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Clin Plastic Surg 31 (2004) 125–140
Current concepts in the embryology and genetics of
cleft lip and cleft palate
Mary L. Marazita, PhD, FACMGa,b,c,*, Mark P. Mooney, PhDd,e
aCenter for Craniofacial and Dental Genetics, Department of Oral and Maxillofacial Surgery, Division of Oral Biology,
School of Dental Medicine, University of Pittsburgh, Pittsburgh, PA 15219, USAbDepartment of Human Genetics, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, PA 15261, USA
cDepartment of Psychiatry, School of Medicine, University of Pittsburgh, Pittsburgh, PA 15261, USAdDepartments of Oral Medicine & Pathology, and Orthodontics, School of Dental Medicine, University of Pittsburgh,
Pittsburgh, PA 15219, USAeSchool of Arts and Sciences, University of Pittsburgh, Pittsburgh, PA 15219, USA
Clefts of the lip and palate (CL/P) are the most productive counseling. Furthermore, a better under-
common craniofacial birth defects and are among the
most common of all birth defects, with birth preva-
lence ranging from 1 in 500 to 1 in 2000 depending
on the population. Although the severity of orofacial
cleft anomalies varies, multidisciplinary treatment is
often necessary and may include craniofacial surgery;
specialized dental and orthognathic treatment; speech
and hearing intervention; and educational, psychologi-
cal, and social assessment and intervention. The mul-
tidisciplinary nature of cleft care was realized even in
the first recorded surgical repair of a cleft lip (in the
annals of the Chin dynasty in China, about A.D. 390
[1])—detailed postoperative instructions were listed
for optimal results.
Orofacial clefts represent a significant public
health problem due to the significant lifelong mor-
bidity and complex etiology of these disorders. The
extensive psychological, surgical, speech, and dental
involvement emphasize the importance of under-
standing the underlying causes of CL/P to optimize
treatment planning, to predict the long-term course
of any affected individual’s development, to improve
recurrence risk estimation, and to provide pre-re-
0094-1298/04/$ – see front matter D 2004 Elsevier Inc. All right
doi:10.1016/S0094-1298(03)00138-X
* Corresponding author. Suite 500, Cellomics Building,
100 Technology Dr., Pittsburgh, PA 15219.
E-mail address: marazita@sdmgenetics.pitt.edu
(M.L. Marazita).
standing of the embryology and genetics of orofacial
clefting is crucial for the development of a biologi-
cally relevant orofacial cleft classification system
[2–4]. The recent identification of specific genes
involved in syndromic and nonsyndromic orofacial
clefting lays the groundwork for cleft classification
based on specific genetic mutations and timing of
craniofacial development rather than on postnatal cra-
niofacial morphology and anatomy [2–5].
This article presents a brief overview of current
concepts in normal and abnormal craniofacial embry-
ology, genetic etiologies of orofacial clefting, and
gene-development interactions that may produce oro-
facial clefts. We encourage the readers to consult more
comprehensive works for additional discussion of
these topics [2–17].
Embryonic development
Early gene expression and signaling molecules in
development
To understand pathologic development, it is fun-
damental to understand and appreciate the complex-
ities of normal development. Genes control early
embryonic development through the production of
transcription factors that can be translated into struc-
tural, regulatory, or enzymatic proteins [10]. These
s reserved.
Table 1
Signaling and growth factors
Factor Abbreviation Derivation Action
Bone morphogenetic proteins BMPs (1–7) Pharyngeal arches;
frontonasal mass
Mesoderm induction; dorso-ventral organizer;
skeletogenesis; neurogenesis
Brain-derived neurotrophic factor BDNF Neural tube Stimulates dorsal root ganglia anlagen
Epidermal growth factor EGF Various organs;
salivary glands
Stimulates proliferation and differentiation of
many cell types
Fibroblastic growth
factors (1–19)
FGFs Various organs and
organizing centers
Neural and mesoderm induction.
Stimulates proliferation of fibroblasts,
endothelium, myoblasts, osteoblasts
Hepatocyte growth factor HGF Pharyngeal arches Cranial motor axon growth; angiogenesis
Homeodomain proteins Hox-a, Hox-b,
PAX
Genome Craniocaudal and dorsoventral patterning
Insulin-like growth
factors 1 and 2
IGF-1
IGF-2
Sympathetic
chain ganglia
Stimulates proliferation of fat and connective
tissues and metabolism
Interleukin-2, Interleukin-3,
Interleukin-4
IL-2, IL-3, IL-4 White blood cells Stimulates proliferation of T-lymphocytes;
hematopoietic growth-factor; B-cell growth factor
Lymphoid enhancer factor 1 Lef1 Neural crest;
mesencephalon
Regulates epithelial–mesenchymal interactions
Nerve growth factor NGF Various organs Promotes axon growth and neuron survival
Platelet-derived growth factor PDGF Platelets Stimulates proliferation of fibroblasts, neurons,
smooth muscle cells, and neuroglia
Sonic hedgehog SHH Various organs Neural plate and craniocaudal
patterning, chondrogenesis
Transcriptional factors TFs Intermediate gene
in mesoderm
induction casade
Stimulates transcription of actin gene
Transforming growth factor-a TGF-a Various organs Promotes differentiation of certain cells
Transforming growth factor-b(Activin A, Activin B)
TGF-b Various organs Mesoderm induction; potentiates or inhibits
responses to other growth factors
Vascular endothelial
growth factor
VEGF Smooth muscle cells Stimulates angiogenesis
Wingless WNT Genome Pattern formation; organizer
From Sperber GH. Craniofacial development. Hamilton, Ontario: B.C. Decker; 2001; with permission.
