Preaxial polydactyly: a model for defective long-range regulation in congenital abnormalities

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Point mutations in the long-range, limb-specific regulatory element of the SHH gene are responsible for the human limb abnormality called preaxial polydactyly (PPD). Disruptions of regulatory elements in developmental genes are a small but increasingly significant class of mutations responsible for congenital defects. Identifying regulatory elements that might reside hundreds of kilobases from their relevant genes is difficult but rendered possible by the emerging field of comparative genomics. Genetic analysis of PPD highlights the notion that regulatory mutations might generate phenotypes distinct from any of those identified for coding region mutations.

Introduction

The exquisite pattern of skeletal elements that compose our fingers and toes is a ready target for defects that can highjack developmental mechanisms and lead to congenital abnormalities. Congenital abnormalities of the limbs are easily ascertained and, in many cases, are not associated with other more adverse defects, thus leading to the clinical description of a number of different limb-dysmorphologies. One of the most frequently observed human limb malformations is preaxial polydactyly (PPD) — including PPD Type 2 and triphalangeal thumb-polysyndactyly syndrome (TPTPS) — which was mapped to human chromosome 7q36 [1, 2, 3, 4, 5]. Patients with PPD have extra digits on the sides of their thumb or great toe (Figure 1). However, the severity is highly variable, manifesting as mild triphalangeal thumb to the more severe duplication of digits and tibial aplasia. Three mutant mouse strains with limb abnormalities similar to PPD (Figure 1) map to proximal mouse chromosome 5, and are now known to be the counterpart to the human condition [6]. The mutations, Sasquatch (Ssq) [7], Hemimelic extratoe (Hx) [8] and M100081 [9], all result in preaxial supernumerary digits.

The initial clue to the molecular basis of PPD was inferred from studies in these mouse mutants. Normally, the signalling molecule Sonic Hedgehog (SHH) is expressed in the mesenchyme along the posterior margin of the limb in a region called the zone of polarising activity (ZPA) (Figure 1). In the mutants, Shh was expressed in an additional, ectopic domain [7, 10] along the opposite anterior margin of the mutant limb buds. The polydactyly phenotype was strikingly similar to limb buds produced in classic embryological studies first performed forty years ago. In the 1960s, Saunders and Gasseling [11] grafted the ZPA of early embryonic chick wing buds into the anterior margin of similarly staged buds and they produced supernumerary digits that were induced in a mirror image configuration. More recent studies [12] show that SHH, on its own, accounts for the digit duplication activity. Hence, the ectopic expression of Shh is a crucial step in the advent of PPD.

Here, we discuss the regulation of Shh, and the role played by mutations that redirect expression. We attempt to put these into the context of developmental gene structural organization and consider other mutations that, similarly, might disrupt developmental expression.

Section snippets

Comparative genomics and identification of regulatory elements

The Ssq mouse mutation manifests as preaxial extra digits as a result of the integration of a transgene [7]. Using the transgene sequence as the molecular landmark, the insertion site was located to a region physically linked to Shh but residing within an intron of a gene called Lmbr1, which is 1 Mb away [6]. To distinguish between disruption of a Shh regulator or an affect on Lmbr1, a genetic cistrans test was designed that convincingly showed that the Ssq mutation — and, by inference, PPD —

Developmental regulatory gene organization

Developmental regulatory genes, including Shh, are expressed in diverse embryonic domains under precise temporal instructions. A complex network of regulatory elements might control these spatiotemporal aspects of gene expression [26, 27]. Identification of the regulatory network components is obscured by the complexity of gene structure and the lack of obvious structural confines within developmental genes. Again, comparative genomics across several vertebrate genomes is helping to highlight

Nature of mutations in developmental genes

Having identified the functional ZRS, a search for mutations was initiated [24••]. Subsequently, point mutations were identified (Figure 2) within the ZRS of patients from three different PPD families. In addition, the two mouse polydactyly mutations, Hx [24••] and M100081 [9], have point mutations within the ZRS. PPD is, therefore, the result of subtle structural changes that activate mis-expression at an ectopic embryonic site (Figure 1). This phenotype is conspicuously different from null

Conclusions

These are the early days of the post-genome era, in which the long range organization of the genome is just beginning to be understood. In addition, the participation of long-range regulatory elements in congenital defects is now being realized. The most obvious cases of transcriptional mis-regulation are associated with chromosomal rearrangements. The more daunting task is the identification of small deletions or mutations in single, cis-acting elements, as exemplified by Shh involvement in

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

Acknowledgements

We would like to thank Rob Watson and Alison Hill for critically reading the manuscript.

References (42)

  • T. Sagai et al.

    Phylogenetic conservation of a limb-specific, cis-acting regulator of Sonic hedgehog (Shh)

    Mamm Genome

    (2004)
  • I. Blanc et al.

    Unusual pattern of Sonic hedgehog expression in the polydactylous mouse mutant Hemimelic extra-toes

    Int J Dev Biol

    (2002)
  • J.W. Saunders et al.

    Ectodermal–mesenchymal interactions in the origin of limb symmetry

  • R.D. Riddle et al.

    Sonic hedgehog mediates the polarizing activity of the ZPA

    Cell

    (1993)
  • J.W. Thomas et al.

    Comparative analyses of multi-species sequences from targeted genomic regions

    Nature

    (2003)
  • K.A. Frazer et al.

    Cross-species sequence comparisons: a review of methods and available resources

    Genome Res

    (2003)
  • D. Boffelli et al.

    Comparative genomics at the vertebrate extremes

    Nat Rev Genet

    (2004)
  • Mouse Genome Sequencing Consortium: Initial sequencing and comparative analysis of the mouse genome. Nature 2002,...
  • International Chicken Genome Sequencing Consortium: Sequence and comparative analysis of the chicken genome provide...
  • S. Aparicio et al.

    Detecting conserved regulatory elements with the model genome of the Japanese puffer fish, Fugu rubripes

    Proc Natl Acad Sci USA

    (1995)
  • H. Marshall et al.

    A conserved retinoic acid response element required for early expression of the homeobox gene Hoxb-1

    Nature

    (1994)
  • Cited by (0)

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