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Alcohol Clin Exp Res ; 45(10): 1965-1979, 2021 10.
Article En | MEDLINE | ID: mdl-34581462

BACKGROUND: Alcohol exposure during the gastrulation stage of development causes the craniofacial and brain malformations that define fetal alcohol syndrome. These malformations, such as a deficient philtrum, are exemplified by a loss of midline tissue and correspond, at least in part, to regionally selective cell death in the embryo. The tumor suppressor protein Tp53 is an important mechanism for cell death, but the role of Tp53 in the consequences of alcohol exposure during the gastrulation stage has yet to be examined. The current studies used mice and zebrafish to test whether genetic loss of Tp53 is a conserved mechanism to protect against the effects of early developmental stage alcohol exposure. METHODS: Female mice, heterozygous for a mutation in the Tp53 gene, were mated with Tp53 heterozygous males, and the resulting embryos were exposed during gastrulation on gestational day 7 (GD 7) to alcohol (two maternal injections of 2.9 g/kg, i.p., 4 h apart) or a vehicle control. Zebrafish mutants or heterozygotes for the tp53zdf1  M214K mutation and their wild-type controls were exposed to alcohol (1.5% or 2%) beginning 6 h postfertilization (hpf), the onset of gastrulation. RESULTS: Examination of GD 17 mice revealed that eye defects were the most common phenotype among alcohol-exposed fetuses, occurring in nearly 75% of the alcohol-exposed wild-type fetuses. Tp53 gene deletion reduced the incidence of eye defects in both the heterozygous and mutant fetuses (to about 35% and 20% of fetuses, respectively) and completely protected against alcohol-induced facial malformations. Zebrafish (4 days postfertilization) also demonstrated alcohol-induced reductions of eye size and trabeculae length that were less common and less severe in tp53 mutants, indicating a protective effect of tp53 deletion. CONCLUSIONS: These results identify an evolutionarily conserved role of Tp53 as a pathogenic mechanism for alcohol-induced teratogenesis.

Abnormalities, Drug-Induced/etiology , Craniofacial Abnormalities/etiology , Ethanol/adverse effects , Fetal Alcohol Spectrum Disorders/metabolism , Tumor Suppressor Protein p53/metabolism , Abnormalities, Drug-Induced/metabolism , Animals , Craniofacial Abnormalities/metabolism , Female , Male , Mice , Pregnancy , Teratogenesis , Zebrafish
Int J Mol Sci ; 22(17)2021 Aug 27.
Article En | MEDLINE | ID: mdl-34502207

The complexity of skeletal pathologies makes use of in vivo models essential to elucidate the pathogenesis of the diseases; nevertheless, chondrocyte and osteoblast cell lines provide relevant information on the underlying disease mechanisms. Due to the limitations of primary chondrocytes, immortalized cells represent a unique tool to overcome this problem since they grow very easily for several passages. However, in the immortalization procedure the cells might lose the original phenotype; thus, these cell lines should be deeply characterized before their use. We immortalized primary chondrocytes from a Cant1 knock-out mouse, an animal model of Desbuquois dysplasia type 1, with a plasmid expressing the SV40 large and small T antigen. This cell line, based on morphological and biochemical parameters, showed preservation of the chondrocyte phenotype. In addition reduced proteoglycan synthesis and oversulfation of glycosaminoglycan chains were demonstrated, as already observed in primary chondrocytes from the Cant1 knock-out mouse. In conclusion, immortalized Cant1 knock-out chondrocytes maintained the disease phenotype observed in primary cells validating the in vitro model and providing an additional tool to further study the proteoglycan biosynthesis defect. The same approach might be extended to other cartilage disorders.

Acid Anhydride Hydrolases/physiology , Chondrocytes/pathology , Craniofacial Abnormalities/pathology , Dwarfism/pathology , Glycosaminoglycans/metabolism , Joint Instability/pathology , Ossification, Heterotopic/pathology , Phenotype , Polydactyly/pathology , Animals , Cell Line, Transformed , Chondrocytes/metabolism , Craniofacial Abnormalities/etiology , Craniofacial Abnormalities/metabolism , Dwarfism/etiology , Dwarfism/metabolism , Joint Instability/etiology , Joint Instability/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Ossification, Heterotopic/etiology , Ossification, Heterotopic/metabolism , Polydactyly/etiology , Polydactyly/metabolism
Development ; 148(17)2021 09 01.
Article En | MEDLINE | ID: mdl-34383890

Neural crest cells (NCCs) within the mandibular and maxillary prominences of the first pharyngeal arch are initially competent to respond to signals from either region. However, mechanisms that are only partially understood establish developmental tissue boundaries to ensure spatially correct patterning. In the 'hinge and caps' model of facial development, signals from both ventral prominences (the caps) pattern the adjacent tissues whereas the intervening region, referred to as the maxillomandibular junction (the hinge), maintains separation of the mandibular and maxillary domains. One cap signal is GATA3, a member of the GATA family of zinc-finger transcription factors with a distinct expression pattern in the ventral-most part of the mandibular and maxillary portions of the first arch. Here, we show that disruption of Gata3 in mouse embryos leads to craniofacial microsomia and syngnathia (bony fusion of the upper and lower jaws) that results from changes in BMP4 and FGF8 gene regulatory networks within NCCs near the maxillomandibular junction. GATA3 is thus a crucial component in establishing the network of factors that functionally separate the upper and lower jaws during development.

