Vomer Flaps Support Velopharyngeal Competency, Fistula Free Repair, and 3D Construction of Normal Anatomy in Patients with Cleft Palate.

Cleft palate repair comprises the surgical creation of a congenitally nonexistent normal anatomy, to establish physiological function by moving tissues into their normal anatomical positions. In patients with isolated incomplete (IICP) or submucous (SMCP) cleft palate, the vomer is usually not completely attached to the palatal plate in the midline. This condition, which is visible through surgical access radiologically or via endoscope, is often disregarded during hard palate repair. This can lead to "hypernasality" despite a well-functioning velopharyngeal mechanism. The general practice of hard palate repair by suturing merely the nasal layers together separates the oral and nasal cavities. However, without incorporation of the vomer, it is impossible to build two separate nasal floors on the left and right sides. We consider that achieving normal speech and separation of the nasal cavities are mutually dependent and have to be considered equally. METHODS: We described hard palate repair involving the vomer for construction of both nasal floors. We presented the occlusal relationship, hypernasality, and fistula rates in 37 patients operated on between January 1, 2017 and June 30, 2018. RESULTS: One child presented minimal hypernasality; all others had normal resonance/voice. Fistula rate was zero, and no cross bites were observed. CONCLUSIONS: The implicit connection between the inner nose, resonance/voice, and prevention of fistulae has not yet been acknowledged. The correct usage of vomer flaps in IICP and SMCP creating separate nasal floors supports the velopharyngeal competency, avoids fistula formation, and should be incorporated regularly, like in other cleft forms.

Presurgical Alignment of Bilateral Cleft Segments With 3D Simulation Under Special Consideration of the Vomer: A Technical Note.

BACKGROUND: The main goal of presurgical orthopedics (PSO) for patients with bilateral cleft lip and palate is to correct the protruded and/or twisted premaxilla. However, PSO is associated with the risk of uncontrolled development of the vomer, which has received little attention to date. SOLUTION: We present a removable orthodontic device that can be used to keep or align the vomer and the premaxilla in the midline during preoperative molding of cleft segments independently and 3 dimensionally.

Customized Pre-Epiglottic Baton Plate-A Practical Guide for Successful, Patient-Specific, Noninvasive Treatment of Neonates With Robin Sequence.

OBJECTIVE: Despite its efficiency and benefits in treating patients with Robin sequence (RS), the pre-epiglottic baton plate (PEBP) is not widely used. However, its acceptance might improve with specific defined parameters for indication and proper design of the velar extension. We present our 13-year, single-center experience in treating infants with RS using PEBP, focusing on the description and insertion of an endoscopically guided PEBP design along with its complications and limitations. DESIGN AND INNOVATION: We recommend PEBP as primary treatment for RS, suggesting a new approach of design adjustment based on endoscopic findings of multilevel upper airway obstruction. SETTING: Department of cleft lip and palate. PATIENTS: Infants with isolated or syndromic RS, period 2010 to 2019. INTERVENTIONS: Pre-epiglottic baton plate treatment, intravelar veloplasty, and hard palate closure after initial PEBP treatment. RESULTS: We treated 132 infants (isolated RS, 111; syndromic RS, 21) with PEBP. All infants with isolated RS were discharged within an average of 8 days of PEBP therapy. For them, no tracheotomy or tongue-lip adhesion procedures were needed. Only 4 of the 20 infants discharged with a nasogastric tube needed it for >2 weeks. Intravelar veloplasty and palate closure were performed after 3 and 6 months of initiating PEBP treatment, respectively. CONCLUSIONS: Application of an orthodontic device in RS therapy has not been accepted worldwide. We hope that our learning curve and recommendations about PEBP will help the implementation of this highly effective and nonsurgical treatment option.

Further evidence for deletions in 7p14.1 contributing to nonsyndromic cleft lip with or without cleft palate.

