Role of Angiogenesis-Related Genes in Cleft Lip/Palate: Review of the Literature

International Journal of Pediatric Otorhinolaryngology 78 (2014) 1579–1585

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International Journal of Pediatric Otorhinolaryngology

journal homepage: www.elsevier.com/locate/ijporl

Review Article Role of angiogenesis-related genes in cleft lip/palate: Review of the literature

C. Franc¸ois-Fiquet a,b,c,*, M.L. Poli-Merol a, P. Nguyen b, E. Landais d, D. Gaillard d, M. Doco-Fenzy b,d a Department of Pediatric Surgery, American Memorial Hospital, CHU Reims, France b EA 3801 Laboratory Champagne Ardenne University, SFR CAP sante´ Reims-Amiens, Reims, France c Department of Plastic and Reconstructive Surgery, Hopital Maison Blanche, CHU Reims, France d Genetics Department, Hoˆpital Maison Blanche, CHU Reims, France

ARTICLE INFO ABSTRACT

Article history: Objectives: Cleft lip and cleft palate (CLP) are the most common congenital craniofacial anomalies. They Received 24 May 2014 have a multifactorial etiology and result from an incomplete fusion of the facial buds. Two main Received in revised form 30 July 2014 mechanisms,acting alone orinteracting with each other, were evidenced inthisfusion defect responsible for Accepted 1 August 2014 CLP: defective tissue development and/or defective apoptosis in normal or defective tissues. The objective of Available online 12 August 2014 this work was to study the implication and role of angiogenesis-related genes in the etiology of CL/P. Methods: Our methodological approach included a systematic and thorough analysis of the genes Keywords: involved in CL/P (syndromic and non-syndromic forms) including previously identiﬁed genes but also Cleft lip genes that could potentially be angiogenesis-related (OMIM, Pub Med).We studied the interactions of Cleft palate Gene these different genes and their relationships with potential environmental factors. Genetic Results: TGFb, FGA, PDGFc, PDGFRa, FGF, FGFR1, FGFR2 growth factors as well as MMP and TIMP2 Angiogenesis proteolytic enzymes are involved in the genesis of CLP (P>L). Furthermore, 18 genes involved in CLP also Etiologic interact with angiogenesis-related genes. Discussion: Even if the main angiogenesis-related genes involved in CLP formation are genes participating in several biological activities and their implication might not be always related to angiogenesis defects, they nevertheless remain an undeniably important research pathway. Furthermore, their interactions with environmental factors make them good candidates in the ﬁeld of CLP prevention. ß 2014 Elsevier Ireland Ltd. All rights reserved.

Contents

1. Introduction ...... 1580 2. Materials and methods ...... 1580 3. Results (Tables 1 and 2)...... 1581 4. Discussion ...... 1581 4.1. TGF pathways ...... 1581 4.1.1. TGFA...... 1581 4.1.2. TGFb ...... 1582 4.2. PDGFC and PDGFRa...... 1583 4.3. FGF and FGFR ...... 1583 4.4. VEGF...... 1583 4.5. Proteolytic enzymes: MMP and TIMP2 ...... 1584 5. Conclusion ...... 1584 Acknowledgements ...... 1584 References ...... 1584

* Corresponding author at: Department of Pediatric surgery, American Memorial Hospital, 47 rue Cognacq Jay, 51092 Reims, France. Tel.: +33 682873893; fax: +33 326784318; mobile: +33 06 82 87 38 93. E-mail addresses: [email protected], carolinefi[email protected] (C. François-Fiquet). http://dx.doi.org/10.1016/j.ijporl.2014.08.001 0165-5876/ß 2014 Elsevier Ireland Ltd. All rights reserved. 1580 C. François-Fiquet et al. / International Journal of Pediatric Otorhinolaryngology 78 (2014) 1579–1585

