RESUMEN
Fibroblast growth factor (FGF)/extracellular signal-regulated kinase (ERK) signaling plays a crucial role in anterior-posterior (A-P) axial patterning of vertebrate embryos by promoting posterior development. In our screens for novel developmental regulators in Xenopus embryos, we identified Fam3b as a secreted factor regulated in ectodermal explants. Family with sequence similarity 3 member B (FAM3B)/PANDER (pancreatic-derived factor) is a cytokine involved in glucose metabolism, type 2 diabetes, and cancer in mammals. However, the molecular mechanism of FAM3B action in these processes remains poorly understood, largely because its receptor is still unidentified. Here we uncover an unexpected role of FAM3B acting as a FGF receptor (FGFR) ligand in Xenopus embryos. fam3b messenger RNA (mRNA) is initially expressed maternally and uniformly in the early Xenopus embryo and then in the epidermis at neurula stages. Overexpression of Xenopus fam3b mRNA inhibited cephalic structures and induced ectopic tail-like structures. Recombinant human FAM3B protein was purified readily from transfected tissue culture cells and, when injected into the blastocoele cavity, also caused outgrowth of tail-like structures at the expense of anterior structures, indicating FGF-like activity. Depletion of fam3b by specific antisense morpholino oligonucleotides in Xenopus resulted in macrocephaly in tailbud tadpoles, rescuable by FAM3B protein. Mechanistically, FAM3B protein bound to FGFR and activated the downstream ERK signaling in an FGFR-dependent manner. In Xenopus embryos, FGFR activity was required epistatically downstream of Fam3b to mediate its promotion of posterior cell fates. Our findings define a FAM3B/FGFR/ERK-signaling pathway that is required for axial patterning in Xenopus embryos and may provide molecular insights into FAM3B-associated human diseases.
Asunto(s)
Citocinas/fisiología , Desarrollo Embrionario/fisiología , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Proteínas de Xenopus/fisiología , Xenopus laevis/embriología , Animales , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Células HEK293 , HumanosRESUMEN
Orofacial clefts (OFCs) are the most common congenital craniofacial disorders and cause serious problems with the appearance, orofacial function and mental health of the patients. The fibroblast growth factor (FGF) signaling pathway is critical for several aspects of craniofacial development and loss-of-function mutations of coding genes for multiple FGFs and FGFRs can lead to OFCs. We recently characterized FAM3B as a novel ligand of FGF signaling, which, through binding to FGFRs and activating downstream ERK, regulates craniofacial development in Xenopus. In this study, we identify two rare variants in FAM3B (p.Q61R and p.D128G) via target region sequencing of FAM3B on 144 unrelated sporadic patients with non-syndromic OFCs (NSOFCs). Bioinformatic analysis predict that these two variants are likely to be damaging and biochemical experiments show that these two variants weaken the FGF ligand activity of FAM3B by decreasing its expression and thus secretion. In summary, our results indicate that FAM3B is a novel candidate gene for NSOFCs in humans.
Asunto(s)
Labio Leporino , Fisura del Paladar , Humanos , Labio Leporino/genética , Fisura del Paladar/genética , Ligandos , Mutación , Factores de Crecimiento de Fibroblastos/genética , Proteínas de Neoplasias/genética , Citocinas/genéticaRESUMEN
BACKGROUND: Multiple supernumerary teeth, combined with numerous impacted teeth, can lead to various malocclusions, posing significant treatment challenges. While certain genes associated with syndromic cases of multiple supernumerary and impacted teeth have been identified, the etiologies of non-syndromic cases still largely remain elusive. CASE PRESENTATION: Here, we report a treatment of a 12-year-old boy who presented with 10 supernumerary teeth and 6 impacted teeth, accompanied by a genetic analysis to explore the underlying etiology. During the treatment, fifteen teeth were extracted, and various skilled techniques, including the closed-eruption technique and the application of by-pass arches, were utilized. Post-treatment, traction was successful for all the impacted teeth, without any tooth mobility or reduction in gingival height. Space closure, well-aligned teeth, and excellent functional occlusion were achieved. Furthermore, comprehensive genetic analysis was conducted through whole-exome sequencing on the patient and his parents, which revealed a potential link between the patient's numerous supernumerary teeth and abnormal mineralization. Notably, the p.Ser496Pro variant in the TCF7L2 gene was identified as a potential candidate variant in this patient. CONCLUSIONS: Overall, our findings not only report the treatment of a rare case involving multiple supernumerary and impacted teeth but also offer valuable insights into the molecular basis of supernumerary teeth.
