**3. Classification**

It is often found in epidemiological studies that CL/P and CPO are classified as either "syndromic" or "nonsyndromic." Cases of "nonsyndromic" CL/P and CPO are further categorized as isolated—those without an underlying syndrome or additional, nonsecondary malformations—or multiple—those that have additional malformations that do not form a recognizable syndrome. These distinctions are important epidemiologically, for identifying homogenous subgroups of cases, and clinically, for informing prognosis, recurrence risk, diagnosis, and treatment plan.

#### **3.1. Syndromic**

Individuals with "syndromic" CL/P or CP present with patterns of malformations and/or symptomatology that form a recognizable syndrome of known or unknown origin; hence, the CL/P or CP is part of a syndrome. Recognition of these syndromes is essential for assessing the risks faced by the child, providing the necessary treatment, and counseling the parents. Because the prevalence of associated anomalies varies across different populations of individuals with OFC, better understanding of the epidemiology of these anomalies could aid in the proper identification and characterization of the syndrome, leading to better care for the individual. Syndromes associated with OFC for which the underlying cause is known include chromosomal abnormalities, such as trisomy 13 or 18, Mendelian disorders such as Van der Woude Syndrome and teratogenic exposure.

A guideline for identifying syndromes in individuals with CL/P or CP is outlined by Venkatesh as follows [8]:


#### **3.2. Multiple**

The multiple subset of CL/P and CPO includes those cases that are not a part of a recognizable syndrome and have major other malformations which may involve, but are not limited to, the eye, ear, head, neck, respiratory tract, gastrointestinal tract, and musculoskeletal system [5, 9]. Cases of "multiple nonsyndromic" CL/P and CPO may be classified as such simply by virtue of unrecognized syndromes or undocumented teratogenic exposures. Furthermore, wide variation exists in the classification of associated anomalies in cases of OFC [10].

#### **3.3. Isolated**

Cases of CL/P and CPO that are classified as "isolated" do not have an underlying syndrome or other secondary malformations. Most epidemiological studies of CL/P and CPO focus on those cases that are isolated in hopes to further gain insight into associations.

### **4. Etiology**

Development of the head and face represents one of the most intricate events during embryonic development, synchronized by a network of transcription factors and signaling molecules together with proteins conferring cell polarity and cell-cell interactions. In mammals, the facial region develops from the facial primordia, which consists of the lateral and medial nasal prominences arising from the frontonasal process and the maxillary and mandibular processes arising from the first branchial arch. As demonstrated in **Figure 1**, fusion of medial nasal and maxillary prominences gives rise to the lip and primary palate, while fusion of separate palatal processes arising from the maxillary prominence gives rise to the secondary palate and occurs later during embryogenesis. These processes are known to be dependent, in part, on the migration and differentiation of neural crest cells from the neuroectoderm into the branchial arches [11].

Disturbance of this closely controlled cascade can result in a facial cleft where these facial primordia ultimately fail to meet and fuse or form the proper structures. Historically, OFCs have been classified as either CL/P or CPO [13, 14]. This broad subdivision is consistent with both the distinct developmental origins of the lip/primary palate and the secondary palate and the distinct cellular and genetic etiologies described for CL/P and CPO; cleft palate may occur secondary to or independently from cleft lip. However, there is some epidemiologic evidence suggesting that cleft lip only has distinct etiologic features from cleft lip with palate and should be classified accordingly [15, 16].

 **Figure 1.** Schematic diagrams depicting human craniofacial development and formation of the secondary palate [12]. (a) By the fourth week of embryonic development, neural crest cells have migrated into the craniofacial region to form the frontonasal prominence, paired maxillary processes and the paired mandibular processes. (b) Formation of the nasal pits by the fifth week of embryogenesis divides the frontonasal prominence into paired medial and lateral nasal processes. (c) By the end of the sixth week of embryonic development, the medial nasal processes have merged with one another and with the maxillary processes to form the upper lip and primary palate, whereas the lateral nasal processes form the alae of the nose. The mandibular processes fuse together to form the lower jaw. (d) The secondary palate develops from the maxillary processes as bilateral outgrowths which grow vertically down the side of the tongue during the sixth week of embryogenesis. (e) During the seventh week of embryonic development, the palatal shelves elevate to a horizontal position above the tongue, make contact with one another and begin to fuse. (f) Fusion of the secondary palatal shelves with one another and with the primary palate and nasal septum is completed by the tenth week of embryogenesis. Figure is adapted from [12] © (2009) John Wiley and Sons Ltd.

