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Unraveling Human Cleft Lip and Palate ResearchDepartments of Oral Biology and Pediatric Dentistry and Center for Craniofacial and Dental Genetics, School of Dental Medicine, and Department of Human Genetics, Graduate School of Public Health, University of Pittsburgh, 614 Salk Hall, Pittsburgh, PA 15261, USA; arv11{at}dental.pitt.edu
The focus of this work is to highlight the most recent advances in the understanding of cleft lip and palate occurrence. Information regarding research on long-term outcomes, genes and their interactions with other genes, and gene-environment interactions is compiled to provide the reader with a critical and up-to-date overview on the current knowledge of the etiology of cleft lip and palate. Recent epidemiological evidence strongly suggests that individuals born with clefts have a shorter lifespan and may have a higher incidence of cancer and psychological disorders. IRF6 has been shown to be an important contributor to cleft lip and palate, but the functional variant leading to the defect has not yet been defined. Inactivation of MSX1 and genes in the FGF family has also been shown to lead to cleft lip and palate. In addition, missense mutations in several candidate genes may cause cleft lip and palate, but definitive evidence regarding the biological consequences of these mutations is yet to be unraveled. Maternal cigarette smoking increases the risk of a baby born with clefts, in particular when the mother carries the GSTT1-null variants. The latest approaches in cleft research include the analysis of several additional phenotypical features of the population, with the goal of increasing the statistical power of genetics studies.
Key Words: birth defect • craniofacial • cleft lip • cleft palate • FGF • IRF6 • mutation • MSX1 • smoking
There is no doubt that the cleft lip and palate field has shown the most progress regarding the understanding of its genetic etiology, compared with other complex birth defects. Non-syndromic or isolated cleft lip, with or without cleft palate, occurs in a wide geographic distribution, with an average birth prevalence of 1/700. However, Northern Europeans, Asians, Native Americans, and Aboriginal Australians are more commonly affected by cleft lip accompanying cleft palate. In contrast, Africans and those of African descent have more instances of cleft lip only (reviewed by Mossey and Little, 2002). The most recent estimates suggest that anywhere from 3 to 14 genes contribute to cleft lip and palate (Schliekelman and Slatkin, 2002). Although there is still much to be learned, the possibility of significantly improving genetic counseling estimates for isolated cleft lip and palate has never been so close. In this review, the genetic findings on cleft lip and palate are interpreted in the light of a future when genetic testing may aid the clinical evaluation of families carrying genetic variants that increase the risk for having a baby with this birth defect.
Not long ago, researchers hoping to study the genetics of cleft lip and palate were haunted by reviews stating that "babies born with clefts can be treated in their first year of life and understanding the genetics contribution to clefts will not change the outcome of these cases". Recent work has suggested that this was a gross underestimation of the consequences of being born with facial clefts. Individuals born with clefts have a shorter lifespan, with increased risk for all major causes of death, when compared with individuals born without clefts (Christensen et al., 2004). Contributing to these higher mortality rates are probably psychiatric disorders and cancer. Facial clefts increase the risk of hospitalization for psychiatric diseases in adults (Christensen and Mortensen, 2002). Also, an increased occurrence of breast and brain cancer among adult females born with oral clefts, and an increased occurrence of primary lung cancer among adult males born with oral clefts have been reported (Bille et al., 2005). Psychiatric disorders can be interpreted under the assumption that the development of the brain and that of the face are intimately related in both normal and pathologic conditions, and suggest that abnormal brain development might accompany an abnormality in facial development (Nopoulos et al., 2007). Animal models have shown that forebrain development and facial development are linked. Molecular signaling in the forebrain regulates the establishment of a signaling center in the face, and thus controls its subsequent morphogenesis. The molecular dialogue that exists between these tissues is essential for patterned outgrowth of the middle and upper face (Marcucio et al., 2005). It appears that defects in signaling within the forebrain can lead to a wide variety of craniofacial malformations, including cleft lip and palate. The possibility that the same genetic variation can contribute to a birth defect (cleft lip and palate) and also contribute to cancer later in life is another fascinating biological scenario. Parents of children born with cleft lip and palate were shown to have a higher risk of cancer—in particular, lymphomas and leukemia (Zhu et al., 2002). Also, two families with hereditary diffuse gastric cancer were reported, segregating CDH1/E-cadherin mutations with cleft lip and palate (Frebourg et al., 2006). It is clear that an improved understanding of the genetics of cleft lip and palate can help improve counseling for families with a possible higher risk for clefts, and can also provide new insight into research in cancer and psychiatric disorders genetics.