Asunto(s)
Diente Impactado , Diente Supernumerario , Humanos , Diente Supernumerario/genética , Diente Impactado/genética , Masculino , Niño , Extracción Dental , Secuenciación del ExomaRESUMEN
OBJECTIVES: Mammalian palatogenesis is a highly regulated morphogenetic process to form the intact roof of the oral cavity. Long noncoding RNAs (lncRNAs) and mRNAs participate in numerous biological and pathological processes, but their roles in palatal development and causing orofacial clefts (OFC) remain to be clarified. METHODS: Palatal tissues were separated from ICR mouse embryos at four stages (E10.5, E13.5, E15, and E17). Then, RNA sequencing (RNA-seq) was used. Various analyses were performed to explore the results. Finally, hub genes were validated via qPCR and in situ hybridization. RESULTS: Starting from E10.5, the expression of cell adhesion genes escalated in the following stages. Cilium assembly and ossification genes were both upregulated at E15 compared with E13.5. Besides, the expression of cilium assembly genes was also increased at E17 compared with E15. Expression patterns of three lncRNAs (H19, Malat1, and Miat) and four mRNAs (Cdh1, Irf6, Grhl3, Efnb1) detected in RNA-seq were validated. CONCLUSIONS: This study provides a time-series expression landscape of mRNAs and lncRNAs during palatogenesis, which highlights the importance of processes such as cell adhesion and ossification. Our results will facilitate a deeper understanding of the complexity of gene expression and regulation during palatogenesis.
Asunto(s)
Labio Leporino , Fisura del Paladar , ARN Largo no Codificante , Ratones , Animales , Perfilación de la Expresión Génica/métodos , ARN Largo no Codificante/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratones Endogámicos ICR , Fisura del Paladar/genética , Mamíferos/genética , Mamíferos/metabolismo , Proteínas de Unión al ADN/genética , Factores de Transcripción/genéticaRESUMEN
OBJECTIVES: Non-syndromic cleft palate only (NSCPO) is one of the most common craniofacial birth defects with largely undetermined genetic etiology. It has been established that Grainyhead-like 3 (GRHL3) plays an essential role in the pathogenesis of NSCPO. This study aimed to identify and verify the first-reported GRHL3 variant underlying NSCPO among the Chinese cohort. METHODS: We performed whole-exome sequencing (WES) on a Chinese NSCPO patient and identified a rare variant of GRHL3 (p.Arg391His). A validated deleterious variant p.Arg391Cys was introduced as a positive control. Zebrafish embryos injection, reporter assays, live-cell imaging, and RNA sequencing were conducted to test the pathogenicity of the variants. RESULTS: Zebrafish embryos microinjection demonstrated that overexpression of the variants could disrupt the normal development of zebrafish embryos. Reporter assays showed that Arg391His disturbed transcriptional activity of GRHL3 and exerted a dominant-negative effect. Interestingly, Arg391His and Arg391Cys displayed distinct nuclear localization patterns from that of wild-type GRHL3 in live-cell imaging. Bulk RNA sequencing suggested that the two variants changed the pattern of gene expression. CONCLUSIONS: In aggregate, this study identified and characterized a rare GRHL3 variant in NSCPO, revealing the critical role of Arginine 391 in GRHL3. Our findings will help facilitate understanding and genetic counseling of NSCPO.