1. Introduction - Significant increase of this gene abnormality compared to the general population Clefts of the lip-palate (CLP) are age-old and multi-ethnic - Existence of functional studies on these genes pertaining to CL/P pathologies. They are the most common congenital craniofacial (gene expression studies or animal models studies) deformities, resulting from incomplete fusion of the facial buds at The search yielded 1118 relevant articles [17/07/2013]. the beginning of pregnancy. In spite of their frequency and age-old For each gene: nature, the etiology of this pathology has only been partially We collected the following general data: explained to this day. However, it has been recognized that several factors are involved. Genetic and environmental factors as well as OMIM reference their interactions have been implied in CLP etiology. Mechanisms Gene localization that could potentially explain this incomplete fusion of the facial buds Its size are quite complex. Two main mechanisms, acting independently or in Its role, its function (when it was clearly identified) association, have been identified: tissue development defect and/or apoptosis defect that can act on normal or defective tissues. Then we analyzed the implication of these genes in CL/P based The term angiogenesis was used for the first time in 1935 by on: Hertig to describe the growth of blood vessels within the placenta. Folkman used the term again in 1972 to describe a neovascular- Analysis of the type of cleft (CL, CP, CL/P) correlated to this gene ization associated with solid tumor growth. Nowadays, angiogen- Analysis of the syndromic or non-syndromic type esis has been correlated to several mechanisms during Analysis of the type of genetic abnormalities highlighted (e.g. embryogenesis and postnatal periods. mutation, deletion, polymorphism) and/or genetic techniques The objective of this work was to investigate the role of used angiogenesis-related genes in the study of CLP etiology. A search for potential interactions with other genes or signaling pathways 2. Materials and methods A search for an interaction with the environment, as well as the agent responsible (e.g. tobacco, alcohol, toxic agent, vitamin Our research was conducted on several databases and the two deficit) main ones were: A search for family studies or maternal studies described A search for animal models described - OMIM (Online Mendelian Inheritance in Man): with the Analysis of the implication of this gene in CL/P, while following keywords ‘‘Cleft, Cleft lip, Cleft palate, Cleft lip palate, underlining the possibility that the implication of this gene might lip, palate’’. We unveiled 331 OMIM references for the item ‘‘cleft be uncertain or its role indirect. lip, cleft palate’’, including 314 corresponding really to CL/P. And finally, we collected references on the gene in relation with - PubMed: with the following keywords ‘‘Cleft, Cleft lip, Cleft CL/P. palate, Cleft lip palate, gene, genetics, mutation’’.

A ﬁrst selection based on the abstracts led to discarding articles Thanks to the data collected on the genes function, we selected that did not focus on cleft lip or cleft palate, when an article did in from all the genes (potentials or involved) those who had an fact underline genetics and cleft lip/palate it was selected and read impact on the angiogenic mechanisms. in its entirety. The genes we considered relevant had to meet one or We also focused on the interactions and signaling pathways. more of the following criteria: Thus, we looked for the following interactions:

- Description in the article of a deleterious genetic abnormality - Between the angiogenic genes. Meaning, angiogenic genes (underlining a mutation or deleterious gene rearrangement in a involved in CL/P and other angiogenic genes not known for patient). their involvement in CL/P. - Recurrence in several articles of the same observation or the - Between an angiogenic gene linked to CL/P and other genes not same subject related to the angiogenesis but linked to CL/P.

Table 1 Overview of relevant genes in CL/P genesis.

Gene Phenotype Genetic Gene or pathway Environmental Familial or Animal interaction interaction maternal study study Cleft Non-syndromic Syndromic

FGF8 CL/P Cleft and Polymorphism variants, SHH Mouse hypogonadotropic mutation (de novo hypogonadism missense mutation) FGF 9 CP (animal), CLP Gene–gene interaction test FGF18 X FGFR1 x Kallmann Mutation, Deletion X FGFR2 CL/P Apert, crouzon Deletion X MMP3 CL+/-P X Polymorphism variants, TIMP2? gene–gene interaction test PDGF-C CP, CL+/-P x Linkage X Mouse PDGFRa CP x Mutation PAX1 X X Mouse, zebraﬁsh TGFA CL P x Polymorphism, linkage, X X Mouse mutation TGFB3 CP X Polymorphism X X Dog, mouse TIMP2 CL P x Polymorphism MMP3 VEGF CP Di george TBX1 Rats, rabbits C. Franc¸ois-Fiquet et al. / International Journal of Pediatric Otorhinolaryngology 78 (2014) 1579–1585 1581

Table 2 Role and function of relevant genes in CL/P genesis.

