Abstract

Split-hand/split-foot malformation (SHFM), also known as ectrodactyly, is a congenital limb malformation, characterized by a deep median cleft of the hand and/or foot due to the absence of the central rays. SHFM may occur as an isolated entity or as part of a syndrome. Both forms are frequently found in association with chromosomal rearrangements such as deletions or translocations. Detailed studies of a number of mouse models for ectrodactyly have revealed that a failure to maintain median apical ectodermal ridge (AER) signalling is the main pathogenic mechanism. A number of factors complicate the identification of the genetic defects underlying human ectrodactyly: the limited number of families linked to each SHFM locus, the large number of morphogens involved in limb development, the complex interactions between these morphogens, the involvement of modifier genes, and the presumed involvement of multiple genes or long-range regulatory elements in some cases of ectrodactyly. So far, the only mutations known to underlie SHFM in humans have been found in the TP63 gene. The identification of novel human and mouse mutations for ectrodactyly will enhance our understanding of AER functions and the pathogenesis of ectrodactyly.

CLINICAL FEATURES OF SHFM

SHFM is a limb malformation involving the central rays of the autopod (hand/foot). Although some authors use ectrodactyly to denote any absence deformity of the distal limbs and reserve SHFM for the typical malformation, others use it synonymously with SHFM. We shall here use the terms interchangeably. SHFM may present with syndactyly, median clefts of the hands and feet, and aplasia and/or hypoplasia of the phalanges, metacarpals and metatarsals. In severe cases, the hands and feet have a lobster claw-like appearance. However, the severity of SHFM is highly variable (Fig. 1A). In mildly affected patients, SHFM may be limited to syndactyly and several instances of non-penetrance have been documented. Clinical variability not only exists between patients, but also between limbs of a single individual (Fig. 1A).

A large number of human gene defects can cause SHFM. The most common mode of inheritance is autosomal-dominant with reduced penetrance. Anticipation has been suggested in some families (1). Autosomal-recessive (2) and X-linked forms (3) occur more rarely and other cases of SHFM are caused by chromosomal deletions and duplications. In addition to the EEC syndrome and related disorders, many syndromes comprising SHFM have been reported, but their status as independent entities is often uncertain (4).

Ectrodactyly has been observed in other species, including dogs (5), cats (6), cows (7), chickens (8), frogs and toads (9), mice (10), rabbits (11), marmosets (12) and West Indian manatees (13). Several mouse mutants have ectrodactyly, and some of these mutations may also be involved in human ectrodactyly (Tables 1 and 2, and text below).

THE ROLE OF THE APICAL ECTODERMAL RIDGE IN LIMB DEVELOPMENT

The developmental patterning of the limbs results from gradients of signalling molecules in three spatial dimensions: proximo-distal (shoulder-finger direction), antero-posterior (thumb-little finger direction), and dorso-ventral (back-palm direction) (Fig. 1B). For correct development, three specialized cell clusters are of primary importance: the apical ectodermal ridge (AER), the progress zone (PZ), and the zone of polarizing activity (ZPA). These groups of cells produce signalling molecules that determine the fate of neighbouring cells by instructing them to remain undifferentiated, to proliferate, or to differentiate into a particular cell type.

Failure to initiate the AER leads to truncations of all skeletal elements of the limb (stylopod, zeugopod, autopod). This was first demonstrated by studies in which the AER was surgically excised (14,15). Since SHFM only affects the autopod, this probably reflects a failure to maintain the normal function of the AER (Fig. 1B). Genetic defects, as well as environmental factors, may cause ectrodactyly by interfering with AER function or maintenance. For instance, treatment of pregnant rats with retinoic acid induces limb malformations, including ectrodactyly, by inducing AER cell death (16). Other environmental factors that are known to induce ectrodactyly in rodents include cadmium, hydroxyurea, cytarabine, methotrexate, ethanol, caffeine, cocaine, valproic acid, acetazolamide and methoxyacetic acid (17).