M.L. Marazita, M.P. Mooney / Clin Plastic Surg 31 (2004) 125–140126
growth factors and morphogens (Table 1) then target
specific embryonic cell populations and their signal
transduction pathways, resulting in the progressive
differentiation, migration, shape changes (morphoge-
netic movements), and programmed cell death (apo-
ptosis) of these cells. These specific activities bring
different groups of embryonic cells into close prox-
imity with each other where inductive biochemical
and biomechanical interactions between these cell
groups may cause certain cell populations to differ-
entiate on their own, even without the continued
presence of the inducing tissue [13]. The molecular
regulation of such interactions and the mechanisms
by which ‘‘pattern’’ development occurs within a
population of cells gives rise to different tissue types
and individual structures, such as bones, muscles,
and teeth.
Although the presence, concentration gradients,
and diffusion patterns of growth factors and signaling
molecules are essential for normal morphogenesis,
intercellular communication and selective permeabil-
ity of cell membranes also act to control and regulate
development. Growth factors stimulate cell prolifer-
ation, differentiation, and permeability through two
general mechanisms. One mechanism involves cer-
tain growth factors (eg, steroids, retinoic acid, and
thyroxin) passing through the plasma cell membrane,
binding with specific receptors, and acting directly on
the genes to alter their function. The second mecha-
nism involves certain other growth factors (eg, fibro-
blast growth factors [FGFs], transforming growth
factor-beta superfamily [TGF-bs], and epidermal
growth factor [EGF]) binding with specific cell sur-
face receptors, activating intracellular signaling path-
Fig. 1. Signaling factors and target genes at different locations and stages of development. (From Johnston MC, Bronsky PT.
Craniofacial embryogenesis: abnormal developmental mechanisms. In: Mooney MP, Siegel MI, editors. Understanding
craniofacial anomalies: the etiopathogenesis of craniosynostosis and facial clefting. New York: John Wiley and Sons; 2002.
p. 61–124; with permission.)
M.L. Marazita, M.P. Mooney / Clin Plastic Surg 31 (2004) 125–140 127
ways (eg, Smad, map-kinase), and eventually causing
gene activation by paracrine activation (Fig. 1). Di-
rect gene activation (the first mechanism) uses ‘‘long
distance’’ endocrine signaling, which is typically more
systemic, potent, of longer duration, and less suscep-
tible to interruption and insult compared with ‘‘short-
distance’’ and localized paracrine signaling. Growth
factors and molecules that function via endocrine
signaling typically are powerful morphogens and
are more potent inducers of craniofacial malforma-
tions [13].
Many signaling molecules (and their receptors)
may be substituted for one another and are present
throughout life. They change their function in the
presence of different concentrations or classes of
growth factors or receptors [10,13]. This redundancy
may account, in part, for the developmental ‘‘plastic-
ity’’ noted during embryogenesis and evolution
[6,7,13]. Gene-controlled, growth factor-induced cell
migrations and cell fusions (fusomorphogenesis) are
essential to organogenesis and normal embryonic
growth [10,18]. Interruptions in these processes typi-
cally produce embryonic death or congenital malfor-
mations [10,13,14].
Germ layer differentiation, neurulation, and midline
malformations
Once the parental sex cells unite and reestablish
the haploid state, the zygote and later the embryo
(1 and 2 weeks postconception) initiate a rapid flurry
of cell growth and differentiation, directed in part by
homeobox genes [10,19]. From this rapidly prolifer-
ating blastocyst develops two distinct germ cell layers
(the embryonic or bilaminar disc stage) by 2 weeks
postconception. During week 3 postconception, the
bilaminar disc is converted into a trilaminar disc
through the process of gastrulation while still under
the direction of homeobox genes [10,19]. It is from
these three primary germ layers (the endoderm, the
mesoderm, and the ectoderm) that the basis of all
subsequent tissue and organ formation arises [9,10].
Hall [6,7,20] suggests that the neural crest cells are a
fourth germ layer in vertebrates.
During week 3 postconception, the neural plate is
derived from the neuroectoderm and extends along
the burgeoning long axis of the disc, forming the
bilateral neural folds and neural tube [10,21]. This is
referred to as the process of neurulation and helps to
M.L. Marazita, M.P. Mooney / Clin P128
determine embryonic polarity (ie, head and tail ends)
[10,21–23]. PAX6, Sonic Hedge-Hog (SHH), and
FGF signaling are involved with neurulation and
with eye formation during this stage (see Table 1
for a summary of genes involved) [10,13,22,23].
The brain and developing placodes are essential for
driving cephalogenesis [5,10,13,24]. Problems in
development during this time may result in midline
neurologic and craniofacial malformations such as
holoprosencephaly (single cavity forebrain), cyclo-
plegias, neural tube defects, and midline orofacial
clefts [5,9,10,13].