The most remarkable result of the latest cleft lip and palate research is the demonstration of an association between variations in the IRF6 locus and isolated cleft lip and palate (Zucchero et al., 2004). When mutated, IRF6 leads to Van der Woude and popliteal pterygium syndromes (Online Mendelian Inheritance in Man #119300 and #119500, respectively), which are disorders that can clinically resemble an isolated cleft lip and palate (Kondo et al., 2002). The association between IRF6 and cleft lip and palate has been independently replicated in many populations (reviewed by Vieira et al., 2007b) and expanded to isolated tooth agenesis, another phenotypic feature of Van der Woude syndrome and the most common congenital anomaly in humans (Vieira et al., 2007a). It is remarkable that the same gene locus appears to contribute to phenotypes varying from very rare syndromic forms of clefting (frequency of 1 to 100,000 to 200,000 live births) to the more common isolated forms of clefting (frequency 1 to 500 to 2000) to the very common tooth agenesis phenotype (frequency 1 to 10 to 100), since these defects were part of the same clinical spectrum (Fig. 1  ).
Attributable fraction calculations performed in the work that originally reported the association between cleft lip and palate and IRF6 (Zucchero et al., 2004) suggested that IRF6 contributes to 12% of total cases of isolated cleft lip and palate. Although wrongly interpreted by the media at that time as a "new genetic testing for cleft lip and palate", this finding suggested that the eventual identification of the functional variant(s) in the IRF6 locus would finally provide a robust marker to test for individual risks for cleft lip and palate. However, the IRF6 contribution is likely to occur in the background of other genes, and parents capable of producing a child who carries both copies of the susceptibility IRF6 allele(s) would have their risk for having a baby with cleft lip and palate increased from the recurrence risk of 3 to 5% to a risk of 9%. It is unlikely that families would react very differently to this small, but important, change in risk estimates (Chakravarti, 2004). Identifying the causative IRF6 variant(s) has been a challenging problem akin to finding a needle in a haystack. The latest attempt to identify the "needle" used a method to test the association of cleft lip and palate with multiple single nucleotide polymorphism (SNP) markers in the IRF6 locus, to subsequently nominate a set of "risk-haplotype-tagging alleles", testing for both maternal and child effects, as well as imprinting (Shi et al., 2007b). The data from the original report, which demonstrated an association between cleft lip and palate and IRF6, were used (Zucchero et al., 2004). The results suggested a maternal risk haplotype that included 14 SNPs, 13 of which were also indicated in the risk haplotype from the offspring analysis. However, at each of the 13 SNPs identified in common, the maternal-risk-tagging allele was opposite to the one nominated by the offspring analysis. After excluding imprinting as an explanation, the authors suggested that the same haplotype could be protective against clefting in the child if carried by the mother and deleterious if carried by her fetus. Although at first this phenomenon seems implausible, the authors suggested that genes in the mother and those in the fetus are potentially doing very different things during fetal development. This dual effect would allow such haplotypes to be preserved in the population, because a benefit would offset the obvious survival-limiting detriment. During prehistoric times, most babies born with clefts of the lip and palate would have been unable to nurse properly and would have died of starvation or abandonment. Any haplotype that increases the risk of clefting would consequently require a compensatory mechanism to explain its persistence. Despite these clear difficulties, the identification of IRF6 as a major contributor to cleft lip and palate provides a promising lead for the identification of other genes linked to this birth defect and for elucidation of the mechanisms of environmental exposure (Chakravarti, 2004).