The AER, which is located at the distal rim of the developing limb bud, is crucial for the formation and identity of digits. Signals from the AER allow the underlying mesenchymal cells of the PZ to maintain their proliferative activity (1822). A number of key players in the AER are known. These include fibroblast growth factors (FGFs), bone morphogenetic proteins (BMPs), WNT signalling molecules, and homeobox-containing proteins, such as MSX1 and MSX2 (Fig. 2). AER formation is induced by mesodermal signalling to the overlying ectoderm. Molecules involved in this process include FGF10, its receptor FGFR2 (23), and BMPs, which control the ectodermal expression of MSX transcription factor genes (24). FGFs fulfil two major functions. They maintain limb outgrowth by inducing proliferation of mesenchymal cells in the PZ and they maintain Sonic Hedgehog (SHH) expression in the ZPA. Important signalling molecules involved in the latter are BMPs, whose activity is modulated by SHH signalling from the ZPA, through Formin (FMN) and Gremlin (GRE) (2527). Several FGFs are restricted to the AER: FGF4, FGF8, FGF9 and FGF17. These AER-FGFs are crucial for limb development. In mice, simultaneous conditional ablation of Fgf4 and Fgf 8 is compatible with normal AER initiation, but defective gene expression in the underlying mesenchyme. The AER itself is maintained until embryonic day E11.25, when it begins to degenerate. The Fgf4 and Fgf 8 double knockouts have aplasia of both proximal and distal limb elements, which may be explained by a reduction of mesenchymal cells in the limb bud (19).

MOUSE MUTANTS WITH AER DISRUPTION

The Dlx5/6 knockout mouse

The tandem Dlx5 and Dlx6 genes are homologues of the Drosophila Distal-less (Dll) gene. They encode homeobox-containing proteins involved in head and limb development and both are expressed in the AER (28,29). Targeted disruption of either Dlx5 or Dlx6 does not cause a limb phenotype, whereas simultaneous disruption of these genes results in axial skeletal, craniofacial and inner ear defects. The limbs show a typical SHFM phenotype with absence of the central rays and a deep cleft between the remaining digits. Adjacent digits are often misshapen or fused to phalanges or metacarpals/metatarsals. These abnormalities have reduced penetrance in the forelimbs and complete penetrance in the hindlimbs (30,31).

In Dlx5/6 double mutant mice, a progressive loss of median AER cells starts prior to E11.5. This is accompanied by normal Shh expression, but severely reduced Fgf 8 levels. No increase in apoptosis is observed, suggesting a principal role of Dlx5 and Dlx6 in promoting proliferation in the median AER (30).

The Dactylaplasia mouse

Increased cell death in the AER underlies the absence of the central digits in the Dactylaplasia (Dac) genetic mouse mutant (31). Dac is a semi-dominant mutant which displays missing central digits in the fore- and hindlimbs of heterozygous animals and monodactyly in homozygous animals. In Dac/+ mice, only the central portion of the AER is degenerated at day E10.5, while Dac/Dac mice have a disruption of both the central and the anterior part of the AER (17,32). The loss of the AER leads to a reduction in cell proliferation in the sub-ridge mesenchyme at E11.5. This constitutes direct genetic evidence for the existence of an AER maintenance activity that is distinct from AER induction and differentiation.

Early speculations marked Fgf 8 as a strong positional candidate gene for the Dactylaplasia phenotype, but mutations could not be detected in this gene (17). In a subsequent study, two independent Dac mutations were fine-mapped and eventually cloned (33). One mutation is an insertion in the dactylin gene, which encodes a member of the F-box/WD40-repeat protein family. Owing to this mutation, transcripts are produced with a frameshift in the coding region of the fifth WD40 repeat, suggesting that disruption of the dactylin gene causes SHFM in mice. However, the other mutation is also an insertion, which neither affects the amount nor the integrity of the dactylin transcript. Therefore, it cannot be excluded that these Dac alleles disrupt long-range regulatory sequences from one or more other genes. The latter has been reported for SHH in another limb malformation, preaxial polydactyly (34).

