Re-focusing on Agnathia-Otocephaly complex.

OBJECTIVES: Agnathia-otocephaly complex is a rare condition characterized by mandibular hypoplasia or agnathia, ear anomalies (melotia/synotia) and microstomia with aglossia. This severe anomaly of the first branchial arch is most often lethal. The estimated incidence is less than 1 in 70.000 births, with etiologies linked to both genetic and teratogenic factors. Most of the cases are sporadic. To date, two genes have been described in humans to be involved in this condition: OTX2 and PRRX1. Nevertheless, the overall proportion of mutated cases is unknown and a significant number of patients remain without molecular diagnosis. Thus, the involvement of other genes than OTX2 and PRRX1 in the agnathia-otocephaly complex is not unlikely. Heterozygous mutations in Cnbp in mice are responsible for mandibular and eye defects mimicking the agnathia-otocephaly complex in humans and appear as a good candidate. Therefore, in this study, we aimed (i) to collect patients presenting with agnathia-otocephaly complex for screening CNBP, in parallel with OTX2 and PRRX1, to check its possible implication in the human phenotype and (ii) to compare our results with the literature data to estimate the proportion of mutated cases after genetic testing. MATERIALS AND METHODS: In this work, we describe 10 patients suffering from the agnathia-otocephaly complex. All of them benefited from array-CGH and Sanger sequencing of OTX2, PRRX1 and CNBP. A complete review of the literature was made using the Pubmed database to collect all the patients described with a phenotype of agnathia-otocephaly complex during the 20 last years (1998-2019) in order (i) to study etiology (genetic causes, iatrogenic causes…) and (ii), when genetic testing was performed, to study which genes were tested and by which type of technologies. RESULTS: In our 10 patients' cohort, no point mutation in the three tested genes was detected by Sanger sequencing, while array-CGH has allowed identifying a 107-kb deletion encompassing OTX2 responsible for the agnathia-otocephaly complex phenotype in 1 of them. In 4 of the 70 cases described in the literature, a toxic cause was identified and 22 out the 66 remaining cases benefited from genetic testing. Among those 22 patients, 6 were carrying mutation or deletion in the OTX2 gene and 4 in the PRRX1 gene. Thus, when compiling results from our cohort and the literature, a total of 32 patients benefited from genetic testing, with only 34% (11/32) of patients having a mutation in one of the two known genes, OTX2 or PRRX1. CONCLUSIONS: From our work and the literature review, only mutations in OTX2 and PRRX1 have been found to date in patients, explaining around one third of the etiologies after genetic testing. Thus, agnathia-otocephaly complex remains unexplained in the majority of the patients, which indicates that other factors might be involved. Although involved in first branchial arch defects, no mutation in the CNBP gene was found in this study. This suggests that mutations in CNBP might not be involved in such phenotype in humans or that, unlike in mice, a compensatory effect might exist in humans. Nevertheless, given that agnathia-otocephaly complex is a rare phenotype, more patients have to be screened for CNBP mutations before we definitively conclude about its potential implication. Therefore, this work presents the current state of knowledge on agnathia-otocephaly complex and underlines the need to expand further the understanding of the genetic bases of this disorder, which remains largely unknown. CLINICAL RELEVANCE: We made here an update and focus on the clinical and genetic aspects of agnathia-otocephaly complex as well as a more general review of craniofacial development.

Bone Regeneration of a 3D-Printed Alloplastic and Particulate Xenogenic Graft with rhBMP-2.

This study aimed to evaluate the bone regeneration capacity of a customized alloplastic material and xenograft with recombinant human bone morphogenetic protein-2 (rhBMP-2). We prepared hydroxyapatite (HA)/tricalcium phosphate (TCP) pure ceramic bone blocks made using a 3D printing system and added rhBMP-2 to both materials. In eight beagle dogs, a total of 32 defects were created on the lower jaws. The defective sites of the negative control group were left untreated (N group; 8 defects), and those in the positive control group were filled with particle-type Bio-Oss (P group; 12 defects). The defect sites in the experimental group were filled with 3D-printed synthetic bone blocks (3D group; 12 defects). Radiographic and histological evaluations were performed after healing periods of 6 and 12 weeks and showed no significant difference in new bone formation and total bone between the P and 3D groups. The 3D-printed custom HA/TCP graft with rhBMP-2 showed bone regeneration effects similar to that of particulate Bio-Oss with rhBMP-2. Through further study and development, the application of 3D-printed customized alloplastic grafts will be extended to various fields of bone regeneration.

