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1.
PLoS One ; 15(1): e0227287, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31914153

RESUMO

Tooth agenesis is one of the most common developmental anomalies in humans and can affect dental occlusion and speech pronunciation. Research has identified an association between mutations in MSX1, PAX9, EDA, AXIN2, WNT10A, WNT10B and LRP6 and human tooth agenesis. Two unrelated individuals with non-syndromic tooth agenesis and their families were enrolled in this study. Using Sanger sequencing of the candidate genes, we identified two novel mutations: a missense mutation c.572 T>C and a frameshift mutation c.590_594 dup TGTCC, which were both detected in the homeodomain of MSX1. After identifying the mutations, structural modeling and bioinformatics analysis were used to predict the resulting conformational changes in the MSX1 homeodomain. Combined with 3D-structural analysis of other MSX1 mutations, we propose that there is a correlation between the observed phenotypes and alterations in hydrogen bond formation, thereby potentially affecting protein binding.


Assuntos
Anodontia/genética , Fator de Transcrição MSX1/genética , Adolescente , Análise Mutacional de DNA , Feminino , Mutação da Fase de Leitura , Humanos , Fator de Transcrição MSX1/química , Masculino , Modelos Estruturais , Mutação de Sentido Incorreto , Linhagem , Conformação Proteica em alfa-Hélice/genética , Domínios Proteicos/genética , Adulto Jovem
2.
Arch Oral Biol ; 109: 104556, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31568994

RESUMO

OBJECTIVES: To investigate the association of MSX1 rs12532 polymorphism with the risk of nonsyndromic unilateral complete cleft lip and palate (NSCLP) and tooth agenesis. MATERIALS AND METHODS: The study is comprised of 384 individuals divided into 4 groups: group 1, patients with unilateral complete NSCLP and premolar agenesis (n = 57); group 2, patients with unilateral NSCLP without tooth agenesis (n = 117); group 3, patients with premolar agenesis without oral cleft (n = 53) and group 4 (n = 157), a control group with individuals without tooth agenesis and oral cleft. Genotyping of rs12532 was carried out with Taqman chemistry, and associations were investigated using logistic regression analyses. RESULTS: Overall rs12532 allele and genotype distributions revealed no significant differences between the groups of NSCLP or tooth agenesis. CONCLUSION: Although our results are consistent with a lack of association of MSX1 rs12532 and the risk of unilateral NSCLP and tooth agenesis, further studies with additional SNPs and a more diverse ethnic cohort are warranted.


Assuntos
Fenda Labial/genética , Fissura Palatina/genética , Fator de Transcrição MSX1/genética , Adolescente , Adulto , Alelos , Anodontia , Estudos de Casos e Controles , Criança , Feminino , Humanos , Masculino , Polimorfismo de Nucleotídeo Único , Adulto Jovem
3.
Biomed Res Int ; 2019: 2183720, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31781599

RESUMO

The etiology of hypodontia is complex, in which both genetic and environmental factors can be related. The main objective of our study was to contribute to elucidating the genetic background of nonsyndromic hypodontia (NSH). In this order, we selected 97 NSH subjects (70 females and 27 males) from patients referred to orthodontic treatment, and we matched to each NSH subject a control by age and sex. DNA was obtained from epithelial cells from the oral mucosa. Genotyping of the PAX9 (rs4904155 and rs61754301), MSX1 (rs8670 and rs12532), and AXIN2 (rs2240308) SNPs was performed by using TaqMan SNP Genotyping Assays on a real-time PCR system. Single-nucleotide polymorphisms (SNPs) were studied for the whole NSH group and for frontal and lateral agenesis NSH subjects separately. Our results showed that the variant genotype (p=0.0008, OR = 2.9, 95% CI = 1.58-5.3) and variant T allele (p=0.0002, OR = 2.65, 95% CI = 1.6-4.39) of the MSX1 rs8670 SNP increased the risk of hypodontia in the studied population when the whole NSH group was compared with controls. The variant genotype of the MSX1 rs8670 SNP was the most frequent in frontal agenesis; meanwhile in the lateral agenesis NSH group, the AXIN2 rs2240308 SNP showed a higher frequency of the variant genotype, with a trend towards statistical significance. In conclusion, the results of the present study showed that the variant genotype and variant T allele of the MSX1 rs8670 SNP increased the risk of hypodontia in the studied population. The presence of the variant A allele of AXIN2 rs2240308 is associated with frontal agenesis but not with lateral agenesis.