The Tp63 knockout mouse

Mice deficient in functional p63 exhibit striking limb, craniofacial and ectodermal abnormalities, including the absence of skin and its derivatives (35,36). The forelimbs of Tp63 knockout mice are severely malformed, lacking the radius and the complete autopod, while the hindlimbs do not develop at all. As AER remnants are observed at E11, the origin of these limb defects is not a failure to initiate the AER. Therefore, p63 is thought to function as an AER maintenance factor, or more generally speaking, as a factor that preserves the proliferative activity in specialized ectodermal cells. In line with this, the absence of skin in these Tp63-deficient mice is caused by a lack of regenerative proliferation of the basal stem cells of the epidermis (36,37). The precise mechanisms and pathways by which p63 executes this function remain to be established. Although p63 is able to transactivate many of the same target genes as p53, it is not known whether these genes are of any relevance to normal limb development. A few AER-specific target genes of p63 were recently identified: Jag1, Jag2, and REDD1 (38,39). Interestingly, Jag2 mutations give rise to a limb phenotype in the syndactylism mouse (40,41). Even less is known about the factors that control expression of p63 and the various p63-isoforms that exist. Recent studies in zebrafish have established that one of the two alternative p63 promoters, the ΔN-p63 promoter, is a direct target of Bmp signalling during neural development (42). Perhaps Tp63 is also a target of BMP-signalling during control of the integrity of the AER in limb bud outgrowth.

Mice deficient in cholesterol-modified Shh

Shh is highly expressed in the ZPA. Shh signalling from the ZPA is the primary determinant of anterior–posterior polarity. The long-range signalling effects of Shh (over a few 100 microns) are brought about by the addition of a cholesterol group to an N-terminal cleavage product of Shh (43). Shh signalling is crucial in maintaining the integrity and function of the AER, which is underscored by the effects of mutations in genes from the Shh pathway, such as Formin, Twist and Gli3. Mutation of these genes gives rise to morphogenetic changes of the AER, predominantly resulting in abnormalities of the distal limb structures.

A mouse mutant with absence of central rays was recently reported (44). In this mutant, ectrodactyly is caused by a defect in the cholesterol-modified form of Shh. This reduced the long-range signalling abilities of Shh, leaving only short range signalling intact in the posterior portion of the developing limb. Such mice lack digits 2 and 3. Mice that lack Shh completely have only a single digit, possibly a first digit. This observation is consistent with a model in which the first digit develops independent of Shh, under the influence of Bmp2. Development of the posterior digits depends on Shh short-range signalling and the central rays require combined Shh and Bmp signalling (27,44).

The Lrp6 knockout mouse

The Wnt signalling pathway is implicated in a number of developmental processes, including dorso-ventral patterning of the limb. Activation of the pathway occurs through the binding of the Wnt ligand to its Frizzled receptor and either the Lrp5 or Lrp6 co-receptor. This triggers activation of Dishevelled, β-catenin and the LEF/TCF transcription factor (45).

Mice deficient in the Lrp6 (low-density lipoprotein receptor-related protein 6 ) co-receptor gene exhibit a number of developmental abnormalities. These include neural tube defects, microphthalmia and axial skeletal, urogenital and limb anomalies (46). The limb defects are variable: digit 5 is consistently absent, but some mice lack additional posterior digits. In addition, the radius may be missing and the ulna malformed. Interestingly, a strongly reduced Shh expression and a subsequent failure to maintain the AER precede aberrant development in Lrp6−/− limbs. A similar failure of AER maintenance is seen upon ectopic expression of Dkk1 (Dickkopf 1) in limb buds, leading to absent and fused digits. Dkk1 is an inhibitor of Wnt signalling, which acts through repression of Lrp6 (47). Since at least one of the Wnts (Wnt7a) can induce Shh expression in the posterior mesenchyme, the absence of Lrp6 may result in perturbed Wnt signalling downstream of the Frizzled receptor and hence aberrant induction of Shh expression. Shh is engaged in a feedback loop that activates Fgf4 in the AER (Fig. 2) (25). Thus, as in mice deficient in cholesterol-modified Shh, disturbance of the Shh-Fgf4 pathway is likely to underlie the ectrodactyly phenotype in Lrp6−/− mice.

HUMAN LOCI FOR ISOLATED SHFM

SHFM1 (chromosome 7q21; OMIM 183600)

A number of cases of either isolated SHFM or syndromic forms of ectrodactyly are associated with chromosomal aberrations involving the 7q21–q22 region (4857). Families with SHFM and sensorineural deafness also show linkage to this locus (58). Extensive analysis of the locations of the deletions has narrowed down the SHFM1 critical region to 1.5 Mb and six breakpoints have been found within a 700 kb region (59,60). Three candidate genes are located in the common deletion interval: DLX5, DLX6 and DSS1 (61).