Gene Name Localization OMIM Size (bp) Role/function

FGF8 FIBROBLAST GROWTH FACTOR 8; FGF8 10q24.32 600483 5.941 Fibroblast growth factors are secreted proteins that ANDROGEN-INDUCED GROWTH FACTOR; interact with FGF tyrosine kinase receptors to AIGF mediate growth and development. The temporal and spatial patterns of FGF8 gene expression suggest that FGF8 is involved in gastrulation, regionalization of the brain, and organogenesis of the limb and face as an embryonic epithelial factor FGF 9 FIBROBLAST GROWTH FACTOR 9 13q12.11 600921 33.426 Peptide regulatory factors that act through distinct tyrosine kinase receptors and are involved in various biologic processes during embryogenesis and adult life, including implantation, morphogenesis, angiogenesis, and possibly tumorigenesis FGFR1 FIBROBLAST GROWTH FACTOR RECEPTOR 1; 8p11.23-p11.22 136350 57.697 Role in regulating cellular responses elicited by FMS-LIKE TYROSINE KINASE 2; FLT2; FMS- acidic FGF stimulation. Is essential for the normal LIKE GENE; FLG formation of the organ of Corti FGFR2 FGF RECEPTOR; PROTEIN TYROSINE KINASE, 10q26.13 176943 120.129 The fibroblast growth factor receptors comprise a RECEPTOR-LIKE, 14; TK14 family of related but individually distinct tyrosine kinase receptors MMP3 MATRIX METALLOPROTEINASE 3 11q23 185250 8.007 Is a proteoglycanase closely related to collagenase with a wide range of substrate specificities. It is a secreted metalloprotease produced predominantly by connective tissue cells. Together with other metalloproteases, it can synergistically degrade the major components of the extracellular matrix. Stromelysin is capable of degrading proteoglycan, fibronectin, laminin, and type IV collagen, but not interstitial type I collagen PDGF-C PLATELET-DERIVED GROWTH FACTOR C 4q32.1 608452 210.941 Major mitogens and stimulants of motility in mesenchymal cells, such as fibroblasts and smooth muscle cells, and also act on other cell types, including capillary endothelial cells and neurons PDGFRa PLATELET-DERIVED GROWTH FACTOR 5q32 173490 69.151 PDGFRA promoter linked to a luciferase reporter. RECEPTOR, ALPHA.PDGFR2 PAX1 acts as a transcriptional activator of the PDGFRA gene in differentiated human embryonal carcinoma cells TGFA TRANSFORMING GROWTH FACTOR 2p13.3 190170 10.914 Transforming growth factors (TGFs) are biologically active polypeptides that reversibly confer the transformed phenotype on cultured cells. TGF-alpha shows about 40% sequence homology with epidermal growth factor (EGF) and competes with EGF for binding to the EGF receptor, stimulating its phosphorylation and producing a mitogenic response TGFB3 TRANSFORMING GROWTH FACTOR, BETA-3 14q24.3 190230 24.893 Beta-type transforming growth factors are polypeptides that act hormonally to control the proliferation and differentiation of multiple cell types. TIMP2 TISSUE INHIBITOR OF METALLOPROTEINASE 2 17q25 188825 72.414 Selective affinities for members of the matrix metalloproteinase family VEGF VASCULAR ENDOTHELIAL GROWTH FACTOR 6p21.1 192240 16.304 Vascular endothelial growth factor is a heparin- binding growth factor specific for vascular endothelial cells that is able to induce angiogenesis in vivo

3. Results (Tables 1 and 2) 4. Discussion

The literature did highlight the involvement of angiogenic Growth factors involved in angiogenesis and correlated to CL/P processes in the formation of CLP via growth factors: are TGFA, TGFb, PDGFc and PDGFRa, FGF and FGFR. We will also look at the place and eventual role of the VEGG. TGFA, TGFb, PDGF C and Ra, FGF, FGFR1, FGFR2 (Tables 1 and 2) 4.1. TGF pathways

