Chad A. Perlyn

RAB23 Mutations in Carpenter Syndrome Imply an Unexpected Role for Hedgehog Signaling in Cranial-Suture Development and Obesity

Carpenter syndrome is a pleiotropic disorder with autosomal recessive inheritance, the cardinal features of which include craniosynostosis, polysyndactyly, obesity, and cardiac defects. Using homozygosity mapping, we found linkage to chromosome 6p12.1-q12 and, in 15 independent families, identified five different mutations (four truncating and one missense) in RAB23, which encodes a member of the RAB guanosine triphosphatase (GTPase) family of vesicle transport proteins and acts as a negative regulator of hedgehog (HH) signaling. In 10 patients, the disease was caused by homozygosity for the same nonsense mutation, L145X, that resides on a common haplotype, indicative of a founder effect in patients of northern European descent. Surprisingly, nonsense mutations of Rab23 in open brain mice cause recessive embryonic lethality with neural-tube defects, suggesting a species difference in the requirement for RAB23 during early development. The discovery of RAB23 mutations in patients with Carpenter syndrome implicates HH signaling in cranial-suture biogenesis--an unexpected finding, given that craniosynostosis is not usually associated with mutations of other HH-pathway components--and provides a new molecular target for studies of obesity.

Long-term outcome of autogenous rib graft nasal reconstruction.

&NA; Whereas reconstruction of the hypoplastic nose with rib grafting is common, the long‐term outcomes of nasal growth and aesthetics are unknown. This study assessed nasal morphometrics, patient satisfaction, and the perception of nasal appearance by others up to 15 years after nasal reconstruction using cantilevered autogenous chondro‐osseous rib grafting with rigid internal fixation in children. Records of all patients who received nasal rib grafts between 1983 and 1998 by one senior surgeon were reviewed. Patients in this study were operated on before their late teens and had greater than 1‐year follow‐up including serial photographic documentation. Nasal growth was determined by comparing anthropometric measurements preoperatively, perioperatively, and postoperatively. Patient satisfaction was determined through a questionnaire that addressed memory, donor‐site morbidity, and nasal perception. Independent, blinded skilled observers who reviewed frontal and lateral photographs of the preoperative, perioperative, and postoperative intervals assessed nasal aesthetics. Thirty‐two patients who underwent 38 rib graft reconstructions of the nasal dorsum and tip at an average age of 8.8 years constitute the study population. Six patients underwent secondary augmentation. The average interval between initial nasal reconstruction and evaluation for this study was 7.9 years. Comparative anthropometric measurements before and after surgery documented increases in both tip projection (2.3 percent) and nasal length (3.0 percent) and a decrease in nasolabial angle (1.9 percent). Patient satisfaction interview response rate was 100 percent of those whom we were able to contact (28 of 32). The average age at interview was 17.2 years. Most patients recalled the operation and denied recollection of pain. Donor‐site long‐term morbidity was not an issue for 86 percent of patients. Sixty‐four percent of patients remembered their preoperative nasal appearance and 89 percent of these preferred the postoperative change and were not concerned with nasal scars or texture. Almost two‐thirds of the patients had fixation screws removed from the nasal dorsum because of skin erosion, easy palpability, or visibility. Although several patients expressed a desire to make minor additional changes to their nose, only one of these elected offered presurgical consultation and none have had such surgery. The postoperative nasal appearance compared with that preoperatively was rated as improved for 66.3 percent of responses, 26.5 percent as unchanged, and 7.2 percent as deteriorated. Cantilevered autogenous chondro‐osseous rib graft reconstruction of the nasal dorsum is an effective means of reconstruction for the hypoplastic nose in childhood with respect to morphometric measurements, patient self‐perception, and the assessment of nasal appearance by others. (Plast. Reconstr. Surg. 108: 1895, 2001.)

The Craniofacial Phenotype of the Crouzon Mouse: Analysis of a Model for Syndromic Craniosynostosis Using Three-Dimensional MicroCT

