© 1999 Oxford University Press
Human Molecular Genetics, 1999, Vol. 8, No. 10 Review 1853–1859
The many faces and factors of orofacial clefts
Brian C. Schutte1 and Jeffrey C. Murray1,2,+
1Department
of Pediatrics and 2Department of Biological Sciences, Department of Preventive Medicine and
Environmental Health and Department of Pediatric Dentistry, University of Iowa, Iowa City, IA 52242-1083, USA
Received April 26, 1999; Revised and Accepted June 28, 1999
Orofacial clefts are congenital structural anomalies of the lip and/or palate that affect ∼1/1000 live births. Their
frequent occurrence as well as their extensive psychological, surgical, speech and dental involvement
emphasize the importance of understanding the underlying causes. The etiology of orofacial clefts is complex,
including multiple genetic and environmental factors. Rare forms, where they occur as one component of
multiple congenital anomaly syndromes, have Mendelian or teratogenic origins; the non-syndromic forms of
orofacial clefts are more common and are likely due to secondary gene–environment interactions. Recent
advances in both molecular and quantitative approaches have begun to identify the genes responsible for the
rare syndromic forms of cleft and have also identified both candidate genes and loci for the more common and
complex non-syndromic variants. Animal models, in particular the mouse, have also contributed greatly to an
understanding of these disorders. This review describes genes that are involved in orofacial clefts in humans
and animal models and explores genetic approaches to identifying additional genes and gene–environment
interactions that constitute the many factors of orofacial clefts.
INTRODUCTION
Cleft lip and palate is a major congenital structural anomaly that
is notable for significant lifelong morbidity and complex
etiology. The prevalence of orofacial clefts varies from 1/500 to
1/2500 births depending on geographic origin (1), racial and
ethnic backgrounds (2,3) and socioeconomic status (4). In many
parts of the world orofacial clefts go unrepaired. Individuals who
do have their clefts repaired undergo surgical, speech, dental and
psychological therapies. These outcomes, along with the
relatively high prevalence of orofacial clefts, emphasize the
importance of understanding the underlying causes of orofacial
clefts.
The causes of orofacial clefts are complex, involving both
genetic and environmental factors. That genes play an almost
deterministic role in the development of normal craniofacial
structures is evident from observations of monozygotic twins,
where the majority are phenotypically indistinguishable. At the
same time, this genetic program is exquisitely sensitive to postconception disturbances in genes or the environment, as
evidenced by the identification of numerous teratogens that lead
to orofacial clefts. Thus, the complex etiology of clefts affords
ample opportunities to identify genes, explore gene–
environment interactions and learn more about human
embryology and its disturbances.
FACIAL DEVELOPMENT
Normal facial development begins with migrating neural crest
cells that combine with mesodermal cells to establish the facial
primordia. The growth of facial primordia from undifferentiated
mesenchymal cells into the finely detailed structures of the
mature head and face is largely determined genetically. These
processes are known to be dependent on a spectrum of signaling
molecules, transcription factors and growth factors. A subset of
genes already shown to play an important role in the
development of the head and with particular relevance to
development of the lips and palate is listed in Table 1.
Additional growth and signaling factors (5) that play a role in
facial development include JAGGED1, sonic hedgehog,
patched, CREB-binding protein, GLI3, FGFR1, CASK, treacle
and FGFR2. Other transcription factors involved include DLX5/
6 and PAX3. Extracellular matrix proteins such as COL2A1,
COL1A2, COL11A2, PIGA, αV integrin, glypican 3, fibrillin
and aggrecan are essential as well. This ever-expanding catalog
of molecules highlights the complex genetics of facial clefts.
When the structure or expression of these genes is altered, a
cleft of some type may occur (Fig. 1). Orofacial clefts can be
divided into four groups: non-syndromic cleft lip with or without
cleft palate, non-syndromic cleft palate only, syndromic cleft lip
with or without cleft palate and syndromic cleft palate only. The
term ‘non-syndromic’ is restricted to cleft cases where the
affected individuals have no other physical or developmental
anomalies and no recognized maternal environmental exposures
(6). Cleft cases are further divided by which palate is affected.