Asunto(s)
Labio Leporino , Fisura del Paladar , Animales , Labio Leporino/genética , Fisura del Paladar/genética , Fisura del Paladar/patología , Proteínas de Unión al ADN/genética , Predisposición Genética a la Enfermedad , Polimorfismo de Nucleótido Simple , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Pez Cebra/genética , Pez Cebra/metabolismoRESUMEN
The Hedgehog (HH) signaling plays key roles in embryogenesis and organogenesis, and its dysfunction causes a variety of human birth defects. Orofacial cleft (OFC) is one of the most common congenital craniofacial defects, and its etiology is closely related to mutations in multiple components in the HH pathway, including the PTCH1 receptor. A quantity of PTCH1 variants have been associated with OFC, but the pathogenicity and underlying mechanism of these variants have not been functionally validated. In our previous studies, we identified two PTCH1 variants (A392V and R945X) in two families with hereditary OFC. Here we explore the functional consequences of these two variants. In zebrafish embryos, microinjection of wild type PTCH1 mRNA causes curved body axis and craniofacial anomalies. In contrast, microinjection of A392V and R945X PTCH1 mRNAs results in much milder phenotypes, suggesting these two variants are loss-of-function mutations. In mammalian cells, A392V and R945X mutations reverse the inhibitory effect of PTCH1 on HH signaling. Biochemically, the two mutants PTCH1 show lower expression levels and shortened half-life, indicting these mutations decrease the stability of PTCH1. A392V and R945X mutations also appear to cause PTCH1 to localize away from vesicles. Taken together, our findings indicate that A392V and R945X variants are loss-of-function mutations that disrupt the function of PTCH1 and thus cause dysregulation of HH signaling, leading to the pathogenesis of OFC.
Asunto(s)
Labio Leporino , Fisura del Paladar , Receptor Patched-1 , Proteínas de Pez Cebra , Animales , Humanos , Labio Leporino/genética , Fisura del Paladar/genética , Proteínas Hedgehog/genética , Pez Cebra/genética , Proteínas de Pez Cebra/genética , Receptor Patched-1/genéticaRESUMEN
OBJECTIVES: Unilateral complete cleft lip and palate (UCCLP) is one of the most severe clinical subtypes among cleft lip and palate (CLP), making repair surgery and subsequent orthodontic treatment particularly challenging. Presurgical nasoalveolar molding (PNAM) has shown conflicting and heterogeneous results in the treatment of UCCLP patients, raising questions about whether the diversity in alveolar anatomical morphology among these patients plays a role in the effectiveness of PNAM treatment. MATERIALS AND METHODS: We collected 90 digital maxillary models of infants with UCCLP and performed mathematical clustering analysis, including principal component analysis (PCA), decision tree modeling, and area under the ROC Curve (AUC) analysis, to classify alveolar morphology and identify key measurements. We also conducted clinical evaluations to assess the association between the alveolar morphology and CLP treatment outcomes. RESULTS: Using mathematical clustering analysis, we classified the alveolar morphology into three distinct types: average form, horizontal form, and longitudinal form. The decision tree model, AUC analysis, and comparison analysis revealed that four measurements (Trans ACG-ACL, ML length, MG length and Inc length) were essential for clustering the alveolar morphology of infants with UCCLP. Furthermore, the blinded clinical evaluation indicated that UCCLP patients with alveolar segments of horizontal form had the lowest treatment outcomes. CONCLUSION: Overall, our findings establish a novel quantitative classification system for the morphology of alveolar bone in infants with UCCLP and suggest that this classification may be associated with the outcomes of CLP treatment. CLINICAL RELEVANCE: The multidisciplinary CLP team should thoroughly evaluate and classify the specific alveolar morphology when administering PNAM to infants with UCCLP.
Asunto(s)
Labio Leporino , Fisura del Paladar , Lactante , Humanos , Labio Leporino/cirugía , Fisura del Paladar/cirugía , Nariz , Cuidados Preoperatorios/métodosRESUMEN
Unilateral complete cleft lip and palate (UCCLP) is one of the most severe clinical subphenotypes among nonsyndromic cleft lip and/or palate (NSCL/P), that complicates surgical repair operations. Presurgical nasoalveolar molding (PNAM) is a technique used to reshape the nose, lip and alveolar bone of infants with UCCLP before surgery (the modified Mohler rotation advancement cheiloplasty and two flap palatoplasty), with the potential to facilitate surgical repair. However, the effectiveness of PNAM treatment is still a matter of debate. In this paper, the 3Shape scanning system and 3dMD stereophotography were used to assess the short-term and long-term effects of PNAM treatment on the dental arch morphology and nasolabial features of patients with UCCLP, respectively. The findings indicated that PNAM treatment negatively affects both short-term and long-term dental arch shape compared to the treatment without PNAM, particularly in terms of limiting the transverse width of the maxillary canine-to-midline. Regarding the nasal and labial symmetry, PNAM improves the symmetry of the nasal alae in patients over 7 years old and the symmetry of the lip in patients under 7 years old. Moreover, UCCLP patients who received PNAM treatment exhibited a shorter and wider shape of the nostril on the cleft side compared to those without PNAM treatment. In clinical practice, the multidisciplinary team should carefully consider the advantages and disadvantages of the outcomes of PNAM treatment when treating infants with cleft lip and palate.