BACKGROUND: Nonsyndromic cleft with or without cleft palate (nsCL/P) is a common birth defect. Although genome-wide association studies (GWAS) have identified numerous risk variants, a considerable fraction of the genetic heritability remains unknown. The aim of the present study was to replicate a previous finding that de novo deletions in a 62 kb region of chromosome 7p14 are a risk factor for nsCL/P, using an independent cohort. METHODS: Data from a published case-control GWAS cohort of 399 patients and 1318 controls were used. Copy number variant (CNV) detection in the 62 kb candidate region of 7p14 was performed using QuantiSNP. Putative CNVs in probands were verified and validated by quantitative polymerase chain reaction. Segregation analyses were performed in family members for whom DNA was available. RESULTS: Within the 62 kb candidate region, a deletion of 7.4 kb showed association with nsCL/P (13/387 cases, 20/1300 controls, plowest = 0.024, odds ratio = 2.22). In all families with a sporadic case (n = 3), the deletion occurred de novo. In multiplex families, both incomplete segregation and incomplete penetrance were observed. CONCLUSION: The present data support the hypothesis that deletions at 7p14 are a common risk factor for nsCL/P. Genome-wide CNV analyses in nsCL/P cohorts are warranted to explore the functional relevance of these deletions and their contribution to nsCL/P, and to determine exact breakpoints. Birth Defects Research (Part A) 106:767-772, 2016. © 2016 Wiley Periodicals, Inc.

Sequencing the GRHL3 Coding Region Reveals Rare Truncating Mutations and a Common Susceptibility Variant for Nonsyndromic Cleft Palate.

Nonsyndromic cleft lip with/without cleft palate (nsCL/P) and nonsyndromic cleft palate only (nsCPO) are the most frequent subphenotypes of orofacial clefts. A common syndromic form of orofacial clefting is Van der Woude syndrome (VWS) where individuals have CL/P or CPO, often but not always associated with lower lip pits. Recently, ∼5% of VWS-affected individuals were identified with mutations in the grainy head-like 3 gene (GRHL3). To investigate GRHL3 in nonsyndromic clefting, we sequenced its coding region in 576 Europeans with nsCL/P and 96 with nsCPO. Most strikingly, nsCPO-affected individuals had a higher minor allele frequency for rs41268753 (0.099) than control subjects (0.049; p = 1.24 × 10(-2)). This association was replicated in nsCPO/control cohorts from Latvia, Yemen, and the UK (pcombined = 2.63 × 10(-5); ORallelic = 2.46 [95% CI 1.6-3.7]) and reached genome-wide significance in combination with imputed data from a GWAS in nsCPO triads (p = 2.73 × 10(-9)). Notably, rs41268753 is not associated with nsCL/P (p = 0.45). rs41268753 encodes the highly conserved p.Thr454Met (c.1361C>T) (GERP = 5.3), which prediction programs denote as deleterious, has a CADD score of 29.6, and increases protein binding capacity in silico. Sequencing also revealed four novel truncating GRHL3 mutations including two that were de novo in four families, where all nine individuals harboring mutations had nsCPO. This is important for genetic counseling: given that VWS is rare compared to nsCPO, our data suggest that dominant GRHL3 mutations are more likely to cause nonsyndromic than syndromic CPO. Thus, with rare dominant mutations and a common risk variant in the coding region, we have identified an important contribution for GRHL3 in nsCPO.

Nonsyndromic cleft lip with or without cleft palate: Increased burden of rare variants within Gremlin-1, a component of the bone morphogenetic protein 4 pathway.

BACKGROUND: The genes Gremlin-1 (GREM1) and Noggin (NOG) are components of the bone morphogenetic protein 4 pathway, which has been implicated in craniofacial development. Both genes map to recently identified susceptibility loci (chromosomal region 15q13, 17q22) for nonsyndromic cleft lip with or without cleft palate (nsCL/P). The aim of the present study was to determine whether rare variants in either gene are implicated in nsCL/P etiology. METHODS: The complete coding regions, untranslated regions, and splice sites of GREM1 and NOG were sequenced in 96 nsCL/P patients and 96 controls of Central European ethnicity. Three burden and four nonburden tests were performed. Statistically significant results were followed up in a second case-control sample (n = 96, respectively). For rare variants observed in cases, segregation analyses were performed. RESULTS: In NOG, four rare sequence variants (minor allele frequency < 1%) were identified. Here, burden and nonburden analyses generated nonsignificant results. In GREM1, 33 variants were identified, 15 of which were rare. Of these, five were novel. Significant p-values were generated in three nonburden analyses. Segregation analyses revealed incomplete penetrance for all variants investigated. CONCLUSION: Our study did not provide support for NOG being the causal gene at 17q22. However, the observation of a significant excess of rare variants in GREM1 supports the hypothesis that this is the causal gene at chr. 15q13. Because no single causal variant was identified, future sequencing analyses of GREM1 should involve larger samples and the investigation of regulatory elements.