Body Patterning , Face/embryology , GATA3 Transcription Factor/metabolism , Animals , Branchial Region/cytology , Branchial Region/embryology , Branchial Region/metabolism , Cell Death , Cell Proliferation , Craniofacial Abnormalities/embryology , Craniofacial Abnormalities/genetics , Craniofacial Abnormalities/metabolism , Embryo, Mammalian , GATA3 Transcription Factor/genetics , Gene Expression Regulation, Developmental , Mandible/cytology , Mandible/embryology , Maxilla/cytology , Maxilla/embryology , Mice , Morphogenesis , Neural Crest/cytology , Neural Crest/embryology , Neural Crest/metabolism
Genes (Basel) ; 12(7)2021 06 30.
Article En | MEDLINE | ID: mdl-34209401

The neural crest is a multipotent cell population that develops from the dorsal neural fold of vertebrate embryos in order to migrate extensively and differentiate into a variety of tissues. A number of gene regulatory networks coordinating neural crest cell specification and differentiation have been extensively studied to date. Although several publications suggest a common role for microRNA-145 (miR-145) in molecular reprogramming for cell cycle regulation and/or cellular differentiation, little is known about its role during in vivo cranial neural crest development. By modifying miR-145 levels in zebrafish embryos, abnormal craniofacial development and aberrant pigmentation phenotypes were detected. By whole-mount in situ hybridization, changes in expression patterns of col2a1a and Sry-related HMG box (Sox) transcription factors sox9a and sox9b were observed in overexpressed miR-145 embryos. In agreement, zebrafish sox9b expression was downregulated by miR-145 overexpression. In silico and in vivo analysis of the sox9b 3'UTR revealed a conserved potential miR-145 binding site likely involved in its post-transcriptional regulation. Based on these findings, we speculate that miR-145 participates in the gene regulatory network governing zebrafish chondrocyte differentiation by controlling sox9b expression.

Gene Expression Regulation, Developmental , Gene Regulatory Networks , MicroRNAs/genetics , Neural Crest/cytology , Organogenesis , Zebrafish Proteins/metabolism , Zebrafish/growth & development , Animals , Cell Differentiation , Craniofacial Abnormalities/etiology , Craniofacial Abnormalities/metabolism , Craniofacial Abnormalities/pathology , Neural Crest/metabolism , Pigmentation Disorders/etiology , Pigmentation Disorders/metabolism , Pigmentation Disorders/pathology , Zebrafish/genetics , Zebrafish Proteins/genetics
Environ Toxicol Pharmacol ; 87: 103700, 2021 Oct.
Article En | MEDLINE | ID: mdl-34237469

Glyphosate [N-(phosphonomethyl)glycine] is the active ingredient in widely used broad-spectrum herbicides. Even though the toxicity mechanism of this herbicide in vertebrates is poorly understood, evidence suggests that glyphosate is an endocrine disruptor capable of producing morphological anomalies as well as cardiotoxic and neurotoxic effects. We used the zebrafish model to assess the effects of early life glyphosate exposure on the development of cartilage and bone tissues and organismal responses. We found functional alterations, including a reduction in the cardiac rate, significant changes in the spontaneous tail movement pattern, and defects in craniofacial development. These effects were concomitant with alterations in the level of the estrogen receptor alpha osteopontin and bone sialoprotein. We also found that embryos exposed to glyphosate presented spine deformities as adults. These developmental alterations are likely induced by changes in protein levels related to bone and cartilage formation.

Bone and Bones/drug effects , Craniofacial Abnormalities/chemically induced , Glycine/analogs & derivatives , Herbicides/toxicity , Teratogens/toxicity , Animals , Bone and Bones/abnormalities , Craniofacial Abnormalities/metabolism , Craniofacial Abnormalities/veterinary , Embryo, Nonmammalian/abnormalities , Embryo, Nonmammalian/drug effects , Embryo, Nonmammalian/metabolism , Estrogen Receptor alpha/metabolism , Female , Fish Proteins/metabolism , Glycine/toxicity , Heart Rate/drug effects , Locomotion/drug effects , Male , Osteopontin/metabolism , Sialoglycoproteins/metabolism , Zebrafish/abnormalities , Zebrafish/metabolism
Nat Commun ; 12(1): 4050, 2021 06 30.
Article En | MEDLINE | ID: mdl-34193871

