Gökhan Tunçbilek

Meta-Analysis of 13 Genome Scans Reveals Multiple Cleft Lip/Palate Genes with Novel Loci on 9q21 and 2q32-35

Isolated or nonsyndromic cleft lip with or without cleft palate (CL/P) is a common birth defect with a complex etiology. A 10-cM genome scan of 388 extended multiplex families with CL/P from seven diverse populations (2,551 genotyped individuals) revealed CL/P genes in six chromosomal regions, including a novel region at 9q21 (heterogeneity LOD score [HLOD]=6.6). In addition, meta-analyses with the addition of results from 186 more families (six populations; 1,033 genotyped individuals) showed genomewide significance for 10 more regions, including another novel region at 2q32-35 (P=.0004). These are the first genomewide significant linkage results ever reported for CL/P, and they represent an unprecedented demonstration of the power of linkage analysis to detect multiple genes simultaneously for a complex disorder.

Disruption of ALX1 Causes Extreme Microphthalmia and Severe Facial Clefting: Expanding the Spectrum of Autosomal-Recessive ALX-Related Frontonasal Dysplasia

We present an autosomal-recessive frontonasal dysplasia (FND) characterized by bilateral extreme microphthalmia, bilateral oblique facial cleft, complete cleft palate, hypertelorism, wide nasal bridge with hypoplasia of the ala nasi, and low-set, posteriorly rotated ears in two distinct families. Using Affymetrix 250K SNP array genotyping and homozygosity mapping, we mapped this clinical entity to chromosome 12q21. In one of the families, three siblings were affected, and CNV analysis of the critical region showed a homozygous 3.7 Mb deletion containing the ALX1 (CART1) gene, which encodes the aristaless-like homeobox 1 transcription factor. In the second family we identified a homozygous donor-splice-site mutation (c.531+1G > A) in the ALX1 gene, providing evidence that complete loss of function of ALX1 protein causes severe disruption of early craniofacial development. Unlike loss of its murine ortholog, loss of human ALX1 does not result in neural-tube defects; however, it does severely affect the orchestrated fusion between frontonasal, nasomedial, nasolateral, and maxillary processes during early-stage embryogenesis. This study further expands the spectrum of the recently recognized autosomal-recessive ALX-related FND phenotype in humans.

ALX4 dysfunction disrupts craniofacial and epidermal development

Genetic control of craniofacial morphogenesis requires a complex interaction of numerous genes encoding factors essential for patterning and differentiation. We present two Turkish families with a new autosomal recessive frontofacial dysostosis syndrome characterized by total alopecia, a large skull defect, coronal craniosynostosis, hypertelorism, severely depressed nasal bridge and ridge, bifid nasal tip, hypogonadism, callosal body agenesis and mental retardation. Using homozygosity mapping, we mapped the entity to chromosome 11p11.2-q12.3 and subsequently identified a homozygous c.793C-->T nonsense mutation in the human ortholog of the mouse aristaless-like homeobox 4 (ALX4) gene. This mutation is predicted to result in a premature stop codon (p.R265X) of ALX4 truncating 146 amino acids of the protein including a part of the highly conserved homeodomain and the C-terminal paired tail domain. Although the RNA is stable and not degraded by nonsense-mediated RNA decay, the mutant protein is likely to be non-functional. In a skin biopsy of an affected individual, we observed a hypomorphic interfollicular epidermis with reduced suprabasal layers associated with impaired interfollicular epidermal differentiation. Hair follicle-like structures were present but showed altered differentiation. Our data indicate that ALX4 plays a critical role both in craniofacial development as in skin and hair follicle development in human.

Audiologic and Tympanometric Findings in Children With Cleft Lip and Palate

OBJECTIVE To evaluate the otologic and audiologic status of 50 children with repaired cleft lip, cleft palate, or both in Hacettepe University, Ankara, Turkey. DESIGN Audiometric and tympanometric evaluation of 100 ears in 50 children were performed. Hearing levels < or = 15 dB and middle ear pressures between -50 to +50 decaPascals were considered to be normal. Results were examined according to cleft type and laterality. The least and most affected frequencies were calculated. A simple evaluation of speech characteristics including nasal resonance, nasal air escape, and errors of articulation was also performed. RESULTS Sixty-three of the 100 ears had normal hearing status, whereas 40 had normal middle ear pressures. No evidence was found to suggest that individual cleft type and laterality of the ear had any effect on hearing loss or middle ear disease. Two-thirds of the patients had normal or acceptable degree of language skills. CONCLUSION The final hearing status of patients with cleft palate is a result of a combination of surgical correction, developmental factors, and treatment of middle ear disease. Early and aggressive ventilation tube placement is the standard of cleft care in many countries. Our long-term hearing outcome is relatively good in a population not treated with routine insertion of ventilation tubes. The majority of patients also have satisfactory speech. Patients with cleft palate should have close follow-up for middle ear disease, but further research is warranted to determine the aggressive usage of ventilation tubes.