The oculo-auriculo-fronto-nasal syndrome (OAFNS) - description of a rare and complex craniofacial deformity and its interdisciplinary management before school age.

Craniofacial clefts are certainly among the most challenging congenital malformations with respect to functional, aesthetic and psychosocial consequences. The aetiology is still under discussion, recent molecular genetic findings suggest defects in the ciliary function of neural crest cells during facial development. The severity of craniofacial clefting is known to be extremely variable. Different classifications have been proposed however nomenclature is not uniform. If vertical, median craniofacial clefting of fronto-naso-maxillary structures is accompanied by auriculo-mandibular malformations the term oculo-auriculo-fronto-nasal syndrome (OAFNS) has been proposed. Extreme craniofacial abnormalities have to be expected in this rare disorder. Adequate correction is a surgical challenge and interventions have to be adapted individually to patient's needs with respect to general condition, age and growth. This case report describes both the underlying pathology as well as the interdisciplinary management of a female patient from birth to 6 years of age affected by this rare combination of vertical craniofacial clefting and bilateral auriculo-mandibular dysplasia.

Genome-wide association study identifies two susceptibility loci for nonsyndromic cleft lip with or without cleft palate.

We conducted a genome-wide association study for nonsyndromic cleft lip with or without cleft palate (NSCL/P) in 401 affected individuals and 1,323 controls, with replication in an independent sample of 793 NSCL/P triads. We report two new loci associated with NSCL/P at 17q22 (rs227731, combined P = 1.07 x 10(-8), relative risk in homozygotes = 1.84, 95% CI 1.34-2.53) and 10q25.3 (rs7078160, combined P = 1.92 x 10(-8), relative risk in homozygotes = 2.17, 95% CI 1.32-3.56).

Genome-wide linkage scan of nonsyndromic orofacial clefting in 91 families of central European origin.

Orofacial clefts are among the most common of all congenital disorders. Nonsyndromic cases of cleft lip with or without cleft palate (NSCL/P) and cleft palate only (NSCPO) are considered to have a multifactorial etiology which involves both genetic and environmental factors. We present the results of a genome-wide linkage scan in 91 families of central European descent with nonsyndromic orofacial clefts (NSC). The sample included 74 NSCL/P families, 15 NSCPO families, and 2 mixed families (a total of 217 affected and 230 unaffected individuals were genotyped). We genotyped 542 microsatellite markers (average intermarker distance = 6.9 cM). Multipoint nonparametric linkage analysis was performed using Allegro 2.0f. In addition to the factors investigated in previous genome-wide linkage analyses, we searched for sex-specific susceptibility loci, loci demonstrating parental imprinting and loci that are shared by NSCL/P and NSCPO. Several genomic regions likely to contain susceptibility loci for NSC were identified at the level of nominal significance. Some of these overlap with regions identified in previous studies. Suggestive evidence of linkage was obtained for the loci 4q21-q26 and 1p31-p21, with the chromosome 1 locus showing a male-specific genetic effect. Our study has identified promising chromosomal regions for the identification of NSC-associated genes, and demonstrates the importance of performing detailed statistical analyses which take into account complex genetic mechanisms such as sex-specific effects and genomic imprinting. Further research in large patient samples is necessary to identify factors common to NSCL/P and NSCPO.

Transforming growth factor-beta receptor type 1 (TGFBR1) is not associated with non-syndromic cleft lip with or without cleft palate in patients of Central European descent.

