Molecular Genetics of Human Congenital Limb Malformations


Congenital limb malformations are observed in approximately 1 in 500 people making them one of the most frequent birth defects. Increasing numbers of genes are being identified that when functionally perturbed result in human limb abnormalities. The functions of many of these genes have been investigated in the two main models of limb development – the chick and the mouse. Limb defects are often part of complex syndromes that affect other structures in the body. This reflects the multiple roles that many genes play during embryonic development. Recently, an emerging theme is that the disruption of cis‐regulatory enhancers controlling expression of important developmental genes can lead to limb‐specific birth defects. Future research focusing on how the human limb develops will increase our understanding of the molecular and genetic basis of congenital defects.

Key Concepts:

  • Chick and mouse embryology has contributed to our knowledge of limb development and the basis of human limb defects.

  • Many key patterning genes found in mouse and chick research cause human limb defects when mutated.

  • Limb defects are often part of complex syndromes.

  • Defects which affect only the limb are often caused by disruption of cis‐regulatory enhancers.

  • Future work on how the human limb develops will further our understanding of congenital defects.

Keywords: embryo; limb development; congenital limb defect; human; hedgehog; patterning; signalling; genetic

Figure 1.

A comparison of upper limb development in chick, mouse and human. (a) The limb bud is first visible at day 3, day 9.5, and day 24, in the chick, mouse and human, respectively. This corresponds to Hamburger–Hamilton (HH) stage 19 in the chick and Carnegie stage 11 in the human. The hand plate is observed by day 4.5 (HH 26), day 12 and day 33 (Carnegie 14), and shortly after this, precartilage condensation begins (day 5 (HH 27), day 12.5 and day 37 (Carnegie 15/16)), giving rise to the skeletal digit primordia at day 4.5 (HH 32), day 14 and day 45, in the chick, mouse and human, respectively. Apoptosis of the interdigital spaces results in the sculpting of free digits by day 10 (HH 36), day 16 and day 53 (Carnegie 21). Digit identity is numbered. (b) A schematic representation of the period of expression of Shh, Fgf8, and Wnt7a. In both chick (green) and mouse (blue), Shh and Fgf8 are expressed for approximately 60 and 72 h, respectively. It is unknown for how long these molecules are expressed in human (red). ?, indicates a lack of human gene expression data. Wnt7a is expressed throughout these stages of limb development in all three species. (c) Developmental axes of the limb.

Figure 2.

Examples of human congenital limb defects. (a) Tetra‐amelia, the absence of limbs. (b) Acheiropodia, the absence of distal extremities. (c) Preaxial polydactyly, extra digits on the anterior side of the hand. (d) Postaxial polydactyly, extra digits on the posterior side of the hand. (e) Triphalangeal thumb, a thumb with three phalanges. (f) Mirrored polydactyly, a duplicated set of digits. (g) Syndactyly, fusion of the digits. (h) Hypodactyly, fewer than five digits. (i) Ectrodactyly, the absence of middle digits. See Table for genetic causes. Images are reproduced by permission from the National Human Genome Research Institute ( (images b–h) and from Wikipedia ( (images a and i). © Public domain.

Figure 3.

The initiation of limb bud development. HoxC genes expressed along the rostro‐caudal axis of the developing embryo restrict Tbx5 expression to the prospective forelimb region (Nishimoto et al., ). Tbx5 is upstream of Fgf10, and is involved in an epithelial‐mesenchymal transition that generates the limb bud progenitor cells from the coelomic epithelium. Fgf8 in the intermediate mesoderm induces the expression of Wnt2b in the lateral plate mesoderm, which is also upstream of Fgf10. Fgf10 switches on Wnt3a expression (in chick) in the apical ectodermal ridge (AER, thick orange line), which in turn induces Fgf8 in the AER. Wnt10a also plays a role in maintaining the expression of Fgf8 in the AER. Finally, AER‐FGF8 contributes to the expression of Fgf10 in the mesenchyme, thus completing a feedback loop and driving limb bud outgrowth.

Figure 4.

A schematic representation of the upstream enhancer regions of SHH. (a) The zone of polarising activity regulatory sequence (ZRS) lies approximately 800 kb upstream of the SHH coding sequence, located in intron 5 of the gene LMBR1. The ZRS, and other enhancers controlling the expression of SHH, are shown as red bars. A deletion of exon 4 (indicated) causes the condition acheiropodia, while duplication of the ZRS sequence leads to formation of extra digits. (b) A magnified view of the 750–800 bp ZRS highlighting the point mutations that cause limb defects in human (black bars). The binding site positions of ETS1/GABPα (blue ovals) and ETV4/5 (green ovals) have also been indicated. Reproduced with permission from Anderson et al. (). © Elsevier.

Figure 5.

The maintenance of limb bud outgrowth. HAND2 and HoxD proteins induce Shh expression in the polarising region (green), which is later maintained by Wnt7a. Shh induces expression of the BMP antagonist, Gremlin1. This maintains expression of FGFs (principally Fgf4) in the apical ectodermal ridge (AER, thick orange line), which would otherwise be inhibited by BMPs. Ridge‐derived FGFs maintain expression of Shh in the polarising region.



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Further Reading

Ferretti P, Copp A, Tickle C and Moore G (2006) The Limbs; Embryos, Genes and Birth Defects, 2nd edn, pp. 123–166. Chichester: John Wiley & Sons, Ltd.

Schoenwolf G, Bleyl S, Brauer P and Francis‐West P (2009) Development of the Limbs; Larsen's Human Embryology, 4th edn, pp. 617–644. Philadelphia: Churchill Livingstone Elsevier.

Zuniga A, Zeller R and Probst S (2012) The molecular basis of human congenital limb malformations. Wiley Interdisciplinary Reviews: Developmental Biology 1(6): 803–822.

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Pickering, Joseph, and Towers, Matthew(Sep 2014) Molecular Genetics of Human Congenital Limb Malformations. In: eLS. John Wiley & Sons Ltd, Chichester. [doi: 10.1002/9780470015902.a0025686]