The superfamily of TGF growth factors and their receptors play The role of the VEGF remains to be defined an essential liaison role in craniofacial development. A large part of and proteases: our knowledge on the role of TGF pathway in CL/P is based on animal models and in vivo studies. MMP/TIMP2. 4.1.1. TGFA These genes interact with one another but also with the other TGFA was one of the first genes reported to be correlated to non- 18 genes involved in CL/P (Fig. 1). syndromic clefts [1–3]. It is strongly expressed during palatal 1582[(Fig._1)TD$IG] C. François-Fiquet et al. / International Journal of Pediatric Otorhinolaryngology 78 (2014) 1579–1585

Fig. 1. Synthesis of the articulation between the various angiogenic genes involved in CL/P genesis. fusion and promotes extracellular matrix (ECM) biosynthesis. [14] and the transformation of mesenchymal cells into epithelial Furthermore, TGFA alleles are among the rare genetic factors that cells [15]. showed important interactions with environmental factors such as - The TGFb-induced apoptosis through various pathways: SMAD, maternal smoking and vitamin nutritional factors [4–8]. DAXX and FasL-Fas-Caspase pathways [16]. It shows a 40% sequence homology with the epidermal - Angiogenesis. TGFb and other components of this signaling growth factor (EGF) and competes with its EGFR receptor. pathway bear a central and essential role in angiogenesis. TGFb The latter is involved in craniofacial development and palate pathway abnormalities are known for promoting cardiovascular formation [9]. It was in fact specifically studied, to no avail, in defects [17]. the framework of its implication in non-syndromic CL/P/ [10]. - ECM synthesis in endothelial cells and vascular cells of smooth A study in a mouse model underlined the role of TGFA as MMP2 muscles. during the embryonic phase. MMP2 promotes the regulation of cell - Maintaining vascular homeostasis. migration and tissue remodeling. In homozygous Ph/Ph mice - Some types of cancers (lung, digestive, oral cavity and thyroid (deletion of the PDGFR alpha subunit), the authors observed a TGFA cancers), Marfan syndrome and Loeys–Dietz syndrome. defect associated with the decreased expression of MMP2 and MT- MMP leading to craniofacial and cardiovascular development Each isoform was incriminated in more specific clinical pictures abnormalities (whereas the in vitro adjunction of TGFA increased such as: MMP2 expression) [11]. - TGF-b1 was reported to be involved in hematopoiesis and 4.1.2. TGFb endothelial differentiation [18]. The TGFb signaling pathway plays a major role in the regulation - TGF-b2 in the cardiac, pulmonary, craniofacial (especially eyes of cellular growth, cell differentiation and development of a large and ears), urogenital system and limb development. array of biological systems. Generally, this pathway is activated by - TGF-b3 in palatogenesis and pulmonary development. a ligand-induced serine/threonine kinase receptor oligomerization and the cytoplasmic phosphorylation of Smad2 and Smad3 However, all isoforms (1-2-3) play an essential role throughout signaling molecules for the TGFb pathway or Smad 1/5/8 signaling palatogenesis and are expressed during the entire palate formation molecules for the BPM pathway. [19–21]. The TGFb family is made of more than 30 proteins (including This signaling pathway interacts with some other signaling BMP, TGF cytokines, and activins) involved in many different pathways or genes involved in CP such as: biological processes. Even though the three TGF isoforms b1, b2, b3 are greatly - Wnt/catenin pathway. He et al. have underlined that the Wnt/ preserved across species [12] and in spite of the 71–76% sequence catenin pathway controls TGFb3 expression [22]. identity, these isoforms have specific activities that cannot be - MMP pathway [23]. compensated by other members of this TGFb family [13]. - FOXE1 gene. FOXE1 gene mutations are responsible for the TGF b1, b2, b3 are involved in: Bamforth–Lazarus syndrome (thyroid and craniofacial defects including cleft palate). Studies in mice have reported its - The cell cycle by regulating cell proliferation, cell survival and cell regulating role for TGFb3 and MSX1 [24]. differentiation, including differentiation of embryonic stem cells - SKI gene. C. François-Fiquet et al. / International Journal of Pediatric Otorhinolaryngology 78 (2014) 1579–1585 1583

