Clefts Of Secondary Palate Research Articles

Conditional inactivation of Sox9: a mouse model for campomelic dysplasia.

Campomelic dysplasia (CD), a semilethal human skeletal malformation syndrome with XY sex reversal, is caused by heterozygous mutations in the SRY-related transcription factor gene SOX9 (Foster et al., 1994; Wagner et al., 1994). The mouse Sox9 gene, located on distal chromosome 11, is expressed during chondrogenesis (Wright et al., 1995), and is a key regulator of cartilage differentiation (Bi et al., 1999) and testis development (Vidal et al., 2001). Sox9 is expressed in a variety of embryonic tissues (Ng et al., 1997), suggesting further functions for Sox9/SOX9 during organogenesis. Indeed, CD patients show malformations in nonskeletal organs such as brain, heart, and kidneys (Houston et al., 1983; Mansour et al., 1995). Recently, Sox9 / mutant mice have been generated by a classical gene targeting approach and were shown to phenocopy the skeletal malformations observed in CD (Bi et al., 2001). However, because Sox9 / mice die shortly after birth, it was not possible to generate a stable Sox9 mutant mouse line. To circumvent this problem and to provide a tool for an in-depth analysis of Sox9 functions in skeletal development and the formation of other organs, we have generated a conditional Sox9 mutant allele in mice using the Cre/loxP system (Nagy, 2000; Le and Sauer, 2001) (Fig.1). Five correctly targeted embryonic stem (ES) cell clones were identified, and, following successful deletion of the neo cassette by transient in vitro expression of Cre recombinase (Taniguchi et al., 1998), two clones were microinjected into C57BL/6 blastocysts. Germline transmission was obtained for both clones and two Sox9-flox mouse lines have been established by backcrossing to C57BL/6 mice. Both Sox9 homozygous male and female mice are viable and fertile and do not show any obvious phenotype. To test for conditional inactivation of Sox9 in vivo, Sox9 /flox mice were crossed to Cre deleter mice which ubiquitously express the Cre recombinase (Schwenk et al., 1995) (Fig 2a). In Sox9 ; Cre–transgenic animals, designated as Sox9 / , exon 2 encoding half of the DNA-binding high-mobility group (HMG) domain and exon 3 encoding the entire transactivation domain are deleted (Fig. 2a, b). Because the transactivation domain is essential for SOX9 function (Sudbeck et al., 1996), and because truncated HMG domains cannot bind to DNA (Giese et al., 1991), our strategy most likely results in a functional null allele of the Sox9 gene. Complete recombination of the loxP-flanked Sox9 allele was demonstrated by allele-specific PCR analysis of Sox9 / mutants (Fig. 2c). Sox9 / mice died shortly after birth, presumably because of severe respiratory failure caused by a cleft secondary palate. Skeletal staining of newborn Sox9 / mutants revealed bone malformations typically observed in CD patients (Fig. 2 and data not shown). For example, the scapula was hypoplastic and malformed and the deltoid tuberosity of the humerus was missing (Fig. 2e). In addition, premature mineralization occurred in many skeletal elements, including the tail vertebrae (Fig. 2g). This is in agreement with the recently proposed role for Sox9 in regulating the rate of hypertrophic chondrocyte differentiation (Bi et al., 2001). Taken together, our analysis revealed that the skeletal phenotype of conditional Sox9 / mutants is very similar to that described for Sox9 / mutants (Bi et al., 2001). The different genetic background of mice used in the two studies may account for minor differences we observed in the severity of the skeletal phenotypes. The availability of tissue-specific or inducible Cre-transgenic mouse strains will make the conditional Sox9 allele a valuable tool not only to understand cartilage differentiation and skeletogenesis but also to investigate Sox9 functions during the development of a variety of other organs during mammalian development.

Craniofacial dysmorphogenesis including cleft palate in mice with an insertional mutation in the discs large gene.