The investigation of genetic forms of juvenile neurodegeneration could shed light on the causative mechanisms of neuronal loss. Schinzel-Giedion syndrome (SGS) is a fatal developmental syndrome caused by mutations in the SETBP1 gene, inducing the accumulation of its protein product. SGS features multi-organ involvement with severe intellectual and physical deficits due, at least in part, to early neurodegeneration. Here we introduce a human SGS model that displays disease-relevant phenotypes. We show that SGS neural progenitors exhibit aberrant proliferation, deregulation of oncogenes and suppressors, unresolved DNA damage, and resistance to apoptosis. Mechanistically, we demonstrate that high SETBP1 levels inhibit P53 function through the stabilization of SET, which in turn hinders P53 acetylation. We find that the inheritance of unresolved DNA damage in SGS neurons triggers the neurodegenerative process that can be alleviated either by PARP-1 inhibition or by NAD + supplementation. These results implicate that neuronal death in SGS originates from developmental alterations mainly in safeguarding cell identity and homeostasis.

Abnormalities, Multiple/pathology , Carrier Proteins/metabolism , Craniofacial Abnormalities/pathology , DNA Damage , Hand Deformities, Congenital/pathology , Heredodegenerative Disorders, Nervous System/pathology , Intellectual Disability/pathology , Mutation , Nails, Malformed/pathology , Neural Stem Cells/pathology , Nuclear Proteins/metabolism , Tumor Suppressor Protein p53/antagonists & inhibitors , Abnormalities, Multiple/genetics , Abnormalities, Multiple/metabolism , Carrier Proteins/genetics , Cells, Cultured , Craniofacial Abnormalities/genetics , Craniofacial Abnormalities/metabolism , Hand Deformities, Congenital/genetics , Hand Deformities, Congenital/metabolism , Heredodegenerative Disorders, Nervous System/genetics , Heredodegenerative Disorders, Nervous System/metabolism , Humans , Intellectual Disability/genetics , Intellectual Disability/metabolism , Nails, Malformed/genetics , Nails, Malformed/metabolism , Neural Stem Cells/metabolism , Nuclear Proteins/genetics , Organoids
Int J Mol Sci ; 22(12)2021 Jun 16.
Article En | MEDLINE | ID: mdl-34208498

CYP1B1 loss of function (LoF) is the main known genetic alteration present in recessive primary congenital glaucoma (PCG), an infrequent disease characterized by delayed embryonic development of the ocular iridocorneal angle; however, the underlying molecular mechanisms are poorly understood. To model CYP1B1 LoF underlying PCG, we developed a cyp1b1 knockout (KO) zebrafish line using CRISPR/Cas9 genome editing. This line carries the c.535_667del frameshift mutation that results in the 72% mRNA reduction with the residual mRNA predicted to produce an inactive truncated protein (p.(His179Glyfs*6)). Microphthalmia and jaw maldevelopment were observed in 23% of F0 somatic mosaic mutant larvae (144 hpf). These early phenotypes were not detected in cyp1b1-KO F3 larvae (144 hpf), but 27% of adult (four months) zebrafish exhibited uni- or bilateral craniofacial alterations, indicating the existence of incomplete penetrance and variable expressivity. These phenotypes increased to 86% in the adult offspring of inbred progenitors with craniofacial defects. No glaucoma-related phenotypes were observed in cyp1b1 mutants. Transcriptomic analyses of the offspring (seven dpf) of cyp1b1-KO progenitors with adult-onset craniofacial defects revealed functionally enriched differentially expressed genes related to extracellular matrix and cell adhesion, cell growth and proliferation, lipid metabolism (retinoids, steroids and fatty acids and oxidation-reduction processes that include several cytochrome P450 genes) and inflammation. In summary, this study shows the complexity of the phenotypes and molecular pathways associated with cyp1b1 LoF, with species dependency, and provides evidence for the dysregulation of extracellular matrix gene expression as one of the mechanisms underlying the pathogenicity associated with cyp1b1 disruption.

Craniofacial Abnormalities/genetics , Craniofacial Abnormalities/metabolism , Cytochrome P-450 CYP1B1/genetics , Cytochrome P-450 CYP1B1/metabolism , Extracellular Matrix/genetics , Genetic Association Studies , Lipid Metabolism/genetics , Animals , Extracellular Matrix/metabolism , Gene Expression Profiling , Gene Expression Regulation , Gene Knockout Techniques , Genetic Predisposition to Disease , Mice, Transgenic , Zebrafish
Sci Rep ; 11(1): 12175, 2021 06 09.
Article En | MEDLINE | ID: mdl-34108542