#### **5. Genetics**

• Physical examination: measurement of weight, length or height, and occipitofrontal circumference, identification of anomalies of eyes, ears, heart, extremities, and also to look for

The multiple subset of CL/P and CPO includes those cases that are not a part of a recognizable syndrome and have major other malformations which may involve, but are not limited to, the eye, ear, head, neck, respiratory tract, gastrointestinal tract, and musculoskeletal system [5, 9]. Cases of "multiple nonsyndromic" CL/P and CPO may be classified as such simply by virtue of unrecognized syndromes or undocumented teratogenic exposures. Furthermore, wide variation exists in the classification of associated anomalies in cases of

Cases of CL/P and CPO that are classified as "isolated" do not have an underlying syndrome or other secondary malformations. Most epidemiological studies of CL/P and CPO focus on

Development of the head and face represents one of the most intricate events during embryonic development, synchronized by a network of transcription factors and signaling molecules together with proteins conferring cell polarity and cell-cell interactions. In mammals, the facial region develops from the facial primordia, which consists of the lateral and medial nasal prominences arising from the frontonasal process and the maxillary and mandibular processes arising from the first branchial arch. As demonstrated in **Figure 1**, fusion of medial nasal and maxillary prominences gives rise to the lip and primary palate, while fusion of separate palatal processes arising from the maxillary prominence gives rise to the secondary palate and occurs later during embryogenesis. These processes are known to be dependent, in part, on the migration and differentiation of neural crest cells from the neuroectoderm into

Disturbance of this closely controlled cascade can result in a facial cleft where these facial primordia ultimately fail to meet and fuse or form the proper structures. Historically, OFCs have been classified as either CL/P or CPO [13, 14]. This broad subdivision is consistent with both the distinct developmental origins of the lip/primary palate and the secondary palate and the distinct cellular and genetic etiologies described for CL/P and CPO; cleft palate may occur secondary to or independently from cleft lip. However, there is some epidemiologic

those cases that are isolated in hopes to further gain insight into associations.

• Documentation by photographs of all affected individuals and first-degree relatives.

associated preauricular tags, lip pits, and epicanthal folds.

• Necessary laboratory and radiological evaluations.

6 Designing Strategies for Cleft Lip and Palate Care

**3.2. Multiple**

OFC [10].

**3.3. Isolated**

**4. Etiology**

the branchial arches [11].

Both genetic and environmental factors have been shown to influence the risk of CL/P and CPO. Approximately 70% of all cases of CL/P and 50% of cases of CPO are designated as nonsyndromic [17], with the rest comprised of a wide range of malformation syndromes with known genetic and/or cellular etiologies. A summary of syndromic forms of CL/O and CPO in which the underlying genetic mutation has been elucidated is provided by Dixon et al. (**Table 1**; see original article for references) [18].



**Cleft type Syndrome Gene**

Cleft palate only Oculofaciocardiodental *BCOR*

Familial gastric cancer and CLP *CDH1* Craniofrontonasal *EFNB1* Roberts *ESCO2* Holoprosencephaly *GLI2* "Oro-facial-digital" *GLI3* Hydrolethalus *HYLS1* Van der Woude/popliteal pterygium *IRF6* X-linked mental retardation and CL/P *PHF8* Gorlin *PTCH1* CLP—ectodermal dysplasia *PVRL1* Holoprosencephaly *SHH* Holoprosencephaly *SIX3* Branchio-oculo-facial *TFAP2A* Holoprosencephaly *TGIF* Ectrodactyly-ectodermal dysplasia-clefting *TP73L* Ankyloblepharon-ectodermal dysplasia-clefting *TP73L* Tetra-amelia with CLP *WNT3*

CHARGE *CHD7* Lethal and Escobar multiple pterygium *CHRNG* Stickler type 1 *COL2A1* Stickler type 2 *COL11A1* Stickler type 3 *COL11A2* Desmosterolosis *DHCR24* Smith-Lemli-Opitz *DHCR7* Miller *DHODH* Craniofrontonasal *EFNB1* Kallmann *FGFR1* Crouzon *FGFR2* Apert *FGFR2* Otopalatodigital types 1 and 2 *FLNA* Larsen syndrome; atelosteogenesis *FLNB* Hereditary lymphedema-distichiasis *FOXC2*