The discs large (Dlg) protein, or synapse-associated protein 97 (SAP97), is a member of the membrane-associated guanylate kinase family of multidomain scaffolding proteins which recruits transmembrane and signaling molecules to localized plasma membrane sites. Murine dlg is the homologue of the Drosophila dlg tumor suppressor gene. The loss of dlg function in Drosophila disrupts cellular growth control, apicobasal polarity, and cell adhesion of imaginal disc epithelial cells, resulting in embryonic lethality. In this study, we isolated a mutational insertion in the murine dlg locus by gene trapping in totipotent embryonic stem cells. This insertion results in a truncated protein product that contains the N-terminal three PSD-95/DLG/ZO-1 domains of Dlg fused to the LacZ reporter and subsequently lacks the src homology 3 (SH3), protein 4.1 binding, and guanylate kinase (GUK)-like domains. The Dlg-LacZ fusion protein is expressed in epithelial, mesenchymal, neuronal, endothelial, and hematopoietic cells during embryogenesis. Mice homozygous for the dlg mutation exhibit growth retardation in utero, have hypoplasia of the premaxilla and mandible, have a cleft secondary palate, and die perinatally. Consistent with this phenotype, Dlg-LacZ is expressed in mesenchymal and epithelial cells throughout palatal development. Our genetic and phenotypic analysis of dlg mutant mice suggests that protein-protein interactions involving the SH3, protein 4.1 binding, and/or GUK-like domains are essential to the normal function of murine Dlg within craniofacial and palatal morphogenesis.

Oronasal fistula repair

Oronasal fistula is a relatively common complication associated with maxillary canine tooth extraction, problematic healing of maxillectomy, and repair of secondary cleft palate in small animals. Regardless of the clinical scenario associated with oronasal fistula, therapy requires surgical treatment. Principles for surgical repair of oronasal fistula include development of mucosal flaps with excellent vascular supply to transpose over the defect to restore continuity of the nasal and oral cavities. The specific surgical technique may vary but includes either single or double mucosal flaps. Oronasal fistula refractory to multiple attempts at surgical repair may be obturated by using a prosthodontic device.

Isolated cleft palate in mice with a targeted mutation of the LIM homeobox gene lhx8.

Formation of the mammalian secondary palate is a highly regulated and complex process whose impairment often results in cleft palate, a common birth defect in both humans and animals. Loss-of-function analysis has linked a growing number of genes to this process. Here we report that Lhx8, a recently identified LIM homeobox gene, is expressed in the mesenchyme of the mouse palatal structures throughout their development. To test the function of Lhx8 in vivo, we generated a mutant mouse with a targeted deletion of the Lhx8 gene. Our analysis of the mutant animals revealed a crucial role for Lhx8 in palatogenesis. In Lhx8 homozygous mutant embryos, the bilateral primordial palatal shelves formed and elevated normally, but they often failed to make contact and to fuse properly, resulting in a cleft secondary palate. Because development of other craniofacial structures appeared normal, the impaired palatal formation in Lhx8-mutant mice was most likely caused by an intrinsic primary defect in the mesenchyme of the palatal shelves. The cleft palate phenotype observed in Lhx8-mutant mice suggests that Lhx8 is a candidate gene for the isolated nonsyndromic form of cleft palate in humans.

IKK1-deficient mice exhibit abnormal development of skin and skeleton.

IkappaB kinases (IKKs) IKK1 and IKK2 are two putative IkappaBalpha kinases involved in NF-kappaB activation. To examine the in vivo functions of IKK1, we generated IKK1-deficient mice. The mutant mice are perinatally lethal and exhibit a wide range of developmental defects. Newborn mutant mice have shiny, taut, and sticky skin without whiskers. Histological analysis shows thicker epidermis, which is unable to differentiate. Limbs and tail are wrapped inside the skin and do not extend properly out of the body trunk. Skeleton staining reveals a cleft secondary palate, split sternebra 6, and deformed incisors. NF-kappaB activation mediated by TNFalpha and IL-1 is diminished in IKK1-deficient mouse embryonic fibroblast (MEF) cells. The IKK complex in the absence of IKK1 is capable of phosphorylating IkappaBalpha and IkappaBbeta in vitro. Our results support a role for IKK1 in NF-kappaB activation and uncover its involvement in skin and skeleton development. We conclude further that the two related kinases IKK1 and IKK2 have distinct functions and can not be substituted for each other's functions.