Apart from the relationship with other signaling pathways, pathway lead to CL/P [50,51]. PDGFc interacts with WT1 and EGR1. TGFb seems to be influenced by some environmental factors such SUMO1 and PDGFc are both influenced by environmental risk factors. as folic acid [3,6,25]. PDGFRa (OMIM 173490) Platelet-derived Growth Factor Receptor Mice without the transforming growth factor-beta-3 gene alpha, also plays a major role in palatogenesis, craniofacial and (TGFb3) present with a cleft palate related to an incomplete fusion neural crest development [52,53]. Mutations in PDGFRa can be the of the facial buds due adhesion defect. An interesting fact is that sole factor responsible for CP [54]. exogenous application of TGFb3 in chicken with cleft palate Besides clefts PDGFRa has also been linked to: induces palate fusion [26]. Furthermore, the TGF b3 signaling pathway is at the heart of palatal medial edge seam (MES) - Neural tube defects in embryogenesis [55]. Thus, in murine disintegration, which independently yet sequentially, triggers cell models alterations of the PDGFRa gene expression triggers cycle arrest, cell migration and apoptosis during advances stages of congenital neural tube defects (spina bifida). Mutations in the palate development [27]. PAX1 gene identified in neural tube closure defects are In humans, genetic variations of TGFb3 were associated with responsible for PDGFRa gene deregulation. non-syndromic clefts in different populations [3,6,28–36]. - Cancer. Xie et al. underlined the fact GLI (SHH pathway mediator) SfaN1 polymorphism was studied in intron 5 of TGFb3 stimulated PDGFRc thus triggering the Ras/Erk pathway and (IVS5 + 104A > G). Frequency of the G allele is significant in the promoted cell proliferation in basal-cell carcinoma [56]. Korean population of non-syndromic CLP (OR 15.92%) [37]. Regarding, TGFb1–b2, Zhu et al. studied the expression of 4.3. FGF and FGFR 28869 genes in vivo on cultures of human embryonic palate mesenchymal cells (from an aborted human fetus during palatal The FGF regulation pathway also plays a major role in shelf elevation period before palate fusion) after treatment by craniofacial development. FGF receptors are activated by extracel- TGFb1–b2–b3, by microarray analysis [20,21]. The authors lular FGF ligands and help regulate cell differentiation, prolifera- highlighted 566 modified transcripts including 234 that had tion and migration. This pathway is essential for the induction and biological cell functions (growth, proliferation, development, migration of neural crests as well as regulating interactions morphogenesis, movement, cell cycle and apoptosis). Among between epithelium and mesenchymal cells during the facial bud these 234 genes, 11 of them were identified as palatogenesis- fusion. Most ligands of the FGF pathway and FGF R1 and R2 are related genes (PDGFC, PIGA, RUNX1, SNAI1, SMAD3, TGF b1, TGF b2, expressed during the initial phase of facial development [57]. TGF bR1, EDN1, INHBA and LHX8) [20,21]. By regulating the in vivo This pathway bears close relationships with other pathways such expression of 234 genes, TGFb1–b2 highlights that the TGFb as SHH, Wnt, BMP, TGFb, SOX, EGF, MSX and DLX, which are also pathway is probably similar to a great maestro in synchronizing involved in craniofacial development [58]. FGF ligands and receptors key moments of the palatogenesis process. bear a chronological and topographical expression. Thus, FGF18 is expressed at the level of the frontonasal process and most 4.2. PDGFC and PDGFRa specifically in the area where the fusion of lateral and medial nasal buds occurs. FGFR1, FGFR2, FGFR3, FGF10 and FGF18 are expressed PDGFC and PDGFRa (its receptor) are key regulators of embryonic during the various stages of secondary palate development, development and are essential during palatogenesis [38–45]. especially for the palatal epithelium during palatal fusion. Their PDGFC (OMIM 608452), Platelet Derived Growth Factor C, inactivation leads to CP. However, the action mechanism underlying FGF remains difficult to ascertain. Studies in mice lacking individual - PDGFC is mitogenic [46] and stimulates fibroblasts, smooth FGF-ligands do not report more CL, even though CP cases are muscle cells, capillary endothelial cells and neurons [47]. observed. According