Asunto(s)
Labio Leporino , Fisura del Paladar , Lactante , Humanos , Niño , Labio Leporino/cirugía , Fisura del Paladar/cirugía , Modelado Nasoalveolar , Arco Dental , Proceso Alveolar , Cuidados Preoperatorios/métodos , NarizRESUMEN
INTRODUCTION: Orthodontic tooth movement (OTM) is the core component of orthodontic treatment and is increasingly popular for treating malocclusions. In this study, we aimed to investigate the role of apolipoprotein E (ApoE) in OTM. METHODS: Thirty patients treated with transmission straight wire technology were selected and longitudinally tracked at 2 different stages of orthodontic treatment (initial 2 months and 12 months of orthodontic treatment). Total saliva was collected and analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Western blotting was used to detect the difference in ApoE expression in the saliva samples of the 2 groups. The expression of ApoE was further verified by immunohistochemical staining in a mouse model of tooth movement. RESULTS: The results of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry showed significant differences in the components of the salivary peptides in the 2 groups and peptides with a molecular weight of 2010.7 Da were predicted to be ApoE by database analysis. Western blotting further verified a significant difference in the expression of salivary ApoE in the 2 groups. In addition, an OTM model was successfully constructed in mice. The immunohistochemical staining results showed that ApoE expression significantly increased after force loading in the OTM model. CONCLUSIONS: This study indicated that ApoE participated in and played a role during OTM in patients treated with transmission straight wire technology. This relationship might be related to alveolar bone reconstruction and root resorption. The results provide new ideas for research on the mechanism of tooth movement using precision medicine based on saliva detection.
Asunto(s)
Resorción Radicular , Técnicas de Movimiento Dental , Animales , Apolipoproteínas , Apolipoproteínas E/genética , Biomarcadores , Humanos , Ratones , OsteoclastosRESUMEN
NELL-1 is a secreted protein that was originally found to be upregulated in pathologically fusing and fused sutures in non-syndromic unilateral coronal synostosis patients. Apart from the ability of NELL-1 to promote osteogenesis in long and craniofacial bones, NELL-1 reportedly inhibits the formation of several benign and malignant tumors. We previously identified a novel transcript of Nell-1 that lacked a calcium-binding epidermal growth factor (EGF)-like domain compared with full-length Nell-1; this new transcript was named Nell-1-ΔE. Three obvious structural differences between these two isoforms were revealed by homology modeling. Furthermore, the recombinant Nell-1-ΔE protein, but not the full-length Nell-1 protein, inhibited cell migration in vitro. However, full-length Nell-1 and Nell-1-ΔE proteins were present in similar subcellular locations and displayed similar expression patterns in both the intracellular and extracellular spaces. The results from the co-immunoprecipitation and liquid chromatography/tandem mass spectrometry analyses using two cell lines demonstrated that Nell-1-ΔE but not full-length Nell-1 interacted with enolase-1 in the extracellular spaces of both cell lines. The results of wound healing assays using ENO-1-overexpressing cells treated with full-length Nell-1/Nell-1-ΔE suggested that Nell-1-ΔE inhibited cell migration by interacting with ENO-1. Our study indicated that the novel transcript Nell-1-ΔE, but not full-length Nell-1, might be a candidate tumor suppressor factor for basic research and clinical practice.
Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Movimiento Celular , Glicoproteínas/metabolismo , Mutación , Osteogénesis , Fosfopiruvato Hidratasa/metabolismo , Animales , Proteínas de Unión al Calcio/genética , Células Cultivadas , Glicoproteínas/genética , Células HEK293 , Humanos , Ratones , Fosfopiruvato Hidratasa/genéticaRESUMEN
Non-syndromic cleft lip with or without cleft palate (NSCLP) is the most common congenital craniofacial malformation, and its harmful influence on affected individuals is apparent. Despite many studies, the causative genes and their mechanisms are not completely clear. We recruited a Han Chinese NSCLP family and explored the causative variant in this pedigree. We performed whole-exome sequencing on two patients. Bioinformatics screening and analysis were used to identify the mutation. We also performed species conservation analysis, mutation function predictions, and homology protein modelling to evaluate the influence of the mutation. We identified a rare mutation in interferon regulatory factor 6 (IRF6) (c.26G>A; p.Arg9Gln) as a candidate of causative mutation. This mutation was predicted to be deleterious. The codon is conserved in many species. The residue change caused by this mutation would affect the structure of IRF6 to a degree. Our study suggested that the rare IRF6 variant is probably the pathogenic mutation in this family. Our result adds evidence that IRF6 variants play a role in the aetiology of orofacial clefts.