Neural crest cell formation, migration, and
differentiation
The ectodermal-derived cells that are found in the
margins of the bilateral neural folds and the transition
zone between the neuroectoderm and epidermis are
referred to as neural crest cells [7,10,13,25–27]. Neu-
ral crest cells migrate as mesenchyme into the devel-
oping embryonic processes of the head and neck
region during neural tube closure (4 weeks postcon-
ception). The pluripotent neural crest stem cells give
rise to a tremendous diversity of cell and tissue types
(eg, neural, pigment, skeletal, connective tissue, car-
diac, dental, and endocrine cells) [6,7,9,10,13,
25–28]. Hall [6,7,20] has suggested that neural crest
cells should be considered a fourth germ layer in
craniate vertebrates because mesoderm and neural
crest cells give rise to a diversity of embryonic meso-
derm. Hall further argues that if mesoderm qualifies
as a secondary germ layer (it is derived secondarily
from ectoderm), then so do neural crest cells.
Neural crest cells migrate in a segmental pattern,
predetermined in part by interactions with hindbrain
neuromeric segments called rhombomeres and par-
axial mesoderm segments called somatomeres (Fig. 2)
[7,10,13]. The neural crest segments migrate into the
developing pharyngeal arches and provide the pre-
cursors of cartilage, bone, muscles, and connective
tissues of the head and neck. The timing and extent of
neural cell migration and differentiation is dependent
on a complex patterning of inductive homeobox gene
(HOX, MSX) signaling between the neural crest and
adjacent neural tube, lateral plate mesoderm, and
epidermis (Fig. 2 and Table 1) [7,10,13,25–27]. De-
ficiencies in neural crest tissue migration or prolif-
eration produce a varied and extensive group of
craniofacial malformations referred to as neurocristo-
pathies, which include von Recklingshausen neuro-
fibromatosis, hemifacial microsomia, orofacial clefts,
and DiGeorge and Treacher Collin syndromes [5,8,
10,13,25,29].
Craniofacial development
The primitive craniofacial complex forms during
week 4 postconception after neural crest tissue migra-
tion, early brain vesicle enlargement, and cranio-
caudal and lateral trunk folding of the trilaminar
disc. Trilaminar disc folding helps incorporate the
endoderm into the body, which in part forms the
mucoepithelial lining of the stomodeum and primitive
oral cavity [9,10,21,30]. A series of inductive events
between the prosencephalon, mesencephalon, and
rhombencephalon and the neural crest tissue that
migrates into the craniofacial complex and pharyngeal
arch apparatus (Fig. 3) helps to form the five facial
prominences (the frontonasal and the bilateral maxil-
lary and mandibular prominences) (Fig. 4) [9,10,
26,31]. It is the differentiation, growth, and eventual
fusion of these prominences that forms the definitive
face. The movement and destination of neural crest
tissues into the facial primordia are controlled in part
by a number of gene families, including (see Table 1)
regulatory homeobox genes (HOXa-1, HOXa-2,
HOXb-1, HOXb-3, and HOXb-4), the SSH gene,
the OTX gene (orthodentical homeobox), the GSC
gene (goosecoid), DLX genes (Drosophila distal-less
homeobox), MSX genes (muscle segment homeo-
box), LHX genes (LIM homeobox), and PRRX genes
(paired-related homeobox) [11,17].
Primary palatogenesis
Normal development. The primary palate is defined
as the portions of the facial primordia that initially
separate the oral and nasal cavities and include the
portions of the medial and lateral nasal processes of
the frontonasal process and the portion of the maxil-
lary processes that contribute to the separation of the
cavities (Fig. 4) [11,13,32]. Normal primary palato-
genesis involves a series of local molecular and
cellular events that are closely timed. Spatial and
biomechanical changes associated with craniofacial
growth must occur in sequence within a critical period
in development (in humans during week 5 to week 7
postconception). The primary palate initially forms
around the developing olfactory placodes with the
rapid proliferation of the lateral epithelium and un-
derlying mesenchyme. These events are controlled in
part by FGFs (FGF8 and FGFR2), bone morphoge-
netic proteins (BMP4 and BMP7), SHH, and retinoic
acid [5,13,32]. Diewert et al [32] have shown that in
human and rodent embryos, as the facial prominences
enlarge around the nasal pits to form the premaxillary
region, growth of supporting brain and craniofacial
components change facial morphology and can affect
the timing, the location, and the extent of contact
lastic Surg 31 (2004) 125–140
Fig. 2. Schematic presentation of the inductive, segmental relationships of different anatomical components in the developing
embryonic head and neck. (A) An overlay of all inductive components showing approximate spacial relationships. (B) Axial and
central nervous system structures. (C) Neural crest cells. (D) Paraxial mesoderm and somatomeres. (E) Arteries and
cardiovascular system. (F) Pharynx and endoderm derived structures. (Modified from Noden DM. Cell movements and control of
patterned tissue assembly during craniofacial development. J Craniof Genet Dev Biol 1991;11:192–213; with permission.) [83]
Fig. 3. The homeotic gene complex of Drosophila (HOM) has been duplicated more or less intact in four different complexes
on different chromosomes of the mouse and human. The same head-to-tail (rostrocaudal) sequence in the chromosomes has been
preserved and corresponds roughly to rostrocaudal gene expression in the neural plate (tube) and neural crest, which is derived
from the neural plate (tube). The newer terminology is used for individual genes, with the older terminology used for mice in
parentheses. Depending on chromosomal positioning, the genes are arranged in paralogous groups 1 through 5 (and beyond) and,
in general, these genes are expressed in a sequential overlapping cascade with one or more active genes being added every two
neuromeres. (Modified from Noden DM. Cell movements and control of patterned tissue assembly during craniofacial de-
velopment. J Craniof Genet Dev Biol 1991;11:192–213; with permission.)
