket epiphysis on the left), small second, fourth and fifth distal phalanges, hypoplastic fifth nails, and very short third to fifth metacarpals. (K,L) Feet of V.2, showing absent fourth and fifth distal phalanges and nails, small second and third distal phalanges with hypoplastic nails, small middle phalanges, and short third to fifth metatarsals. (M,N) Feet of VI.2, showing absent fifth distal phalanges and nails, small first to fourth distal phalanges, hypoplastic second to fourth nails, small middle phalanges, short third to fifth metatarsals, and small middle cuneiforms with overgrowth of the second metatarsals.

 


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  		Fig. 3. The novel point mutation in HOXD13. (A,B) Electropherograms showing the wild-type (A) and mutant (B) alleles (subcloned) in V.7. (C) PCR amplification of genomic DNA using a forward primer specific for the mutant allele yielded a 100 bp product in seven affected family members but no product in an unaffected family member or a control individual.

 


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  		Fig. 4. Transcriptional activity of HOXD13, HOXD13(I47L) and HOXD13(IQN) at the HCR. P19 cells were transiently transfected with 10 µg of pTHCR reporter construct and the indicated quantities of expression constructs. Bars represent the mean luciferase activity±s.e.m. of at least six independent experiments. (A) Transcriptional activity mediated by increasing amounts of pSG-HOXD13, pSG-HOXD13(I47L) or pSG-HOXD13(IQN) assayed separately. (B) Transcriptional activity mediated by 1.0 µg pSG-HOXD13 in the presence of increasing amounts of pSG-HOXD13(I47L) or pSG-HOXD13(IQN), with the activity mediated by increasing amounts of pSG-HOXD13 shown for comparison. (C) Sequence of the HCR element, with potential Hox core binding motifs (5'-TAAT-3' or 5'-TTAT-3') on the forward strand and reverse strands over- and underlined respectively. The single 5'-TTAC-3' motif is highlighted in grey.

 


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  		Fig. 7. Phenotypes produced by misexpressing HOXD13, HOXD13(I47L) and HOXD13(IQN) in developing chick limbs. Frontal views of stage 32-34 hindlimbs stained with Alcian Blue, showing the injected right limb on the right and the uninjected opposite limb on the left. Ti, tibia; Fi, fibula; t, tibiale; f, fibulare. (A) Embryo injected with RCAS-HOXD13, showing normal phalanges but mild shortening of the proximal cartilages, especially the femur and tibia, resulting in an abnormal articulation between the fibula and fibulare (arrowhead). (B,C) Embryos injected with RCAS-HOXD13(I47L), showing normal phalanges (the embryo in C was the only one out of 38 with a rudimentary extra digit (asterisked), but marked shortening of the proximal cartilages, particularly the tibia, which has a rounded morphology, and extra cartilages in the zeugopod (arrowheads). (D,E) Embryos injected with RCAS-HOXD13(IQN), showing normal phalanges but marked shortening of the proximal cartilages, especially the femur and tibia (again resulting in an abnormal articulation between the fibula and fibulare; upper arrowheads), and also the metatarsals (lower arrowheads).

 


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  		Fig. 8. Overexpression of HOXD13 upregulates expression of chick EphA7, but overexpression of HOXD13(I47L) does not. Wholemounts of stage 28 chick embryos expressing HOXD13, HOXD13(I47L) or HOXD13(IQN) in the right hindlimb. Embryos were hybridised in situ with an antisense chick EphA7 probe. Chick EphA7 is expressed in the perichondrium of the digital condensations. This expression is increased in the right limbs overexpressing HOXD13 (B), but unaltered in limbs overexpressing HOXD13(I47L) (D) or HOXD13(IQN) (F). In A,C,E, the respective contralateral uninjected limbs are shown.

 

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© The Company of Biologists Ltd 2003