CHARGE syndrome without colobomas: Ophthalmic findings.

To report ophthalmic findings of patients without colobomas, and with a clinical and molecular diagnosis of CHARGE Syndrome. Retrospective study of ophthalmic findings in 67 CHARGE patients-clinically confirmed diagnosis with positive CHD7 mutation-seen in the Ophthalmology department of Cincinnati Children's Hospital Medical Center between January 1, 2008 through September 25, 2018. Criteria for inclusion in this study was absence of any form of a coloboma in either eye. In our cohort, all patients had a positive CHD7 mutation, in addition to a clinical diagnosis. 19.4% (13/67) of CHARGE patients did not have a coloboma in either eye. 69.2% (9/13) had strabismus, 76.9% (10/13) had a refractive error that warranted refractive correction, 23.1% (3/13) had amblyopia, 38.5% (5/13) had nasolacrimal duct obstruction, 30.8% (4/13) had dry eye syndrome and exposure keratopathy, 15.4% (2/13) had ptosis, 15.4% (2/13) had blepharitis, 15.4% (2/13) had Cortical Visual Impairment, 7.7% (1/13) of patients had optic nerve drusen, 7.7% (1/13) had Marcus Gunn Jaw Winking, and 7.7% (1/13) with an eyelid nevus. There are numerous ophthalmic findings in individuals with CHARGE Syndrome without colobomas. No study to date has evaluated the ophthalmic findings in CHD7 positive CHARGE patients without colobomas. These findings need to be assessed and treated to ensure optimal vision in the CHARGE patient population. Absence of coloboma does not rule out a diagnosis of CHARGE syndrome, and if there is a clinical suspicion, clinical confirmation then genetic testing would be warranted.

DLX4 is associated with orofacial clefting and abnormal jaw development.

Cleft lip and/or palate (CL/P) are common structural birth defects in humans. We used exome sequencing to study a patient with bilateral CL/P and identified a single nucleotide deletion in the patient and her similarly affected son­c.546_546delG, predicting p.Gln183Argfs*57 in the Distal-less 4 (DLX4) gene. The sequence variant was absent from databases, predicted to be deleterious and was verified by Sanger sequencing. In mammals, there are three Dlx homeobox clusters with closely located gene pairs (Dlx1/Dlx2, Dlx3/Dlx4, Dlx5/Dlx6). In situ hybridization showed that Dlx4 was expressed in the mesenchyme of the murine palatal shelves at E12.5, prior to palate closure. Wild-type human DLX4, but not mutant DLX4_c.546delG, could activate two murine Dlx conserved regulatory elements, implying that the mutation caused haploinsufficiency. We showed that reduced DLX4 expression after short interfering RNA treatment in a human cell line resulted in significant up-regulation of DLX3, DLX5 and DLX6, with reduced expression of DLX2 and significant up-regulation of BMP4, although the increased BMP4 expression was demonstrated only in HeLa cells. We used antisense morpholino oligonucleotides to target the orthologous Danio rerio gene, dlx4b, and found reduced cranial size and abnormal cartilaginous elements. We sequenced DLX4 in 155 patients with non-syndromic CL/P and CP, but observed no sequence variants. From the published literature, Dlx1/Dlx2 double homozygous null mice and Dlx5 homozygous null mice both have clefts of the secondary palate. This first finding of a DLX4 mutation in a family with CL/P establishes DLX4 as a potential cause of human clefts.