Fig. 4. Stages of facial formation at (A) 4 weeks, (B) 5 weeks, (C) 6 weeks, and (D) 7 weeks. (From Sperber GH. Craniofacial
development. Hamilton, Ontario: B.C. Decker; 2001; with permission.)
M.L. Marazita, M.P. Mooney / Clin Plastic Surg 31 (2004) 125–140 131
between the facial prominences. At the same time, the
forebrain elevates as the cranial base angle decreases,
the medial nasal region narrows, and the maxilla
grows forward to meet the medial and lateral nasal
prominences that relocate with growth of the fore-
brain (see Fig. 4). The upper lip is completed on either
side of the globular prominence (see Fig. 4) by fusing
with the freely projecting medial nasal prominences
of the frontonasal prominence [33]. Such fusion
requires critically timed coordination of growth be-
tween the processes, exact spatial localization, and
apoptosis (or further differentiation) of the epithelium
that forms the transient nasal bridge or fin between
the two processes [32]. The degradation of the un-
derlying nasal fin allows for the uninterrupted move-
ment of mesenchymal cells between the medial and
lateral components of the upper lip by 7 weeks post-
conception (see Fig. 4) [9,10,13,32]. Abnormal de-
velopment of this epithelium may be involved with
clefts of the primary palate.
Additional structures in the primary palate include
the dentition, alveolar and basal bone of the primary
palate, and labial musculature. Typically, four tooth
buds start to develop in the primary palate, anterior to
M.L. Marazita, M.P. Mooney / Clin Plastic Surg 31 (2004) 125–140132
the incisive fissure, about 4 weeks postconception
[11]. Tooth bud formation is dependent on a large
number of genes (PAX9, MSX1, SHH, DLX, WNT)
and growth factors (nerve growth factor, FGF, and
BMPs), which are expressed in the oral ectoderm and
underlying neural crest tissue [11]. Around 7 weeks
postconception, myogenic mesenchyme, derived from
the sixth somite, migrates into the lip primordia with
accompanying branches of the facial nerve (CNVII)
[9,10,33,34]. Ossification of the primary palate be-
gins around the 8 weeks postconception in the me-
dial nasal prominence and continues laterally to the
maxillary process [11,35].
Orofacial clefting of the primary palate. Many de-
fects in the orofacial tissues that form the primary
palate and surround and support the sensory units are
expressed morphologically as failures of facial promi-
nence merging or fusion resulting in clefts [5,10,11,
13,32]. These defects can be classified as those that
affect the midline (median facial clefts) and those that
occur laterally (lateral facial clefts). Median facial
defects occur early and probably relate closely to the
initial events directing morphogenesis of the anterior
Fig. 5. Defects of orofacial development. (A) Unilateral cleft lip.
cleft lip and nasal defect. (Modified from Sperber GH. Craniofa
with permission.)
midline tissue of the trilaminar disc [5]. Lateral facial
clefts can be conceptualized as defects resulting from
abnormal events usually occurring later in develop-
ment once the facial primordia are in place. It is un-
likely that median and lateral facial cleft defects
are simply the result of single genetic aberrations be-
cause normal craniofacial development results from
many genes inhibiting or enhancing the expression
of others; thus, identifying specific cleft mechanisms
has been difficult.
Additional structures that can be affected by pri-
mary palatal clefting include the dentition, alveolar
and basal bone of the primary palate, and the labial
musculature. Primary palatal clefting occurs most
commonly between the primary and secondary pal-
ates at the incisive fissure that separates the lateral
incisors and canine teeth. Individuals with clefts of
the primary palate may present with dental displace-
ment or dental agenesis from premaxillary hypoplasia
[11]. Labial defects typically involve discontinuity of
the circumoral musculature and reduced lip muscle
volume in cleft embryos and fetuses [33,34]. Re-
cent work from our laboratory [36,37] has detected
subclinical orbicularis oris muscle anomalies visual-
(B) Bilateral cleft lip. (C) Oblique facial cleft. (D) median
cial development. Hamilton, Ontario: B.C. Decker; 2001;
M.L. Marazita, M.P. Mooney / Clin Plastic Surg 31 (2004) 125–140 133
ized using ultrasonography in the ‘‘unaffected’’ rela-
tives of cleft probands. Such morphologies may result
from an initial mesenchymal deficiency during pri-
mary palatogenesis. Embryos and fetuses with clefts
of the primary palate also show delayed ossification
and decreased volume of the premaxilla and anterior
basal bone of the maxilla compared with age-matched
control subjects [35,38]. Such bony morphologies
may result from an initial mesenchymal deficiency
during primary palatogenesis or from later bone re-
sorption due to a lack of functional forces on the pri-
mary palate [39–41].
Secondary palatogenesis
Normal development. The secondary palate is de-
fined as the portions of the facial primordia posterior
to the primary palate and includes the two lateral
palatal processes that project medially from the max-
illary processes. The primordia of the secondary pal-
ate forms the hard (bony) palate, the soft palate (the
velum), the alveolar and basal bone of the maxillae,
and the associated dentition posterior to the incisive
fissure (Fig. 6) [12,13,32].
As with primary palatogenesis, closure and fusion
of the secondary palate requires a complex interaction
of palatal shelf movements, critically timed coordi-
nation of growth between the processes, and apopto-
sis (or further differentiation) of the epithelium along
medial margins of the palatal shelves [2,12,13,32].