Genetics and embryology suggest that clefts of the primary
(hard) palate that involve the lip and/or palate are different in
mechanism from clefts that affect the secondary (soft) palate (7).
At the present time, most studies suggest that ∼70% of cleft lip
with or without cleft palate (CL/P) cases are non-syndromic (8).
The remaining 30% of syndromic cases can be subdivided into
chromosomal anomalies, >300 different recognizable
+To whom correspondence should be addressed at: Department of Pediatrics, University of Iowa, 200 Hawkins Drive, W229-1 GH, Iowa City, IA 52242-1083,
USA. Tel: +1 319 335 6897; Fax: +1 319 335 6970; Email: [email protected]
1854 Human Molecular Genetics, 1999, Vol. 8, No. 10 Review
Table 1. Genes regulating development of the head, in particular of the lip and palate
Gene
Typea
Featureb
TGFΑ
GF
L/P
END1
SF
M
Mousec
KO
Humand
Key references
LD
23,24
Linkage
88
RARΑ
SF
P/M
TG/EXP
LD, linkage
27
TGFB
GF
L/P
KO, EXP
LD
26,28
SKI
GF
L/P/M
KO, EXP
MSX1
HD
L/P
KO, EXP
DLX 1/2
HD
P/M
EXP
89
LD, linkage
26
90
PITX2
HD
P/M
KO, EXP
PAX9
HD
P/M
KO, EXP
Rieger
91
AP2
TF
L/P/M
KO, EXP
Linkage
42
TTF2
TF
P
KO
Thyroid dysgenesis
93,94
92
aGF,
growth factor; HD, homeodomain; SF, signaling factor; TF, transcription factor.
lip; P, palate; M, maxilla and/or mandible.
cKO, knockout; TG, transgene; EXP, expression.
dLD, linkage disequilibrium.
bL,
Mendelian syndromes (http://www.ncbi.nlm.nih.gov/Omim/ ),
teratogens and uncategorized syndromes. Recent reviews of
birth defects secondary to chromosomal anomalies identified
five regions in which there was a significantly higher frequency
of clefting associated with either specific deletions (4p16–14,
4q31–35 or 1q25) or duplications (3p26–21 or 10p15–11) than
would be expected from the background frequency (9,10).
Nonetheless, it is also apparent that deletions or duplications of
portions of every chromosomal arm, including the X
chromosome, have been associated with clefts, suggesting that
many genes are involved in facial development and that cleft lip
and palate can represent a common end-point for disruption of
facial processes (9,10). It is likely that in the future CL/P will be
subdivided further based on underlying genetic etiologies or
better phenotypic descriptors. Some attempts at clinical
distinction already include associations with hypoplasia of the
orbicularis oris muscle or handedness (11).
GENETICS OF OROFACIAL CLEFTS
The study of orofacial clefts has a rich history in human genetics
and provides a model for complex disease study in general. An
inherited component for clefts was first widely recognized
through the work of Fogh-Andersen in his thesis of 1942 (12).
Genetic factors in clefting are now well established through
segregation analysis (13,14) as well as through twin studies (15).
Additional genetic linkage and association studies are now being
used to identify these genetic factors.
Although genetic linkage studies of CL/P have been limited
by insufficient numbers of families and genotyping resources, a
few efforts using candidate genes or loci have been reported.
Studies (reviewed in refs 6,16) using from 1 to 40 families have
suggested loci for clefts on chromosomes 2, 4, 6, 17 and 19.
Linkage has been excluded at these same loci in other data sets.
These inconsistent linkage results reflect the small number of
families studied and probable locus heterogeneity. Thus, while
the studies are useful for preliminary data, they need to be
replicated on far larger sample sets. Only loci on 6p have
consistently shown linkage to CL/P, beginning with studies
from Denmark (17) and subsequently in Italy (18–20).