Skeletal anomaly assessment in diploid and triploid juvenile Atlantic salmon (Salmo salar L.) and the effect of temperature in freshwater.

Triploid Atlantic salmon tend to develop a higher prevalence of skeletal anomalies. This tendency may be exacerbated by an inadequate rearing temperature. Early juvenile all-female diploid and triploid Atlantic salmon were screened for skeletal anomalies in consecutive experiments to include two size ranges: the first tested the effect of ploidy (0.2-8 g) and the second the effect of ploidy, temperature (14 °C and 18 °C) and their interaction (8-60 g). The first experiment showed that ploidy had no effect on skeletal anomaly prevalence. A high prevalence of opercular shortening was observed (average prevalence in both ploidies 85.8%) and short lower jaws were common (highest prevalence observed 11.3%). In the second experiment, ploidy, but not temperature, affected the prevalence of short lower jaw (diploids > triploids) and lower jaw deformity (triploids > diploids, highest prevalence observed 11.1% triploids and 2.7% diploids) with a trend indicating a possible developmental link between the two jaw anomalies in triploids. A radiological assessment (n = 240 individuals) showed that at both temperatures triploids had a significantly (P < 0.05) lower number of vertebrae and higher prevalence of deformed individuals. These findings (second experiment) suggest ploidy was more influential than temperature in this study.

Directed Bmp4 expression in neural crest cells generates a genetic model for the rare human bony syngnathia birth defect.

Congenital bony syngnathia, a rare but severe human birth defect, is characterized by bony fusion of the mandible to the maxilla. However, the genetic mechanisms underlying this birth defect are poorly understood, largely due to limitation of available animal models. Here we present evidence that transgenic expression of Bmp4 in neural crest cells causes a series of craniofacial malformations in mice, including a bony fusion between the maxilla and hypoplastic mandible, resembling the bony syngnathia syndrome in humans. In addition, the anterior portion of the palatal shelves emerged from the mandibular arch instead of the maxilla in the mutants. Gene expression assays showed an altered expression of several facial patterning genes, including Hand2, Dlx2, Msx1, Barx1, Foxc2 and Fgf8, in the maxillary and mandibular processes of the mutants, indicating mis-patterned cranial neural crest (CNC) derived cells in the facial region. However, despite of formation of cleft palate and ectopic cartilage, forced expression of a constitutively active form of BMP receptor-Ia (caBmprIa) in CNC lineage did not produce the syngnathia phenotype, suggesting a non-cell autonomous effect of the augmented BMP4 signaling. Our studies demonstrate that aberrant BMP4-mediated signaling in CNC cells leads to mis-patterned facial skeleton and congenital bony syngnathia, and suggest an implication of mutations in BMP signaling pathway in human bony syngnathia.

Distinct populations within Isl1 lineages contribute to appendicular and facial skeletogenesis through the ß-catenin pathway.

Isl1 expression marks progenitor populations in developing embryos. In this study, we investigated the contribution of Isl1-expressing cells that utilize the ß-catenin pathway to skeletal development. Inactivation of ß-catenin in Isl1-expressing cells caused agenesis of the hindlimb skeleton and absence of the lower jaw (agnathia). In the hindlimb, Isl1-lineages broadly contributed to the mesenchyme; however, deletion of ß-catenin in the Isl1-lineage caused cell death only in a discrete posterior domain of nascent hindlimb bud mesenchyme. We found that the loss of posterior mesenchyme, which gives rise to Shh-expressing posterior organizer tissue, caused loss of posterior gene expression and failure to expand chondrogenic precursor cells, leading to severe truncation of the hindlimb. In facial tissues, Isl1-expressing cells broadly contributed to facial epithelium. We found reduced nuclear ß-catenin accumulation and loss of Fgf8 expression in mandibular epithelium of Isl1(-/-) embryos. Inactivating ß-catenin in Isl1-expressing epithelium caused both loss of epithelial Fgf8 expression and death of mesenchymal cells in the mandibular arch without affecting epithelial proliferation and survival. These results suggest a Isl1→ß-catenin→Fgf8 pathway that regulates mesenchymal survival and development of the lower jaw in the mandibular epithelium. By contrast, activating ß-catenin signaling in Isl1-lineages caused activation of Fgf8 broadly in facial epithelium. Our results provide evidence that, despite its broad contribution to hindlimb mesenchyme and facial epithelium, the Isl1-ß-catenin pathway regulates skeletal development of the hindlimb and lower jaw through discrete populations of cells that give rise to Shh-expressing posterior hindlimb mesenchyme and Fgf8-expressing mandibular epithelium.