During week 8 postconception, the palatal shelves ro-
tate from a vertical position surrounding the tongue
and elevate into horizontal approximation [12,13,32],
with a slight delay in this process noted in female
embryos [42]. Rapid palatal shelf elevation is thought
to result from a number of mechanisms, including de-
Fig. 6. Cleft palate variations. (A) Bifid uvula. (B) Unilateral cleft p
GH. Craniofacial development. Hamilton, Ontario: B.C. Decker; 2
velopmental changes in the connective tissue matrix
and associated glycosaminoglycans of the shelves
leading to hydration, swelling, and rapid elevation; a
change in shelf vascularity leading to increased tissue
fluid pressure and turgor; rapid differential mitotic
growth of the shelf mesenchyme; and movements
of the tongue, facial, and suprahyoid musculature
leading to cranial flexion, swallowing and mandibular
depression, tongue withdrawal from the cleft, and
hence shelf closure [10–13,32]. FGF8 and SHH
expression are found along the medial edge of the
maxillary prominence and presumably are involved in
growth and elevation of the palatal shelves [13]. Once
the palatal shelves are elevated and approximated,
adhesive contact, seam fusion along the medial edges,
and apoptosis of the epithelium are essential for
normal secondary palatogenesis. An increased ex-
pression of the cell adhesion molecule syndecan is
seen during shelf elevation. An increased expression
of TGF-b3 and N-cadherin is also seen along the
medial margins of the palatal shelves, both of which
may cause epithelial apoptosis and differentiation
[12,13,43,46,47].
Before shelf elevation, the tongue-mandibular
complex is small relative to the nasomaxillary com-
plex. The tongue is positioned immediately ventral to
the cranial base, and the head posture is flexed against
the thorax. At the time of palatal shelf elevation, the
tongue and mandible extend beneath the caudal por-
tion of the primary palate, the nasomaxillary complex
lifts up and back relative to the body, and the palatal
shelves elevate above the tongue to occupy the oro-
nasal cavity space. As closure of the secondary palate
progresses, the prominence of mandible increases
and the tongue, attached to the anterior region of
alate and lip. (C) Bilateral cleft palate and lip. (From Sperber
001; with permission.)
M.L. Marazita, M.P. Mooney / Clin Plastic Surg 31 (2004) 125–140134
Meckel’s cartilage via the genioglossus and genio-
hyoid muscles, also becomes positioned forward in
the oral cavity [32].
Normal fusion of the palatal shelves and primary
palate produces a relatively flat, unarched roof, and
the lines of fusion are seen in the adult skull as the
incisive fissure and midpalatal suture. Ossification of
the palate proceeds from the lateral palatal shelves
and the premaxilla during week 8 postconception.
Myogenic mesenchymal tissue from the first and
fourth pharyngeal arches migrates into the soft palate
and fauces, which accounts for multiple innervation
of the regional musculature—the tensor veli palatini
muscle by the trigeminal nerve (CNV2) and levator
veli palatini and other muscles by the vagus nerve
(CNX) [9–12,30].
Orofacial clefting of the secondary palate. Defects
of the secondary palate are expressed morphologi-
cally as failures of elevation, failures of contact and
adhesion, or failures of fusion resulting in clefts [5,10,
11,13,32]. In humans and in animal models for cleft
palate, wide clefts usually result when shelves remain
in the vertical position, whereas narrow clefts usually
indicate elevated shelves that failed to contact and
fuse or that failed to fuse even if contact was made
[32,44–47]. Major factors shown to limit shelf con-
tact include delayed shelf movement to the horizontal
position, reduction in palatal shelf size, deficient
extracellular matrix accumulation, delayed achieve-
ment of mandibular prominence, head extension (thus
an increase in facial vertical dimension), abnormal
craniofacial morphology, abnormal first arch devel-
opment, increased tongue obstruction of shelf move-
ment secondary to mandibular retrognathia, growth
retardation or chondrodysplasia in Meckel’s cartilage
and increased tongue obstruction to shelf movement
and palatal closure, and amniotic sac rupture leading
to severely constricted fetal head and body posture
[13,32].
Genetic etiologies of orofacial clefting
In this section we summarize the current evidence
regarding genetic etiologies for cleft lip and cleft pal-
ate. Orofacial clefts can occur as part of Mendelian
syndromes, as part of the phenotype resulting from
chromosomal anomalies, or as the result of prenatal
exposure to certain teratogens. Orofacial clefts dem-
onstrate remarkable differences in frequency by gen-
der and laterality. There is an approximate 2:1 ratio
of males to females for CL/P, although slightly
more females than males have CP. Within unilateral
clefts, the ratio of left-sided to right-sided clefts is
also about 2:1. Orofacial cleft birth prevalence shows
a wide range, from about 1/500 births to about
1/2000, depending on population; in general, Asian
and Amerindian populations have the highest fre-
quencies, and African-derived populations have the
lowest frequencies.
Over 300 syndromes exist in which orofacial
clefts are part of the phenotype; about half of these
are due to Mendelian inheritance of alleles at a single
genetic locus. Much progress has been made in recent
years in delineating Mendelian disorders and in gene
discovery of such disorders (refer to the Online
Mendelian Inheritance in Man database available
on the NCBI web site [48] for a catalog of such
disorders). However, only a small portion of indi-
viduals with orofacial clefts has a known etiology
[16,49]. The majority of orofacial clefts are non-
syndromic and are considered complex traits. Given
the public health importance of orofacial clefts [50],
many etiologic studies have been conducted of non-
syndromic orofacial clefts, and many environmental
and genetic factors have been implicated [10,11,13,
32,51,52].