Assuntos
Anodontia/genética , Proteína Axina/genética , Fator de Transcrição MSX1/genética , Fator de Transcrição PAX9/genética , Adolescente , Adulto , Alelos , Anodontia/fisiopatologia , Feminino , Estudos de Associação Genética , Predisposição Genética para Doença , Genótipo , Humanos , Masculino , Polimorfismo de Nucleotídeo Único/genética , Adulto Jovem
4.
Organogenesis ; 15(2): 55-67, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31240991

RESUMO

Previous studies indicated that the elevated mesenchymal Wnt/ß-catenin signaling deprived dental mesenchyme of odontogenic fate. By utilizing ex vivo or pharmacological approaches, Wnt/ß-catenin signaling in the developing dental mesenchyme was suggested to suppress the odontogenic fate by disrupting the balance between Axin2 and Runx2. In our study, the Osr2-creKI; Ctnnb1ex3f mouse was used to explore how mesenchymal Wnt/ß-catenin signaling suppressed the odontogenic fate in vivo. We found that all of the incisor and half of the molar germs of Osr2-creKI; Ctnnb1ex3fmice started to regress at E14.5 and almost disappeared at birth. The expression of Fgf3 and Msx1 was dramatically down-regulated in the E14.5 Osr2-creKI; Ctnnb1ex3f incisor and molar mesenchyme, while Runx2transcription was only diminished in incisor mesenchyme. Intriguingly, in the E14.5 Osr2-creKI; Ctnnb1ex3f incisor epithelium, the expression of Noggin was activated, while Shh was abrogated. Similarly, the Wnt and BMP antagonists, Ectodin and Noggin were also ectopically activated in the E14.5 Osr2-creKI; Ctnnb1ex3f molar epithelium. Recombination of E13.5 Osr2-creKI; Ctnnb1ex3f molar mesenchyme with E10.5 and E13.5 WT dental epithelia failed to develop tooth. Taken together, the mesenchymal Wnt/ß-catenin signaling resulted in the loss of odontogenic fate in vivo not only by directly suppressing odontogenic genes expression but also by inducing Wnt and BMP antagonists in dental epithelium.


Assuntos
Proteínas Morfogenéticas Ósseas/antagonistas & inibidores , Epitélio/metabolismo , Mesoderma/metabolismo , Boca/metabolismo , Dente/embriologia , Via de Sinalização Wnt , Animais , Proliferação de Células , Sobrevivência Celular , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Feminino , Fator 3 de Crescimento de Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Genótipo , Fator de Transcrição MSX1/metabolismo , Masculino , Camundongos , Dente Molar/metabolismo , Odontogênese , Organogênese , Transdução de Sinais , Proteína Wnt1/antagonistas & inibidores , Proteína Wnt1/metabolismo , beta Catenina/metabolismo
5.
Eur J Endocrinol ; 181(2): 103-119, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31200363

RESUMO

Context: Congenital hypogonadotropic hypogonadism (CHH) is a rare condition caused by GnRH deficiency. Several genes have been associated with the pathogenesis of CHH, but most cases still remain without a molecular diagnosis. The advent of next-generation sequencing (NGS) has allowed the simultaneous genotyping of several regions, faster, making possible the extension of the genetic knowledge of CHH. Objective: Genetic characterization of a large cohort of Brazilian CHH patients. Design and patients: A cohort of 130 unrelated patients (91 males, 39 females) with CHH (75 normosmic CHH, 55 Kallmann syndrome) was studied using a panel containing 36 CHH-associated genes. Results: Potential pathogenic or probably pathogenic variants were identified in 43 (33%) CHH patients. The genes ANOS1, FGFR1 and GNRHR were the most frequently affected. A novel homozygous splice site mutation was identified in the GNRH1 gene and a deletion of the entire coding sequence was identified in SOX10. Deleterious variants in the IGSF10 gene were identified in two patients with reversible normosmic CHH. Notably, 6.9% of the patients had rare variants in more than one gene. Rare variants were also identified in SPRY4, IL17RD, FGF17, IGSF1 and FLRT3 genes. Conclusions: This is a large study of the molecular genetics of CHH providing new genetic findings for this complex and heterogeneous genetic condition. NGS has been shown to be a fast, reliable and effective tool in the molecular diagnosis of congenital CHH and being able to targeting clinical genetic testing in the future.


Assuntos
Hipogonadismo/congênito , Hipogonadismo/genética , Mutação , Adulto , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Brasil/epidemiologia , Proteínas de Transporte/genética , Estudos de Coortes , Análise Mutacional de DNA , Feminino , Predisposição Genética para Doença , Testes Genéticos , Glicoproteínas/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Hipogonadismo/diagnóstico , Hipogonadismo/epidemiologia , Imunoglobulinas/genética , Síndrome de Kallmann/diagnóstico , Síndrome de Kallmann/epidemiologia , Síndrome de Kallmann/genética , Fator de Transcrição MSX1/genética , Masculino , Proteínas de Membrana/genética , Fatores de Transcrição Otx/genética , Linhagem , Receptores de Grelina/genética , Ribonucleoproteínas/genética , Adulto Jovem
6.
Oral Dis ; 25(6): 1492-1501, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31132300