The murine orthologue of DSS1 (Deleted in Split-hand/Split-foot malformation 1) is expressed in the branchial arches, genital tubercle and the developing limb bud. In the limb bud, expression is first detected throughout the mesenchyme, but not in the ectoderm. At later stages, expression is confined to the distal mesenchyme and ultimately to the interdigital mesenchyme only. Targeted deletion of both Dlx5 and Dlx6, but not either one alone, results in typical ectrodactyly in addition to inner ear and severe craniofacial defects (30). The combined homozygous Dlx5/6−/− mice thus recapitulate the dominantly inherited limb and ear canal defects in SHFM1 (54). Dss1 expression is normal in these mutant mice, suggesting that this gene does not contribute to the phenotype.

SHFM1 patients do not have mutations in the coding regions of DLX5, DLX6 or DSS1 (61). One possible explanation for this is that mutations of long distance transcriptional control elements cause SHFM1.

SHFM2 (chromosome Xq26; OMIM 313350)

Only a single SHFM family has been reported with X-chromosomal inheritance of isolated ectrodactyly (3). The patients from a large inbred Pakistani family exhibited mono- or bidactylous hands and typical ectrodactyly of the feet. Of a total of 36 individuals with the full expression, 33 were males and only three were female. A presumed X-chromosomal inheritance of the SHFM phenotype led to a more extensive clinical examination of the obligate heterozygous females. In about half of these heterozygous females, mild malformations of the hands and/or feet were observed (3). Cytogenetic studies ruled out the possibility of translocations or X-chromosomal rearrangements in this family. Linkage analysis then mapped the SHFM2 locus to chromosome region Xq26 (62). Possible candidate genes in the region include FGF13 and TONDU. The latter is homologous to the Drosophila wing-development gene vestigial (63).

SHFM3 (chromosome 10q24; OMIM 600095)

A third locus for isolated SHFM was mapped to a 2 cM region on chromosome 10q24–q25 (59,6466). The Dactylaplasia (Dac) mouse is considered a model for human SHFM3 as the human 10q24 region is homologous to the Dac locus on mouse chromosome 19. Yet no mutations in human Dactylin have been reported to date and other positional candidates should not be excluded. Expansion or contraction of a CGG trinucleotide repeat in HOX11 might explain the anticipation observed in some SHFM3 families. However, mutations in this gene could not be identified (1). A number of further positional candidates are located in 10q24, including FGF8, SUFU and BTRC, but no mutations have been found in the former two genes (1,67). BTRC is the human orthologue of Drosophila Slimb (for supernumerary limbs), an F-box/WD40 repeat protein. Interestingly, Slimb is a regulator of both the Shh and the Wnt signalling pathways (68).

SHFM4 (chromosome 3q27; OMIM 605289)

Up to now, the 3q27 locus is the only SHFM locus for which the causative gene has been identified. Mutations underlying SHFM4 have been found in the TP63 gene, which encodes a homologue of the tumour-suppressor p53. Despite this homology, p63 plays a key role in embryonic development, rather than in tumour suppression. The TP63 gene encodes at least six different isoforms (69). Some of these act as transcriptional activators like p53, whereas some other isoforms have repressive activity towards p53- and p63-driven gene expression. The dominant-negative isoform ΔN-p63α is specifically found in the AER and in the basal cell layer of a number of epithelia, including the epidermis of the skin (69). Mutations in the TP63 gene were first identified in patients with ectrodactyly-ectodermal dysplasia-clefting (EEC) syndrome (70). Later, TP63 mutations were reported in patients with isolated ectrodactyly (71,72) as well as in a number of other syndromes (see below). In total, seven TP63 mutations have been identified in non-syndromic SHFM families and isolated patients (71,72). Four of these are uniquely found in SHFM: two missense mutations in the DNA binding domain (K193E and K194E), a splice site mutation that predicts an amino acid insertion in the DNA binding domain, and two nonsense mutations that predict carboxy-terminal truncations of three and eight amino acids, respectively. The two other SHFM mutations, R280C and R280H, have also been encountered many times in EEC syndrome (Fig. 3) (72).