Genome‐scan for loci involved in cleft lip with or without cleft palate in consanguineous families from Turkey

Cleft lip with or without cleft palate (CL/P) is a common congenital anomaly, with birth prevalence ranging from 1/500 to 1/1,000. A number of genetic loci have shown positive linkage or association results in European Caucasian populations. The purpose of the current study was to assess whether any of those loci have positive results in Turkish Caucasian CL/P families, and to perform a 10 cM genome scan to identify other regions potentially containing cleft susceptibility loci. Eighteen affected individuals with consanguineous parents were identified as part of our on‐going studies of orofacial clefts in Ankara, Turkey. Genotyped were 383 genome‐scan markers, and 70 additional markers, including markers in six candidate loci or regions on chromosomes 2, 4, 6, 14, 17, and 19 (TGFA, D4S175, F13A1, TGFB3, D17S250, and APOC2) that have been implicated in other studies of families with orofacial clefting. LOD scores (two point and multiple point) and family‐based association statistics (TDT) were calculated between each of the markers and CL/P. For the LOD score calculations, an autosomal recessive model was assumed for the inheritance of CL/P. Of the six candidate markers, significant TDT results were obtained with TGFA (P = 0.05). The most statistically significant multipoint results from the linkage genome scan were between putative genes controlling risk of CL/P and regions on chromosomes 4, 10, 12, and 15 (maximum multipoint HLODs of 1.25, 1.30, 2.73, and 1.28 respectively). These results demonstrate the power of small numbers of families with inbred probands to detect linkage and association.

Evaluation of velopharyngeal insufficiency with magnetic resonance imaging and nasoendoscopy.

&NA; Several radiological methods have been utilized to assess velopharyngeal function. The more recent imaging technique, magnetic resonance imaging (MRI), which has a number of advantages over radiographic and computed tomographic imaging, has been used rarely for evaluating velopharyngeal insufficiency. In this study, 5 normal volunteers and 10 patients with surgically repaired cleft palate were examined with MRI using midsagittal, coronal, and axial images. Nasoendoscopy was also performed to complete and confirm the diagnoses. Complete and tight closure of the velopharynx and full backward and upward movement of the soft palate was observed in volunteers. In coronal images, medial movement of lateral pharyngeal walls could also be seen. Despite this, patients with surgically repaired cleft palate had some degree of motion of the soft palate, ranging from no movement to maximal movement. In most of the patients, short soft palates with restricted motion was seen. MRI visualizes the velopharyngeal sphincter in all planes and provides high‐resolution images of the soft tissues. Objective measurements can be made as well. In this study, MRI and nasoendoscopy were used together in the diagnosis of velopharyngeal insufficiency and gave satisfactory results. Özgür F, Tunçbilek G, Cila A. Evaluation of velopharyngeal insufficiency with magnetic resonance imaging and nasoendoscopy. Ann Plast Surg 2000;44:8‐13

Blood loss and transfusion rates during repair of craniofacial deformities.

Surgical procedures for correction of craniofacial deformities resulted in unavoidable and extensive blood loss in small children and infants. Almost all of the patients undergoing these procedures will undergo a blood transfusion either during or immediately after the operation. A retrospective review of 30 patients who underwent craniofacial surgery was performed in this study to determine the magnitude of transfusion required for craniofacial surgery, document transfusion morbidity, and identify variables associated with the transfusion. The mean estimated blood loss was 566.8 mL, the mean intraoperative transfusion was 394.8 mL, the mean postoperative transfusion was 103.2 mL, and the mean total transfusion was 505 mL. The mean operative time was 450 minutes, the mean preoperative hemoglobin and the mean postoperative hemoglobin before hospital discharge were 11.6 g/dL and 10.3 g/dL, respectively. Craniofacial surgical procedures involve extensive scalp dissection and calvarial and facial bone osteotomies in patients with a low total blood volume. Every medical and surgical strategy for minimizing the need for blood transfusion should be considered.

Solvent-dehydrated calvarial allografts in craniofacial surgery.