Objective: To characterize the craniofacial phenotype of a mouse model for Crouzon syndrome by a quantitative analysis of skull morphology in mutant and wild-type mice and to compare the findings with skull features observed in humans with Crouzon syndrome. Methods: MicroCT scans and skeletal preparations were obtained on previously described Fgfr2C342Y/+ Crouzon mutant mice and wild-type mice at 6 weeks of age. Three-dimensional coordinate data from biologically relevant landmarks on the skulls were collected. Euclidean Distance Matrix Analysis was used to quantify and compare skull shapes using these landmark data. Results: Obliteration of bilateral coronal sutures was observed in 80% of skulls, and complete synostosis of the sagittal suture was observed in 70%. In contrast, fewer than 40% of lambdoid sutures were found to be fully fused. In each of the 10 Fgfr2C342Y/+ mutant mice analyzed, the presphenoid-basisphenoid synchondrosis was fused. Skull height and width were increased in mutant mice, whereas skull length was decreased. Interorbital distance was also increased in Fgfr2C342Y/+ mice as compared with wild-type littermates. Upper-jaw length was shorter in the Fgfr2C342Y/+ mutant skulls, as was mandibular length. Conclusion: Skulls of Fgfr2C342Y/+ mice differ from normal littermates in a comparable manner with differences between the skulls of humans with Crouzon syndrome and those of unaffected individuals. These findings were consistent across several regions of anatomic interest. Further investigation into the molecular mechanisms underlying the anomalies seen in the Crouzon mouse model is currently under way.

Effect of distraction osteogenesis of the mandible on upper airway volume and resistance in children with micrognathia.

&NA; Children with craniofacial anomalies often have compromise of the upper airway, a condition with potential for morbidity and mortality. In children with microretrognathia, the diminutive size and retruded position of the mandible reduces the size of the oropharynx, thereby predisposing to glossoptosis and airway obstruction. Although several authors have reported successful use of mandibular distraction osteogenesis to alleviate this type of upper airway obstruction, the physiologic relationship between changes in mandibular shape, size, and position and upper airway dynamics remains undefined. The purpose of this study was to develop methodologies to quantitatively evaluate upper airway dynamics in children with micrognathia both before and after mandibular distraction osteogenesis. The patient population consisted of four children with micrognathia who had successfully undergone upper airway stabilization by bilateral mandibular distraction osteogenesis. The data used were digitally archived computed tomographic scan data from highresolution, thin‐slice head computed tomographic scans obtained before and after mandibular distraction. Upper airway evaluation was performed in two ways: static and dynamic. Static analysis consisted of computer quantification of predistraction and postdistraction mandibular and upper airway volumes using Analyze imaging software. Dynamic analysis consisted of fabrication of rigid stereolithographic hollow cast models of the upper airway produced from computed tomographic scan data. Models were used for characterization of upper airway resistance and flow patterns as related to respiration. After distraction osteogenesis, mandibular total volume increased 32, 32, 18, and 25 percent (mean, 27 percent) and upper airway volume increased by 20, 31, 23, and 71 percent (mean, 37 percent). A significant decrease in flow resistance, both inspiratory and expiratory, was observed in the patient with the greatest upper airway volume increase (71 percent) after distraction. After distraction, the inspiratory resistance was diminished by 51 percent and the expiratory resistance diminished by 85 percent. However, the three patients with more modest upper airway volume increases of 20 to 31 percent demonstrated no statistically significant change in flow resistance after distraction. Results of this study support the conclusion that distraction osteogenesis of the micrognathic mandible increases the volume of the upper airway, roughly paralleling the increase in mandibular volume. In the biomechanical airway model studied, upper airway volume expansion has been shown to be able to decrease the flow resistance over the length of the airway, presumably secondary to an increase in the average cross‐sectional area. The artificial rigidity of the stereolithographic “airway” compared with the elasticity of the human upper airway may account for the insensitivity of this model to smaller but clinically significant airway changes. (Plast. Reconstr. Surg. 109: 1809, 2002.)

Analysis of a gain-of-function FGFR2 Crouzon mutation provides evidence of loss of function activity in the etiology of cleft palate

Cleft palate is a common birth defect in humans and is a common phenotype associated with syndromic mutations in fibroblast growth factor receptor 2 (Fgfr2). Cleft palate occurred in nearly all mice homozygous for the Crouzon syndrome mutation C342Y in the mesenchymal splice form of Fgfr2. Mutant embryos showed delayed palate elevation, stage-specific biphasic changes in palate mesenchymal proliferation, and reduced levels of mesenchymal glycosaminoglycans (GAGs). Reduced levels of feedback regulators of FGF signaling suggest that this gain-of-function mutation in FGFR2 ultimately resembles loss of FGF function in palate mesenchyme. Knowledge of how mesenchymal FGF signaling regulates palatal shelf development may ultimately lead to pharmacological approaches to reduce cleft palate incidence in genetically predisposed humans.