Several private missense mutations (in the vast majority in single cases, i.e., private families) have been reported in candidate genes for clefts [TGFB3 (Lidral et al., 1998); MSX1 (Jezewski et al., 2003; Suzuki et al., 2004; Vieira et al., 2005; Tongkobpetch et al., 2006); FOXE1, GLI2, JAG2, LHX8, MSX2, SKI, SPRY2, and TBX10 (Vieira et al., 2005); PVRL1 (Avila et al., 2006); PTCH (Mansilla et al., 2006); PVR and PVRL2 (Warrington et al., 2006); RYK (Watanabe et al., 2006); FGFs (Riley et al., 2007); and TBX22 (Marçano et al., 2004)]. TBX22 mutations, which are embryologically distinct from isolated cleft lip and palate, were found in isolated cleft palate cases.
The missense mutations reported in the candidate genes listed above do not clearly segregate in the families. Variable expression and incomplete penetrance have been suggested as possible scenarios. In many instances, parent DNA was not available for testing. These limitations make it impossible to suggest potential functional consequences, and these variants could very well be rare, functionally neutral, changes. However, several of the reported mutations are affecting conserved sites in other mammals, may disrupt exonic splicing enhancer sequences, and were not found in between 400 and 2000 control chromosomes (Vieira et al., 2005). These mutations could lead to reduced expression of the genes during development and, consequently, to cleft lip and palate (Fig. 2
The MSX1 gene is shown in Fig. 3 . For MSX1, it has been proposed that mutations in this gene alone could contribute to as many as 2% of total cleft lip and palate cases (Jezewski et al., 2003; Tongkobpetch et al., 2006). However, the mutation identification effort was unable to find another inactivating mutation such as the one that causes autosomal-dominant cleft lip and palate-oligodontia syndrome (Online Mendelian Inheritance in Man #106600). One MSX1 missense mutation was thought to be disease-causing, based on early statistical evidence. The MSX1 P147Q mutation was first reported in a Vietnamese population (Suzuki et al., 2004), and this mutation was thought to have arisen from a founder individual, based on the analysis of shared haplotypes in unrelated Vietnamese families. This mutation was later found in two Filipino families (Vieira et al., 2005), after the screening of 1468 cleft lip and palate cases from the Philippines. Although the mutation was not found in a pan-ethnic panel of 1671 individuals, it did not segregate with the cleft phenotype, and low penetrance was proposed to explain this evidence. Finally, a study with Thai people showed that the frequency of the MSX1 P147Q mutation can be as high as 8% in Thailand, suggesting that this variant by itself is not pathogenic (Tongkobpetch et al., 2006). Perhaps the most interesting case related to the MSX1 P147Q saga is that of one Vietnamese child who was born with a sporadic cleft lip and palate and carried two MSX1 mutations: G98E and P147Q, one from each parent (Suzuki et al., 2004). One can argue that MSX1 P147Q can be pathogenic in association with additional MSX1 mutations. Variation in MSX1 appears to contribute to a variety of ectodermal-derived traits. In part, this may be the consequence of independent gene-gene interactions, as well as alterations in specific sites of the gene (Fig. 4, Table).