SHFM5 (chromosome 2q31; OMIM 606708)

Patients with deletions of chromosome region 2q24–q31 exhibit a number of abnormalities, including microcephaly, mental retardation, micrognatia, low-set ears, and limb abnormalities (73,74). Only some patients with such deletions present with typical ectrodactyly (7577). Recent studies have revealed that the 2q24.3–q31 region can be subdivided into three distinct loci for limb abnormalities. Patients with deletions of the 2q24.3 locus exhibit a typical wide gap between the first and second toes in combination with flexion deformity of the fingers. Deletions of 2q31 removing the HOXD10-HOXD13 and EVX2 genes, as well as mutations in HOXD13 cause synpolydactyly (SPD) (7880). Finally, Goodman et al. (80) have suggested that a locus for ectrodactyly is present between these two loci. This putative ectrodactyly locus is situated centromeric to the HOXD gene cluster, between the EVX2 gene and microsatellite marker D2S294 at 2q31. This region also contains the ‘digit enhancer’ that controls the expression levels of the distal HOXD genes (HOXD10–HOXD13) (81). Candidate genes located in the critical SHFM5 interval include DLX1 and DLX2, two homeobox genes expressed in the AER and the PZ. However, despite their role in limb development, heterozygous or homozygous knock-out mice of Dlx1, Dlx2, or both do not show any limb abnormalities at all (82). A comprehensive search for 2q31 microdeletions in additional patients with isolated SHFM should confirm the SHFM5 locus and facilitate its fine-mapping.

HUMAN ECTRODACTYLY SYNDROMES

Ectrodactyly is frequently seen in combination with other congenital anomalies. Such ectrodactyly syndromes may be caused by genetic factors or by exposure of the embryo to environmental factors. More than 50 syndromes and associations are distinguished in the London Dysmorphology Database (4). Several of these represent single case reports and neither their molecular basis nor their status as independent syndromes is known. A selection of recurrent pattern syndromes is listed in Table 1. Syndromes in which ectrodactyly is associated with other abnormalities can occur when two or more genes are affected by a chromosomal rearrangement. This explains the association of SHFM with other congenital anomalies in patients with deletions in 2q31 or 7q21. In contrast, syndromic ectrodactyly may also be the result of single gene defects. The most common and best known human SHFM syndrome is EEC syndrome, which is caused by missense mutations in the TP63 gene (70,72). EEC syndrome is characterized by ectrodactyly, ectodermal dysplasia and clefting of the lip/palate. A number of similar disorders that are also caused by TP63 mutations include limb-mammary syndrome (LMS) (83), acro-dermato-ungual-lacrimal-tooth (ADULT) syndrome (84,85), and Hay–Wells syndrome (86). Interestingly, a clear genotype–phenotype correlation is observed for p63 mutations. EEC syndrome mutations cluster in the DNA binding domain of p63, while Hay–Wells syndrome, which does not comprise limb defects, is caused by mutations in the SAM protein–protein interaction domain (86). The patterns of multiple abnormalities in patients with TP63 gene mutations allow the identification of specific domains within this gene that are most relevant for the limb phenotype (Fig. 3). Two mutations are of particular interest: R280C and R280H. Strikingly, the phenotypic consequences of these mutations appear to be consistent within families, giving rise to either the full-blown EEC syndrome or to non-syndromic SHFM. In the latter case, reduced penetrance of the R280 mutation has been observed. These observations suggest the involvement of genetic modifiers in these families (8789). Thus, careful evaluation of specific mutations, such as R280C and R280H, may pave the way to the identification of genetic modifiers. A paradigm for the role of modifier genes in ectrodactyly is offered by the Dac mouse, a model for human SHFM3. The Dactylaplasia phenotype only develops in mice homozygous for the mdac modifier gene, located at mouse chromosome 13. This region is homologous to the human chromosome 9q22.31 region. Interestingly, ectrodactyly occurs in at least four unrelated patients with autosomal recessive Robinow syndrome (90) (Balci et al., in preparation). These patients have homozygous loss-of-function mutations in the ROR2 gene, which maps to this putative 9q22 modifier locus for SHFM3 (91,92). Perhaps, mdac is an allele of the Ror2 gene, which has a crucial role in the chondrocyte lineage in late limb development (93). Other genes of interest on 9q22.31 include GAS1, a regulator of FGF8/FGF10 (94), and PTCH, a modulator of SHH signalling (44).