Med14 cooperates with brg1 in the differentiation of skeletogenic neural crest.

BACKGROUND: An intricate gene regulatory network drives neural crest migration and differentiation. How epigenetic regulators contribute to this process is just starting to be understood. RESULTS: We found that mutation of med14 or brg1 in zebrafish embryos resulted in a cluster of neural crest cell-related defects. In med14 or brg1 mutants, neural crest cells that form the jaw skeleton were specified normally and migrated to target sites. However, defects in their subsequent terminal differentiation were evident. Transplantation experiments demonstrated that med14 and brg1 are required directly in neural crest cells. Analysis of med14; brg1 double mutant embryos suggested the existence of a strong genetic interaction between members of the Mediator and BAF complexes. CONCLUSIONS: These results suggest a critical role for Mediator and BAF complex function in neural crest development, and may also clarify the nature of defects in some craniofacial abnormalities.

Ocular manifestations of 22q11.2 microduplication.

PURPOSE: To report a new ocular manifestation of the dup22q11 syndrome and explore involved genes that may offer insight to mechanisms of pathogenesis. DESIGN: Case series. PARTICIPANTS: Two male patients with this syndrome diagnosed with dup22q11.2. METHODS: Medical records were reviewed. Duplication was detected in the oligo-single nucleotide polymorphism chromosomal microarray and duplicated genes within the segment where determined by literature and database review. Potential associations between the ophthalmologic manifestations and their physiopathology were investigated. MAIN OUTCOME MEASURES: Microarray results and identification of candidate genes within the duplicated segment. RESULTS: Our patients demonstrate previously unreported findings of dup22q11.2, including Marcus Gunn jaw winking, Duane's retraction syndrome, and other abnormal eye movements consistent with a congenital cranial dysinnervation disorder (CCDD), retinal vascular tortuosity, and primary infantile glaucoma. The duplicated segment in case 1 includes SNAP29, which could be linked with the development of retinal vascular tortuosity, and MAPK1, which seems to play a role in axonal development through the semaphorin pathway, which may serve as a candidate gene for CCDD. In case 2, the CLDN5 gene is within the duplicated segment. CLDN5 could be involved in the pathophysiology of glaucoma. CONCLUSIONS: Our cases expand the ocular phenotype for duplication of 22q11 and serve to identify potential candidate genes for the development of CCDD, retinal vascular tortuosity, and glaucoma.

OTX2 mutations contribute to the otocephaly-dysgnathia complex.

BACKGROUND: Otocephaly or dysgnathia complex is characterised by mandibular hypoplasia/agenesis, ear anomalies, microstomia, and microglossia; the molecular basis of this developmental defect is largely unknown in humans. METHODS AND RESULTS: This study reports a large family in which two cousins with micro/anophthalmia each gave birth to at least one child with otocephaly, suggesting a genetic relationship between anophthalmia and otocephaly. OTX2, a known microphthalmia locus, was screened in this family and a frameshifting mutation was found. The study subsequently identified in one unrelated otocephalic patient a sporadic OTX2 mutation. Because OTX2 mutations may not be sufficient to cause otocephaly, the study assayed the potential of otx2 to modify craniofacial phenotypes in the context of known otocephaly gene suppression in vivo. It was found that otx2 can interact genetically with pgap1, prrx1, and msx1 to exacerbate mandibular and midline defects during zebrafish development. However, sequencing of these loci in the OTX2-positive families did not unearth likely pathogenic lesions, suggesting further genetic heterogeneity and complexity. CONCLUSION: Identification of OTX2 involvement in otocephaly/dysgnathia in humans, even if loss of function mutations at this locus does not sufficiently explain the complex anatomical defects of these patients, suggests the requirement for a second genetic hit. Consistent with this notion, trans suppression of otx2 and other developmentally related genes recapitulate aspects of the otocephaly phenotype in zebrafish. This study highlights the combined utility of genetics and functional approaches to dissect both the regulatory pathways that govern craniofacial development and the genetics of this disease group.

