In a previous study we were able to identify mutations in HOXD13 in a dominantly inherited condition called synpolydactyly (OMIM 186000, SPD) (Muragaki et al. 1996). Synpolydactyly or syndactyly type II consists of syndactyly between the third and fourth fingers, with digit duplication in the syndactylous web. The feet are affected with syndactyly of the fourth and fifth toes and postaxial polydactyly. Incomplete penetrance and variability both between and within affected families is common. The mutations identified in SPD lead to an in frame expansion of a 15-residue polyalanine tract encoded by an imperfect trinucleotide repeat sequence by 7, 8, 9, 10 or, in one family, by 14 additional residues. In an extensive study analyzing a total of 20 families with 99 affected individuals, we were able to demonstrate (Goodman et al. 1997) a highly significant increase in the penetrance and severity of phenotype with increasing expansion size.

Synpolydactyly phenotype with mutation in HOXD13 (+10 Ala expansion) in heterozygous (left) and homozygous (hand and foot) individuals.

The triplet expansions observed in SPD represent a new class of triplet repeat mutations where a relatively short expansion of a homopolymeric tract results in disease. Subsequently, disease causing polyalanine tract expansions were reported in other human conditions, supporting the concept that such expansions may have a common pathomechanism. Similar repeat expansions coding for polyglutamine stretches are known to cause a variety of neurodegenerative disorders such as Huntington disease.

Inactivation of Hoxd13 in the mouse results in a mild phenotype that is strikingly different from that of humans with SPD. These mice merely have a shortening of digits. It was therefore speculated that the polyalanine tract expansions lead to a dominant negative effect, possibly by interfering with other (Hox-) genes. This concept was supported by publications by D. Duboules group. In these studies the authors had inactivated several paralogous Hox genes. Inactivation of Hoxd11, Hoxd12 and Hoxd13 together result in a synpolydactyly phenotype comparable to human SPD (Zakany and Duboule 1996). In addition, inactivation of one additional allele of Hoxa13 causes severe brachydactyly without polydactyly comparable to the human homozygous phenotype (Zakany et al. 1997). Thus, the limb expressed Hox genes of the A- and D-cluster control both the size and number of digits in a dose dependant manner. The polyalanine tract expansions are likely to work in a dominant negative fashion by interfering with other Hox genes.

A mouse mutant has been described that should serve as an excellent model for SPD. This mutant (spdh) exhibits synpolydactyly in a very similar fashion as in human SPD. Like in the human condition, the underlying mutation is an expansion of the N-terminal polyalanine tract, supporting the concept that his type of mutation causes the SPD phenotype (Johnson et al. 1998).

Skeletons of wt (right) and spdh mutant (left) fore limbs. Note brachydactyly and polydactyly in mutant.

In mouse the spdh mutation results in abnormal limb patterning, defective chondrocyte differentiation, missing joint development and in a drastic reduction in proliferation. Cell experiments revealed, that the spdh protein is transported into the nucleus like the wt, while constructs that have longer expansions of the alanine-repeat (+14 and +21) accumulate in the cytoplasm. Interestingly, the protein accumulations increased with rising number of alanines. This could be an explanation for the increase of penetrance and severity of the phenotype in SPD patients. There is strong evidence that phenotype variability of Hoxd13 mutation is influenced by additional modifiers. The dominant negative effect could be a result of altered behaviour of mutant Hoxd13 concerning DNA- binding, protein-protein-interactions and protein-misfolding. Our aim is to elucidate the pathomechanism of SPD variability by identifying the both modifiers and affected pathway and patterning mechanisms during embryonic development.