FGF genes have been proposed to contribute to as many as 3% of total cleft lip and palate cases. One de novo mutation in FGF8 and one nonsense mutation in FGFR1 can certainly be assumed as etiologic mutations (Riley et al., 2007). FGFR1 mutations have also been described in patients with Kallmann syndrome with cleft lip and palate and/or tooth agenesis (Online Mendelian Inheritance in Man #147950). The FGF8 de novo mutation was predicted, by structural analysis, to be a loss-of-function mutation. The FGFR1 nonsense mutation was identified in a person who could be re-assessed and in whom additional features of Kallmman syndrome (hypogonadotropic hypogonadism, anosmia, and tooth agenesis) were identified. The parents were tested, and the mutation was found in the father, who was born with cleft lip and palate, but had no apparent additional features of Kallmann syndrome. This family illustrates that a subset of isolated cleft lip and palate cases may be syndromes with variable expression. In the aggregate, mutations in several candidate genes for clefts may contribute to as many as 6% of total isolated cleft lip and palate cases (Vieira et al., 2005). However, with the likely exception of the two inactivating mutations in FGF8 and FGFR1, definitive conclusions regarding all other mutations can still not be drawn, and formal genetic screening for these mutations is still not justified. Future studies should focus on testing the potential functional consequences of these changes, which would provide more conclusive evidence regarding the etiologic role of these specific variants, and possibly a panel of genes that could be screened for risk assessment purposes.
A likely scenario is that genetic variation in more than one gene is needed to cause isolated cleft lip and palate. Lip and palate formations are the consequence of several processes that involve cell proliferation, cell differentiation, cell adhesion, and apoptosis. In theory, failure anywhere in these processes can lead to clefts. Therefore, cleft of the lip and palate can be caused by lack of growth (insufficient cell differentiation and/or proliferation), failure in fusion (lack of cell adhesion and/or excess of apoptosis), or both. Consequently, "interactive" genes can be genes regulating cell differentiation, proliferation, adhesion, or apoptosis. Statistical evidence of gene interaction leading to clefts has been reported for MSX1 (a transcription repressor) and TGFB3 (involved in cell differentiation) (Vieira et al., 2003), and for MSX1 and TGFA (a growth factor) (Jugessur et al., 2003). In both studies, the evidence of interaction was related to carrying two copies of the MSX1 risk allele.
MSX1 shows bidirectional transcription, with the expression of an antisense RNA partially complementary to the protein coding sense RNA (Blin-Wakkach et al., 2001) (Fig. 3 Maternal genotypes vs. infant genotypes have also been studied, in the context that some predisposing factors leading to a birth defect could be related to the mother’s genetic background. The studies involving maternal effects on cleft lip and palate have focused on genes involved in detoxification and maternal folate intake (see GENE-ENVIRONMENT INTERACTIONS, below). It is reasonable to propose that genetic variation of the mother, combined with genetic variation in the fetus, could increase the risk of cleft lip and palate. The only study that has shown some data supporting this assumption reported statistical evidence that MTHFR (which catalyzes the conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate) maternal genotypes interacted with the child’s BCL3 (a proto-oncogene and transcription activator) genotypes in cleft lip and palate cases (Gaspar et al., 2004). The work with tooth agenesis has also suggested possible interactions that may be relevant to cleft lip and palate. Statistical evidence of interactions between IRF6 (transcription factor) and MSX1, and IRF6 and TGFA has been reported (Vieira et al., 2007a). The identification of interactive genes will be a crucial step toward providing relevant clinical information to families inquiring about risks for having a baby with cleft lip and palate.
Maternal smoking and folic acid intake are the two main factors under investigation that appear to modify genetic risks for cleft lip and palate. Maternal cigarette smoking increases the risk of the baby’s having cleft lip and palate (Shi et al., 2007a). Attributable fraction calculations suggest that maternal smoking contributes to 4% of the total cleft lip and palate cases and 12% of bilateral cleft lips and palates (Honein et al., 2007). The suggested interaction between genetic variation in TGFA and maternal smoking leading to cleft lip and palate has not been confirmed by the latest investigations (reviewed by Zeiger et al., 2005). Interaction between maternal smoking and fetal inheritance of a GSTT1-null deletion was shown to be significant in two independent cleft lip and palate populations (Fig. 5 ). GSTT1 catalyzes the conjugation of reduced glutathione, an anti-oxidant that protects cells from toxins such as free radicals, which are induced by smoking. Analysis of the data indicated that if a pregnant woman smokes 15 cigarettes or more per day, the chances of her GSTT1-lacking fetus developing cleft lip and palate increase nearly 20-fold (Shi et al., 2007a).
