Craniofacial Phenotypes and Genetics of DiGeorge Syndrome

The 22q11.2 deletion is one of the most common genetic microdeletions, affecting approximately 1 in 4000 live births in humans. A 1.5 to 2.5 Mb hemizygous deletion of chromosome 22q11.2 causes DiGeorge syndrome (DGS) and velocardiofacial syndrome (VCFS). DGS/VCFS are associated with prevalent cardiac malformations, thymic and parathyroid hypoplasia, and craniofacial defects. Patients with DGS/VCFS manifest craniofacial anomalies involving the cranium, cranial base, jaws, pharyngeal muscles, ear-nose-throat, palate, teeth, and cervical spine. Most craniofacial phenotypes of DGS/VCFS are caused by proximal 1.5 Mb microdeletions, resulting in a hemizygosity of coding genes, microRNAs, and long noncoding RNAs. TBX1, located on chromosome 22q11.21, encodes a T-box transcription factor and is a candidate gene for DGS/VCFS. TBX1 regulates the fate of progenitor cells in the cranial and pharyngeal apparatus during embryogenesis. Tbx1-null mice exhibit the most clinical features of DGS/VCFS, including craniofacial phenotypes. Despite the frequency of DGS/VCFS, there has been a limited review of the craniofacial phenotypes of DGC/VCFS. This review focuses on these phenotypes and summarizes the current understanding of the genetic factors that impact DGS/VCFS-related phenotypes. We also review DGS/VCFS mouse models that have been designed to better understand the pathogenic processes of DGS/VCFS.

There have been some excellent reviews on genetics and cardiovascular anomalies of DGS/VCFS [3,[11][12][13]. However, information on the craniofacial anomalies of DGS/VCFS is limited. This review focuses on these phenotypes and summarizes the current understanding of the genetic factors that impact DGS/VCFS-related phenotypes. We also review DGS/VCFS mouse models that have been designed to better understand the pathogenic processes of DGS/VCFS.

Craniofacial Phenotypes of Patients with DGS/VCFS
Patients with DGS/VCFS manifest craniofacial anomalies involving the cranium, cranial base, jaws, pharyngeal muscles, ear-nose-throat, palate, teeth, and cervical spine ( Figure S1, Tables 1 and 2). Frequently observed craniofacial phenotypes include velopharyngeal insufficiency (27-92%), enamel hypomineralization (39-41%), hearing loss (33-39%), platybasia (50-91%), and cervical spine anomalies (75%) ( Table 1). Delayed development of the hyoid bone has also been reported [14,15].   In addition to morphological anomalies, infants and young children with DGS/VCFS often exhibit a high prevalence of functional difficulties in feeding and speech/language associated with cleft palate, laryngeal anomalies, and velopharyngeal dysfunction [37]. Even after cleft palate closure, children with DGS/VCFS sometimes present communication disorders related to speech-language problems, such as articulation disorders of speech sounds and vocal disorders [37]. They exhibit slower language acquisition than those with other disorders that may be associated with abnormal muscle development.

TBX1 Gene
The proximal deletion of 1.5 Mb on the 22q11.2 locus includes TBX1 (Figure 1). TBX1 is considered a candidate gene of DGS/VCFS because haploinsufficiency of TBX1 leads to the typical phenotypes of DGS/VCFS, conotruncal anomaly face syndrome (OMIM #217095), and tetralogy of Fallot (OMIM #187500) ( Table 3). Identical mutations in TBX1 present among patients resulted in distinct phenotypes, suggesting that genetic and epigenetic changes or environmental factors are involved in the clinical phenotypes [5]. The coding variants in the T-box and C-terminal domains of TBX1 showed high combined annotationdependent depletion (CADD) scores (Table S1); however, further investigation is required to confirm that the variants cause DGS/VCFS and how they impact the phenotypes.

DiGeorge Syndrome Critical Region (DGCR)
DGCR8, DGCR6, and DGCR6L map to the commonly deleted 1.5 Mb region in DGS/VCFS ( Figure 1). DGCR8 is a nuclear miRNA-binding protein required for miRNA biogenesis. Dgcr8 haploinsufficiency in mice reduces the expression of miRNAs in the brain [45]. DGCR6 and DGCR6L genes encode a protein with a sequence similar to the Drosophila gonadal [46] (Figure S2B). In a chicken model, targeting DGCR6 function resulted in a vascular phenotype [47]. Attenuation of DGCR6 affects the expression of three genes localized within the 1.5 Mb region, upregulating the expression of TBX1 and UFD1 and reducing the expression of HIRA in the heart and pharyngeal arches of the chicken embryos [47]. Thus, the haploinsufficiency of DGCR8 or DGCR6 may be linked to DGS/VCFS phenotypes when targeting DGS/VCFS-related genes and miRNAs.