Association studies have also been used to examine candidate
genes in CL/P. Association studies have the advantage over
linkage in that they use the large number of cases that occur in
isolation without affected relatives (21). In addition, association
studies exploit a wealth of literature in developmental biology
that identifies specific genes expressed during critical phases of
lip or palate formation (22). Ardinger et al. (23) first reported a
role for transforming growth factor-α (TGFΑ) as contributing to
CL/P. Since that time, five studies have confirmed this result
and, although several other studies have failed to replicate the
association, a recent meta-analysis supports a role for TGFA as
a modifying factor in cleft lip and palate (24). Other genes/loci
showing association include D4S192, MSX1, TGFB3 and
RARΑ (16,25–29), with only the TGFΒ3 finding replicated. A
summary of human studies is shown in Table 2.
ANIMAL MODELS
Candidate loci for CL/P have also been proposed based on
spontaneously arising or transgenic mouse models with clefts.
While there are many mouse mutants that include clefts of the lip
or palate as part of the phenotype, the best candidates for human
clefting are those in which clefts appear without other
abnormalities, including Clf1, Clf2, Cps-1, Dcp-1 and Dcp-2. Two
genome-wide searches for susceptibility loci in the mouse have
also been performed. One used the A strain derivative A/WySn to
identify two loci for cleft susceptibility, Clf1 and Clf2 (30,31). A
second scan used teratogen susceptibility in the AXB/BXA inbred
strains and identified 16 susceptibility regions, including one near
Msx1 (32).
Random insertions and targeted knockouts in the mouse have
now been generated for >10 years and 30 of these are listed in
the transgenic database (http://tbase.jax.org ) as including cleft
lip and/or palate. Although, initially, transgene phenotypes
appeared to support a role for certain genes in cleft causation, it
is now apparent that clefts are a frequent end-point of knockout
and insertion experiments. For a gene to be a strong cleft
Human Molecular Genetics, 1999, Vol. 8, No. 10 Review 1855
Figure 1. A photomontage of children with various forms of orofacial clefts. (A) Right-sided unilateral cleft lip and palate; (B) left-sided unilateral cleft lip only;
(C) bilateral cleft lip and palate with right lower paramedian lip pit, indicative of Van der Woude syndrome; (D) isolated cleft of the soft palate.
candidate, it must result in a clefting phenotype in the transgene
and be expressed at a critical time and in a tissue relevant to lip
and palate development. The three best examples to date are the
Msx1, Tgfb3 and Ap-2 knockouts, in which gene expression
supports a role in craniofacial development and the knockouts
result in clefts.
For Msx1, two independent knockouts (33,34) result in 100%
cleft palate and Msx1 is expressed in developing craniofacial
structures (35), including the palate (P. Sharpe, personal
communication). MSX1 is deleted in cases of the human 4p–
syndrome, which commonly includes clefts. In the case of
Tgfb3, two independent knockouts result in the phenotype of
cleft palate (36,37). Expression data (38,39) and recent work
showing that exogenous TGFβ3 can induce palate fusion in the
chicken, where the palate is normally cleft (40), further support
a role for TGFΒ3 in clefting.
1856 Human Molecular Genetics, 1999, Vol. 8, No. 10 Review
Table 2. Studies of candidate genes for clefting
Gene
Locus
Linkagea
Associationa
TDTa
Other datab
TGFΑ
2p13
–
++/–
+/–
EXP
23,24,26
MSX1
4p16
–
+
+/–
CH/KO/EXP
26,95
N/Ac
4q31
+/–
+/–
–
–
25,96
N/Ac
6p23
++/–
–
+/–
CH/KO
17–19
References
TGFΒ3
14q24
–
++/–
+/–
KO/EXP
26,28
RARΑ
17q21
+/–
+/–
+/–
TG/EXP
27,97–99
BCL3
19q13
+/–
+/–
+/–
CH
26,29,100
a–,
one or more negative studies; +, single positive study; ++, more than one positive study.
chromosome deletion (recurrent) or translocation; KO, mouse knockout; TG, transgene; EXP,
expression.
cNo candidate genes have been identified at this map locus.
bCH,
Knockouts of the retinoic acid-dependent transcription factor
Ap-2 resulted in extensive craniofacial and more generalized
structural disruptions (41). Recently, chimeric knockouts for Ap-2
(42) suggest a more specific role for Ap-2 in clefting. Ap-2 also lies
near the site of two balanced translocations at 6p that have CL/P
phenotypes (43,44).