Craniofacial dysmorphism is associated with thousands of genetic and environmental disorders. Delineation of salient facial characteristics can guide clinicians towards a correct clinical diagnosis and understanding the pathogenesis of the disorder. Abnormal facial shape might require craniofacial surgical intervention, with the restoration of normal shape an important surgical outcome. Facial anthropometric growth curves or standards of single inter-landmark measurements have traditionally supported assessments of normal and abnormal facial shape, for both clinical and research applications. However, these fail to capture the full complexity of facial shape. With the increasing availability of 3D photographs, methods of assessment that take advantage of the rich information contained in such images are needed. In this article we derive and present open-source three-dimensional (3D) growth curves of the human face. These are sequences of age and sex-specific expected 3D facial shapes and statistical models of the variation around the expected shape, derived from 5443 3D images. We demonstrate the use of these growth curves for assessing patients and show that they identify normal and abnormal facial morphology independent from age-specific facial features. 3D growth curves can facilitate use of state-of-the-art 3D facial shape assessment by the broader clinical and biomedical research community. This advance in phenotype description will support clinical diagnosis and the understanding of disease pathogenesis including genotype-phenotype relations.

Abnormalities, Multiple/pathology , Craniofacial Abnormalities/pathology , Face/pathology , Imaging, Three-Dimensional/methods , Models, Statistical , Muscular Atrophy/pathology , Abnormalities, Multiple/genetics , Abnormalities, Multiple/metabolism , Adolescent , Adult , Aged , Aged, 80 and over , Anthropometry , Case-Control Studies , Child , Child, Preschool , Craniofacial Abnormalities/genetics , Craniofacial Abnormalities/metabolism , Face/abnormalities , Female , Follow-Up Studies , Growth Charts , Humans , Infant , Male , Middle Aged , Muscular Atrophy/genetics , Muscular Atrophy/metabolism , Phenotype , Prognosis , Young Adult
Genes (Basel) ; 12(5)2021 05 13.
Article En | MEDLINE | ID: mdl-34068038

Cells use membrane-bound carriers to transport cargo molecules like membrane proteins and soluble proteins, to their destinations. Many signaling receptors and ligands are synthesized in the endoplasmic reticulum and are transported to their destinations through intracellular trafficking pathways. Some of the signaling molecules play a critical role in craniofacial morphogenesis. Not surprisingly, variants in the genes encoding intracellular trafficking machinery can cause craniofacial diseases. Despite the fundamental importance of the trafficking pathways in craniofacial morphogenesis, relatively less emphasis is placed on this topic, thus far. Here, we describe craniofacial diseases caused by lesions in the intracellular trafficking machinery and possible treatment strategies for such diseases.

Craniofacial Abnormalities/genetics , Vesicular Transport Proteins/metabolism , Animals , Craniofacial Abnormalities/metabolism , Humans , Protein Transport , Secretory Pathway , Vesicular Transport Proteins/genetics
Toxicology ; 458: 152843, 2021 06 30.
Article En | MEDLINE | ID: mdl-34186166

Adverse outcome pathway (AOP) is a conceptual framework that links a molecular initiating event (MIE) via intermediate key events (KEs) with adverse effects (adverse outcomes, AO) relevant for risk assessment, through defined KE relationships (KERs). The aim of the present work is to describe a linear AOP, supported by experimental data, for skeletal craniofacial defects as the AO. This AO was selected in view of its relative high incidence in humans and the suspected relation to chemical exposure. We focused on inhibition of CYP26, a retinoic acid (RA) metabolizing enzyme, as MIE, based on robust previously published data. Conazoles were selected as representative stressors. Intermediate KEs are RA disbalance, aberrant HOX gene expression, disrupted specification, migration, and differentiation of neural crest cells, and branchial arch dysmorphology. We described the biological basis of the postulated events and conducted weight of evidence (WoE) assessments. The biological plausibility and the overall empirical evidence were assessed as high and moderate, respectively, the latter taking into consideration the moderate evidence for concordance of dose-response and temporal relationships. Finally, the essentiality assessment of the KEs, considered as high, supported the robustness of the presented AOP. This AOP, which appears of relevance to humans, thus contributes to mechanistic underpinning of selected test methods, thereby supporting their application in integrated new approach test methodologies and strategies and application in a regulatory context.

Adverse Outcome Pathways , Craniofacial Abnormalities/metabolism , Tretinoin/metabolism , Animals , Azoles/toxicity , Cytochrome P450 Family 26/antagonists & inhibitors , Female , Gene Expression Regulation/drug effects , Humans , Male , Mice , Neural Crest/abnormalities , Neural Crest/drug effects , Risk Assessment
Sci Rep ; 11(1): 11295, 2021 05 28.
Article En | MEDLINE | ID: mdl-34050248