Tbx1
Craniofacial structures with DGS/VCFS phenotypes are derivatives of the head mesenchyme and the first and second pharyngeal arches [62]. Tbx1 is expressed in the mesoderm, ectoderm, and endoderm of the pharyngeal apparatus and head mesenchyme between embryonic day (E)9.5 and E11.5 in mice [62,63]. At E12.5, Tbx1 is expressed in the oral epithelium, the myogenic core of the tongue, incisor tooth buds, pharyngeal muscles,

Tbx1
Craniofacial structures with DGS/VCFS phenotypes are derivatives of the head mesenchyme and the first and second pharyngeal arches [62]. Tbx1 is expressed in the mesoderm, ectoderm, and endoderm of the pharyngeal apparatus and head mesenchyme between embryonic day (E)9.5 and E11.5 in mice [62,63]. At E12.5, Tbx1 is expressed in the oral epithelium, the myogenic core of the tongue, incisor tooth buds, pharyngeal muscles, and otic vesicle epithelium [63,64]. Tbx1-null mice exhibit the most clinical features of DGS/VCFS, while Tbx1 +/− mice exhibit no significant craniofacial phenotypes (Tables 2, 5 and S2). Information about ocular phenotypes in Tbx1-mutant mice is limited ( Table 2), although these anomalies in patients with DGS/VCFS have been reported [16,17]. The Cre/loxP system has been used with Tbx1 conditional knockout mice to examine the tissue-specific function of TBX1 in craniofacial development (Table 5).

Cleft Palate
During palatogenesis, the palatal shelves develop bilaterally from the internal parts of the maxillary prominences and fuse above the tongue to form an intact oral cavity roof [67,68]. Because the palate consists of a bone-lined hard palate and a bone-free soft palate, cleft palate phenotypes include incomplete and submucosal cleft palates [67,68]. Ablation of Tbx1, which is expressed in the epithelium of the palatal shelves, results in abnormal intraoral epithelial fusions between the palatal shelves and the mandible, resulting in various degrees of the cleft palate phenotype (complete, incomplete, and submucosal cleft palate) [30,34,69]. Expression of Pax9, whose mutations lead to cleft palate and tooth agenesis [70], is downregulated in the palatal shelves and pharyngeal region of Tbx1-null embryos [34,71]. In Tbx1-null palatal shelves, muscle-and bone-related genes are downregulated, whereas neuron-and collagen biosynthesis-related genes are upregulated [72].

Abnormalities in Craniofacial Bones
Tbx1-null mice display craniofacial bone abnormalities, including persistently open fontanelles, micrognathia, a short clavicle, a hypoplastic zygomatic arch, and the absence of the hyoid bone (Tables 2 and S2). Conditional deletion of Tbx1 in the mesoderm or osteochondral progenitors recapitulates the calvarial and mandibular phenotypes of Tbx1-null mice [35,66], suggesting that Tbx1 is required for morphogenesis and ossification of craniofacial bones. Although Tbx1 expression has not been reported in the neural crest, conditional deletion of Tbx1 here results in a hypoplastic hyoid bone [35] (Tables 2 and 5). These results indicate that Tbx1 is required for the morphogenesis and ossification of mesoderm-and neural crest-derived membranous bones, although malformations observed in most neural crest-derived bones of Tbx1-null mice are secondary defects induced by non-neural crest cells [35,66]. Interestingly, abnormalities in membranous bones observed in Tbx1-null mice are similar to those of cleidocranial dysplasia (OMIM #119600 and #216330) in humans, exhibiting hypoplastic membranous bones, including abnormal neurocranial morphology, a short clavicle, a hypoplastic zygomatic arch, and hyoid bone [73][74][75]. Cleidocranial dysplasia (OMIM #119600) is caused by heterozygous mutations in RUNX2, which encodes a master transcription factor for osteoblast differentiation [74,75]. Since ablation of Tbx1 affects Runx2 expression in calvarial bones, and TBX1 overexpression induces Runx2 expression in vitro [35], TBX1 may act upstream of Runx2 by maintaining cell populations that express Runx2 at the onset of bone development. In addition, TBX1 could be a candidate gene for recessive inheritance of cleidocranial dysplasia (OMIM #216330).

Abnormalities in the Cranial Base and Cervical Spine
The spheno-occipital synchondrosis (SOS) in the cranial base is a vital growth center for the skull (reviewed in [76]). TBX1 is expressed in the mesoderm-derived cartilage primordium of the SOS and basioccipital bones, and Tbx1 deletion in the mesoderm induces malformed basioccipital bones and precocious ossified SOS. This indicates that Tbx1 is an essential regulator of chondrocyte differentiation and subsequent ossification at the SOS [36]. TBX1 inhibits the transcriptional activity of RUNX2 in vitro as well as the expression of RUNX2 target genes in SOS [36]. Tbx1-null mice also exhibit endochondral bone abnormalities in the atlas, axis, and xiphoid process [6,35]. There is potential to examine the phenotypes of cranial synchondroses in DGS/VCFS patients, as abnormalities in the SOS and basioccipital bones may induce cranial phenotypes of DGS/VCFS, such as dolichocephaly, basilar impression, and platybasia.