Many genes control early embryonic development
through the production of transcription factors that
can be translated into structural, regulatory, or enzy-
matic proteins [10] (see Table 1); therefore, it is not
surprising that scientists have long felt that orofacial
clefts have a familial basis. The first published de-
scription of a family with several affected members
was in 1757 [53]. Charles Darwin [54] pointed out a
publication of ‘‘the transmission during a century of
hare-lip with a cleft-palate’’ by Sproule [55] describ-
ing the author’s family. Since those early publications,
many statistical analyses of family datasets have been
undertaken to better understand the inheritance pat-
terns of orofacial clefting [56]. The multifactorial
threshold model was proposed to explain many of
the features of nonsyndromic orofacial clefts (such as
the altered gender ratio); however, the predictions of
that specific model could be rejected when tested in
several populations. In the early years of the 20th
century, several seminal works were published regard-
ing the inheritance patterns of orofacial clefts, but
until recently progress has been slow in determining
the exact genes involved. Segregation analyses [56]
and statistical analyses of familial recurrence risk
patterns [57] are consistent with hypotheses of ma-
jor locus involvement or relatively few loci (on the or-
der of 3–14 loci [57]) interacting to cause orofacial
clefts. With statistical evidence that orofacial cleft
family patterns were consistent with genetic inheri-
tance, several groups began linkage and association
M.L. Marazita, M.P. Mooney / Clin Plastic Surg 31 (2004) 125–140 135
studies to identify the genes contributing to the famil-
iality of orofacial clefts.
Etiologic insights from embryology
Most of the environmental and genetic factors im-
plicated in orofacial clefting of the primary palate
[10,11,13,32,51,52] are postulated to produce clefts
by interrupting facial prominence merging or fusion.
A failure of normal disintegration of the nasal fin
or inadequate mesenchymal migration between the
maxillary and medial nasal processes results in bilat-
eral or unilateral clefting of primary palate (ie, lip or
maxillary alveolus) (Fig. 5). Embryonic face shape
[32,58] has also been shown to be related clefts of
the primary palate. Mouse embryos from strains ge-
netically predisposed to primary palatal clefting (the
A/J, A/WySn, and CL/Fr strains) had medial nasal
prominences that were more medially convergent
than normal strain embryos, resulting in decreased
contact with the lateral nasal prominences and a
greater chance of failure of consolidation of tissues.
Embryonic face shape has also been shown to be a
causal factor in genetic predisposition to cleft lip in
mice [59]. Strains susceptible to spontaneous clefts
of the primary palate had a significantly smaller dis-
tance between the nasal pits, different orientation of
medial nasal prominences, a reduction (or absence)
of epithelial activity throughout the developmental
period of primary-palate fusion, and hypoplasia of
the lateral nasal prominences compared with control
strains [13,32]. Embryonic face shape, as a predispo-
sition for primary palatal clefting, may also help ex-
plain the observed ethnic (Asian derived > European
derived > African derived [60]) and gender differ-
ences (males 2:1 over females) in the frequencies of
primary palatal clefting [25,61,62].
There are also clues from our understanding of
embryology with implications for the etiology of the
secondary palate. Hypothesized mechanisms include
abnormal TGF-b isoforms in cleft palate individuals
[63]; unusually wide faces (especially in Asian pop-
ulations), which could move palatal shelves further
apart and prevent adhesion and fusion [64–66] and
which could partly explain the ethnic variability in
palatal clefting (Asian derived > European derived >
African derived) [25,61,62]; tongue-tie (which could
inhibit protrusion of the tongue during shelf eleva-
tion) in a familial form of cleft palate in Iceland [30];
macroglossias in MZ twins discordant for cleft palate
[25]; and a small mandible, as in Pierre Robin se-
quence [13].
When clefts of the primary and secondary palates
are present together (cleft lip plus cleft palate), failure
of secondary palatal closure is thought to occur as a
by-product of the primary palate cleft because of the
resulting alterations in the tongue and palatomaxillary
relationships [67,68].
Chromosomal anomalies
Orofacial clefting is seen as part of the phenotype
in a wide variety of types of chromosomal rearrange-
ments of many chromosomes, including trisomies,
duplications, deletions, micro-deletions, or cryptic
rearrangements [69,70]. Rearrangements that can in-
clude clefts of the primary palate (F the secondary
palate) include deletions of 4p (Wolf-Hirschhorn syn-
drome), 4q or 5p (Cri-du-chat syndrome); duplica-
tions of 3p, 10p, and 11p; and trisomy 13 or 18 (and
trisomy 9 mosaic) [69,70]. Clefts of the secondary
palate alone are seen with deletions of 4q and 7p;
duplications of 3p, 7p, 7q, 8q, 9q, 10p, 11p, 14q, 17q,
19q; and trisomy 9 or 13 [69,70].
The role of micro-deletions and other cryptic
rearrangements in orofacial cleft etiology has recently
been recognized [16]. Such small rearrangements are
notable in cleft etiology because they are often
transmitted within families, unlike the larger rear-
rangements that are more likely to be de novo. Micro-
deletions of 22q11.2 are now known to be the
common etiology for at least three clinically classi-
fied syndromes with clefts of the secondary palate as
a frequent feature (DiGeorge syndrome, velocardio-
facial syndrome, and conotruncal anomaly face syn-
drome; for more details see Gorlin et al [8]).
Single gene etiologies
Almost 300 syndromes have been described in
which a cleft of the lip or palate is a feature [4,8].