RESUMO

OBJECTIVE: Non-syndromic cleft lip, with or without cleft palate (NSCL/P), is a common craniofacial birth defect, the risk of which is influenced from multiple genetic loci. Association study outcomes between single nucleotide polymorphisms (SNPs) near the muscle segment homeobox gene 1 (MSX1) and NSCL/P have been inconsistent. This compels a meta-analysis to obtain more precise estimates. METHODS: From 15 publications, we examined 12 SNPs under six groups (SG), based on linkage disequilibrium. Pooled odds ratios and 95% confidence intervals were calculated under the standard genetic models. The pooled effects were subjected to subgroup, outlier, sensitivity, and funnel plot (publication bias) analyses. RESULTS: Three of the six SGs showed significant associations. SG1 and SG4 effects indicated reduced risks. SG1 outcomes were attributed to outlier treatment, which the Asian outcomes validated. In contrast, increased risks were observed in SG3. All these significant outcomes were deemed robust by sensitivity analysis with no evidence of publication bias. CONCLUSIONS: Our study shows eight MSX1 SNPs associated with risk of NSCL/P. SG1 and SG4 carriers are protected (up to 23%), but SG3 carriers are 1.3-fold susceptible. Outlier treatment unmasked the significant associations in SG1. Non-heterogeneity and robustness helped elevate the level of evidence in our significant findings.


Assuntos
Fenda Labial/genética , Fissura Palatina/genética , Fator de Transcrição MSX1/genética , Genes Homeobox , Marcadores Genéticos , Predisposição Genética para Doença , Genótipo , Humanos , Polimorfismo de Nucleotídeo Único
7.
J Orthod ; 46(1): 14-19, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-31056064

RESUMO

OBJECTIVE: Maxillary canines are the second-most commonly impacted teeth. About two-thirds of the impacted maxillary canines are palatally impacted. Studies in the past have shown that 40% of cases with palatal impaction of maxillary canines presented with agenesis of third molars. Sporadic agenesis of third molars have been associated with polymorphisms in the MSX1 and PAX9 genes. The present study aims at evaluating the association between polymorphisms of PAX9, MSX1 and palatally impacted canines in a random population sample. DESIGN AND SETTING: Fifty individuals with palatally impacted maxillary canines and 50 gender and age-matched controls were included in this study. METHODS: Single nucleotide polymorphisms (SNPs), rs12532 of MSX1 and rs2073247 of PAX9, were genotyped using polymerase chain reaction and restriction fragment length polymorphism. The significance of the differences among the groups was assessed by odds ratio and Chi-squared test with a 95% confidence interval. RESULTS: Single nucleotide polymorphisms rs12532 [MSX1] and rs 2073247 [PAX9] showed a statistically significant association with palatal impaction of maxillary canines. In addition, the combined presence of the AG/CT genotypes of these genes in an individual caused a significant increase in the risk for palatal impaction. CONCLUSION: These results suggest that the rs12532 and rs2073247 polymorphisms of genes MSX1 and PAX9 are positively associated with palatal impaction of maxillary canines. Future studies investigating various other SNPs of these genes in a larger sample of different populations could provide clinching details.


Assuntos
Fator de Transcrição MSX1 , Fator de Transcrição PAX9 , Dente Impactado , Dente Canino , Humanos , Maxila , Dente Serotino , Razão de Chances , Dente Impactado/genética
8.
Endocrinology ; 160(7): 1631-1644, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31125045

RESUMO

Endometrial stromal cells differentiate to form decidual cells in a process known as decidualization, which is critical for embryo implantation and successful establishment of pregnancy. We previously reported that bone morphogenetic protein 2 (BMP2) mediates uterine stromal cell differentiation in mice and in humans. To identify the downstream target(s) of BMP2 signaling during decidualization, we performed gene-expression profiling of mouse uterine stromal cells, treated or not treated with recombinant BMP2. Our studies revealed that expression of Msx2, a member of the mammalian Msx homeobox gene family, was markedly upregulated in response to exogenous BMP2. Interestingly, conditional ablation of Msx2 in the uterus failed to prevent a decidual phenotype, presumably because of functional compensation of Msx2 by Msx1, a closely related member of the Msx family. Indeed, in Msx2-null uteri, the level of Msx1 expression in the stromal cells was markedly elevated. When conditional, tissue-specific ablation of both Msx1 and Msx2 was accomplished in the mouse uterus, a dramatically impaired decidual response was observed. In the absence of both Msx1 and Msx2, uterine stromal cells were able to proliferate, but they failed to undergo terminal differentiation. In parallel experiments, addition of BMP2 to human endometrial stromal cell cultures led to a robust enhancement of MSX1 and MSX2 expression and stimulated the differentiation process. Attenuation of MSX1 and MSX2 expression by small interfering RNAs greatly reduced human stromal differentiation in vitro, indicating a conservation of their roles as key mediators of BMP2-induced decidualization in mice and women.