*ACTB*

Cleft lip +/− cleft palate Autosomal dominant developmental malformations, deafness, and dystonia

8 Designing Strategies for Cleft Lip and Palate Care

**Table 1.** Clefting syndromes in which the mutated gene has been identified. Adapted from Ref. [18].

In contrast, nonsyndromic CL/P is complex and multifactorial in origin. Both genetic and environmental risk factors have been shown to influence the probability of occurrence. Furthermore, there is evidence that the presence of environmental factors—in particular, maternal smoking—modulates the risk conferred by genetic factors and vice-versa, complicating the genetic analysis of nonsyndromic forms of CLP [19]. As such, multifactorial models of inheritance which allow for the evaluation of these risk factors both independently and in interaction with each other are preferred.

Association studies such as candidate gene studies, which test correlation between a phenotype and prespecified genes of interest, and genome-wide association studies (GWAS), which identify genetic variations across entire genomes that are associated with a phenotype, have been used to evaluate a variety of genetic polymorphisms associated with nonsyndromic OFC. Genes that have been examined through these studies for associations with nonsyndromic OFC exhibit a range of functions, including growth, DNA transcription, nutrient metabolism, immunity, and oncogenesis. A few such genes are described here.

#### **5.1. Growth factors**

Transforming growth factor alpha (TGF-α) is a growth factor encoded by the *TGFA* gene that serves as a ligand for the epidermal growth factor receptor, which is involved in cell proliferation, differentiation, and development [20]. The first association study of genes associated with CL/P found an association with *TFGA* [21]; however, evidence of this linkage since then has been mixed [22, 23]. *TGFA* is currently viewed as a modifier, rather than a necessary or sufficient determinant, of risk for OFC.

Proteins in the transforming growth factor beta (TGF-β) family bind various TGF-β receptors leading to recruitment and activation of the SMAD family of transcription factors. TGF-β is involved in processes including apoptosis, modulation of immune cell function, and wound healing; disruption of TGF-β has been implicated in cancer, Loeys-Dietz syndrome, and other conditions [20]. Knockout experiences in mice have shown the *TGFB3* gene to be associated with OFC [24, 25], and subsequent association studies have identified these results in humans [26].

#### **5.2. Transcription factors**

The *MSX1* gene, which is a part of the homeobox gene family, codes for a protein that is involved in transcriptional regulation during embryogenesis as well as limb pattern formation, craniofacial development (in particular odontogenesis), and tumor growth inhibition [20]. This gene has been implicated in the development of cleft in several candidate gene studies, and may even account for 1–2% of all isolated cases of OFC [27].

Interferon regulatory factor 6 (IRF6) is a transcription factor protein that is involved in early development, especially of tissue in the head and face [20]. Mutations of the *IRF6* gene at 1q32 causes Van der Woude syndrome, a Mendelian-inherited disorder which induces CL/P or CPO and accounts for about 2% of all CL/P cases [28, 29]. The overlap between phenotypic presentation of Van der Woude syndrome and isolated CL/P motivated further study into the role of *IRF6* in development of OFC. Variation at *IRF6* has been found to be strongly associated with CL/P and may account for up to 12% of the genetic contribution to CL/P at the population level [30–32]. Furthermore, the discovery of *ILF6* as a risk factor for CL/P served as an important example of elucidating genetic variants associated with cases of nonsyndromic OFC, which are often excluded from genetic analyses [33].

#### **5.3. Nutrient metabolism**

Deficient maternal folate intake has long been implicated in risk of OFC in children, leading to suggestions that mutations of the enzyme 5,10-methyltetranhydrofolate reductase (MTHFR), which catalyzes the synthesis of 5-methylenetetrahydrofolate, play a role in the etiology of cases of nonsyndromic CL/P [34]. However, results from several association studies evaluating the role of *MTHFR* mutations in CL/P have been conflicting [35–37].

Retinoic acid plays an important role during development. Its functions, mediated by retinoic acid receptor alpha (RAR-α), include regulation of development, differentiation, apoptosis, granulopoeisis, as well as transcription of genes involved in the circadian rhythm [20]. Transgenic and knockout mice studies have additionally proposed a role in facial development [38]. Mutations of the *RARA* gene have been associated with development of OFC [39].