CONCLUSIONS

As observed in the mouse models for ectrodactyly, disturbances in AER signalling appear to be the major cause of SHFM. More specifically, ectrodactyly develops due to a failure to maintain median AER activity, either through increased cell death, or through reduced cell proliferation. This AER defect does not occur in the very earliest stages of limb development, since that would result in more severe limb malformations that are not limited to the autopod. The observation that defects in genes involved in antero-posterior signalling (Shh), dorso-ventral signalling (Lrp6, Dkk1), or proximo-distal signalling (Tp63, Dlx5/6) can all cause ectrodactyly demonstrates that signalling pathways acting in each of the three spatial dimensions are closely linked. For instance, the dorsalizing factor Wnt7a positively regulates posterior Shh expression, which in turn induces Fgf4 expression in the AER through the Shh–Fgf4 feedback loop (25). Although in humans TP63 is currently the only gene identified, a similar diverse array of SHFM genes is likely to exist. The existence of autosomal dominant, autosomal recessive, and X-linked inheritance and the extreme genetic heterogeneity of non-syndromic and syndromic ectrodactyly underlines this assumption. Thus, many more SHFM loci and genes remain to be discovered. The identification of the genetic defects underlying SHFM will aid in deciphering the molecular processes that govern normal AER function, as well as the pathogenesis of ectrodactyly and other limb malformations.

ACKNOWLEDGEMENTS

We thank Ronald Roepman for help with the preparation of the figures. The work in our laboratory is supported by a grant from the Dutch Foundation for Scientific Research (NWO), grant 903-42-190 (to H.v.B.).

*

To whom correspondence should be addressed. Tel: +31 243616696; Fax: +31 243540488; Email: h.vanbokhoven@antrg.umcn.nl

Figure 1. The ectrodactyly phenotype and underlying AER defect. (A) Clinical variability of ectrodactyly. (B) Normal development of the autopod (top) and ectrodactyly malformation (bottom). Ectrodactyly is caused by a failure to maintain median AER activity (red) in the developing limb bud (left), leading to the absence of the central rays (right). (Future) positions of digits 1–5 are indicated. AER, apical ectodermal ridge; PZ, progress zone; ZPA, zone of polarizing activity.

Figure 1. The ectrodactyly phenotype and underlying AER defect. (A) Clinical variability of ectrodactyly. (B) Normal development of the autopod (top) and ectrodactyly malformation (bottom). Ectrodactyly is caused by a failure to maintain median AER activity (red) in the developing limb bud (left), leading to the absence of the central rays (right). (Future) positions of digits 1–5 are indicated. AER, apical ectodermal ridge; PZ, progress zone; ZPA, zone of polarizing activity.

Figure 2. Signalling pathways in the developing limb bud. Failure to maintain the AER or defective AER signalling underlie SHFM. Correct signalling in the anterior and posterior apical ectodermal ridge (AER; light grey), but not in the median AER (yellow), may explain the relatively normal development of the anterior and posterior digits, respectively, while the median digits either develop very poorly or do not form at all. The positions of the AER (light grey and yellow), underlying progress zone (PZ; dark grey), and zone of polarizing activity (ZPA; brown) are indicated. Numbers 1–5 refer to the future positions of digits 1–5, respectively. Directions of the three-dimensional axes are indicated. Protein products from positional candidate genes for isolated SHFM are highlighted in red. Other molecules are shown in blue. Dorsally and ventrally expressed proteins are depicted in lighter and darker blue, respectively. Inhibitory and stimulatory effects are indicated with bars and arrows, respectively.

Figure 2. Signalling pathways in the developing limb bud. Failure to maintain the AER or defective AER signalling underlie SHFM. Correct signalling in the anterior and posterior apical ectodermal ridge (AER; light grey), but not in the median AER (yellow), may explain the relatively normal development of the anterior and posterior digits, respectively, while the median digits either develop very poorly or do not form at all. The positions of the AER (light grey and yellow), underlying progress zone (PZ; dark grey), and zone of polarizing activity (ZPA; brown) are indicated. Numbers 1–5 refer to the future positions of digits 1–5, respectively. Directions of the three-dimensional axes are indicated. Protein products from positional candidate genes for isolated SHFM are highlighted in red. Other molecules are shown in blue. Dorsally and ventrally expressed proteins are depicted in lighter and darker blue, respectively. Inhibitory and stimulatory effects are indicated with bars and arrows, respectively.