The range of congenital malformations associated with cleft lip and palate

The congenital malformations associated to cleft lip and cleft palate cover a wide range of pathologies with incidence and acuteness that vary according to the authors. In the 284 patients followed and/or referred to the Cleft Consultation of Dona Estefânia Hospital, 78 had associated malformations (27.5%). In three cases there was consanguinity in parents. A familial incidence of cleft and/or other congenital malformations was found in 13 and 10 patients respectively. Thirty patients had malformative syndromes. In the remaining 48, 127 associated congenital malformations (M.C.A.) were identified, in accordance with the Smith criteria, 81 major and 46 minor. The most frequent associated malformations are those of the face (25.9%) and of the cardiovascular system (16.5%). The multiple anomalies (of various systems) are the most frequent (47.9%), followed by the isolated anomaly (29.1%) and the multiple of one system (22.9%). In what concerns the association of syndromes with the type of cleft, primary, secondary or total palate, those of the secondary were the most frequent, particularly that of the Pierre Robin sequence (S.P.R.)--19 in 36 secondary cleft palate.

Problems associated with Le Fort I osteotomy for secondary cleft palate skeletal deformity

Problems associated with Le Fort I osteotomy for secondary cleft palate skeletal deformity

Pax9-deficient mice lack pharyngeal pouch derivatives and teeth and exhibit craniofacial and limb abnormalities.

Pax genes have been shown to play important roles in mammalian development and organogenesis. Pax9, a member of this transcription factor family, is expressed in somites, pharyngeal pouches, mesenchyme involved in craniofacial, tooth, and limb development, as well as other sites during mouse embryogenesis. To analyze its function in vivo, we generated Pax9 deficient mice and show that Pax9 is essential for the development of a variety of organs and skeletal elements. Homozygous Pax9-mutant mice die shortly after birth, most likely as a consequence of a cleft secondary palate. They lack a thymus, parathyroid glands, and ultimobranchial bodies, organs which are derived from the pharyngeal pouches. In all limbs, a supernumerary preaxial digit is formed, but the flexor of the hindlimb toes is missing. Furthermore, craniofacial and visceral skeletogenesis is disturbed, and all teeth are absent. In Pax9-deficient embryos tooth development is arrested at the bud stage. At this stage, Pax9 is required for the mesenchymal expression of Bmp4, Msx1, and Lef1, suggesting a role for Pax9 in the establishment of the inductive capacity of the tooth mesenchyme. In summary, our analysis shows that Pax9 is a key regulator during the development of a wide range of organ primordia.

Combined composite correction of orthognathic, nasal and cosmetic deformities

Many patients who present for orthognathic surgical correction have additional profile problems such as the position of the brow, the nasal anatomy, zygomatic hypoplasia, or positional chin problems independent of mandibular form. It is customary to assess the overall form and relationship of the facial structures but in the past surgical correction was carried out in separate stages. In other situations, for example craniofacial deformities, secondary cleft lip and palate anomalies, a single stage correction has been the goal. This has been aided by advances in anesthesia such as hypotension, autologous transfusions are now available and the surgical experience gained in other areas allows a reduction in operating time, thus single stage procedures have become the rule. In addition to this, it seems logical that all aspects of the deformity which is, in effect, inter-related should be corrected at the same time. Thus, composite corrections of the total facial region are practiced routinely, and this approach has given no cause for regret. In this paper, patients with a long-term follow-up will be presented.

Cleft palate in mice with a targeted mutation in the gamma-aminobutyric acid-producing enzyme glutamic acid decarboxylase 67.

The functions of neurotransmitters in fetal development are poorly understood. Genetic observations have suggested a role for the inhibitory amino acid neurotransmitter gamma-aminobutyric acid (GABA) in the normal development of the mouse palate. Mice homozygous for mutations in the beta-3 GABAA receptor subunit develop a cleft secondary palate. GABA, the ligand for this receptor, is synthesized by the enzyme glutamic acid decarboxylase. We have disrupted one of the two mouse Gad genes by gene targeting and also find defects in the formation of the palate. The striking similarity in phenotype between the receptor and ligand mutations clearly demonstrates a role for GABA signaling in normal palate development.

Embryologic features of term fetuses and newborns in CL/Fr mice with special reference to cyanosis.