Assuntos
Proteína Morfogenética Óssea 2/metabolismo , Implantação do Embrião/fisiologia , Endométrio/metabolismo , Proteínas de Homeodomínio/metabolismo , Fator de Transcrição MSX1/metabolismo , Transdução de Sinais/fisiologia , Animais , Proteína Morfogenética Óssea 2/farmacologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Endométrio/citologia , Endométrio/efeitos dos fármacos , Feminino , Proteínas de Homeodomínio/genética , Humanos , Fator de Transcrição MSX1/genética , Camundongos , Camundongos Knockout , Células Estromais/citologia , Células Estromais/efeitos dos fármacos , Células Estromais/metabolismo
9.
Stem Cell Reports ; 12(5): 1159-1177, 2019 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-31031189

RESUMO

Anamniotes, rodents, and young humans maintain neural stem cells in the ependymal zone (EZ) around the central canal of the spinal cord, representing a possible endogenous source for repair in mammalian lesions. Cell diversity and genes specific for this region are ill defined. A cellular and molecular resource is provided here for the mouse and human EZ based on RNA profiling, immunostaining, and fluorescent transgenic mice. This uncovered the conserved expression of 1,200 genes including 120 transcription factors. Unexpectedly the EZ maintains an embryonic-like dorsal-ventral pattern of expression of spinal cord developmental transcription factors (ARX, FOXA2, MSX1, and PAX6). In mice, dorsal and ventral EZ cells express Vegfr3 and are derived from the embryonic roof and floor plates. The dorsal EZ expresses a high level of Bmp6 and Gdf10 genes and harbors a subpopulation of radial quiescent cells expressing MSX1 and ID4 transcription factors.


Assuntos
Células-Tronco Embrionárias/metabolismo , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica no Desenvolvimento , RNA/genética , Medula Espinal/metabolismo , Células-Tronco/metabolismo , Animais , Células-Tronco Embrionárias/citologia , Células Ependimogliais/citologia , Células Ependimogliais/metabolismo , Feminino , Humanos , Fator de Transcrição MSX1/genética , Fator de Transcrição MSX1/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Microscopia de Fluorescência , Pessoa de Meia-Idade , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , RNA/metabolismo , Medula Espinal/citologia , Nicho de Células-Tronco , Células-Tronco/citologia , Adulto Jovem
10.
Biochimie ; 162: 55-65, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30959082

RESUMO

The homeoprotein Msx1 plays a critical role in embryonic patterning, particularly to maintain myogenic progenitor cell fate. However, the mechanisms underlying Msx1-mediated inhibition of myogenesis remain largely unknown. Here, we show that Msx1 cooperates with the protein kinase C-related kinase 1 (Pkn1), a member of the protein kinase C-related kinase family, to prevent the terminal differentiation of myogenic precursor cells. In mouse C2C12 cells, Pkn1 knockout partly impaired Msx1-mediated inhibition of myogenic differentiation, indicating a role for Pkn1 in this process. Furthermore, we found that Pkn1 was required for Msx1 enrichment at the promoter of Myf5, a myogenic regulatory gene. In Pkn1 knockout cells, this reduced Msx1 enrichment at the Myf5 promoter coincided with attenuated repression of Myf5 transcription. Together with our observation that Msx1 and Pkn1 were associated in a protein complex, these findings strongly suggest that Msx1 cooperates with Pkn1 to down-regulate Myf5 and, therefore, prevent the differentiation of myogenic precursor cells. Collectively, our data provide key insights into the mechanisms underlying Msx1 function in the prevention of myogenic differentiation.


Assuntos
Fator de Transcrição MSX1/metabolismo , Desenvolvimento Muscular , Mioblastos Esqueléticos/fisiologia , Fator Regulador Miogênico 5/metabolismo , Proteína Quinase C/metabolismo , Animais , Diferenciação Celular , Regulação da Expressão Gênica no Desenvolvimento , Células HEK293 , Humanos , Camundongos , Mioblastos Esqueléticos/citologia , Fator Regulador Miogênico 5/genética , Regiões Promotoras Genéticas , Proteína Quinase C/genética
11.
Am J Obstet Gynecol ; 221(1): 46.e1-46.e16, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30826341

RESUMO

BACKGROUND: Maternal-embryonic crosstalk between the endometrium and the preimplantation embryo is required for normal pregnancy. Our previous results demonstrated that maternal microRNAs secreted into the endometrial fluid, specifically miR-30d, act as a transcriptomic regulator of the preimplantation embryo by the maternal intrauterine environment. OBJECTIVE: To investigate the reproductive and fetal effects of murine miR-30d deficiency at the maternal-embryonic interface according to the origin of its maternal or embryonic default. STUDY DESIGN: A miR-30d knockout murine model was used as the animal model to investigate the impact of maternal and/or embryonic origin of miR-30d deficiency on embryonic implantation and fetal development. Wild-type and miR-30d knockout pseudopregnant mice were used to study the effect of miR-30d deficiency on the receptivity markers by means of real-time quantitative polymerase chain reaction, immunofluorescence, and western blotting. We assessed receptivity markers and implantation rates in 6 different transfer conditions in which embryos obtained from wild-type, knockout, and knockout embryos pretreated with a miR-30d analog were transferred into either wild-type or knockout pseudopregnant females. The impact of miR-30d deficiency on fetal development was evaluated by analyzing the implantation sites and resorbing sites under physiological conditions at days 5, 6, 8, and 12 of pregnancy. Fetal growth was evaluated by analyzing fetuses and placentas at days 12 and 16 of pregnancy. RESULTS: Maternal miR-30d deficiency induced a significant downregulation of endometrial receptivity markers. In wild-type recipients, miR-30d knockout embryos had poorer implantation rates than wild-type embryos (48.86 ± 14.33% vs 75.00 ± 10.47%, respectively, P = .0061). In miR-30d knockout recipients, the lowest implantation rate was observed when knockout embryos were transferred compared to wild-type embryos (26.04 ± 7.15% and 49.71 ± 8.59%, respectively, P = .0059). A positive correlation (r = 0.9978) was observed for maternal leukemia inhibitor factor expression with implantation rates. Further, the course of gestation was compromised in miR-30d knockout mothers, which had smaller implantation sites, greater rates of resorption, and fetuses with smaller crown-rump length and fetal/placental weight ratio. CONCLUSION: Our results demonstrate that maternal and/or embryonic miR-30d deficiency impairs embryonic implantation and fetal development in the animal model. This finding adds a novel miRNA dimension to the understanding of pregnancy and fetal growth restriction in humans.