Asunto(s)
Encéfalo/anomalías , Labio Leporino/genética , Fisura del Paladar/genética , Factores Reguladores del Interferón/genética , Encéfalo/fisiopatología , Labio Leporino/fisiopatología , Fisura del Paladar/fisiopatología , Femenino , Predisposición Genética a la Enfermedad , Humanos , Masculino , Mutación , Linaje , Secuenciación del ExomaAsunto(s)
Labio Leporino , Fisura del Paladar , Humanos , Proteómica , Polimorfismo de Nucleótido SimpleRESUMEN
OBJECTIVES: Cleft lip and/or palate (CL/P) is the most common craniofacial congenital disease, and it has a complex aetiology. This study aimed to identify the causative gene mutation of a Han Chinese family with CL/P. SUBJECTS AND METHODS: Whole exome sequencing was conducted on the proband and her mother, who exhibited the same phenotype. A Mendelian dominant inheritance model, allele frequency, mutation regions, functional prediction and literature review were used to screen and filter the variants. The candidate was validated by Sanger sequencing. Conservation analysis and homology modelling were conducted. RESULTS: A heterozygous missense mutation c.1175C>T in the PTCH1 gene predicting p.Ala392Val was identified. This variant has not been reported and was predicted to be deleterious. Sanger sequencing verified the variant and the dominant inheritance model in the family. The missense alteration affects an amino acid that is evolutionarily conserved in the first extracellular loop of the PTCH1 protein. The local structure of the mutant protein was significantly altered according to homology modelling. CONCLUSIONS: Our findings suggest that c.1175C>T in PTCH1 (NM_000264) may be the causative mutation of this pedigree. Our results add to the evidence that PTCH1 variants play a role in the pathogenesis of orofacial clefts.
Asunto(s)
Labio Leporino/genética , Fisura del Paladar/genética , Receptor Patched-1/genética , Pueblo Asiatico , Estudios de Casos y Controles , Femenino , Frecuencia de los Genes , Humanos , Mutación Missense , Linaje , FenotipoRESUMEN
Orofacial clefts (OFCs) are the most common congenital craniofacial disorders, of which the etiology is closely related to rare coding variants. Filamin B (FLNB) is an actin-binding protein implicated in bone formation. FLNB mutations have been identified in several types of syndromic OFCs and previous studies suggest a role of FLNB in the onset of non-syndromic OFCs (NSOFCs). Here, we report two rare heterozygous variants (p.P441T and p.G565R) in FLNB in two unrelated hereditary families with NSOFCs. Bioinformatics analysis suggests that both variants may disrupt the function of FLNB. In mammalian cells, p.P441T and p.G565R variants are less potent to induce cell stretches than wild type FLNB, suggesting that they are loss-of-function mutations. Immunohistochemistry analysis demonstrates that FLNB is abundantly expressed during palatal development. Importantly, Flnb-/- embryos display cleft palates and previously defined skeletal defects. Taken together, our findings reveal that FLNB is required for development of palates in mice and FLNB is a bona fide causal gene for NSOFCs in humans.