Craniofacial surgery almost always requires the use of bone grafting. Although autografts are the standard procedure for bone grafting, it is sometimes not possible to harvest bone, and autografts have particular risks. The use of allograft bone provides a reasonable alternative to meet the need for graft material. Solvent dehydration is a multistage procedure in which human cadaveric bone is processed by osmotic exchange baths and gamma sterilization. This processing avoids the risk of infection transmission, decreases antigenicity, and does not weaken the mechanical properties of the bone. Solvent-dehydrated, gamma-irradiated human calvarial bone allografts were used for reconstruction of craniofacial deformities in 24 patients between 1988 and 2002. Resorption of the allografts and results of the surgical intervention were evaluated with plain radiographs and three-dimensional computed tomography 12 months after surgery, in 21 patients. Serologic tests for human immunodeficiency virus-1 antibody, hepatitis B surface antigen, and hepatitis C antigen were also performed. Biopsy specimens were taken from the allografts. Average follow-up in this group was 30 months (range, 8 to 60 months), and results of serologic tests were negative in all patients. Seventy-one percent of the patients (15 of 21) showed no resorption, with partial and complete allograft fusion. One patient had nearly total graft loss and the remaining five patients had 10 to 25 percent graft resorption. Rigid fixation of the allograft, contact with the dura and periosteum, and prevention of dead spaces around the allograft are the most important factors in achieving a satisfactory result. In solvent-dehydrated bone allografts, sterilization and antigenic tissue cleaning are achieved after several steps with a minimal dose of radiation. The result is a nonantigenic, sterile mechanical scaffold that can tolerate external forces. Although autografts are the standard in craniofacial surgery, solvent-dehydrated calvarial bone allografts produced successful results in selected cases.

Skeletal and Dental Stability After Maxillary Distraction With a Rigid External Device in Adult Cleft Lip and Palate Patients

PURPOSE To evaluate skeletal and dental stability in adult cleft lip and palate patients treated with a rigid external distraction system at the end of distraction and during the postdistraction period. PATIENTS AND METHODS Lateral cephalograms of 7 patients were obtained before distraction, at the end of distraction, and during the postdistraction period. The mean age before distraction was 21.56 +/- 4.73 years. The mean follow-up was 37.3 +/- 12.4 months. RESULTS The assessment of findings showed that skeletal maxillary sagittal movement was achieved in a superoanterior direction. The maxillary depth angle and effective maxillary length increased significantly (2 degrees and 9 mm, respectively) after distraction, whereas the palatal plane angle increased by 8 degrees , resulting in an anterior movement of the maxilla with a counterclockwise rotation. The lower facial height showed no significant changes after distraction. The sagittal movement of the upper incisors and the angulation of the upper first molars increased significantly (4.5 mm and 5.5 degrees , respectively). During the postdistraction period, the maxilla showed a slight relapse (22%). The effective maxillary length decreased by 2 mm. The palatal plane angle almost returned to its original position, showing 7 degrees of clockwise rotation. The lower facial height remained stable. The upper incisors moved anteriorly and the upper first molars showed a significant mesioangular change during follow-up. CONCLUSIONS After distraction, significant maxillary advancement was achieved with a counterclockwise rotation. The upper incisors moved labially, and the upper first molars angulated mesially. After 3 years, a 22% relapse rate was seen in the maxilla. The counterclockwise rotation of the maxilla was returned to its original position. The upper incisors moved more anteriorly.

Mutations in the interleukin receptor IL11RA cause autosomal recessive Crouzon-like craniosynostosis

We have characterized a novel autosomal recessive Crouzon‐like craniosynostosis syndrome in a 12‐affected member family from Antakya, Turkey, the presenting features of which include: multiple suture synostosis, midface hypoplasia, variable degree of exophthalmos, relative prognathism, a beaked nose, and conductive hearing loss. Homozygosity mapping followed by targeted next‐generation sequencing identified a c.479+6T>G mutation in the interleukin 11 receptor alpha gene (IL11RA) on chromosome 9p21. This donor splice‐site mutation leads to a high percentage of aberrant IL11RA mRNA transcripts in an affected individual and altered mRNA splicing determined by in vitro exon trapping. An extended IL11RA mutation screen was performed in a cohort of 79 patients with an initial clinical diagnosis of Crouzon syndrome, pansynostosis, or unclassified syndromic craniosynostosis. We identified mutations segregating with the disease in five families: a German patient of Turkish origin and a Turkish family with three affected sibs all of whom were homozygous for the previously identified IL11RA c.479+6T>G mutation; a family with pansynostosis with compound heterozygous missense mutations, p.Pro200Thr and p.Arg237Pro; and two further Turkish families with Crouzon‐like syndrome carrying the homozygous nonsense mutations p.Tyr232* and p.Arg292*. Using transient coexpression in HEK293T and COS7 cells, we demonstrated dramatically reduced IL11‐mediated STAT3 phosphorylation for all mutations. Immunofluorescence analysis of mouse Il11ra demonstrated specific protein expression in cranial mesenchyme which was localized around the coronal suture tips and in the lambdoidal suture. In situ hybridization analysis of adult zebrafish also detected zfil11ra expression in the coronal suture between the overlapping frontal and parietal plates. This study demonstrates that mutations in the IL11RA gene cause an autosomal recessive Crouzon‐like craniosynostosis.