A MODEL FOR THE PHARMACOLOGICAL TREATMENT OF CROUZON SYNDROME

OBJECTIVE Crouzon syndrome is caused by mutations in fibroblast growth factor receptor 2 (FGFR2) leading to constitutive activation of receptors in the absence of ligand binding. The syndrome is characterized by premature fusion of the cranial sutures that leads to abnormal cranium shape, restricted brain growth, and increased intracranial pressure. Surgical remodeling of the cranial vault is currently used to treat affected infants. The purpose of this study was to develop a pharmacological strategy using tyrosine kinase inhibition as a novel treatment for craniosynostotic syndromes caused by constitutive FGFR activation. METHODS Characterization of cranial suture fusion in Fgfr2 mutant mice, which carry the most common Crouzon mutation, was performed using micro-computed tomographic analysis from embryogenesis through maturation. Whole calvarial cultures from wild-type and Fgfr2 mice were established and cultured for 2 weeks in the presence of dimethyl sulfoxide control or PD173074, an FGFR tyrosine kinase inhibitor. Paraffin sections were prepared to show suture morphology and calcium deposition. RESULTS In untreated Fgfr2 cultures, the coronal suture fused bilaterally with loss of overlap between the frontal bone and parietal bone. Calvaria treated with PD173074 (2 micromol/L) showed patency of the coronal suture and were without evidence of any synostosis. CONCLUSION We report the successful use of PD173074 to prevent in vitro suture fusion in a model for Crouzon syndrome. Further studies are underway to develop an in vivo treatment protocol as a novel therapeutic modality for FGFR associated craniosynostotic syndromes.

Computational mouse atlases and their application to automatic assessment of craniofacial dysmorphology caused by the Crouzon mutation Fgfr2C342Y

Crouzon syndrome is characterized by premature fusion of sutures and synchondroses. Recently, the first mouse model of the syndrome was generated, having the mutation Cys342Tyr in Fgfr2c, equivalent to the most common human Crouzon/Pfeiffer syndrome mutation. In this study, a set of micro‐computed tomography (CT) scannings of the skulls of wild‐type mice and Crouzon mice were analysed with respect to the dysmorphology caused by Crouzon syndrome. A computational craniofacial atlas was built automatically from the set of wild‐type mouse micro‐CT volumes using (1) affine and (2) non‐rigid image registration. Subsequently, the atlas was deformed to match each subject from the two groups of mice. The accuracy of these registrations was measured by a comparison of manually placed landmarks from two different observers and automatically assessed landmarks. Both of the automatic approaches were within the interobserver accuracy for normal specimens, and the non‐rigid approach was within the interobserver accuracy for the Crouzon specimens. Four linear measurements, skull length, height and width and interorbital distance, were carried out automatically using the two different approaches. Both automatic approaches assessed the skull length, width and height accurately for both groups of mice. The non‐rigid approach measured the interorbital distance accurately for both groups while the affine approach failed to assess this parameter for both groups. Using the full capability of the non‐rigid approach, local displacements obtained when registering the non‐rigid wild‐type atlas to a non‐rigid Crouzon mouse atlas were determined on the surface of the wild‐type atlas. This revealed a 0.6‐mm bending in the nasal region and a 0.8‐mm shortening of the zygoma, which are similar to characteristics previously reported in humans. The most striking finding of this analysis was an angulation of approximately 0.6 mm of the cranial base, which has not been reported in humans. Comparing the two different methodologies, it is concluded that the non‐rigid approach is the best way to assess linear skull parameters automatically. Furthermore, the non‐rigid approach is essential when it comes to analysing local, non‐linear shape differences.

Plasticity of the endocranial base in nonsyndromic craniosynostosis.