Figure 3. Mutations in p63. Two of at least six isoforms of the p63 protein are depicted: TA-p63α and ΔN-p63γ. Mutations causing syndromic SHFM are indicated in black (top), mutations causing isolated SHFM in red (top), and syndromic mutations without SHFM in blue (bottom). The latter all cause Hay–Wells syndrome. Mutations in black cause either EEC syndrome, ADULT syndrome, or LMS. The R280C and R280H mutations can either cause EEC syndrome or isolated ectrodactyly (see text for details). TA, transactivation domain; Iso, isomerization domain; SAM, sterile alpha motif; TID, transactivation inhibitory domain.

Figure 3. Mutations in p63. Two of at least six isoforms of the p63 protein are depicted: TA-p63α and ΔN-p63γ. Mutations causing syndromic SHFM are indicated in black (top), mutations causing isolated SHFM in red (top), and syndromic mutations without SHFM in blue (bottom). The latter all cause Hay–Wells syndrome. Mutations in black cause either EEC syndrome, ADULT syndrome, or LMS. The R280C and R280H mutations can either cause EEC syndrome or isolated ectrodactyly (see text for details). TA, transactivation domain; Iso, isomerization domain; SAM, sterile alpha motif; TID, transactivation inhibitory domain.

Table 1.

Human genetic disorders with ectrodactylya

Disorder Chromosomal location Candidate gene(s)b MIM 
Isolated SHFM    
SHFM1 7q21 DLX5, DLX6, DSS1 183600 
SHFM2 Xq26 FGF13, TONDU 313350 
SHFM3 10q24 Dactylin, SUFU, BTRC 600095 
SHFM4 3q27 TP63 605289 
SHFM5 2q31 DLX1, DLX2 606708 
EEC and related syndromesc    
Acro-dermato-ungual-lacrimal-tooth (ADULT) syndrome 3q27 TP63 103285 
Ectrodactyly-ectodermal dysplasia-cleft lip/palate (EEC) syndrome 3q27 TP63 604292 
Limb-mammary syndrome (LMS) 3q27 TP63 603543 
Other selected SHFM syndromesd    
Acro-renal-mandibular syndrome   200980 
Ectrodactyly-cleft palate (ECP) syndrome  TP63 129830 
Ectrodactyly-ectodermal dysplasia-macular dystrophy (EEM) syndrome   225280 
Ectrodactyly-fibular aplasia/hypoplasia (EFA) syndrome   113310 
Ectrodactyly-polydactyly   225290 
Ectrodactyly-sensorineural hearing loss 7q21 DLX5, DLX6, DSS1 605617 
Gollop–Wolfgang complex (GWC)/monodactylous ectrodactyly-split femur   228250 
Goltz syndrome/focal dermal hypoplasia (FDH) syndrome   305600 
Karsch–Neugebauer syndrome (KNS)   183800 
Limb/pelvis hypoplasia/aplasia syndrome   276820 
Microcephaly-microphthalmia-ectrodactyly-prognathism (MMEP) 6q21 SNX3 601349 
Myelinated nerve fibres-vitreoretinopathy-split hand/foot    
Pfeiffer–Tietze–Welte/sagittal craniostenosis-mental retardation-split hand    
Recessive Robinow syndrome 9q22 ROR2, GAS1, PTCHe 268310 
Split hand/foot malformation-long bone deficiency (SHFLD)   119100 
Split hand-urinary anomalies-spina bifida/diaphragm defects   183802 
Triphalangeal thumbs-brachyectrodactyly   190680 
Ulnar aplasia-lobster claw deformity of feet   314360 
Van Allen–Myhre/ectopia cordis-split hand/foot-skin defects    
Van den Ende/ectrodactyly-congenital heart disease-characteristic facies   601348 
Verloes-Koulischer/oral-acral syndrome   603446 
Disorder Chromosomal location Candidate gene(s)b MIM 
Isolated SHFM    
SHFM1 7q21 DLX5, DLX6, DSS1 183600 
SHFM2 Xq26 FGF13, TONDU 313350 
SHFM3 10q24 Dactylin, SUFU, BTRC 600095 
SHFM4 3q27 TP63 605289 
SHFM5 2q31 DLX1, DLX2 606708 
EEC and related syndromesc    
Acro-dermato-ungual-lacrimal-tooth (ADULT) syndrome 3q27 TP63 103285 
Ectrodactyly-ectodermal dysplasia-cleft lip/palate (EEC) syndrome 3q27 TP63 604292 
Limb-mammary syndrome (LMS) 3q27 TP63 603543 
Other selected SHFM syndromesd    
Acro-renal-mandibular syndrome   200980 
Ectrodactyly-cleft palate (ECP) syndrome  TP63 129830 
Ectrodactyly-ectodermal dysplasia-macular dystrophy (EEM) syndrome   225280 
Ectrodactyly-fibular aplasia/hypoplasia (EFA) syndrome   113310 
Ectrodactyly-polydactyly   225290 
Ectrodactyly-sensorineural hearing loss 7q21 DLX5, DLX6, DSS1 605617 
Gollop–Wolfgang complex (GWC)/monodactylous ectrodactyly-split femur   228250 
Goltz syndrome/focal dermal hypoplasia (FDH) syndrome   305600 
Karsch–Neugebauer syndrome (KNS)   183800 
Limb/pelvis hypoplasia/aplasia syndrome   276820 
Microcephaly-microphthalmia-ectrodactyly-prognathism (MMEP) 6q21 SNX3 601349 
Myelinated nerve fibres-vitreoretinopathy-split hand/foot    
Pfeiffer–Tietze–Welte/sagittal craniostenosis-mental retardation-split hand    
Recessive Robinow syndrome 9q22 ROR2, GAS1, PTCHe 268310 
Split hand/foot malformation-long bone deficiency (SHFLD)   119100 
Split hand-urinary anomalies-spina bifida/diaphragm defects   183802 
Triphalangeal thumbs-brachyectrodactyly   190680 
Ulnar aplasia-lobster claw deformity of feet   314360 
Van Allen–Myhre/ectopia cordis-split hand/foot-skin defects    
Van den Ende/ectrodactyly-congenital heart disease-characteristic facies   601348 
Verloes-Koulischer/oral-acral syndrome   603446 