The objective of this study was to clarify the embryologic features of CL/Fr mice in relation to cyanosis. Orofacial examinations were conducted on 18-day-old term fetuses and newborns. Except for open eyelids in a few cases, all of the external abnormalities in this strain were cleft lip and/or palate (CLP). The CLP incidence in term fetuses was 28.4%. The body and placental weights were not different between CLP(+) and CLP(-) fetuses. Several types of CLP were found; the most frequent was bilateral complete CLP (60%) followed by left complete (16%). Bilateral complete CL, which is the most severe CL type, was associated with poorly developed secondary palate and wide cleft. In newborns, 39.4 % of CLP(+) mice showed cyanosis, where no CLP(-) mice had such signs. The body weight of cyanotic mice with CLP was significantly lighter than that of noncyanotic mice with CLP. Cyanotic mice had severe types of CLP and unelevated palatal shelves. We suggest that mechanical airway obstruction may be one of the causes of cyanosis in newborn CL/Fr mice.

Embryologic Features of Term Fetuses and Newborns in CL/Fr Mice with Special Reference to Cyanosis

The objective of this study was to clarify the embryologic features of CL/Fr mice in relation to cyanosis.Orofacial examinations were conducted on 18-day-old term fetuses and newborns. Except for open eyelids in a few cases, all of the external abnormalities in this strain were cleft lip and/or palate (CLP).The CLP incidence in term fetuses was 28.4%. The body and placental weights were not different between CLP(+) and CLP(-) fetuses. Several types of CLP were found; the most frequent was bilateral complete CLP (60%) followed by left complete (16%). Bilateral complete CL, which is the most severe CL type, was associated with poorly developed secondary palate and wide cleft. In newborns, 39.4 % of CLP(+) mice showed cyanosis, where no CLP(-) mice had such signs. The body weight of cyanotic mice with CLP was significantly lighter than that of noncyanotic mice with CLP. Cyanotic mice had severe types of CLP and unelevated palatal shelves.We suggest that mechanical airway obstruction may be one of the causes of cyanosis in newborn CL/Fr mice.

Perinatal lethality and multiple craniofacial malformations in MSX2 transgenic mice.

MSX2 is a homeodomain transcription factor that has been implicated in craniofacial morphogenesis on the basis of its expression pattern during mouse development and the finding of a missense mutation (P148H) in humans affected with Boston-type craniosynostosis. We have generated transgenic mice carrying a 34 kb DNA fragment encompassing a human MSX2 gene encoding either wild-type or mutant (P148H) MSX2. Inheritance of either transgene resulted in perinatal lethality and multiple craniofacial malformations of varying severity, including mandibular hypoplasia, cleft secondary palate, exencephaly, and median facial cleft, which are among the severe craniofacial malformations observed in humans. Transgenic mice also manifested aplasia of the interparietal bone and decreased ossification of the hyoid. Transgene-induced malformations involved cranial neural-crest derivatives, were characterized by a deficiency of tissue, and were similar to malformations associated with embryonic exposure to ethanol or retinoic acid, teratogens that cause increased cell death. Together with previous observations implicating MSX2 expression in developmentally-programmed cell death, these results suggest that wild-type levels of MSX2 activity may establish a balance between survival and apoptosis of neural crest-derived cells required for proper craniofacial morphogenesis.

Questionnaire evaluation of feeding methods for cleft lip and palate neonates

A questionnaire evaluation was undertaken of feeding methods used by the mothers of 25 neonates with cleft lip and/or palate. Most parents had problems feeding their babies, both with the quantity of food taken and especially with the time taken to feed; even after a period of 2 months over a quarter were still having problems with the quantity of feed and a third had not established a regular feeding pattern. Just over a half needed to change the feeding method from the one with which they started. Twelve of the 25 mothers tried to breast-feed but none of them was successful. The Haberman feeder was the most popular, being used by 18 mothers. An acrylic feeding plate was considered to be helpful by 6 of 11 mothers of babies with complete clefts but by only 1 of 9 mothers of babies with secondary palate clefts. Almost all the mothers were dissatisfied with the information they had received while in hospital and with the back-up when they went home.

Limits of the mandibular symphysis as a donor site for bone grafts in early secondary cleft palate osteoplasty

With the assistance of computed tomography, the bone volume in the symphysis was assessed on 28 historic mandibles with mixed denition, before the eruption of the canines. The quantification gave an average bone volume of 1.0 cm 3, and the values fitted a normal distribution curve. Assuming that there were no appreciable differences in size between the historic and recent mandibles, the mandibular symphysis can theoretically be used as a donor site in early secondary osteoplasty in about 92% of cleft patients to augment a cleft volume of 0.5 cm 3, in 50% for a cleft volume of 1.0 cm 3, and in 18% for a cleft volume of 1.5 cm 3. With cleft volumes larger than 1.5 cm 3, the symphysis can hardly be considered. If, however, a safety margin of 5 mm from the apices of the front teeth is maintained, the chance that the mandibular symphysis will suffice decreases for a 0.5 cm 3 volume to 64%, for a 1.0 cm 3 cleft to 3.5%, and for a 1.5 cm 3 cleft to 0%.

