**6. Epigenetics**

**5.1. Growth factors**

sufficient determinant, of risk for OFC.

10 Designing Strategies for Cleft Lip and Palate Care

often excluded from genetic analyses [33].

**5.3. Nutrient metabolism**

**5.2. Transcription 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

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].

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 stud-

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

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 evaluat-

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

ies, and may even account for 1–2% of all isolated cases of OFC [27].

ing the role of *MTHFR* mutations in CL/P have been conflicting [35–37].

Due to the relative lack of success in identifying causal genetic factors involved in OFC despite the numerous association studies that have been performed, recent attention has been directed toward the role of epigenetic programming, or modifications that do not involve DNA sequencing. Commonly studied epigenetic events include histone modification, chromatin remodeling, posttranscriptional gene alteration via noncoding MicroRNAs, and DNA methylation. MicroRNAs and DNA methylation, in particular, have begun to demonstrate distinct roles in etiologies of OFC.

#### **6.1. MicroRNAs**

While protein-coding genes make up only about 1.2% of the human genome, recent estimates suggest that up to 93% of the human genome codes for RNA transcripts. MicroRNAs (miR-NAs) represent the largest family of such noncoding RNAs in the human genome. They are involved in gene silencing and play important roles in cell and tissue differentiation, including development of the secondary palate [40–43]. miRNAs have been shown to orchestrate many of the processes that are central to palatal morphogenesis, including epithelial-mesenchymal transformation, platelet-derived growth factor (PDGF) and TGF-β signaling, cell migration and proliferation, and collagen synthesis [44–48]. As such, further analysis of miRNA expression and gene networks will be key to elucidating mechanisms of palatal development as well as etiologies of OFC.

#### **6.2. DNA methylation**

DNA methylation, one of the most important epigenetic modifications in mammalian cells, is a process by which methyl groups are added to DNA in order to regulate gene expression. Methylation generally occurs at cytosines within the context of symmetrical CpG dinucleotide sequences, which are often concentrated in regions known as *CpG islands* and found in both gene bodies and promoter regions [49, 50]. Classically, methylation of *CpG islands* at gene promoters is thought to induce silencing of gene transcription; however, *positive* correlation between gene body methylation and gene expression has been observed [51, 52].

DNA methylation was first identified as a potential mediator of palatal development after a series of studies in which DNA demethylating agents were used to induce cleft palate in mice [53–55]. Since then, failures in DNA methylation demonstrated involvement in craniofacial malformations including cleft palate [56, 57]. Despite the current lack of knowledge regarding the epigenetic mechanisms mediating palatal development, evidence strongly indicates that DNA methylation plays a central role in regulating this process, and may perhaps serve as future risk assessment and therapeutic targets for patients with OFC.