Asunto(s)
Encéfalo , Labio Leporino , Fisura del Paladar , Animales , Humanos , Ratones , Encéfalo/anomalías , Labio Leporino/genética , Fisura del Paladar/genética , Filaminas/genética , Mamíferos , MutaciónRESUMEN
BACKGROUND: Hemerocallis citrina Baroni (Huanghuacai), a plant of the genus Hemerocallis in the family Asphodelaceae, is widely planted in China. Based on our survey results, the chemical compounds in the essential oil of the flowers of Hemerocallis citrina Baroni (EOFHCB) and relevant pharmacological activities have never been studied systematically. OBJECTIVE: To preliminarily decipher the pharmacological activities and mechanisms of EOFHCB in the treatment of anxiety disorders by GC-MS, Network Pharmacology, and Molecular docking. METHODS: EOFHCB compositions were identified using GC-MS, and their targets were predicted using Swiss Target Prediction databases. The targets of anxiety disorders were obtained by GeneCards, DisGeNET, and OMIM databases. The STRING database was used to construct the protein-protein interaction networks, and the DAVID database was used to carry out GO enrichment and KEGG pathway enrichment analysis. The EOFHCB-components-targetspathways- anxiety disorders network was constructed by Cytoscape software (Version 3.10.0). Finally, the result was verified by molecular docking. RESULTS: 28 chemical components were identified by GC-MS, including 3-furanmethanol (28.43%), 2-methyl-1-butanol (27.13%), nerolidol (10.62%), and so on, which correspond to 241 potential targets. Several 2440 biological processes, 187 cellular compositions, and 311 molecular functions were enriched by GO enrichment analysis and 174 pathways by KEGG enrichment analysis. The key targets are PTGS 2, SRC, DRD 2, ESR 1, MAOB, and SLC6A4. The most important pathway is the neuroactive ligand-receptor interaction. CONCLUSION: EOFHCB exerts its therapeutic effects on anxiety disorders through multicomponents, multi-targets, and multi-pathways, which provided new ideas and methods for the in-depth research of aromatic Chinese medicine in the treatment of anxiety disorders.
RESUMEN
Orofacial clefts (OFCs) represent the most prevalent congenital craniofacial anomalies, significantly impacting patients' appearance, oral function, and psychological well-being. Among these, non-syndromic OFCs (NSOFCs) are the most predominant type, with the etiology attributed to a combination of genetic and environmental factors. Rare variants of key genes involved in craniofacial development-related signaling pathway are crucial in the occurrence of NSOFCs, and our recent studies have identified PTCH1, a receptor-coding gene in the Hedgehog signaling pathway, as a causative gene for NSOFCs. However, the role of PTCH2, the paralog of PTCH1, in pathogenesis of NSOFCs remains unclear. Here, we perform whole-exome sequencing to explore the genetic basis of 144 sporadic NSOFC patients. We identify five heterozygous variants of PTCH2 in four patients: p.L104P, p.A131G, p.R557H, p.I927S, and p.V978D, with the latter two co-occurring in a single patient. These variants, all proven to be rare through multiple genomic databases, with p.I927S and p.V978D being novel variants and previously unreported. Sequence alignment suggests that these affected amino acids are evolutionarily conserved across vertebrates. Utilizing predictive structural modeling tools such as AlphaFold and SWISS-MODEL, we propose that these variants may disrupt the protein's structure and function. In summary, our findings suggest that PTCH2 may be a novel candidate gene predicted to be associated with NSOFCs, thereby broadening the spectrum of causative genes implicated in the craniofacial anomalies.
Asunto(s)
Labio Leporino , Fisura del Paladar , Receptor Patched-2 , Animales , Humanos , Encéfalo/anomalías , Labio Leporino/genética , Fisura del Paladar/genética , Proteínas Hedgehog/genética , Receptor Patched-2/genética , Transducción de SeñalRESUMEN
In this study, through linkage analysis of a four-generation Chinese family with multiple members afflicted with DGI (type II), we identified a novel missense mutation in DSPP. The mutation was located in exon 2 at the second nucleotide position of the last codon and resulted in a substitution of a proline with a leucine residue (c.50C>T, p.P17L, g.50C>T). To assess the potential effects of this novel mutation, we utilized various bioinformatics analysis programs. The results indicate that the mutation likely affects protein cleavage/trafficking. We also analyzed previously reported mutations of DSPP. In summary, our finding supports that the genomic sequence that corresponds to the P17 residue of DSPP is a mutational hotspot and P17 may be critical for the function of DSPP.