GENE IDENTIFICATION
An alternative approach for identifying genetic factors in nonsyndromic clefting is to study a disorder that exhibits a clear
Mendelian pattern of inheritance and a closely related
phenotype. Of the ∼150 Mendelian disorders that include cleft
lip, 50% are autosomal recessive, 40% autosomal dominant and
10% X-linked. Approximately 30 genes have been cloned from
humans wherein gene disruptions can include a cleft as part of
the phenotype. Gene classes represented in this group include
extracellular matrix proteins (COL2A1, COL11A2 and GPC3),
transcription factors (GLI3, PAX3, SIX3 and SOX9) and cell
signaling molecules (FGFR2, PTCH and SHH). Two mapped
disorders, X-linked cleft palate (OMIM 303400) and Van der
Woude syndrome (OMIM 119300), are particularly attractive
models because their phenotypes are so similar to nonsyndromic forms of clefting.
One of the first birth defect loci mapped using DNA-based
polymorphisms was that for the X-linked cleft palate and
ankyloglossia locus (CPX), mapped by linkage to Xq21–q22
(45). The CPX phenotype is exclusively confined to the palate,
which may be cleft or insufficient, and to the tongue, resulting in
ankyloglossia (tongue-tied). Although the gene associated with
this phenotype is not yet identified, high resolution mapping of
the X chromosome will likely soon disclose its nature (46) and
lead to a better understanding of palate development.
Another good Mendelian model for non-syndromic cleft lip
and palate is the Van der Woude syndrome, an autosomal
dominant disorder characterized by paramedian lip pits of the
lower lip (Fig. 1C), cleft lip with or without cleft palate, isolated
cleft palate alone and occasional hypodontia. The disorder has
no other craniofacial anomalies and is associated with normal
intelligence. The condition received its descriptive eponym
following Anne Van der Woude’s description of the disorder in
1954 (47) but has been recognized as a common form of clefting
with multiple families reported for well over a century (48).
Burdick et al. (49) have reported an extensive summary of
families with the disorder and it is particularly remarkable in that
it is one of the very rare disorders in which clefts of the primary
palate can be found in the same family and segregating with the
identical allele as isolated secondary palate clefts. This suggests
that a very early stage of embryogenesis is likely being affected.
Linkage for Van der Woude syndrome and chromosomal
localization was first suggested by Wienker et al. (50) and by the
report of Bocian and Walker (51) of a large, cytogenetically
visible deletion on the long arm of chromosome 1. In 1990,
genetic linkage was established (52) to 1q32 and, subsequent to
this, linkage has been confirmed by other groups more finely
defined through the use of recombinants (53,54) and
microdeletion patients (55,56) that have confirmed a relatively
narrow region in which the gene is likely to reside. Efforts at
identifying genes within this region are currently under way and
a complete BAC contig and sequence from this region is now
under analysis.
The identification of the Van der Woude gene itself is likely to
contribute greatly to a better understanding of other genes and
gene paths likely to play a role in craniofacial development. A
substantial number of such genes that include other complex
anomalies are shown in Table 1, but Van der Woude syndrome
is of particular interest because the phenotype is so similar, with
the exception of the lip pits, to the appearance of non-syndromic
cleft lip and palate.