Limited in vivo data exist on the dysmorphology of the cranial base in nonsyndromic craniosynostosis. Few studies have documented the effect of calvarial surgery for synostosis on endocranial morphology. Previous work has suggested that the dysmorphology of the endocranial base is diagnostically specific for metopic, sagittal, and unicoronal sutures. The purpose of this study was to further evaluate the endocranial base in infants with nonsyndromic craniosynostosis by testing the hypothesis that the dysmorphology is, to some degree, a secondary deformation rather than a primary malformation. Three questions were addressed: (1) Can individuals reliably identify affected suture‐specific endocranial‐base morphology using standard templates? (2) Does calvarial surgery in infancy for craniosynostosis affect the perception of endocranial‐base morphology? and (3) Does calvarial surgery in infancy for nonsyndromic craniosynostosis normalize the endocranial base? In this study, three‐dimensional volumetric reconstructions from archived computed tomography digital data were processed using the ANALYZE imaging software. Dysmorphology was assessed by nine independent, blinded skilled observers who reviewed two separate sets of images of endocranial bases. Both sets contained images from the same patients: one set contained preoperative images, and the other contained images of the endocranial base 1 year after calvarial surgery. Observers were asked to sort each set into four suture‐specific diagnostic groups: normal, unicoronal, metopic, and sagittal. Each set contained 10 patients with unicoronal synostosis, 10 with metopic synostosis, 10 with sagittal synostosis, and four normal patients. Seventy‐eight percent of the total number of preoperative images were correctly sorted into the suture‐specific diagnostic group, whereas only 55 percent of the total number of postoperative images were correctly matched. With regard to the individual sutures, the results were as follows (data are presented as preoperative accuracy versus postoperative accuracy): metopic, 76 percent versus 44 percent; sagittal, 58 percent versus 34 percent; unicoronal, 100 percent versus 79 percent; and normal, 83 percent versus 72 percent. Although 36 of 306 total images per group (12 percent) actually represented normal patients, the observers called 72 of 306 normal (24 percent) in the preoperative set versus 110 of 306 normal (36 percent) in the postoperative set. In conclusion, (1) the endocranial dysmorphology of nonsyndromic craniosynostosis is recognizably specific to the affected suture; (2) calvarial surgery for nonsyndromic craniosynostosis normalizes the endocranial base qualitatively with regard to the diminished ability of raters to identify the primary pathology; and (3) the documented postoperative changes in endocranial base morphology after calvarial surgery for nonsyndromic craniosynostosis in infancy indicates that a major component of that dysmorphology is a secondary deformity rather than a primary malformation. (Plast. Reconstr. Surg. 108: 294, 2001.)

The craniofacial anomalies archive at St. Louis Children's Hospital: 20 years of craniofacial imaging experience.

&NA; This article describes how the Craniofacial Imaging Laboratory at the Cleft Palate and Craniofacial Deformities Institute, St. Louis Childrens Hospital, Washington University Medical Center, has developed an electronic archive for the storage of computed tomography image digital data that is independent of scanner hardware and independent of units of storage media (i.e., floppy disks and optical disks). The archive represents one of the largest repositories of high‐quality computed tomography data of children with craniofacial deformities in the world. Archiving reconstructed image data is essential for comparative imaging, surgical simulation, quantitative analysis, and use with solid model fabrication (e.g., stereolithography). One tertiary craniofacial centers experience in the establishment and maintenance of such an archive through three generations of storage technology is reported. The current archive is housed on an external 35‐GB hard drive attached to a Windows‐based desktop server. Data in the archive were categorized by specific demographics into groups of patients, number of scans, and diagnoses. The Craniofacial Imaging Laboratory archive currently contains computed tomography image digital data for 1827 individual scans. The earliest scan was done in 1980; the most recently stored scan for the purposes of this report occurred in May of ‘2000. The average number of scans archived per complete year was 94, with a range of 59 to 138. Of the 1827 total scans, 74 percent could be classified into specific diagnostic categories. The majority of the archive (55 percent) is composed of the following five diagnoses: sagittal synostosis (17 percent), unilateral coronal synostosis (11 percent), hemifacial microsomia (10 percent), plagiocephaly without synostosis (10 percent), and metopic synostosis (7 percent). Storage of computed tomography image data in a digital archive currently allows for continuous upgrading of image display and analysis and facilitates longitudinal and cross‐sectional studies, both intramural and extramural. Internet access for clinical and research purposes is feasible, but contingent on protection of patient confidentiality. The future of digital imaging regarding craniofacial computed tomography scan storage and processing is also discussed. (Plast. Reconstr. Surg. 108: 1862, 2001.)

Mechanism of skull suture maintenance and interdigitation

Skull sutures serve as growth centers whose function involves multiple molecular pathways. During periods of brain growth the sutures remain thin and straight, later developing complex fractal interdigitations that provide interlocking strength. The nature of the relationship between the molecular interactions and suture pattern formation is not understood. Here we show that by classifying the molecules involved into two groups, stabilizing factors and substrate molecules, complex molecular networks can be modeled by a simple two‐species reaction–diffusion model that recapitulates all the known behavior of suture pattern formation. This model reproduces the maintenance of thin sutural tissue at early stages, the later modification of the straight suture to form osseous interdigitations, and the formation of fractal structures. Predictions from the model are in good agreement with experimental observations, indicating that the model captures the essential nature of the interdigitation process.