MBD5-associated neurodevelopmental disorder (MAND) is an autism spectrum disorder (ASD) characterized by intellectual disability, motor delay, speech impairment and behavioral problems; however, the biological role of methyl-CpG-binding domain 5, MBD5, in neurodevelopment and ASD remains largely undefined. Hence, we created neural progenitor cells (NPC) derived from individuals with chromosome 2q23.1 deletion and conducted RNA-seq to identify differentially expressed genes (DEGs) and the biological processes and pathways altered in MAND. Primary skin fibroblasts from three unrelated individuals with MAND and four unrelated controls were converted into induced pluripotent stem cell (iPSC) lines, followed by directed differentiation of iPSC to NPC. Transcriptome analysis of MAND NPC revealed 468 DEGs (q < 0.05), including 20 ASD-associated genes. Comparison of DEGs in MAND with SFARI syndromic autism genes revealed a striking significant overlap in biological processes commonly altered in neurodevelopmental phenotypes, with TGFß, Hippo signaling, DNA replication, and cell cycle among the top enriched pathways. Overall, these transcriptome deviations provide potential connections to the overlapping neurocognitive and neuropsychiatric phenotypes associated with key high-risk ASD genes, including chromatin modifiers and epigenetic modulators, that play significant roles in these disease states.

Autism Spectrum Disorder/genetics , DNA-Binding Proteins/genetics , Neurodevelopmental Disorders/genetics , Abnormalities, Multiple/genetics , Abnormalities, Multiple/metabolism , Autism Spectrum Disorder/metabolism , Autistic Disorder/genetics , Cell Differentiation/genetics , Chromosome Deletion , Chromosomes, Human, Pair 2/genetics , Chromosomes, Human, Pair 2/metabolism , Craniofacial Abnormalities/genetics , Craniofacial Abnormalities/metabolism , DNA-Binding Proteins/metabolism , Gene Expression/genetics , Gene Expression Profiling/methods , Humans , Induced Pluripotent Stem Cells/metabolism , Intellectual Disability/genetics , Intellectual Disability/metabolism , Neural Stem Cells/metabolism , Neurodevelopmental Disorders/metabolism , Phenotype , Primary Cell Culture , RNA-Seq , Signal Transduction/genetics , Transcriptome/genetics
Dev Dyn ; 250(8): 1125-1139, 2021 08.
Article En | MEDLINE | ID: mdl-33667029

BACKGROUND: Foxc2 is a member of the winged helix/forkhead (Fox) box family of transcription factors. Loss of function of Foxc2 causes craniofacial abnormalities such as cleft palate and deformed cranial base, but its role during craniofacial development remains to be elucidated. RESULTS: The contributions of Foxc2-positive and its descendant cells to the craniofacial structure at E18.5 were examined using a tamoxifen-inducible Cre driver mouse (Foxc2-CreERT2) crossed with the R26R-LacZ reporter mouse. Foxc2 expression at E8.5 is restricted to the cranial mesenchyme, contributing to specific components including the cranial base, sensory capsule, tongue, upper incisor, and middle ear. Expression at E10.5 was still positively regulated in most of those regions. In situ hybridization analysis of Foxc2 and its closely related gene, Foxc1, revealed that expression domains of these genes largely overlap in the cephalic mesenchyme. Meanwhile, the tongue expressed Foxc2 but not Foxc1, and its development was affected by the neural crest-specific deletion of Foxc2 in mice (Wnt1-Cre; Foxc2fl/fl ). CONCLUSIONS: Foxc2 is expressed in cranial mesenchyme that contributes to specific craniofacial tissue components from an early stage, and it seems to be involved in their development in cooperation with Foxc1. Foxc2 also has its own role in tongue development.

Cell Lineage/genetics , Craniofacial Abnormalities/genetics , Forkhead Transcription Factors/genetics , Gene Expression Regulation, Developmental , Organogenesis/genetics , Animals , Craniofacial Abnormalities/metabolism , Forkhead Transcription Factors/metabolism , Mice , Mice, Transgenic , Neural Crest/embryology , Neural Crest/metabolism
Sci Rep ; 11(1): 4976, 2021 03 02.
Article En | MEDLINE | ID: mdl-33654163

Circumferential skin creases (CSC-KT) is a rare polymalformative syndrome characterised by intellectual disability associated with skin creases on the limbs, and very characteristic craniofacial malformations. Previously, heterozygous and homozygous mutations in MAPRE2 were found to be causal for this disease. MAPRE2 encodes for a member of evolutionary conserved microtubule plus end tracking proteins, the end binding (EB) family. Unlike MAPRE1 and MAPRE3, MAPRE2 is not required for the persistent growth and stabilization of microtubules, but plays a role in other cellular processes such as mitotic progression and regulation of cell adhesion. The mutations identified in MAPRE2 all reside within the calponin homology domain, responsible to track and interact with the plus-end tip of growing microtubules, and previous data showed that altered dosage of MAPRE2 resulted in abnormal branchial arch patterning in zebrafish. In this study, we developed patient derived induced pluripotent stem cell lines for MAPRE2, together with isogenic controls, using CRISPR/Cas9 technology, and differentiated them towards neural crest cells with cranial identity. We show that changes in MAPRE2 lead to alterations in neural crest migration in vitro but also in vivo, following xenotransplantation of neural crest progenitors into developing chicken embryos. In addition, we provide evidence that changes in focal adhesion might underlie the altered cell motility of the MAPRE2 mutant cranial neural crest cells. Our data provide evidence that MAPRE2 is involved in cellular migration of cranial neural crest and offers critical insights into the mechanism underlying the craniofacial dysmorphisms and cleft palate present in CSC-KT patients. This adds the CSC-KT disorder to the growing list of neurocristopathies.