About half of those syndromes are due to Mendelian
inheritance of alleles at a single genetic locus, and
great strides have been made in recent years in
mapping genes for such Mendelian disorders. Analo-
gous to the diversity seen in chromosomal abnormal-
ities leading to clefts, every possible Mendelian
pattern is observed in the syndromes that include
orofacial clefts in their phenotypes. About 50% follow
autosomal recessive inheritance, 40% follow autoso-
mal dominant inheritance, and 10% follow X-linked
inheritance (recessive or dominant). Complications
commonly seen in other Mendelian disorders are also
seen in clefting syndromes, such as reduced pene-
trance, variable expressivity, imprinting, allelic het-
erogeneity, and locus heterogeneity. Some patterns of
anomalies can be due to cytogenetic rearrangements
or Mendelian segregation.
M.L. Marazita, M.P. Mooney / Clin Plastic Surg 31 (2004) 125–140136
Of the 150 Mendelian clefting syndromes, ap-
proximately 30 genes have been cloned [29]. These
genes fall into various classes, including transcription
factors (GLI3, 7p13; PAX3, 2q35—Waardenburg
syndrome; SIX3, 2p21—holoprosencephaly 2; and
SOX9, 17q24.3-q25.1—Camptomelic dysplasia),
extracellular matrix proteins (COL2A1,12q13.1-q
13.2—Stickler syndrome type I; COL11A2, 1p21—
Stickler syndrome type II, and GPC3, Xp22—Simp-
son-Golabi-Behmel syndrome), and cell signaling
molecules (FGFR2, 10q26, Apert-Crouzon syn-
drome; PTCH, 9q22.3—basal cell nevus syndrome;
and SHH, 7q36, holoprosencephaly 3). A full de-
scription of the syndromes that can include an oro-
facial cleft is beyond the scope of this article; please
refer to the online data resources [48] for more
complete details.
One of the major reasons to map and clone genes
for syndromic forms of clefting is to help develop
strategies for delineating the etiology of nonsyndromic
clefting that is by far more common than the syn-
dromic forms. Van der Woude syndrome (VDWS, 1q)
is a clearly Mendelian syndrome that has a phenotype
only slightly more complicated than isolated clefting
(ie, families segregating the VDWS gene exhibit
orofacial clefts [CL/P or CP] paramedian lip pits of
the lower lip, and sometimes hypodontia). VDWS
follows an autosomal dominant inheritance pattern,
with reduced penetrance (individuals carrying the gene
who show no phenotypic features) and variable ex-
pressivity (individuals expressing the phenotype may
have a cleft or lip pits or both, with varying degrees of
severity). Furthermore, VDWS is rare among syn-
dromic forms of clefting in that clefts of the secondary
and primary palates are seen in the same families. The
gene responsible for VDWS (ie, IRF6) has been
recently identified [71] and has shown a strong asso-
ciation with nonsyndromic clefting in a large series of
families from several different populations [72].
Genetic etiologies of nonsyndromic orofacial clefts
Background
Early estimates of the genetic contribution to non-
syndromic orofacial clefts ranged from about 12% to
20%, with the remainder attributed to environmental
factors or gene–environment interactions [73,74]. Es-
timates from more recent studies suggest that about
20% to 50% may be more realistic [49,75–77]. Two
general approaches have been taken to investigate
genetic factors involved in nonsyndromic orofacial
clefting: large scale family studies and linkage/asso-
ciation studies with specific genetic markers.
Statistical segregation analyses of orofacial clefts
investigating primary or secondary cleft palate in
large series of families have consistently resulted in
evidence for genes of major effect [56]. Although one
interpretation of such studies is inheritance at a single
major locus, hypotheses of multiple interacting loci or
genetic heterogeneity cannot be ruled out and were
not explicitly tested in any of the published segrega-
tion analyses to date [56]. Statistical analyses of re-
currence risk patterns [57] have been consistent with
oligenic models with 3 to 14 interacting loci. With
evidence that orofacial cleft family history patterns
are consistent with one or a few loci, there are now
many groups attempting to identify those genes using
the positional cloning approach, beginning with link-
age and association analyses.
Linkage and association studies
The procedures for mapping, cloning, and char-
acterizing genes are now well established, with many
successes for rare Mendelian traits. If nonsyndromic
orofacial clefts can be shown to be linked to or as-
sociated with a marker of known genetic location, it
would be powerful support for a Mendelian genetic
contribution to the etiology. However, only in recent
years have investigators attempted such studies be-
cause nonsyndromic clefting was considered to fol-
low the multifactorial threshold model [78] and thus
would not be amenable to a linkage approach. With
emerging statistical evidence from human family
studies and from knockout mouse experiments in
which one or a few gene(s) can explain clefting etio-
logy, linkage and association studies were launched
in a variety of populations [16,56,57].
Linkage analyses assess the co-segregation of
alleles at a genetic locus of known chromosomal lo-
cation (marker) and a disease locus. Different marker
alleles thus co-segregate with the disease allele in
different families, and the overall frequencies of the
marker alleles calculated from population-based sam-
ples need not vary between affected and control
groups. In this situation, the two loci are said to be
in linkage equilibrium (ie, linked but not associated).
In contrast, if allele frequencies differ significantly
between the affected and control groups, the specific
allele at the marker or candidate locus is said to be
associated with the disease at the population level,
with the most common interpretation of an associa-
tion being linkage disequilibrium. Association meth-
ods are used as an adjunct to linkage approaches for
gene mapping, especially for complex traits [79,80].