OBJECTIVE: Transforming growth factor-beta (TGF-ß) type 1 receptor (also known as activin receptor-like kinase 5, ALK5) is expressed in palatal tissue during embryogenesis. Experimental studies in transgenic mice with a genetic deletion of Alk5 showed that TGF-ß type 1 receptor is required for upper lip and midline fusion of the hard and soft palate. In humans, association of TGF-ß type 1 receptor gene (TGFBR1) and the development of non-syndromic cleft lip with or without cleft palate (NSCL/P) had been observed in a multiethnic sample of Chinese, Philippine, Indian and Turkish families. In order to re-evaluate the relevance of these findings, we carried out a family-based association study among 218 NSCL/P families of Central European descent. METHODS: Genomic DNA was obtained from peripheral blood of 218 complete parent-offspring triads with NSCL/P. The sample comprised 14 patients with cleft lip only (CLO) and 204 patients with cleft lip and palate (CLP). Genotyping and transmission disequilibrium test (TDT) were performed on all 218 triads with a total of 17 tagging single-nucleotide polymorphisms (SNPs). We also performed testing for extended haplotypes and a log-linear model by Weinberg was used to screen parent-of-origin effects. Furthermore the use of estimates for the relative risks (RR) of Weinberg's model was obtained. RESULTS: TDT analysis revealed no significant transmission distortion, neither at the level of individual markers nor at the level of haplotypes. Similarly negative results were obtained when we restricted our analysis to the subgroup of patients with CLP (n=204). Relative risk calculations (RR) of the children's and mothers' genotypes obtained negative results, after correction of p-values for multiple testing. Likewise application of Weinberg's log-linear model did not find any evidence for parent-of-origin effects in our sample. CONCLUSION: Despite the ample evidence supporting the role of TGF-ß type 1 receptor as a critically important and widespread morphogenetic regulator of craniofacial development in murine models, our results do not support TGFBR1 as major risk factor for NSCL/P in patients of Central European descent.

Key susceptibility locus for nonsyndromic cleft lip with or without cleft palate on chromosome 8q24.

We conducted a genome-wide association study involving 224 cases and 383 controls of Central European origin to identify susceptibility loci for nonsyndromic cleft lip with or without cleft palate (NSCL/P). A 640-kb region at chromosome 8q24.21 was found to contain multiple markers with highly significant evidence for association with the cleft phenotype, including three markers that reached genome-wide significance. The 640-kb cleft-associated region was saturated with 146 SNP markers and then analyzed in our entire NSCL/P sample of 462 unrelated cases and 954 controls. In the entire sample, the most significant SNP (rs987525) had a P value of 3.34 x 10(-24). The odds ratio was 2.57 (95% CI = 2.02-3.26) for the heterozygous genotype and 6.05 (95% CI = 3.88-9.43) for the homozygous genotype. The calculated population attributable risk for this marker is 0.41, suggesting that this study has identified a major susceptibility locus for NSCL/P.

Further evidence for the involvement of MYH9 in the etiology of non-syndromic cleft lip with or without cleft palate.

Non-syndromic cleft lip with or without cleft palate (NSCL/P) is one of the most common birth defects and has a multifactorial etiology that includes both genetic and environmental components. MYH9, the gene coding for the heavy chain of non-muscle myosin II, has been considered as a good candidate gene in NSCL/P on the basis of its expression profile during craniofacial morphogenesis. Reports in an Italian sample, as well as in an ethnically mixed North American sample, of a positive association between single-nucleotide polymorphisms in the MYH9 gene and NSCL/P have provided further support for the role of MYH9 in the development of NSCL/P. In the present study, we aimed to replicate these findings by conducting a family-based association study with seven single nucleotide polymorphisms in MYH9 using a sample of 248 NSCL/P patients and their parents. Single marker analysis resulted in a highly significant association for rs7078. In haplotype analysis, the most significant result was obtained for the SNP combination (rs7078; rs2071731; rs739097; rs5995288). Our results thus confirm the potential involvement of MYH9 in the etiology of NSCL/P in our patients of Central European origin, although further studies are warranted to determine its exact pathogenetic role.

Cleft lip and/or palate with monogenic autosomal recessive transmission in Pyrenees shepherd dogs.

OBJECTIVE: To document the genetic background of Pyrenees shepherd dogs as it relates to the incidence of cleft lip and/or cleft palate, to describe the phenotype, and to determine possible candidate genes. DESIGN: Pedigree analysis was performed and blood samples were taken from five affected pups, their siblings, and parents. Seven candidate genes were selected and linkage analysis was performed. Further methods used included sequencing and histology. RESULTS: In 37 litters consisting of 163 pups, we found 47 affected pups in a total population of 2104. The male:female ratio was 1:0.96. Affected pups showed isolated cleft lip and/or cleft palate; no attendant disorders have been reported. Despite a high degree of relationship, two affected pups displayed a cleft palate (- H S H -) and a cleft lip with or without cleft palate (L A -) cleft formation. Histology of affected pups showed that the medial edge epithelium remained intact and did not undergo an epithelial-mesenchymal transformation. There was no evidence for linkage between the trait and TGFb3 or Msx1. Subsequent sequencing excluded the coding sequence of Fst as well. CONCLUSION: Pedigree analysis showed that cleft palate is not genetically distinct from cleft lip with or without cleft palate but is inherited in this breed as a monogenic autosomal recessive trait. Linkage analysis and sequencing excluded TGFb3, Msx1, and Fst as candidate genes. Histology of affected pups showed that the medial edge epithelium is still intact.