Assuntos
Implantação do Embrião/genética , Endométrio/metabolismo , Desenvolvimento Fetal/genética , MicroRNAs/genética , Animais , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Transferência Embrionária , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Fator Inibidor de Leucemia/genética , Fator Inibidor de Leucemia/metabolismo , Fator de Transcrição MSX1/genética , Fator de Transcrição MSX1/metabolismo , Camundongos , Camundongos Knockout , Placenta , Placentação , Gravidez , Reação em Cadeia da Polimerase em Tempo Real , Receptores Estrogênicos/genética , Receptores Estrogênicos/metabolismo , Receptores de Progesterona/genética , Receptores de Progesterona/metabolismo
12.
Clin Oral Investig ; 23(11): 4107-4111, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30809714

RESUMO

OBJECTIVE: Tooth agenesis is one of the most common craniofacial developmental anomalies. In hypodontia, one to five teeth are missing, whereas oligodontia refers to the absence of at least six teeth, excluding the third molars. Mutations in several genes including MSX1, PAX9, AXIN2, and WNT10A have been shown to cause non-syndromic tooth agenesis. Regional odontodysplasia (RO), also known as "ghost teeth," is a rare developmental anomaly of tooth formation affecting both dentitions. Some possible causes of RO have been suggested, yet the etiology remains unknown. Because the phenotypes of both oligodontia and RO co-occur in one Finnish family, the aim here was to investigate the genetic etiology of the two conditions. MATERIALS AND METHODS: A mutation screening of the genes MSX1, PAX9, AXIN2, and WNT10A was performed for the family members of a RO patient and family history of oligodontia. RESULTS: An initiation codon mutation of the PAX9 gene was found in the proband and segregating with oligodontia in the family. CONCLUSIONS: The etiology of regional odontodysplasia (RO) may be genetic and the same genes can be involved both in RO and tooth agenesis. CLINICAL RELEVANCE: Our results give new insights into the etiology of regional odontodysplasia, yet further results are needed.


Assuntos
Anodontia , Odontodisplasia , Fator de Transcrição PAX9 , Anodontia/genética , Códon de Iniciação , Humanos , Fator de Transcrição MSX1 , Mutação , Odontodisplasia/genética , Fator de Transcrição PAX9/genética , Linhagem
13.
Cleft Palate Craniofac J ; 56(3): 363-372, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-29738289

RESUMO

OBJECTIVE: To evaluate the association of transforming growth factor ß3 ( TGFß3), muscle segment homeobox 1 ( MSX1), Metalloproteinases 3 ( MMP3), and MMP9 genes as candidates for nonsyndromic cleft lip and/or palate in an Indian population. DESIGN: Case-control association study, mutational screening, and functional evaluation of obtained mutations. SETTING: Mutational screening of the developmental genes, TGFß3 and MSX1, along with functional evaluation and association of promoter region SNPs-one each in MMP3 and MMP9. PATIENTS, PARTICIPANTS: Two hundred forty five NSCL±P cases from G. S. Memorial Plastic Surgery Hospital and Trauma Center, Varanasi and 201 healthy controls without a family history of congenital malformations from nearby schools, primary health centers, and the university hospital. MAIN OUTCOME MEASURE(S): Sequencing, SSCP, and PCR-RFLP were used for candidate gene screening. MatInspector and electrophoretic mobility shift assay (EMSA) were used to check the differential transcription factor binding of the variants at promoter region. Luciferase assay was used to test the transcriptional potential of the variant, and evaluation of the alternative splice site was carried out using exon-trapping experiment. RESULTS: Metalloproteinases3 -1171 5A/6A was associated with NSCL±P, whereas MMP9 -1562 C/T did not show association. A rare variant in the promoter region of TGFß3 (rs117462711) creates a differential binding site, confirmed by EMSA. Luciferase assay showed 3.7-fold increased expression level in mutant construct. A synonymous change in MSX1 (rs34165410) showed association with NSCL±P, which may create an alternative splice site or lead to low codon usage. Exon-trapping experiment failed to confirm alternative splicing, indicating low codon usage frequency of the mutant affecting the gene function. CONCLUSIONS: TGFß3, MSX1, and MMP3 are candidates for NSCL±P.