ENVIRONMENTAL INTERACTIONS
An environmental component to clefting was recognized when
Warkany et al. (57) associated nutritional deficiencies with cleft
palate. Recognized teratogens that cause clefts include rare
exposures, such as phenytoin, valproic acid and thalidomide,
and also common environmental exposures, such as maternal
alcohol or cigarette use (58) and, more recently, pesticides such
as dioxin (59). The exposures are important in that they can
suggest metabolic pathways whose disruption may play a role in
the development of CL/P.
Epidemiological studies support a role for environmental
factors in clefting, especially in regions of low socioeconomic
status (SES). In the Philippines, three studies (4,60,61) all report
incidences of CL/P of 2/1000 in indigent populations, while
complementary studies show an incidence of 1.2/1000 in native
Filipinos living in areas of higher SES, including Manila (60),
Hawaii (62) and California (2). When SES did not change
Human Molecular Genetics, 1999, Vol. 8, No. 10 Review 1857
through a geographic move, no change in frequency was noted
by Christensen et al. (63). Thus, nutritional or toxic
environmental exposures may contribute directly to as much as
one third of cleft cases and etiologies will be most identifiable in
indigent populations.
Evaluation of gene–environment interaction (64) is still at a
preliminary stage (28,65), but studies have looked at the role of
smoking with TGFΑ acting as a covariate (66–68). An
interaction is suggested, although not confirmed, in all studies
(69,70). Preliminary data (68,70–72) support interactions
between alcohol, nutritional factors and the MSX1 and TGFΒ3
genes, in addition to TGFΑ. Fetal alcohol syndrome can include
clefts of the lip and/or palate as part of the phenotype. Vitamin A
and its congeners, such as Accutane, are known to induce
craniofacial anomalies (73). Folate-metabolizing enzymes are
candidates based on preliminary data that suggest that folic acid
supplementation can reduce the incidence of clefting (74,75).
However, the data remain controversial (76), with no gene
association yet found for methylene tetrahydrofolate reductase
(77), a key player in the folate pathway in neural tube defects.
Pyridoxine (vitamin B6) may also play a role in facial
development and protective effects have been suggested in
studies in rats (78) and humans (79). Enzymatic pathways that
are candidates for variation-induced clefting thus include the
genes for alcohol, vitamin A, vitamin B6 and folate metabolism.
Other risks include environmental estrogens or dioxins
(TCDD) which bind to endogenous nuclear receptors that also
serve as transcription factors (80,81). This activity is mediated
through the aryl hydrocarbon receptor (AhR) and the AhR
nuclear translocator (ARNT) genes, which are expressed in
developing palate (82) and have their expression altered by
dioxins (83). Dioxin and retinoic acids also alter TGFΒ3
expression (84,85) and there are strong teratogenic effects of
dioxins (86) and retinoic acid (87) in the mouse and possibly
human (59,73). Thus, a plausible path for gene–environment
interactions might involve environmental effects (alcohol,
dioxins and estrogens) mediated via the AhR/ARNT and
retinoic acid pathways and disturbing the critical role of TGFΑ
or TGFB3 in lip and palate formation.
SUMMARY
Studies of orofacial clefting are valuable, both for the morbidity
of the defect itself and for providing a model to examine a
complex human birth defect. Identification of specific genes and
environmental factors will immediately provide for better risk
counseling and holds the promise of preventive strategies and
improved therapeutics. As a model for complex traits, cleft lip
and palate is ideal, with a high frequency in global populations,
so that collections of large numbers of isolated and familial cases
will have the power to determine genetic and environmental
etiology.
ACKNOWLEDGEMENTS
We are especially grateful to the many patients and colleagues
who have worked with us over the years, in particular Holly
Ardinger, Ken Buetow, Kaare Christensen, Sandra DaackHirsch, Ed Lammer, Andrew Lidral, Mary Marazita, Ron
Munger, Paul Romitti, Achim Sander, Gary Shaw and Michael
Solursh. The authors’ investigations of cleft lip and palate have
been supported by grants DE08559, DE09170 and DE11948
from the US National Institutes of Health.
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