Asunto(s)
Biología Computacional/métodos , Dentinogénesis Imperfecta/genética , Proteínas de la Matriz Extracelular/genética , Mutación Missense , Fosfoproteínas/genética , Sialoglicoproteínas/genética , Pueblo Asiatico/genética , China , Cromosomas Humanos/genética , Exones , Proteínas de la Matriz Extracelular/metabolismo , Femenino , Ligamiento Genético , Predisposición Genética a la Enfermedad , Haplotipos , Humanos , Masculino , Linaje , Fosfoproteínas/metabolismo , Análisis de Secuencia de ADN/métodos , Sialoglicoproteínas/metabolismoRESUMEN
The Patched 1 (PTCH1) gene encodes a membrane receptor involved in the Hedgehog (Hh) signaling pathway, an abnormal state of which may result in congenital defects or human tumors. In this study, we conducted whole-exome sequencing on a three-generation Chinese family characterized with variable penetrance of orofacial clefts. A rare heterozygous variant in the PTCH1 gene (c.2833C > T p.R945X) was identified as a disease-associated mutation. Structural modeling revealed a truncation starting from the middle of the second extracellular domain of PTCH1 protein. This may damage its ligand recognition and sterol transportation abilities, thereby affecting the Hh signaling pathway. Biochemical assays indicated that the R945X protein had reduced stability compared to the wild-type in vitro. In addition, we reviewed the locations and mutation types of PTCH1 variants in individuals with clefting phenotypes, and analyzed the associations between clefts and locations or types of variants within PTCH1. Our findings provide further evidence that PTCH1 variants result in orofacial clefts, and contributed to genetic counseling and clinical surveillance in this family.
RESUMEN
BACKGROUND: Loss-of-function mutations in interferon regulatory factor-6 (IRF6) are responsible for about 70% of cases of Van Der Woude Syndrome (VWS), an autosomal dominant developmental disorder characterized by pits and/or sinuses of the lower lip and cleft lip, cleft palate, or both. METHODS: We collected a Chinese Han VWS pedigree, performed sequencing and screening for the causal gene mutant. Initially, species conservation analysis and homology protein modeling were used to predict the potential pathogenicity of mutations. To test whether a VWS family-derived mutant variant of IRF6 retained function, we carried out rescue assays in irf6 maternal-null mutant zebrafish embryos. To assess protein stability, we overexpressed reference and family-variants of IRF6 in vitro. RESULTS: We focused on a VWS family that includes a son with bilateral lip pits, uvula fissa and his father with bilateral cleft lip and palate. After sequencing and screening, a frameshift mutation of IRF6 was identified as the potential causal variant (NM.006147.3, c.1088-1091delTCTA; p.Ile363ArgfsTer33). The residues in this position are strongly conserved among species and homology modeling suggests the variant alters the protein structure. In irf6 maternal-null mutant zebrafish embryos the periderm differentiates abnormally and the embryos rupture and die during gastrulation. Injection of mRNA encoding the reference variant of human IRF6, but not of the frame-shift variant, rescued such embryos through gastrulation. Upon overexpression in HEK293FT cells, the IRF6 frame-shift mutant was relatively unstable and was preferentially targeted to the proteasome in comparison to the reference variant. CONCLUSION: In this VWS pedigree, a novel frameshift of IRF6 was identified as the likely causative gene variant. It is a lost function mutation which could not rescue abnormal periderm phenotype in irf6 maternal-null zebrafish and which causes the protein be unstable through proteasome-dependent degradation.
RESUMEN
Cleft lip and/or palate (CL/P) is a most common craniofacial birth defect which has multifactorial etiology. In our study, we aimed to discover the underlying etiological gene variation in a Chinese family diagnosed as non-syndromic CL/P (NSCL/P). The blood sample of the proband and her parents were detected by whole exome sequencing. The Mendelian inheritance pattern, allele frequency, variation location, function analysis and literature search were applied to filtrate and screen the mutation. Besides, the candidates were confirmed by Sanger sequencing. We meanwhile explored the conservative analysis and protein homology simulation. As a result, a start-lost mutation c.1A > GAtg/Gtg in the Frizzled-6 (FZD6) gene predicting p.Met1 was detected. The variation has not been reported before and was predicted to be harmful. The alteration caused missing of two starting amino acids that are evolutionarily conserved for FZD6 protein. Moreover, the specific structure of the mutant protein obviously changed according to the results of the homologous model. In conclusion, the results suggest c.1A > GAtg/Gtg in the FZD6 (NM_001164616) might be the genetic etiology for non-syndromic CL/P in this pedigree. Furthermore, this finding provided new etiologic information, supplementing the evidence that FZD6 is a strong potential gene for CL/P.