Where one or more additional features are involved, clefts are refered to as syndromic. Using a combination of gene targeting technology and traditional developmental techniques in both mouse and chick, significant progress has been made in the identification of numerous genes and gene pathways critical for craniofacial development. Nevertheless, some important findings have recently come from studies involving syndromic forms of the disorder.
The study of these syndromic genes and their molecular pathways will provide a useful and informative route with which to gain a better understanding of human craniofacial pathology.
Disturbance of this tightly controlled cascade can result in a facial cleft where the facial primordia ultimately fail to meet and fuse or form the appropriate structures. Collectively, craniofacial abnormalities are among the most common features of all birth defects. Patients will undergo multiple rounds of surgical repair starting in the first year of life and may continue until 18 or 20 years old.
Frequently, extensive dental and orthodontic treatment, speech and hearing therapy may be required as well as referral for psychotherapy and genetic counselling. It has been reported that CLP occurs more frequently in males, while the sex bias is reversed for CP, which is more common in females 2. CP occurring alone is therefore considered to be etiologically distinct from CLP.
The remaining syndromic cases have additional characteristic features that can be subdivided into categories of chromosomal abnormalities, recognizable Mendelian single gene syndromes, teratogenic effects and various unknown syndromes. Despite this, familial inheritance is complex with simple Mendelian inheritance considered uncommon. As a general model, it is thought that both genes and environmental factors, acting either independently or in combination, are responsible for facial clefting.
While numerous non-genetic risk factors have been identified such as use of anti-epileptic drugs, maternal alcohol or cigarette use 4 , much effort has been concentrated on identifying the genetic contribution.
Many mouse mutants with isolated clefts have been described where the specific gene is known 5 , and these too contribute to the pool of candidate genes.
We can therefore confidently predict that many individual genes, acting either alone or within gene networks, will be responsible for the heterogeneous causality observed in humans. It has been predicted that CLP would best fit an oligogenic model where one or a few major genes were influenced by a small number of modifiers 3 , 6 — 8.
Nevertheless, the intense efforts of current screening programmes have not revealed major risk factors for human clefting, and the identification of causative mutations has remained elusive.
This general failure probably reflects a more complicated and diverse etiology than these studies suggested. It is therefore encouraging that several important risk factors have recently been identified directly from human analyses. The maxillary prominences enlarge and grow towards each other and the nasal prominences. During the sixth to seventh weeks, the nasal prominences merge to form the intermaxillary segment resulting in both the filtrum and primary palate.
This region then fuses to the maxillary prominences, which form the lateral parts of the upper lip 9. The secondary palate is also CNC derived and forms the palatal shelves, which grow out from the maxillary prominences.
In the mouse the palatal shelves first appear at around E A key stage in mouse palatogenesis occurs during E Several genes have been implicated in palatal mesenchymal proliferation such as Msx1 and Lhx8, where CP is seen in the respective null mice due to the palatal shelves failing to meet in the horizontal plane 13 , As a general model, insufficient mesenchyme is believed to be the most common reason for CP in mice 5.
Although several theories exist for how the palatal shelves elevate, the actual mechanisms responsible are unclear. Movement from the vertical to the horizontal is likely to be a consequence of an intrinsic force resulting from increased turgidity through recruitment of water in response to elevated levels of glycosaminoglycans such as hyaluronan This occurs concomitantly with rapid remodelling of the extracellular matrix ECM The shelves require the ability to hinge and maintenance of the appropriate structural shape is important during changes to the ECM and proliferating mesenchyme.
Pax9 mutant mice, for example, exhibit CP due to an abnormal morphology of the palatal shelves. The shelves are shorter and broader than the wild-type, which causes mechanical inhibition of shelf reorientation Following elevation, the medial edge epithelia MEE of the opposing palatine shelves fuse in the midline through interactions of cell adhesion molecules and desmosomes.
The palatal shelves initially contact in the mid portion and then zipper closed towards both the primary palate and the uvula. The resulting epithelial seam is rapidly removed through a combination of programmed cell death, epithelial cell migration and transdifferentiation 18 , Palatogenesis is considered to be complete in mouse by around E Shh plays an important role in the early induction of facial primordia in addition to expression in the palatal MEE Bmp2 and Bmp4 on the other hand, are expressed more specifically within the epithelia and mesenchyme of the palatal shelves.
The Msx1 homeobox gene, which is also expressed in the facial primordia, is required for expression of Bmp2 and Bmp4 in the palatal mesenchyme and Shh in the MEE Fibronectin and collagen III act as modulating factors on hyaluronate expansion during shelf reorientation while collagen IX plays a critical role in signalling epithelial—mesenchymal interactions, appearing in the MEE cell surface just prior to shelf elevation Transcription factors such as the distal-less Dlx , Hox, Gli and T-box families also play key roles in maxillary and mandibular specification and are regulated by Shh, Bmps and Fgf signals Clearly epithelial—mesenchymal interactions are crucial in craniofacial development and specific sites of expression such as the tooth buds may function as inductive signalling centres influencing palate morphogenesis.
Recent evidence suggests that their function in the embryonic palate is at least in part mediated through the Smad signalling system It is also expressed later in the horizontal shelves and MEE, but expression is undetected once the epithelial seam disrupts. Although the palatal shelves otherwise develop normally, they show a marked reduction in the filopodia present on the MEE surface 29 , 30 and show down-regulation of condroitin sulphate proteoglycan on the apical surface of the MEE Both of these are required for efficient MEE adhesion.