Assuntos
Fenda Labial , Fissura Palatina , Fator de Transcrição MSX1/genética , Metaloproteinase 3 da Matriz/genética , Fator de Crescimento Transformador beta3/genética , Grupo com Ancestrais do Continente Asiático , Estudos de Casos e Controles , Humanos , Metaloproteases , Polimorfismo de Nucleotídeo Único
14.
Oral Dis ; 25(3): 646-651, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29969831

RESUMO

Tooth agenesis (TA) is one of the most common developmental anomalies that affects the number of teeth. An extensive analysis of publicly accessible databases revealed 15 causative genes responsible for nonsyndromic TA, along with their signaling pathways in Wnt/ß-catenin, TGF-ß/BMP, and Eda/Edar/NF-κB. However, genotype-phenotype correlation analysis showed that most of the causal genes are also responsible for syndromic TA or other conditions. In a total of 198 different mutations of the 15 genes responsible for nonsyndromic TA, 182 mutations (91.9%) are derived from seven genes (AXIN2, EDA, LRP6, MSX1, PAX9, WNT10A, and WNT10B) compared with the remaining 16 mutations (8.1%) identified in the remaining eight genes (BMP4, DKK1, EDAR, EDARADD, GREM2, KREMEN1, LTBP3, and SMOC2). Furthermore, specificity analysis in terms of the ratio of nonsyndromic TA mutations versus syndromic mutations in each of the aforementioned seven genes showed a 98.2% specificity rate in PAX9, 58.9% in WNT10A, 56.6% in MSX1, 41.2% in WNT10B, 31.4% in LRP6, 23.8% in AXIN2%, and 8.4% in EDA. These findings underscore an important role of the Wnt and Wnt-associated pathways in the genetic etiology of this heterozygous disease and shed new lights on the discovery of novel molecular mechanisms associated with tooth agenesis.


Assuntos
Anodontia/genética , Proteína Morfogenética Óssea 4/genética , Ectodisplasinas/genética , Receptor Edar/genética , Fator de Crescimento Transformador beta/genética , Via de Sinalização Wnt/genética , Animais , Proteína Axina/genética , Proteínas de Ligação ao Cálcio/genética , Proteína de Domínio de Morte Associada a Edar/genética , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteínas de Ligação a TGF-beta Latente/genética , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Fator de Transcrição MSX1/genética , Proteínas de Membrana/genética , Mutação , NF-kappa B/genética , Fator de Transcrição PAX9/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Wnt/genética
15.
Int J Mol Sci ; 19(12)2018 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-30551562

RESUMO

The neural crest (NC) comprises a multipotent cell population that produces peripheral neurons, cartilage, and smooth muscle cells, among other phenotypes. The participation of Hes1 and Msx1 when expressed in mouse embryonic stem cells (mESCs) undergoing NC differentiation is unexplored. In this work, we generated stable mESCs transfected with constructs encoding chimeric proteins in which the ligand binding domain of glucocorticoid receptor (GR), which is translocated to the nucleus by dexamethasone addition, is fused to either Hes1 (HGR) or Msx1 (MGR), as well as double-transgenic cells (HGR+MGR). These lines continued to express pluripotency markers. Upon NC differentiation, all lines exhibited significantly decreased Sox2 expression and upregulated Sox9, Snai1, and Msx1 expression, indicating NC commitment. Dexamethasone was added to induce nuclear translocation of the chimeric proteins. We found that Collagen IIa transcripts were increased in MGR cells, whereas coactivation of HGR+MGR caused a significant increase in Smooth muscle actin (α-Sma) transcripts. Immunostaining showed that activation in HGR+MGR cells induced higher proportions of ß-TUBULIN III⁺, α-SMA⁺ and COL2A1⁺ cells. These findings indicate that nuclear translocation of MSX-1, alone or in combination with HES-1, produce chondrocyte-like cells, and simultaneous activation of HES-1 and MSX-1 increases the generation of smooth muscle and neuronal cells.


Assuntos
Condrócitos/citologia , Fator de Transcrição MSX1/genética , Células-Tronco Embrionárias Murinas/citologia , Miócitos de Músculo Liso/citologia , Crista Neural/citologia , Receptores de Glucocorticoides/genética , Fatores de Transcrição HES-1/genética , Actinas/genética , Animais , Diferenciação Celular , Núcleo Celular/metabolismo , Células Cultivadas , Condrócitos/metabolismo , Colágeno Tipo II/genética , Dexametasona/farmacologia , Fator de Transcrição MSX1/metabolismo , Camundongos , Camundongos Transgênicos , Células-Tronco Embrionárias Murinas/metabolismo , Miócitos de Músculo Liso/metabolismo , Células NIH 3T3 , Crista Neural/metabolismo , Regiões Promotoras Genéticas , Transporte Proteico/efeitos dos fármacos , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Transcrição SOX9/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Fatores de Transcrição da Família Snail/metabolismo , Fatores de Transcrição HES-1/metabolismo
16.
PLoS One ; 13(9): e0202989, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30192788