IRF6 gene variants in Central European patients with non-syndromic cleft lip with or without cleft palate.

Variants in the interferon regulatory factor 6 (IRF6) gene have repeatedly been associated with non-syndromic cleft lip with or without cleft palate (NSCL/P). A recent study has suggested that the functionally relevant variant rs642961 is the underlying cause of the observed associations. We genotyped rs642961 in our Central European case-control sample of 460 NSCL/P patients and 952 controls. In order to investigate whether other IRF6 variants contribute independently to the etiology of NSCL/P, we also genotyped the non-synonymous coding variant V274I (rs2235371) and five IRF6-haplotype tagging single nucleotide polymorphisms (SNPs). A highly significant result was observed for rs642961 (P = 1.44 x 10(-6)) in our sample. The odds ratio was 1.75 [95% confidence interval (CI): 1.38-2.22] for the heterozygous genotype and 1.94 (95% CI: 1.21-3.10) for the homozygous genotype, values that are similar to those reported in a previously published family-based study. Our results thus confirm the involvement of the IRF6 variant, rs642961, in the etiology of NSCL/P in the Central European population. We also found evidence suggestive of an independent protective effect of the coding variant V274I. In order to understand fully the genetic architecture of the IRF6 locus, it will be necessary to conduct additional SNP-based and resequencing studies using large samples of patients.

TGFB3 displays parent-of-origin effects among central Europeans with nonsyndromic cleft lip and palate.

Mice with a deletion of Tgf-beta3 (-/-) and association studies in humans of different ethnicities support the involvement of TGFB3 in the etiology of orofacial clefts. In this study, we investigated the relevance of TGFB3 in the development of cleft lip and palate (CL/P) among 204 triads of central European origin. Transmission-disequilibrium test (TDT) analysis revealed no significant transmission distortions for each marker alone, and none for any possible haplotypes. However, we found strong evidence for parent-of-origin effects, with lower risk of maternal transmission compared with paternal transmission [I (M) = 0.38; confidence interval (CI): 0.17-0.86] of the risk allele T to an affected offspring at marker rs2300607. This is also expressed in an increased risk of heterozygous children having the T allele inherited from the father (R (P) = 3.47; CI: 1.32-9.11). Our data support the involvement of TGFB3 in the development of oral clefts in patients of central European origin.

Follistatin antagonizes transforming growth factor-beta3-induced epithelial-mesenchymal transition in vitro: implications for murine palatal development supported by microarray analysis.

Epithelial-mesenchymal transition (EMT) is involved in normal embryonic development as well as in tumor progression and invasiveness. This process is also known to be a crucial step in palatogenesis during fusion of the bi-lateral palatal processes. Disruption of this step results in a cleft palate, which is among the most frequent birth defects in humans. A number of genes and encoded proteins have been shown to play a role in this developmental stage. The central role is attributed to the cytokine transforming growth factor-beta3 (TGF-beta3), which is expressed in the medial edge epithelium (MEE) already before the fusion process. The MEE covers the tips of the growing palatal shelves and eventually undergoes EMT or programmed cell death (apoptosis). TGF-beta3 is described to induce EMT in embryonic palates. With regard to the early expression of this molecule before the fusion process, it is not well understood which mechanisms prevent the TGF-beta3 producing epithelial cells from undergoing differentiation precociously. We used the murine palatal fusion to study the regulation of EMT. Specifically, we analyzed the MEE for the expression of known antagonists of TGF-beta molecules using in situ hybridization and detected the gene coding for Follistatin to be co-expressed with TGF-beta3. Further, we could show that Follistatin directly binds to TGF-beta3 and that it completely blocks TGF-beta3-induced EMT of the normal murine mammary gland (NMuMG) epithelial cell line in vitro. In addition, we analyzed the gene expression profile of NMuMG cells during TGF-beta3-induced EMT by microarray hybridization, detecting strong changes in the expression of apoptosis-regulating genes.