Human Molecular Genetics Advance Access published online on December 5, 2008
Human Molecular Genetics, doi:10.1093/hmg/ddn410
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A G220V substitution within the N-terminal transcription regulating domain of HOXD13 causes a variant synpolydactyly phenotype
1 University of Modena and Reggio Emilia, Department of Animal Biology, Via Campi 213/D, 41100 Modena, Italy 3 Laboratory of Skeletal Development and Joint Disorders, University of Leuven, Herestraat 49, 3000 Leuven, Belgium 4 Department of Musculoskeletal Sciences, Division of Orthopedics, University of Leuven, Heresstraat 49, 3000 Leuven, Belgium
* Address correspondence to: Vincenzo Zappavigna, Department of Animal Biology, University of Modena and Reggio Emilia, Via G. Campi 213/d, Modena 41100, Italy, Tel. 0039 059 2055537, Fax 0039 059 2055548, E-Mail: zappavigna.vincenzo{at}unimore.it
Received November 10, 2008; Revised December 3, 2008; Accepted December 3, 2008
The 5' members of the HoxD gene cluster (paralogous groups 9-13) are crucial for correct vertebrate limb patterning. Mutations in the HOXD13 gene have been found to cause synpolydactyly (SPD) and other limb malformations in human. We report the identification in a Greek family of variant form of SPD caused by a novel missense mutation that substitutes glycine for valine in position 220 (G220V) of HOXD13. This mutation represents the first substitution of an amino acid located outside of the HOXD13 homeodomain that causes autopodal limb malformations. We have characterized this mutation at the molecular level and found that the G220V substitution causes a significant impairment of the capacity of HOXD13 to bind DNA and regulate transcription. HOXD13(G220V) was found to be deficient in both activating and repressing transcription through HOXD13-responsive regulatory elements. In accordance with these results, a comparison of the activities of HOXD13 and HOXD13(G220V) in vivo using retrovirus-mediated misexpression in developing chick limbs, showed that the G220V mutation impairs the capacity of HOXD13 to perturb the development of proximal limb skeletal elements and to ectopically activate the transcription of the Hand2 target gene. We moreover show that the G220V mutation compromises the stability of the HOXD13 protein within cells and causes its partial accumulation in the cytosol in the form of subtle aggregates. Taken together, our results establish that the G220V substitution does not produce a dominant-negative effect or a gain-of-function, but represents a dominant loss-of-function mutation revealing haploinsufficiency of HOXD13 in human.
2 These authors contributed equally to this work.