Dental Anomalies
Dental abnormalities (single central incisors, enamel hypoplasia, and small teeth) have been reported in many patients [18,28]. Accordingly, in approximately 30% of Tbx1-null mice, the upper incisors are absent [6]. Tbx1 is expressed in the cervical loops, which contain the dental stem cell niche in mice. The cervical loop region of the incisor is either severely reduced or completely absent in Tbx1-null mice, and cultured incisors of Tbx1-null mice are hypoplastic and lack enamel [77]. Ablation of Tbx1 in the epithelium results in smaller teeth than in the wild type, suggesting that TBX1 regulates the proliferation of dental progenitor cells [48].

Vascular Endothelial Growth Factor A (Vegfa)
VEGFA is an essential cytokine in angiogenesis and vascular development during embryogenesis [86]. Vegfa-null neonates exhibit a few aspects of DGS/VCFS-related craniofacial anomalies, including unfused cranial sutures, absent incisors, and short mandibles, as well as cardiovascular abnormalities [54] (Table S2). The deletion of Vegfa in mice reduces Tbx1 expression, and the knockdown of vegfaa/vegfa levels in zebrafish enhances the pharyngeal arch malformations induced by tbx1 knockdown [54]. In humans, low expression of the VEGFA haplotype increases the risk of a cardiac phenotype of DGS/VCFS, indicating that expression levels of VEGFA affect the severity of DGS/VCFS phenotypes [87]. These results suggest that VEGFA modifies DGS/VCFS-related phenotypes by regulating TBX1 expression.

CRK like Proto-Oncogene, Adaptor Protein (Crkl)
CRKL maps to the 2.5 Mb region commonly deleted in DGS/VCFS (Figure 1). Variants in a predicted enhancer of CRKL are significantly associated with the risk of congenital heart defects in DGS/VCFS [94]. Approximately 12% of Crkl-null mice show mild cranial bone defects, such as small cranium and poor membranous ossification of the nasal bones [56]. Compound heterozygosity of Crkl and Tbx1 in mice has revealed that Crkl deletion enhances DGS/VCFS-related abnormalities compared with Tbx1-heterozygous embryos [56], suggesting that Tbx1 and Crkl genes act in the same genetic pathway. CRKL encodes an adaptor protein that promotes the intracellular response of FGF signaling. Crkl +/− ;Fgf8 +/− double heterozygous mice showed DGS/VCFS-related defects [95]. Thus, CRKL mutations cause or modify DGS/VCFS-related phenotypes and/or penetrance as a contiguous gene syndrome. 4.6. Aldehyde Dehydrogenase Family 1, Subfamily A2 (Aldh1a2/Raldh2) Retinoic acid (RA), an active vitamin A derivative, is essential for various developmental processes in vertebrates. High levels of RA act as morphogens that cause phenocopies of DGS/VCFS by downregulating Tbx1 expression in the pharyngeal apparatus [96,97]. RA levels are balanced by the RA-synthesizing enzyme aldehyde dehydrogenase (ALDH) and the Cyp26 RA-catabolizing enzyme [98,99]. Mouse embryos hypomorphic for Aldh1a2/Raldh2 display DGS/VCFS-related cardiovascular, thymic, and parathyroid malformations [57]. Haploinsufficiency of Aldh1a2/Raldh2 results in reduced embryonic synthesis of RA, increased levels of Tbx1, and accelerated recovery from ar-terial growth delay in Tbx1-heterozygous mice [100]. An inhibitor of the Cyp26 enzyme induces a phenocopy of DGS/VCFS in chick embryos [101]. In Tbx1-null mice, upregulated expression of Aldh1a2/Raldh2 and downregulated expression of Cyp26a1 have been observed [71].

Homeobox A3 (Hoxa3)
RA exposure increases the expression of Hoxa3, a gene which encodes a homeobox transcription factor, in the neural tube and pharyngeal apparatus [103]. Interestingly, Hoxa3null neonates show some aspects of the abnormalities of DGS/VCFS [58,104] (Table S2). Thus, HOXA3 may be a genetic modifier of DGS/VCFS-related abnormalities.