Protection of Mice from Teratogen-Induced Cleft Palate by Exogenous Methionine

A major challenge for biomedical research is the reduction and/or prevention of congenital craniofacial abnormalities which can be induced by some extrinsic toxicants such as retinoic acids (e.g. isotretinoin, Accutane) and glucocorticoids (corticosteroid hormones) during embryonic craniofacial morphogenesis. Our present studies using a genetically susceptible mouse strain (B10.A) indicate that the teratogenic actions of exogenous retinoic acid or glucocorticoid in secondary cleft palate induction can be largely reduced or even completely rescued by subsequent administration of methionine. The greatest reduction in frequency of all-trans retinoic acid- or triamcinolone-induced secondary cleft palate was obtained by a single-dose IP administration of methionine at 187 mg/kg to pregnant mice on E13 21 hr. It appears that detrimental toxic effects were not observed in mice treated with this therapeutic level of methionine. Our present findings support the need for further research into the role of exogenous methionine in cleft palate reduction, that will provide a biological rationale for considering methionine as a therapeutic agent.

Dermatography as an adjunctive treatment for cleft lip and palate patients

Purpose: To investigate dermatography as a treatment for cleft lip and palate. Methods: The results of the treatment of 19 cleft lip patients with discoloration of scars and the vermilion border after secondary cleft lip and palate surgery by dermatography and intracicatricial keloidectomy are described. Results: This group of patients has gained self-confidence after treatments, to the extent that their articulation has improved. Conclusion: Dermatography can be applied as an adjuvant treatment with good cosmetic and esthetic results.

Clinico-statistical Investigation of Secondary Cleft Palate Patients

Clinico-statistical Investigation of Secondary Cleft Palate Patients

Msx1 deficient mice exhibit cleft palate and abnormalities of craniofacial and tooth development

The Msx1 homeobox gene is expressed at diverse sites of epithelial-mesenchymal interaction during vertebrate embryogenesis, and has been implicated in signalling processes between tissue layers. To determine the phenotypic consequences of its deficiency, we prepared mice lacking Msx1 function. All Msx1- homozygotes manifest a cleft secondary palate, a deficiency of alveolar mandible and maxilla and a failure of tooth development. These mice also exhibit abnormalities of the nasal, frontal and parietal bones, and of the malleus in the middle ear. Msx1 thus has a critical role in mediating epithelial-mesenchymal interactions during craniofacial bone and tooth development. The Msx1-/Msx1- phenotype is similar to human cleft palate, and provides a genetic model for cleft palate and oligodontia in which the defective gene is known.

Vertical lengthening of the unilateral cleft lip after a triangular flap repair

A triangular flap repair for unilateral cleft lip is stated to have the potential problem of creating a lip that is too long. Although preoperative measurements were performed with calipers, the gap created in the medial segment was directly measured during the procedure, in order to determine the size of the flap more precisely. Ninety patients with complete unilateral cleft lips underwent a triangular flap repair using this method. The symmetry of the Cupid's bow was evaluated at a follow-up period averaging between six years and three months. Sixty-two lips obtained “symmetry” and the remaining 28 cases showed a lip which was “too long”; none of the lips were “too short”. The comparison between 27 primary palate clefts (UCLA) and 63 primary and secondary palate clefts (UCLP) showed no significant difference in the surgical result. The preoperative shortness of the lip on the cleft side was significantly greater in the “too long” group (5.01 ± 0.95 mm) than in the “symmetry” group (4.40 ± 0.87 mm). The width of the flap used was significantly larger in the “too long” group (3.76 ± 0.79 mm) than in the “symmetry” group (3.42 ± 0.69 mm). The triangular flap repair with intraoperative measurements is considered to be beneficial, although preoperative measurements provide an optimal design in most cases. It is suggested that the use of a wider triangular flap results in a lip which tends to be too long in spite of the preoperative shortness on the cleft side.