Cell Movement/drug effects , Craniofacial Abnormalities , Microtubule-Associated Proteins , Neural Crest/metabolism , Neural Stem Cells/metabolism , Animals , Chick Embryo , Craniofacial Abnormalities/genetics , Craniofacial Abnormalities/metabolism , Humans , Induced Pluripotent Stem Cells/metabolism , Microtubule-Associated Proteins/genetics , Microtubule-Associated Proteins/metabolism , Mutation , Syndrome , Zebrafish
Hum Genet ; 140(6): 879-884, 2021 Jun.
Article En | MEDLINE | ID: mdl-33386993

DOORS syndrome is characterized by deafness, onychodystrophy, osteodystrophy, intellectual disability, and seizures. In this study, we report two unrelated individuals with DOORS syndrome without deafness. Exome sequencing revealed a homozygous missense variant in PIGF (NM_173074.3:c.515C>G, p.Pro172Arg) in both. We demonstrate impaired glycosylphosphatidylinositol (GPI) biosynthesis through flow cytometry analysis. We thus describe the causal role of a novel disease gene, PIGF, in DOORS syndrome and highlight the overlap between this condition and GPI deficiency disorders. For each gene implicated in DOORS syndrome and/or inherited GPI deficiencies, there is considerable clinical variability so a high index of suspicion is warranted even though not all features are noted.

Craniofacial Abnormalities/genetics , Glycosylphosphatidylinositols/deficiency , Hand Deformities, Congenital/genetics , Hearing Loss, Sensorineural/genetics , Intellectual Disability/genetics , Membrane Proteins/genetics , Mutation, Missense , Nails, Malformed/genetics , Seizures/genetics , Adolescent , Amino Acid Sequence , Animals , Consanguinity , Craniofacial Abnormalities/metabolism , Craniofacial Abnormalities/pathology , Female , Gene Expression , Glycosylphosphatidylinositols/genetics , Glycosylphosphatidylinositols/metabolism , HEK293 Cells , Hand Deformities, Congenital/metabolism , Hand Deformities, Congenital/pathology , Hearing Loss, Sensorineural/metabolism , Hearing Loss, Sensorineural/pathology , Homozygote , Humans , Infant , Intellectual Disability/metabolism , Intellectual Disability/pathology , Male , Membrane Proteins/deficiency , Nails, Malformed/metabolism , Nails, Malformed/pathology , Seizures/metabolism , Seizures/pathology , Sequence Alignment , Whole Exome Sequencing
Dev Dyn ; 250(4): 527-541, 2021 04.
Article En | MEDLINE | ID: mdl-33165989

BACKGROUND: The hedgehog signaling pathway is critical for developmental patterning of the limb, craniofacial and axial skeleton. Disruption of this pathway in mice leads to a series of structural malformations, but the exact role and critical period of the Hh pathway in the early development of the cranial base have been rarely described. RESULTS: Embryos exposed to vismodegib from E7.5, E9.5, and E10.5 had a higher percentage of cranial base fenestra. The peak incidence of hypoplasia in sphenoid winglets and severe craniosynostosis in cranial base synchondroses was observed when vismodegib was administered between E9.5 and E10.5. Cranial base craniosynostosis results from accelerating terminal differentiation of chondrocytes and premature osteogenesis. CONCLUSIONS: We define the critical periods for the induction of cranial base deformity by vismodegib administration at a meticulous temporal resolution. Our findings suggest that the Hh pathway may play a vital role in the early development of the cranial base. This research also establishes a novel and easy-to-establish mouse model of synostosis in the cranial base using a commercially available pathway-selective inhibitor.