Kat6a/Moz/Myst3 (Lysine Acetyltransferase 6A) and Epigenetic Modifiers
Homozygous mutation of Kat6a/Moz/Myst3, which encodes a histone acetyltransferase, leads to cardiovascular defects seen in DGS/VCFS and reduces Tbx1 expression [59]. Treatment of pregnant mice with a histone demethylase inhibitor reportedly increased the methylation levels of histone H3 lysine K4 (H3K4) and partially rescued the cardiovascular phenotypes of Tbx1-heterozygous mice [105]. TBX1 regulates genes transcribed at a low level by recruiting lysine methyltransferase (KMT2C) and controlling monomethylation of H3K4 (H3K4me1) enrichment on chromatin [105]. In addition, TBX1 transcriptionally targets Wnt5a by interacting with SMARCD1/BAF60a, a component of the SWI/SNF-like BAF chromatin remodeling complex, along with the H3K4 monomethyltransferase SETD7 [106]. Microduplication in KANSL1, which encodes a member of the histone acetyltransferase complex, is associated with heart anomalies in individuals with DGS/VCFS [107]. In T cells of patients with DGS/VCFS, the status of transcriptional activation (H3K4me3 and H3K27ac) is globally increased [108]. Thus, epigenetic changes are involved in DGS/VCFSrelated phenotypes.

Sonic Hedgehog (Shh)
Shh encodes an SHH signaling molecule. In humans, SHH mutations lead to holoprosencephaly 3 (OMIM #142945), microphthalmia with coloboma (OMIM #611638), and single median maxillary central incisor (OMIM #147250). Shh-null embryos exhibit conotruncal and pharyngeal arch artery defects similar to those observed in DGS/VCFS and Tbx1-null embryos [109]. Tbx1 expression is reduced in Shh-null embryos, and ectopic expression of Shh can result in the upregulation of Tbx1, suggesting that Shh is a possible modifier for DGS/VCFS [62,110]. Shh is also required for the expression of the Fox family of transcription factor genes, forkhead box A2 (Foxa2) and forkhead box C2 (Foxc2), in the head mesenchyme and the pharyngeal endoderm [62]. FOXA2 and FOXC2 bind to regulatory regions in the mouse and human TBX1 loci [111].

Discussion
The penetrance and severity of congenital anomalies are related to genetic and environmental factors. Recent studies have revealed the function of TBX1 and modifiers that impact the severity and penetrance of DGS/VCFS. Studies of DGS/VCFS mouse models have provided insights into signaling pathways and genes that interact with TBX1 and/or affect the DGS/VCFS phenotypes. In addition, mouse models with DGS/VCFS may help us to identify additional DGS/VCFS-related phenotypes. For example, there is potential to examine the phenotypes of cranial synchondroses, cranium, zygomatic arches, and pharyngeal muscles in DGS/VCFS patients. We also noted that information about ocular phenotypes in Tbx1-mutant mice is limited, although these anomalies in patients with DGS/VCFS have been reported [16,17]. Crosstalk with key embryonic signals, especially BMP, TGFβ, VEGFA, FGF, RA, and SHH, critically regulates DGS/VCFS-related pharyngeal development. Genes involved in these signaling pathways may modify the phenotypic spectrum of DGS/VCFS. Given the broad spectrum of DGS/VCFS disease phenotypes, other genes essential to craniofacial development could modify the phenotypic spectrum. Genetically engineered mice are useful for studying disease phenotypes; however, ablation of essential genes involved in cardiovascular development may cause early embryonic lethality, which would prevent observation of craniofacial phenotypes. For example, ablation of Ufd1, whose human ortholog has been mapped to the 1.5 Mb region, causes early embryonic lethality before organogenesis in mice [116]. It is also essential to identify novel proteins that interact with TBX1 and examine whether interacting partners may influence the phenotypes of mouse models.

Conclusions
Studies of Tbx1-mutant mice have provided insights into the underlying pathogenesis of DGS/VCFS and the knowledge to diagnose patients with DGS/VCFS. Genes, miRNAs, and epigenetics could change Tbx1 expression. Polymorphisms, variations, and mutations in TBX1 may induce the penetrance and severity of DGS/VCFS-like craniofacial phenotypes. The molecular basis of the variant sequence of TBX1 will further define how TBX1 contributes to the craniofacial and other phenotypes of DGS/VCFS. Since interactions with TBX1 and other molecules in transcriptional complexes or chromatin remodeling are crucial for TBX1 function, identifying and understanding these genetic and epigenetic modifiers individually for each patient may direct therapeutics to minimize the severity.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/jdb10020018/s1, Figure S1: Craniofacial and skeletal phenotypes of DGS/VCFS. Figure S2: Human genes in the proximal deletion of 1.5 Mb on the 22q11.2 locus. Table S1: Craniofacial and skeletal phenotypes of DGS/VCFS and Tbx1-null mice. Table S2: Craniofacial and skeletal phenotypes in mouse models of DGS/VCFS. Table S3: Classification of mouse genes associated with DGS/VCFS. Author Contributions: N.F. contributed to the conceptual idea, performed the database searches, analyzed the data, and wrote the manuscript. All authors have read and agreed to the published version of the manuscript.