RESUMO

Tooth agenesis is one of the most common craniofacial disorders in humans. More than 350 genes have been associated with teeth development. In this study, we enrolled 60 child patients (age 13 to 17) with various types of tooth agenesis. Whole gene sequences of PAX9, MSX1, AXIN2, EDA, EDAR and WNT10a genes were sequenced by next generation sequencing on the Illumina MiSeq platform. We found previously undescribed heterozygous nonsense mutation g.8177G>T (c.610G>T) in MSX1 gene in one child. Mutation was verified by Sanger sequencing. Sequencing analysis was performed in other family members of the affected child. All family members carrying g.8177G>T mutation suffered from oligodontia (missing more than 6 teeth excluding third molars). Mutation g.8177G>T leads to a stop codon (p.E204X) and premature termination of Msx1 protein translation. Based on previous in vitro experiments on mutation disrupting function of Msx1 homeodomain, we assume that the heterozygous g.8177G>T nonsense mutation affects the amount and function of Msx1 protein and leads to tooth agenesis.


Assuntos
Anodontia/genética , Códon sem Sentido , Fator de Transcrição MSX1/genética , Adolescente , Anodontia/patologia , Família , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Modelos Moleculares , Unhas Malformadas , Linhagem
17.
Mol Reprod Dev ; 85(10): 790-801, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30216582

RESUMO

BACKGROUND: Successful implantation of embryos requires endometrial receptivity. Calcitonin is one of the factors influencing the implantation window. This study aimed to evaluate calcitonin effects on endometrial receptivity. To this end, the effects of calcitonin on the implantation window in the ovarian stimulation and the normal ovarian cycle were investigated by the morphological study of the endometrium as well as the expression of MSX.1, HB-EGF, and micro-RNA (miRNA) Let-7a; then the mechanisms of calcitonin effects were studied through the mammalian target of rapamycin (mTOR) and extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathways. MATERIALS AND METHODS: A total of 64 Bulb/c mice were divided into two groups: Normal ovarian cycle and ovarian stimulation. Each group consisted of four subgroups: Ctrl, CT, PP242, and CT + PP242. Calcitonin and PP242 were injected on the fourth day of pregnancy and 24 hr later all the mice were killed. Uterine tissue samples were used for morphological analysis and the endometrial epithelial and the stromal cells were isolated from myometrium for evaluation of gene and protein expression. RESULTS: Ovarian stimulation increased the phosphorylation levels of mTOR and ERK1/2 and the expression of miRNA Let-7a. Calcitonin injection increased the expression of HB-EGF, Msx.1, and miRNA Let-7a in a normal ovarian cycle and in ovarian-stimulated mice. It also increased eukaryotic initiation factor 4E-binding protein 1 and ERK1/2 phosphorylation in normal ovarian cycles. CONCLUSION: Calcitonin improved the receptivity of the uterine endometrium by upregulation of the HB-EGF, Msx.1, and miRNA Let-7a likely through mTOR and ERK1/2 signaling pathway.


Assuntos
Calcitonina/farmacologia , Implantação do Embrião/efeitos dos fármacos , Endométrio/metabolismo , Fator de Crescimento Semelhante a EGF de Ligação à Heparina/biossíntese , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Fator de Transcrição MSX1/biossíntese , MicroRNAs/biossíntese , Serina-Treonina Quinases TOR/biossíntese , Regulação para Cima/efeitos dos fármacos , Animais , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Gravidez
18.
J Alzheimers Dis ; 65(4): 1353-1364, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30124448

RESUMO

BACKGROUND: Weighted co-expression network analysis (WGCNA) is a powerful systems biology method to describe the correlation of gene expression based on the microarray database, which can be used to facilitate the discovery of therapeutic targets or candidate biomarkers in diseases. OBJECTIVE: To explore the key genes in the development of Alzheimer's disease (AD) by using WGCNA. METHODS: The whole gene expression data GSE1297 from AD and control human hippocampus was obtained from the GEO database in NCBI. Co-expressed genes were clustered into different modules. Modules of interest were identified through calculating the correlation coefficient between the module and phenotypic traits. GO and pathway enrichment analyses were conducted, and the central players (key hub genes) within the modules of interest were identified through network analysis. The expression of the identified key genes was confirmed in AD transgenic mice through using qRT-PCR. RESULTS: Two modules were found to be associated with AD clinical severity, which functioning mainly in mineral absorption, NF-κB signaling, and cGMP-PKG signaling pathways. Through analysis of the two modules, we found that metallothionein (MT), Notch2, MSX1, ADD3, and RAB31 were highly correlated with AD phenotype. Increase in expression of these genes was confirmed in aged AD transgenic mice. CONCLUSION: WGCNA analysis can be used to analyze and predict the key genes in AD. MT1, MT2, MSX1, NOTCH2, ADD3, and RAB31 are identified to be the most relevant genes, which may be potential targets for AD therapy.