aThis list is not complete.

bCausative genes in bold.

cHay–Wells syndrome is not listed because it does not comprise ectrodactyly.

dData from the London Dysmorphology Database (4).

eRecessive Robinow syndrome is caused by ROR2 mutations, but it is not clear whether these mutations are also responsible for SHFM in some of these patients.

Table 2.

Mouse mutants with AER defects

Mouse genotype Site of limb expression Limb phenotype AER defect References 
Dlx5−/−Dlx6−/− AER Ectrodactyly From day E11.5 onward median AER expression declines and the median AER becomes thinner (30
mdac/mdac Dac/+ Mesenchyme? Ectrodactyly Failure of median AER maintenance either due to increased AER cell death or reduced AER cell proliferation (17,31
mdac/mdac Dac/Dac Mesenchyme? Monodactyly Failure of median and anterior AER maintenance either due to increased AER cell death or reduced AER cell proliferation (17,31
Tp63−/− AER Forelimbs: monodactyly; hindlimbs: absent Failure of AER maintenance due to loss of regenerative activity in ectodermal cells (34,35
N-Shh/−a ZPA; posterior mesenchyme Ectrodactyly, lacking digits 2 and 3 Absence of Fgf4 expression in anterior AER due to loss of long-range Shh signalling, which normally activates Fgf4 (44
Lrp6−/− N/D Ectrodactyly (variable) Failure of AER maintenance due to loss of Fgf8 expression in median AER (46
Mouse genotype Site of limb expression Limb phenotype AER defect References 
Dlx5−/−Dlx6−/− AER Ectrodactyly From day E11.5 onward median AER expression declines and the median AER becomes thinner (30
mdac/mdac Dac/+ Mesenchyme? Ectrodactyly Failure of median AER maintenance either due to increased AER cell death or reduced AER cell proliferation (17,31
mdac/mdac Dac/Dac Mesenchyme? Monodactyly Failure of median and anterior AER maintenance either due to increased AER cell death or reduced AER cell proliferation (17,31
Tp63−/− AER Forelimbs: monodactyly; hindlimbs: absent Failure of AER maintenance due to loss of regenerative activity in ectodermal cells (34,35
N-Shh/−a ZPA; posterior mesenchyme Ectrodactyly, lacking digits 2 and 3 Absence of Fgf4 expression in anterior AER due to loss of long-range Shh signalling, which normally activates Fgf4 (44
Lrp6−/− N/D Ectrodactyly (variable) Failure of AER maintenance due to loss of Fgf8 expression in median AER (46

aIn contrast to wild-type Shh, N-Shh cannot be cholesterol-modified.

N/D, not determined.

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