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.

Craniofacial abnormalities result from knock down of nonsyndromic clefting gene, crispld2, in zebrafish.

Nonsyndromic cleft lip and palate (NSCLP), a common birth defect, affects 4,000 newborns in the US each year. Previously, we described an association between CRISPLD2 and NSCLP and showed Crispld2 expression in the murine palate. These results suggested that a perturbation in CRISPLD2 activity affects craniofacial development. Here, we describe crispld2 expression and the phenotypic consequence of its loss of function in zebrafish. crispld2 was expressed at all stages of zebrafish morphogenesis examined and localized to the rostral end by 1-day postfertilization. Morpholino knockdown of crispld2 resulted in significant jaw and palatal abnormalities in a dose-dependent manner. Loss of crispld2 caused aberrant patterning of neural crest cells (NCC) suggesting that crispld2 is necessary for normal NCC formation. Altogether, we show that crispld2 plays a significant role in the development of the zebrafish craniofacies and alteration of normal protein levels disturbs palate and jaw formation. These data provide support for a role of CRISPLD2 in NSCLP.

Agnathia-Otocephaly Complex Due to a De Novo Deletion in the OTX2 Gene.

Agnathia-otocephaly complex (AOC) is a rare and usually lethal malformation typically characterized by hypoplasia or the absence of the mandible, ventromedial and caudal displacement of the ears with or without the fusion of the ears, a small oral aperture with or without a tongue hypoplasia. Its incidence is reported as 1 in 70,000 births and its etiology has been attributed to both genetic and teratogenic causes. AOC is characterized by a wide severity clinical spectrum even when occurring within the same family, ranging from a mild mandibular defect to an extreme facial aberration incompatible with life. Most AOC cases are due to a de novo sporadic mutation. Given the genetic heterogeneity, many genes have been reported to be implicated in this disease but to date, the link to only two genes has been confirmed in the development of this complex: the orthodenticle homeobox 2 (OTX2) gene and the paired related homeobox 1 (PRRX1) gene. In this article, we report a case of a fetus with severe AOC, diagnosed in routine ultrasound scan in the first trimester of pregnancy. The genetic analysis showed a novel 10 bp deletion mutation c.766_775delTTGGGTTTTA in the OTX2 gene, which has never been reported before, together with a missense variant c.778T>C in cis conformation.

A 7-year old female with arthrogryposis multiplex congenita, Duane retraction syndrome, and Marcus Gunn phenomenon due to a ZC4H2 gene mutation: a clinical presentation of the Wieacker-Wolff syndrome.

Background: Duane retraction syndrome and arthrogryposis multiplex congenita have an incidence of approximately 1:1500-1:3000 live births. However, the association of these two entities with a Marcus-Gunn might be a rare and, until now, under-recognized clinical presentation of the Wieacker-Wolff Syndrome.Patient and methods: We report a 7-year-old female with dysmorphic features, global developmental delay, arthrogryposis multiplex congenita (AMC), Duane retraction syndrome (DRS), and unilateral Marcus Gunn jaw winking.Results: Whole Exome Sequencing showed a de novo premature stop codon in ZC4H2. Extensive genetic and metabolic work was negative otherwise and Brain MRI showed delayed non-specific myelination abnormalities. She continues to have significant delays but does not have regression, seizures or other neurological complications. She has required a multidisciplinary approach for the management of her multiple contractures.Conclusion: This case confirms ZC4H2 as a cause of syndromic DRS and extends the ZC4H2 phenotype to include Marcus Gunn jaw winking.