to the authors, this could be explained by - It regulates cell proliferation, cell survival and cell migration. embryogenic differences between humans and mice [59]. - It plays an important role in conjunctive tissue growth. FGFR2 is one of FGF receptors. FGF pathway bears an essential - It is involved in extracellular matrix formation and maintenance. role in craniofacial development. Most FGF ligands and FGF R1 and R2 are expressed during the initial phase of facial development It has been linked to certain types of cancers such as papillary [57]. Their inactivation leads to CP. Mutations in FGFR1 and FGFR2 thyroid carcinoma. are observed in syndromic clefts such as Kallmann’s syndrome but PDGFC is part of the cystine-knot growth-factor family, which also Pfeiffer’s syndrome, Apert’s syndrome (FGFR2 mutation in includes VEGF, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, PDGF-A, Apert’s syndrome with 76% of CP) and Crouzon’s syndrome. Studies PDGF-B, PDGF-C, PDGF-D, PIGF-1 and PIGF-2. There is a 25% have shown that Apert’s and Crouzon’s syndromes corresponded homology between VEGF and PDGFC but no relationship has been to allelic variations of the same syndrome. Furthermore, FGF8 was highlighted between the latter and VEGF receptors [47]. identified in syndromic CP with hypogonadism. Conversely, In mice, PDGFC is a powerful mitogen that promotes palatal mutations for FGF and its receptors were only found in rare mesenchymal cell proliferation during the embryonic period. It occurrences for non-syndromic clefts [60–62]. is essential in the morphogenesis of pharyngeal arches. Genetic inactivation of PDGFc (PDGFc/), or teratogenicity inactivation 4.4. VEGF (retinoic acid) [41,42] disrupts MMP [11] and TIF pathways at palate level during the development of pharyngeal arches [48]. The VEGF (Vascular Endothelial Growth Factor) protein plays an In mice, the presence of retinoic acid (dose-dependent) blocks essential role in angiogenesis. As underlined in its name, the VEGF PDGFc and triggers defects in craniofacial development. Conversely, acts essentially on vascular endothelial cells. However, it does have the exogenous adjunction of PDGFc enables the prevention of these an impact on a limited number of other cells, for example during defects. In the presence of retinoic acid (inactive PDGFc) we observe the stimulation of the monocyte/macrophage migration. In vitro, an inhibition in the migration capacity of the mesenchymal cells in VEGF stimulates the proliferation and migration of endothelial the first pharyngeal arch, through MMP2, MMP14 and TIMP2 cells. It also increases microvascular permeability. It is sometimes regulation [42]. even referred as a vascular permeability factor. Five types have PDGFc expression is influenced by FGF and SUMO1 pathways been identified, VEGF-A to VEGF-D as well as PIGF (placental growth [49]. As we will see in the next chapter, alterations to the FGF factor). VEGF-A is known for its role in cancer development. It has 1584 C. François-Fiquet et al. / International Journal of Pediatric Otorhinolaryngology 78 (2014) 1579–1585 not been directly described in relation to CL/P; however two MMPs are generally scarcely expressed in healthy tissues, yet research pathways are relevant for this discussion: their expression increases during physiological or pathological tissue remodeling process with the influence of factors able to - A study in rats and rabbits focusing on the teratogenic effects of modulate their gene expression (e.g. IL-1, TNFa, prostaglandin, cell Sunitinib malate (SU11248, SUTENT1, Pfizer, a potent VEGF lesion). Under these conditions, they are involved in several signaling inhibitor with multi-targeted angiogenic activity) processes such as angiogenesis, tissue reparation, embryogenesis highlighted that this treatment led to the onset of CL and CP or wound healing. MMP2 and MMP9 are especially involved in the in rabbits in doses to 1 mg/kg/day. However, the teratogenic angiogenesis and wound healing processes. MMP3, MMP9 and effect on the lip and palate was not an isolated one [63] it also TIMP2 polymorphisms in CL/P have been described in humans concerned other organs. [66–69]. In animal models, there are some correlations between - Several studies on TBX1 have suggested an indirect