Assuntos
Doença de Alzheimer/genética , Redes Reguladoras de Genes , Predisposição Genética para Doença/genética , Proteínas de Ligação a Calmodulina/genética , Feminino , Perfilação da Expressão Gênica , Humanos , Fator de Transcrição MSX1/genética , Masculino , Entrevista Psiquiátrica Padronizada , Metalotioneína/genética , RNA Mensageiro/metabolismo , Receptor Notch2/genética , Biologia de Sistemas , Proteínas rab de Ligação ao GTP/genética
19.
Stem Cell Res Ther ; 9(1): 221, 2018 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-30134957

RESUMO

BACKGROUND: Tooth agenesis, one of the most common developmental anomalies, can affect the function and esthetics of patients. The aim of the present study was to identify genetic clues for familial tooth agenesis and explore the underlying mechanisms, focusing on the role of human dental pulp stem cells (hDPSCs). METHODS: We applied Sanger sequencing to identify the cause of oligodontia in a Chinese family. DNA transfection and functional analysis in DPSCs was also performed to explore the impact of the identified mutation on this phenotype. RESULTS: In this study, a novel frameshift mutation, the twenty-nucleotide deletion (c.128_147del20, p.Met43Serfsx125), in exon1 of MSX1 was detected in a Chinese family causing autosomal dominant nonsyndromic oligodontia. The mutation cosegregated with the tooth agenesis phenotype in this family. DPSCs transfected with mutant MSX1 plasmid showed decreased capacity of osteo/odontogenic differentiation with a lower expression level of dentin sialophosphoprotein (DSPP) and bone sialoprotein (BSP) compared with those transfected with control MSX1 plasmid. Mechanically, control MSX1 showed nuclear localization while the mutant MSX1 inhibited its nuclear translocation and localized on the cytoplasm to inhibit ERK phosphorylation. Furthermore, we inhibited the ERK pathway using ERK inhibitor (U0126) treatment in control MSX1-transfected DPSCs which could downregulate mineralized nodule formation and the expression of odontogenic genes. CONCLUSION: We demonstrated a novel MSX1 mutation causing familial nonsyndromic oligodontia and mechanically MSX1 regulates odontogenesis through the ERK signaling pathway in human dental pulp stem cells.


Assuntos
Anodontia/genética , Polpa Dentária/metabolismo , Mutação da Fase de Leitura , Fator de Transcrição MSX1/genética , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/genética , Células-Tronco/metabolismo , Adolescente , Adulto , Anodontia/metabolismo , Anodontia/patologia , Butadienos/farmacologia , Diferenciação Celular , Núcleo Celular/metabolismo , Proliferação de Células , Polpa Dentária/patologia , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Família , Feminino , Regulação da Expressão Gênica , Genes Dominantes , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Sialoproteína de Ligação à Integrina/genética , Sialoproteína de Ligação à Integrina/metabolismo , Fator de Transcrição MSX1/metabolismo , Masculino , Pessoa de Meia-Idade , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Nitrilos/farmacologia , Linhagem , Fenótipo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilação/efeitos dos fármacos , Plasmídeos/química , Plasmídeos/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Sialoglicoproteínas/genética , Sialoglicoproteínas/metabolismo , Células-Tronco/patologia , Transfecção
20.
Zhonghua Kou Qiang Yi Xue Za Zhi ; 53(7): 466-469, 2018 Jul 09.
Artigo em Chinês | MEDLINE | ID: mdl-29996364

RESUMO

Objective: To further study the effects of distal-less homeobox gene 5 (Dlx-5) and Msh homeobox 1 (Msx-1) in the pathogenic mechanism of bisphosphonate related osteonecrosis of the jaw (BRONJ) . Methods: Twenty-four SD rats were divided into two groups, the experimental group was injected intraperitoneally with zoledronic acid for 12 weeks (0.2 mg/kg, three times a week), and the control group was injected with saline solution for 12 weeks. The first mandibular molars were extracted after 12 weeks. All of the animals were sacrificed eight weeks after teeth extraction. The BRONJ was diagnosed by gross observation, X-ray examination and histopathlolgical examination. Through real-time PCR, the expression level of Dlx-5 and Msx-1 were detected in the mandible of BRONJ samples and normal samples. Results: X-ray examination and histopathlolgical analysis showed the presence of BRONJ. The results of real-time PCR showed that the expression levels of Dlx-5 were increased (P=0.001) and the expression level of Msx-1 was decreased (P=0.001) in the experimental group compared with the control group. Conclusions: Dlx-5 and Msx-1 genes play roles in the pathogenic mechanism of BRONJ.


Assuntos
Osteonecrose da Arcada Osseodentária Associada a Difosfonatos/genética , Proteínas de Homeodomínio/genética , Fator de Transcrição MSX1/genética , Fatores de Transcrição/genética , Animais , Osteonecrose da Arcada Osseodentária Associada a Difosfonatos/metabolismo , Conservadores da Densidade Óssea , Difosfonatos/administração & dosagem , Genes Homeobox , Proteínas de Homeodomínio/metabolismo , Humanos , Imidazóis/administração & dosagem , Fator de Transcrição MSX1/metabolismo , Mandíbula , Modelos Animais , Dente Molar/cirurgia , Ratos , Ratos Sprague-Dawley , Extração Dentária , Fatores de Transcrição/metabolismo , Ácido Zoledrônico
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