Development of an oligo DNA microarray for the European sea bass and its application to expression profiling of jaw deformity.

BACKGROUND: The European sea bass (Dicentrarchus labrax) is a marine fish of great importance for fisheries and aquaculture. Functional genomics offers the possibility to discover the molecular mechanisms underlying productive traits in farmed fish, and a step towards the application of marker assisted selection methods in this species. To this end, we report here on the development of an oligo DNA microarray for D. labrax. RESULTS: A database consisting of 19,048 unique transcripts was constructed, of which 12,008 (63%) could be annotated by similarity and 4,692 received a GO functional annotation. Two non-overlapping 60mer probes were designed for each unique transcript and in-situ synthesized on glass slides using Agilent SurePrint technology. Probe design was positively completed for 19,035 target clusters; the oligo microarray was then applied to profile gene expression in mandibles and whole-heads of fish affected by prognathism, a skeletal malformation that strongly affects sea bass production. Statistical analysis identified 242 transcripts that are significantly down-regulated in deformed individuals compared to normal fish, with a significant enrichment in genes related to nervous system development and functioning. A set of genes spanning a wide dynamic range in gene expression level were selected for quantitative RT-PCR validation. Fold change correlation between microarray and qPCR data was always significant. CONCLUSIONS: The microarray platform developed for the European sea bass has a high level of flexibility, reliability, and reproducibility. Despite the well known limitations in achieving a proper functional annotation in non-model species, sufficient information was obtained to identify biological processes that are significantly enriched among differentially expressed genes. New insights were obtained on putative mechanisms involved on mandibular prognathism, suggesting that bone/nervous system development might play a role in this phenomenon.

Irreversible effects of retinoic acid pulse on Xenopus jaw morphogenesis: new insight into cranial neural crest specification.

Jaws are formed by cephalic neural crest (CNCCs) and mesodermal cells migrating to the first pharyngeal arch (PA1). A complex signaling network involving different PA1 components then establishes the jaw morphogenetic program. To gather insight on this developmental process, in this study, we analyze the teratogenic effects of brief (1-15 min) pulses of low doses of retinoic acid (RA: 0.25-2 µM) or RA agonists administered to early Xenopus laevis (X.l.) embryos. We show that these brief pulses of RA cause permanent craniofacial defects specifically when treatments are performed during a 6-hr window (developmental stages NF15-NF23) that covers the period of CNCCs maintenance, migration, and specification. Earlier or later treatments have no effect. Similar treatments performed at slightly different developmental stages within this temporal window give rise to different spectra of malformations. The RA-dependent teratogenic effects observed in Xenopus can be partially rescued by folinic acid. We provide evidence suggesting that in Xenopus, as in the mouse, RA causes craniofacial malformations by perturbing signaling to CNCCs. Differently from the mouse, where RA affects CNCCs only at the end of their migration, in Xenopus, RA has an effect on CNCCs during all the period ranging from their exit from the neural tube until their arrival in the PA1. Our findings provide a conceptual framework to understand the origin of individual facial features and the evolution of different craniofacial morphotypes.

A three-generation family with metaphyseal dysplasia, maxillary hypoplasia and brachydactyly (MDMHB) due to intragenic RUNX2 duplication.

Metaphyseal dysplasia with maxillary hypoplasia and brachydactyly (MDMHB) is an autosomal-dominant skeletal dysplasia characterised by metaphyseal flaring of the long bones, enlargement of the medial halves of the clavicles, maxillary hypoplasia, brachydactyly, dental anomalies and mild osteoporosis. To date, only one large French Canadian family and a Finnish woman have been reported with the condition. In both, intragenic duplication encompassing exons 3-5 of the RUNX2 gene was identified. We describe a new, three-generation family with clinical features of MDMHB and an intragenic tandem duplication of RUNX2 exons 3-6. Dental problems were the primary presenting feature in all four affected individuals. We compare the features in our family to those previously reported in MDMHB, review the natural history of this condition and highlight the importance of considering an underlying skeletal dysplasia in patients presenting with significant dental problems and other suggestive features, including disproportionate short stature and/or digital anomalies.