link in the TGFb, PDGF and TGFA receptors and the MMP pathway (MMP13, framework of syndromic cleft palate. The T-box family plays a MMP2 and TIMP2). In animal models, MMPs are also responsible role in the formation of tissues and organs during embryogenesis. for the proteolytic degradation phase of the ECM [11,23].MMP The TBX1 protein is necessary for the arterial development of the pathway is an active player in CL/P physiopathology. heart, face, neck, thymus and thyroid. The coding gene for this protein is located on chromosome 22 in 22q11.21 [64] (Yagi 5. Conclusion [64]). The microdeletions in this locus are also responsible for Digeorge syndrome. In the studies by Yagi, the hypothesis of the The etiopathogenesis of CL/P is still being debated today. embryologic micro-vascular pathology has been brought up. In Advances in genetic research and multiple studies have led authors animals (mice, zebrafish), authors noted that the expression of to conclude that the etiology of CL/P stems from a multifactor the Digeorge syndrome and the expression of TBX1 depended on origin. Furthermore, genetics have been identified has an impor- the VEGF isoforms. Thus, a reduced quantity of VEGF aggravated tant part of this etiology even though today, it is impossible to the arterial malformations induced by the invalidation of TBX1 determine exhaustively which genes and interactions pathways [65]. Furthermore, in humans, the 1154G!A allele of the VEGF is are involved in cleft formation. Our study had for objectives to preferentially correlated to cases with 22q11 deletion bearing define the role of angiogenesis-related genes in CL/P. Based on a severe cardiovascular abnormalities. review of the literature, we highlighted some relevant genes (i.e. genes previously identified or highly suspected of influencing cleft However, the presence of CP, is not constant in Digeorge formation and interacting with angiogenic signaling pathways). syndrome. Among other things, there are abnormalities with Today we know that TGFb, FGA, PDGFc, PDGFRa, FGF, FGFR1, FGFR2 velopharyngeal insufficiency (VPI) without cleft palate. Conversely, growth factors as well as MMP and TIMP2 proteolytic enzymes are several patients have a Digeorge-like phenotype without TBX1 involved in CL/P genesis (P > L). The role of VEGF remains to be deletion. In the framework of this syndrome, some phenotype– precisely determined. Furthermore, 18 genes involved in CL/P genotype refinements remain necessary. Yet, today we cannot be interact also with angiogenesis-related genes. Even if the main definite in stating that VEGF truly has a role in the genesis of CP. It angiogenesis-related genes are linked to lip-palate formation they seems that this gene is only a marker of the vascular abnormalities in also participate in several other biological activities and their Digeorge syndrome. Studies in animal models have focused on involvement is not always a proof of angiogenic defects, yet they vascular abnormalities, but few or no elements have emerged on the remain an essential and precious research target. Furthermore frequency and correlation with the CP phenotypes. It seems essential their interactions with environmental factors make them good to develop study model to specifically analyze the place of VEGF (via candidates for prevention studies. the TBX1) in the genesis of CP, and not only in Digeorge syndrome. Some observations underline the fact that VEGG signaling Conflict of interest pathways play a role in bone development via their activating actions on vascular development during endochondral bone The authors report no conflict of interest. formation, and their regulating actions on the survival and activity of chondrogenic and osteogenic cells. VEGF might have a direct Acknowledgments action on the chondrocytes to protect them from hypoxia. It is also involved in post-fracture bone remodeling via the same mecha- We would like to thank all patients and their families as well as nisms. The bone development capacities of this growth factor the American Memorial Hospital Foundation Inc (Boston, USA) for represent an interesting research way for surgeries relying on bone its continuing advice and support. remodeling (e.g. bone grafts). References

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