Folic acid supplements appear to reduce the risk of a baby having cleft lip and palate by about a third (Wilcox et al., 2007). An ongoing clinical trial is under way in Brazil, and more definitive answers regarding the preventive effects of folic acid supplementation on cleft lip and palate should be available in the next few years (Jeffrey C. Murray, personal communication). Preliminary evidence of gene-multivitamin intake interaction in cleft lip and palate exists. Infants who are homozygous for the variant 1095 NAT1 (an enzyme involved in catabolism of folates) allele, and whose mothers did not take multivitamins during early pregnancy, have a two-fold higher risk of developing cleft lip and palate (Lammer et al., 2004).
The identification of the IRF6 contribution to cleft lip and palate (Zucchero et al., 2004) was a pivotal finding that evaluated 8003 individuals in 1968 families derived from 10 populations from Asia, Europe, and North and South America. Assuming that most genes contributing to cleft lip and palate that remain to be discovered have much smaller effects compared with the IRF6 contribution, it appears that many thousands of samples and families will be needed to unravel their contributions. These numbers would have to grow even more to establish definitive evidence of gene-gene and gene-environment interactions. One may argue that some of the "small effect" genes will never be found. The most recent work on the etiology of cleft lip and palate has focused on increasing the sophistication of the clinical descriptions, rather than aiming to study many thousands of people. The definition of the cleft phenotype used in genetics and epidemiology research was proposed more than 60 years ago: cleft lip with or without cleft palate, and cleft palate only (Fogh-Andersen, 1942). The creation of subphenotypes based on minor clinical features has been suggested to allow for the identification of "unaffected" individuals who in fact could be "carrying" the disease-causing alleles. It has been proposed that occult defects of the superior orbicularis oris muscle may represent a subclinical form of cleft of the lip. Comparisons between unaffected cleft relatives and control individuals showed that relatives have twice as many orbicularis oris muscle discontinuities (Neiswanger et al., 2007). Dental development has also been recently suggested as a tool for the creation of cleft subphenotypes. Individuals with cleft lip and palate present considerably more dental anomalies outside the cleft area than do individuals without clefts. Several subphenotypes based on the associated dental anomalies—such as tooth agenesis, supernumerary teeth, tooth impaction, tooth malposition, and the combination of more than one of these abnormalities—have been proposed (Letra et al., 2007). Brain structure in children with cleft lip and palate has been shown to be abnormal when compared with that in healthy children born without clefts, potentially due to abnormal brain development (Nopoulos et al., 2007). Children born with cleft lip and palate had abnormally small brains, with both cerebrum and cerebellum volumes substantially decreased. The authors also found that pattern of brain abnormalities in adults with cleft lip and palate, suggesting that brain growth and development trajectory are also abnormal in individuals with cleft lip and palate. The evaluation of unaffected cleft relatives would provide relevant information regarding the use of brain structure as another subphenotype for cleft lip and palate.
If one can imagine the etiology of cleft lip and palate as a puzzle that may have as many as 100 pieces, the outstanding recent advances in the cleft lip and palate field have started to label the pieces with some level of certainty (Fig. 6
This work greatly benefited from many discussions with Jeffrey Murray, Mary Marazita, Iêda Orioli, Eduardo Castilla, Brian Schutte, Andrew Lidral, Adriana Modesto, Lina Moreno, Ariadne Letra, and Renato Menezes. Melissa Carp provided administrative support. Diane Abate helped with the figures. Financial support was provided by NIH Grant R21 DE16718 and by the University of Pittsburgh School of Dental Medicine. Received for publication October 2, 2007. Revision received November 26, 2007. Accepted for publication November 27, 2007.
Journal of Dental Research, Vol. 87, No. 2,
119-125 (2008)
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ABSTRACT
INTRODUCTION