Craniofacial Abnormalities/etiology , Hedgehog Proteins/metabolism , Skull Base/abnormalities , Anilides , Animals , Craniofacial Abnormalities/metabolism , Female , Hedgehog Proteins/antagonists & inhibitors , Male , Mice, Inbred ICR , Pyridines
Genes (Basel) ; 11(12)2020 12 09.
Article En | MEDLINE | ID: mdl-33316910

Ring chromosome 8 (r(8)) is one of the least frequent ring chromosomes. Usually, maternal chromosome 8 forms a ring, which can be lost from cells due to mitotic instability. The 8q24 region contains the imprinted KCNK9 gene, which is expressed from the maternal allele. Heterozygous KCNK9 mutations are associated with the imprinting disorder Birk-Barel syndrome. Here, we report a 2.5-year-old boy with developmental delay, microcephaly, dysmorphic features, diffuse muscle hypotonia, feeding problems, motor alalia and noncoarse neurogenic type of disturbance of muscle electrogenesis, partially overlapping with Birk-Barel syndrome phenotype. Cytogenetic analysis of lymphocytes revealed his karyotype to be 46,XY,r(8)(p23q24.3)[27]/45,XY,-8[3]. A de novo 7.9 Mb terminal 8p23.3p23.1 deletion, a 27.1 Mb 8p23.1p11.22 duplication, and a 4.4 Mb intact segment with a normal copy number located between them, as well as a 154-kb maternal LINGO2 gene deletion (9p21.2) with unknown clinical significance were identified by aCGH + SNP array. These aberrations were confirmed by real-time PCR. According to FISH analysis, the 8p23.1-p11.22 duplication was inverted. The ring chromosome originated from maternal chromosome 8. Targeted massive parallel sequencing did not reveal the KCNK9 mutations associated with Birk-Barel syndrome. Our data allow to assume that autosomal monosomy with inactive allele of imprinted gene arising from the loss of a ring chromosome in some somatic cells may be an etiological mechanism of mosaic imprinting disorders, presumably with less severe phenotype.

Craniofacial Abnormalities/genetics , Intellectual Disability/genetics , Muscle Hypotonia/genetics , Child, Preschool , Chromosome Deletion , Chromosomes, Human, Pair 8/genetics , Chromosomes, Human, Pair 8/metabolism , Craniofacial Abnormalities/metabolism , Genomic Imprinting/genetics , Humans , Intellectual Disability/metabolism , Karyotype , Karyotyping/methods , Male , Membrane Proteins/genetics , Mosaicism , Muscle Hypotonia/metabolism , Mutation/genetics , Nerve Tissue Proteins/genetics , Phenotype , Potassium Channels, Tandem Pore Domain/genetics , Ring Chromosomes
PLoS Genet ; 16(12): e1009219, 2020 12.
Article En | MEDLINE | ID: mdl-33382686

Roberts syndrome (RBS) is a rare developmental disorder that can include craniofacial abnormalities, limb malformations, missing digits, intellectual disabilities, stillbirth, and early mortality. The genetic basis for RBS is linked to autosomal recessive loss-of-function mutation of the establishment of cohesion (ESCO) 2 acetyltransferase. ESCO2 is an essential gene that targets the DNA-binding cohesin complex. ESCO2 acetylates alternate subunits of cohesin to orchestrate vital cellular processes that include sister chromatid cohesion, chromosome condensation, transcription, and DNA repair. Although significant advances were made over the last 20 years in our understanding of ESCO2 and cohesin biology, the molecular etiology of RBS remains ambiguous. In this review, we highlight current models of RBS and reflect on data that suggests a novel role for macromolecular damage in the molecular etiology of RBS.

Acetyltransferases/genetics , Chromosomal Proteins, Non-Histone/genetics , Craniofacial Abnormalities/genetics , DNA Damage , Ectromelia/genetics , Hypertelorism/genetics , Acetyltransferases/metabolism , Animals , Chromosomal Proteins, Non-Histone/metabolism , Craniofacial Abnormalities/metabolism , Ectromelia/metabolism , Genomic Instability , Humans , Hypertelorism/metabolism
Biol Open ; 9(12)2020 12 29.
Article En | MEDLINE | ID: mdl-33234702

Frank-Ter Haar syndrome (FTHS, MIM #249420) is a rare skeletal dysplasia within the defective collagen remodelling spectrum (DECORS), which is characterised by craniofacial abnormalities, skeletal malformations and fibrotic soft tissues changes including dermal fibrosis and joint contractures. FTHS is caused by homozygous or compound heterozygous loss-of-function mutation or deletion of SH3PXD2B (Src homology 3 and Phox homology domain-containing protein 2B; MIM #613293). SH3PXD2B encodes an adaptor protein with the same name, which is required for full functionality of podosomes, specialised membrane structures involved in extracellular matrix (ECM) remodelling. The pathogenesis of DECORS is still incompletely understood and, as a result, therapeutic options are limited. We previously generated an mmp14a/b knockout zebrafish and demonstrated that it primarily mimics the DECORS-related bone abnormalities. Here, we present a novel sh3pxd2b mutant zebrafish, pretzel, which primarily reflects the DECORS-related dermal fibrosis and contractures. In addition to relatively mild skeletal abnormalities, pretzel mutants develop dermal and musculoskeletal fibrosis, contraction of which seems to underlie grotesque deformations that include kyphoscoliosis, abdominal constriction and lateral folding. The discrepancy in phenotypes between mmp14a/b and sh3pxd2b mutants suggests that in fish, as opposed to humans, there are differences in spatiotemporal dependence of ECM remodelling on either sh3pxd2b or mmp14a/b The pretzel model presented here can be used to further delineate the underlying mechanism of the fibrosis observed in DECORS, as well as screening and subsequent development of novel drugs targeting DECORS-related fibrosis.This paper has an associated First Person interview with the first author of the article.