This model shows that proteolytic degradation of the ECM is essential for palatal fusion. Overall it is clear that tight control of a cascade of genes is required to complete normal palatogenesis. The four studies do not generally concur on significant or highly suggestive regions, probably reflecting the diverse populations investigated.
An exception to this is a region on 2q, which overlaps between the Chinese study and a subset of the American families. A number of new genome scans have been presented at the American Society of Human Genetics meeting, including a meta-analysis totalling 11 studies Some regions consistent with previous linkage or candidate gene association studies 39 have been highlighted such as 2p13 TGFA , 6p This produces a higher chance of identifying single gene causation.
While these subgroups are referred to as syndromic clefts, it is now becoming apparent that the same genes contribute to the population of non-syndromic clefts, perhaps through variable penetrance or the action of different modifiers. This has been extensively studied as a rare but strongly genetic influence for nonsyndromic CP 40 — In addition to CP or in some cases bifid or absent uvula, the majority of these patients also display ankyloglossia tongue-tie. This minor feature is frequently missed or unreported; however, when noted in addition to X-linked inheritance, it is an important diagnostic marker for CPX.
TBX22 is a recently described member of the T-box containing transcription factor gene family that is conserved throughout metazoan evolution. These genes play essential roles in early development and in particular mesoderm specification.
The first T-box gene, Brachyury or T, was originally identified in mice 51 , where heterozygous animals have short tails and homozygotes lack a notochord and mesoderm posterior to somite 7 Subsequently, a family of T-box proteins have been described including 18 in humans, all characterized by a similar DNA binding domain. In addition to TBX22, several other T-box genes have been implicated in human syndromes, emphasizing their importance in development.
Although no gene deletions have yet been described for TBX22, a variety of point mutations have been identified. These include nonsense, splice site, frameshift and missense changes, with the latter affecting highly conserved residues within the T-box DNA-binding domain It is interesting to note that the GC missense mutation found in a Canadian CPX family 50 , 59 is at the equivalent position within the T-box domain to the G80R change seen in a Holt—Oram syndrome patient This position is predicted to interact with the major groove of target DNA and has been demonstrated to result in loss of DNA binding 61 , Taken together with the X-chromosomal localization, CP in males is likely to result from complete loss of TBX22 function, while haploinsufficient females frequently exhibit a milder phenotype.
In addition to families with clear X-linked inheritance, mutations were also found in smaller families where ankyloglossia is a diagnostic feature 50 , TBX22 expression correlates precisely with the phenotype seen in CPX patients, both in the vertical palatal shelves and the base of the tongue corresponding to the frenulum A similar expression pattern is seen in mouse 63 , 64 and in chick The latter is interesting since birds have a constitutional cleft and one could speculate that Tbx22 expression is important in palatal shelf outgrowth rather than fusion.
Despite the frequent concordance of CP and ankyloglossia in CPX patients, the phenotype can vary even within single families Fig. These include several sporadic patients with no ankyloglossia and a patient with isolated CP but with ankyloglossia only seen in the extended family.
Furthermore, we have recently identified a DNA binding domain missense mutation in a five generation CP family with no evidence of ankyloglossia unpublished data. Positional cloning mapped the locus to 11q23 and mutations were identified in the cell adhesion molecule PVRL1 Nectin-1 , which is expressed in the developing face and palate The same mutation is also present on the Venezuelan mainland, where heterozygosity was found to be a significant risk factor for non-syndromic CLP It will be important to investigate whether PVRL1 mutations contribute to non-syndromic clefts in other geographical locations.
Linkage analysis localized the gene to 1q32—41 and mutations were identified in the interferon regulatory factor 6 gene IRF6 In the mouse, Irf6 expression is restricted to the palatal MEE immediately prior to and during fusion. This markedly overlaps with the site of Tgfb3 expression and may suggest a potential interaction. Whilst sequence analysis in this cohort has not yet identified changes with clear causative function, a second reported study has identified an IRF6 missense mutation in a two-generation apparently non-syndromic CLP family EEC syndrome was mapped to 3q27 and heterozygous mutations were identified in the p63 gene One unusual phenomenon with p63 is that mutation to different parts of the gene can influence the cleft phenotype.
Mutation at the N-terminal end outside of the conserved domains gives rise to CP or no clefting at all. Not only is a larger study warranted but further investigation of downstream targets might be revealing. In particular Jagged2, a ligand in the notch signalling pathway, is known to act downstream of p63 and homozygous mouse knockouts of Jagged2 exhibit CP An MSX1 mutation was reported in a Dutch family with tooth agenesis and a mixture of CLP and CP, providing another rare example of where a single gene, and in this case single mutation, can give rise to a mixed clefting phenotype Subsequently, conflicting reports have been published, some with evidence of linkage or association to either CLP or CP 78 — 81 and some with no association to either 82 , As well as the characteristic hypogonadotropic hypogonadism and anosmia, five of the 13 patients with mutations had clefts of the lip or palate.
Several forkhead genes represent good candidates, not only because of their craniofacial expression pattern but also because mutations give rise to clefts, e.
In addition, two genes have recently been identified through chromosome rearrangements in cleft patients. The use of model organisms and in particular the mouse, has for some time been a rich source of information for craniofacial development. The use of mutant inbred strains to tease out causative genes and provide models is an exceptionally powerful tool.
Clearly we are now in a far better position to address this. These genes will provide tools to study and elucidate the genetic pathways that they function in. This can be combined with a second powerful approach, using the latest generation of expression profiling techniques. In conjunction with the genome sequence and virtually a complete list of genes, detailed information about the genes that are expressed and those that are switched on or off at different stages of craniofacial development can be determined.