Gene mapping studies of orofacial clefts have used
linkage and association methods. Candidate loci or
regions on seven chromosomes (ie, chromosomes 1, 2,
Table 2
Evidence for genes and regions potentially involved in non-
syndromic orofacial clefts
Candidate genesa
Chromosomal
region
Animal
modelsbCandidate
genecGenome
scand
1p36-31 SKI1, LHX8:
K/O, E
MTHFR
(L, A)
L
1q32 IRF6: E IRF6 (A) M
2p13 TGFA: E TGFA (A) L, M
2q35 L, M
3p25 A, M
3q26 TP63: K/O, E L
4p16 MSX1: K/O, E MSX1 (L, A) A
4q31 L
5p15 L, A
6p23 TFAP2A:
K/O, E
F13A1 (L, A) L, M
6q25 L, M
7p13 A, M
7q21 L, A, M
8p21 L, M
8q23 L, M
9q21 L, M
10q25 L
11p12 L, A
12p11 L, A, M
14q12 TGFB3: K/O, E TGFB3 (A) L, M
15q22 L, M
16q L
17p11 L
17q12 KCNJ2: K/O RARA (A) M
18q23
19q13 APOC2/BCL3
(L, A)
L, M
20p12 L, A
Xq21 TBX22: E L
a Candidate genes: genes potentially involved in oro-
facial clefting with evidence from animal models or human
linkage and association studies.b Animal models: genes investigated in animal models
with phenotypes that include clefting. K/O = knockout, E =
expression studies.c Candidate genes/regions: genes and regions with at
least two positive reports of linkage (L) or association (A) in
the literature [16].d Genome scan: regions with positive linkage (L) or
association (A) results with anonymous markers spaced
V10 cM apart throughout the genome (one or more genome
scans [80,81]). M = positive meta-analysis results over all
genome scans.
M.L. Marazita, M.P. Mooney / Clin Plastic Surg 31 (2004) 125–140 137
4, 6, 14, 17, and 19) have positive linkage or asso-
ciation results in CL/P, CP, or both; Table 2 summa-
rizes those candidate genes [16,77]. There are a few
additional loci and chromosomal regions that have
only negative results reported in the literature and are
not presented in detail here. Also, there are many
studies for some loci and few studies for others—this
is not a reflection of the strength of the evidence for
any particular locus; it is merely a reflection of the
interest in particular loci. Table 2 summarizes evidence
from animal models (knock out and expression stud-
ies) for genes on those chromosomes.
Genome-wide scans
In addition to linkage and association studies of
candidate genes for orofacial clefting, genome-wide
scans of large numbers of anonymous markers (ie,
genetic markers of unknown function whose exact
chromosomal location is known) have been con-
ducted [81,82]. Analyses of recurrence risk patterns
[57] suggest that there may be about 3 to 14 genetic
loci involved in nonsyndromic clefts of the primary
palate (F the secondary palate). Given the contradic-
tory results from candidate locus approaches and
given the availability of dense maps of markers,
studies of orofacial clefting are now turning to ge-
nome-wide scans to simultaneously search for multi-
ple regions. Table 2 summarizes those chromosomal
regions with positive results in either or both of
the two published genome scans for nonsyndromic
clefts of the primary palate (F the secondary palate)
[81,82]. Additional genome scans in other popula-
tions and in larger sample sizes are necessary to
confirm these results. Our group has also conducted
a meta-analysis of the published genome scans plus
several other recently completed scans for nonsyn-
dromic clefts of the primary palate (F the secondary
palate) [84]; Table 2 includes a summary of the results
from the meta-analysis (ie, those regions that gave
statistically significant evidence of linkage in the
meta-analysis). There have not been any genome-
wide scans for isolated clefts of the secondary palate.
Summary
Many mechanisms underlying normal and abnor-
mal craniofacial embryogenesis are well understood.
The genetic factors that provoke abnormal develop-
ment and result in orofacial clefts are not clear, but
much progress has occurred in our understanding.
Genes or chromosomal rearrangements on many chro-
mosomes can lead to syndromes that include orofacial
clefts. This diversity in the mechanisms that can lead
to syndromic clefts highlights the fact that the pro-
cesses leading to the development of the oral cavity
and face are complex and sensitive to disturbances at
M.L. Marazita, M.P. Mooney / Clin Plastic Surg 31 (2004) 125–140138
multiple timepoints or within multiple genetic do-
mains. As for nonsyndromic clefting, large-scale
family studies are consistent with one or a few loci
exerting major effects on phenotypic expression, al-
though no single gene has been identified as a ‘‘nec-
essary’’ locus for development of nonsyndromic
clefts. Rather, the emerging consensus is that the
genetic etiology of nonsyndromic clefting is complex,
with several loci showing significant results in at least
some studies. Some of these loci may be genes for
susceptibility to environmental factors, some may be
modifying loci, and some may be ‘‘necessary’’ loci.
Mutations in genes that are now known to control
early development are logical candidate genes for fu-
ture studies of nonsyndromic orofacial clefting. Con-
tinued genetic analyses and developmental studies are
crucial for eventual understanding of the complex
etiology of these common congenital anomalies.
Acknowledgments
The authors thank Dr. Geoffrey H. Sperber for his
critical reading and constructive comments on the
embryology portion of this manuscript. This work
was supported in part by NIH/NIDCR (DE13078,
MPM; DE09886) (MLM).
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