Adaptor Proteins, Signal Transducing/genetics , Collagen/metabolism , Craniofacial Abnormalities/etiology , Craniofacial Abnormalities/metabolism , Drosophila Proteins/genetics , Heart Defects, Congenital/etiology , Heart Defects, Congenital/metabolism , Osteochondrodysplasias/congenital , Adaptor Proteins, Signal Transducing/metabolism , Animals , Craniofacial Abnormalities/pathology , Dermis/metabolism , Dermis/pathology , Developmental Disabilities/etiology , Developmental Disabilities/metabolism , Developmental Disabilities/pathology , Disease Models, Animal , Drosophila Proteins/metabolism , Extracellular Matrix/metabolism , Fibrosis , Gene Editing , Heart Defects, Congenital/pathology , Immunohistochemistry , Mutation , Osteochondrodysplasias/etiology , Osteochondrodysplasias/metabolism , Osteochondrodysplasias/pathology , Phenotype , Zebrafish
Theranostics ; 10(19): 8648-8664, 2020.
Article En | MEDLINE | ID: mdl-32754269

Exosomes, a specific subgroup of extracellular vesicles that are secreted by cells, have been recognized as important mediators of intercellular communication. They participate in a diverse range of physiological and pathological processes. Given the capability of exosomes to carry molecular cargos and transfer bioactive components, exosome-based disease diagnosis and therapeutics have been extensively studied over the past few decades. Herein, we highlight the emerging applications of exosomes as biomarkers and therapeutic agents in the craniofacial and dental field. Moreover, we discuss the current challenges and future perspectives of exosomes in clinical applications.

Craniofacial Abnormalities/diagnosis , Exosomes/genetics , Exosomes/metabolism , Stomatognathic Diseases/diagnosis , Biomarkers/metabolism , Cell Communication , Craniofacial Abnormalities/genetics , Craniofacial Abnormalities/metabolism , Early Diagnosis , Humans , Precision Medicine , Stomatognathic Diseases/genetics , Stomatognathic Diseases/metabolism
Alcohol Clin Exp Res ; 44(10): 1988-1996, 2020 10.
Article En | MEDLINE | ID: mdl-32767777

BACKGROUND: Prenatal alcohol exposure (PAE) is perhaps the most common environmental cause of human birth defects. These exposures cause a range of structural and neurological defects, including facial dysmorphologies, collectively known as fetal alcohol spectrum disorders (FASD). While PAE causes FASD, phenotypic outcomes vary widely. It is thought that multifactorial genetic and environmental interactions modify the effects of PAE. However, little is known of the nature of these modifiers. Disruption of the Hedgehog (Hh) signaling pathway has been suggested as a modifier of ethanol teratogenicity. In addition to regulating the morphogenesis of craniofacial tissues commonly disrupted in FASD, a core member of the Hh pathway, Smoothened, is susceptible to modulation by structurally diverse chemicals. These include environmentally prevalent teratogens like piperonyl butoxide (PBO), a synergist found in thousands of pesticide formulations. METHODS: Here, we characterize multifactorial genetic and environmental interactions using a zebrafish model of craniofacial development. RESULTS: We show that loss of a single allele of shha sensitized embryos to both alcohol- and PBO-induced facial defects. Co-exposure of PBO and alcohol synergized to cause more frequent and severe defects. The effects of this co-exposure were even more profound in the genetically susceptible shha heterozygotes. CONCLUSIONS: Together, these findings shed light on the multifactorial basis of alcohol-induced craniofacial defects. In addition to further implicating genetic disruption of the Hh pathway in alcohol teratogenicity, our findings suggest that co-exposure to environmental chemicals that perturb Hh signaling may be important variables in FASD and related craniofacial disorders.

Craniofacial Abnormalities/chemically induced , Ethanol/adverse effects , Gene-Environment Interaction , Hedgehog Proteins/antagonists & inhibitors , Signal Transduction/drug effects , Zebrafish Proteins/antagonists & inhibitors , Animals , Craniofacial Abnormalities/embryology , Craniofacial Abnormalities/metabolism , Embryo, Nonmammalian/abnormalities , Embryo, Nonmammalian/drug effects , Hedgehog Proteins/metabolism , Piperonyl Butoxide/pharmacology , Teratogens/pharmacology , Zebrafish/abnormalities , Zebrafish/embryology , Zebrafish Proteins/metabolism