Multigenic Delineation of Lower Jaw Deformity in Triploid Atlantic Salmon (Salmo salar L.).

Lower jaw deformity (LJD) is a skeletal anomaly affecting farmed triploid Atlantic salmon (Salmo salar L.) which leads to considerable economic losses for industry and has animal welfare implications. The present study employed transcriptome analysis in parallel with real-time qPCR techniques to characterise for the first time the LJD condition in triploid Atlantic salmon juveniles using two independent sample sets: experimentally-sourced salmon (60 g) and commercially produced salmon (100 g). A total of eleven genes, some detected/identified through the transcriptome analysis (fbn2, gal and gphb5) and others previously determined to be related to skeletal physiology (alp, bmp4, col1a1, col2a1, fgf23, igf1, mmp13, ocn), were tested in the two independent sample sets. Gphb5, a recently discovered hormone, was significantly (P < 0.05) down-regulated in LJD affected fish in both sample sets, suggesting a possible hormonal involvement. In-situ hybridization detected gphb5 expression in oral epithelium, teeth and skin of the lower jaw. Col2a1 showed the same consistent significant (P < 0.05) down-regulation in LJD suggesting a possible cartilaginous impairment as a distinctive feature of the condition. Significant (P < 0.05) differential expression of other genes found in either one or the other sample set highlighted the possible effect of stage of development or condition progression on transcription and showed that anomalous bone development, likely driven by cartilage impairment, is more evident at larger fish sizes. The present study improved our understanding of LJD suggesting that a cartilage impairment likely underlies the condition and col2a1 may be a marker. In addition, the involvement of gphb5 urges further investigation of a hormonal role in LJD and skeletal physiology in general.

Familial Congenital Facial Synkinesis Due to 12q Duplication: A Case Report and Literature Review.

Inverse Marcus Gunn phenomenon is a rare form of congenital facial synkinesis in which jaw movement temporarily elicits ptosis, either unilateral or bilateral. This phenomenon is presumed to result from dysinnervation of facial muscles during development of the nervous system. We describe 2 brothers, both with inverse Marcus Gunn phenomenon in the context of multiple other congenital anomalies, all presumed secondary to a chromosomal abnormality involving 12q duplication and 1p36 deletion. Although a handful of familial cases of congenital facial synkinesis have been previously described, this is the first in which a genetic abnormality has been identified. Of the 4 genetic abnormalities previously described in association with congenital facial synkinesis (based on isolated case reports), 1 also involved duplication at the long arm of chromosome 12. We conclude that duplication of ≥1 of the roughly 44 protein-coding genes in the ∼6.3-Mb overlap region between the previously published case and our 2 patients is a likely genetic cause of congenital facial synkinesis.

Genetic and environmental factors influencing tooth and jaw malformations in rabbits.

Tooth alterations of 281 rabbits from 10 different breeds were evaluated, starting at the age of three weeks until they were fully grown. All rabbits were kept in an outdoor facility, fed with a pelleted diet and had free access to water and hay. The most common finding in 3182 clinical examinations was a gap between the mandibular incisors (38 per cent). Skull X-rays from 4 adult breeding rabbits, 15 offspring with clinical signs of dental disease and 12 clinically healthy siblings were assessed by two different methods using cephalometric distances and anatomical reference lines. Repeatabilities of cephalometric distances were mostly low and no significant associations to tooth health were found. The anatomical reference lines revealed dental findings even in rabbits that were graded as tooth healthy in previous clinical examinations. On the basis of the demonstrated age-dependent development of tooth and jaw malformations a first examination and selection at the age of 12 weeks can be recommended. Also genetic aspects for tooth and jaw malformations were considered. The estimated heritability for brachygnathia superior was 0.254 ± 0.169 for all examinations and 0.105 ± 0.092 comprising the last examination of each rabbit when fully grown.