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Background: Current clinical variant analysis pipelines focus on coding variants and intronic variants within 10-20 bases of an exon-intron boundary that may affect splicing. The impact of newer splicing prediction algorithms combined with in vitro splicing assays on rare variants currently considered Benign/Likely Benign (B/LB) is unknown. Methods: Exome sequencing data from 576 pediatric cancer patients enrolled in the Texas KidsCanSeq study were filtered for intronic or synonymous variants absent from population databases, predicted to alter splicing via SpliceAI (>0.20), and scored as potentially deleterious by CADD (>10.0). Total cellular RNA was extracted from monocytes and RT-PCR products analyzed. Subsequently, rare synonymous or intronic B/LB variants in a subset of genes submitted to ClinVar were similarly evaluated. Variants predicted to lead to a frameshifted splicing product were functionally assessed using an in vitro splicing reporter assay in HEK-293T cells. Results: KidsCanSeq exome data analysis revealed a rare, heterozygous, intronic variant (NM_177438.3(DICER1):c.574-26A>G) predicted by SpliceAI to result in gain of a secondary splice acceptor site. The proband had a personal and family history of pleuropulmonary blastoma consistent with DICER1 syndrome but negative clinical sequencing reports. Proband RNA analysis revealed alternative DICER1 transcripts including the SpliceAI-predicted transcript.Similar bioinformatic analysis of synonymous or intronic B/LB variants (n=31,715) in ClinVar from 61 Mendelian disease genes yielded 18 variants, none of which could be scored by MaxEntScan. Eight of these variants were assessed (DICER1 n=4, CDH1 n=2, PALB2 n=2) using in vitro splice reporter assay and demonstrated abnormal splice products (mean 66%; range 6% to 100%). Available phenotypic information from submitting laboratories demonstrated DICER1 phenotypes in 2 families (1 variant) and breast cancer phenotypes for PALB2 in 3 families (2 variants). Conclusions: Our results demonstrate the power of newer predictive splicing algorithms to highlight rare variants previously considered B/LB in patients with features of hereditary conditions. Incorporation of SpliceAI annotation of existing variant data combined with either direct RNA analysis or in vitro assays has the potential to identify disease-associated variants in patients without a molecular diagnosis.
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BACKGROUND: Using immunomodulatory methods to address the challenging issue of craniofacial bone repair may be a potentially effective approach. The protease inhibitor saquinavir has been shown to inhibit the inflammatory response by targeting the toll-like receptor 4/myeloid differentiation primary response complex. Independently, inhibition of toll-like receptor 4 or myeloid differentiation primary response led to enhanced skull bone repair. Therefore, the authors aimed to investigate the effects of saquinavir on skull bone healing. METHODS: The effects of saquinavir on skull bone healing were assessed by means of gene expression, histology, immunohistochemistry, and tomography in a mouse calvarial defect model. Subsequently, the role of saquinavir in cell viability, migration, and osteogenic and osteoclastogenic differentiation was also evaluated in vitro. RESULTS: One-week saquinavir administration improved skull bone healing based on micro-computed tomographic and histomorphometric analyses. Compared to the vehicle control, 1-week saquinavir treatment (1) enhanced osteoclast infiltration (tartrate-resistant acid phosphatase staining) at day 7, but not at days 14 and 28; (2) induced more CD206 + M2 macrophage infiltration, but not F4/80 + M0 macrophages at days 7, 14, and 28; and (3) elevated osteoclastogenic gene RANKL (quantitative polymerase chain reaction) expression and other osteogenic and cytokine expression. Furthermore, in vitro data showed that saquinavir administration did not influence MC3T3-E1 cell migration or mineralization, whereas higher concentrations of saquinavir inhibited cell viability. Saquinavir treatment also enhanced the osteoclastic differentiation of bone marrow-derived precursors, and partially reversed high-mobility group box 1-driven osteoclastogenesis inhibition and elevated proinflammatory cytokine expression. CONCLUSION: The improved skull bone repair following short-term saquinavir treatment may involve enhanced osteoclastogenesis and modulated inflammatory response following skull injury. CLINICAL RELEVANCE STATEMENT: The authors' work demonstrates improved skull bone healing by short-term application of saquinavir, a drug traditionally used in the treatment of acquired immunodeficiency syndrome. As such, saquinavir may be repurposed for skeletal repair.
Assuntos
Inibidores da Protease de HIV , Saquinavir , Camundongos , Animais , Saquinavir/farmacologia , Saquinavir/metabolismo , Saquinavir/uso terapêutico , Inibidores da Protease de HIV/farmacologia , Inibidores da Protease de HIV/metabolismo , Inibidores da Protease de HIV/uso terapêutico , Receptor 4 Toll-Like/fisiologia , Osteogênese , Crânio/lesõesRESUMO
The ALF transcription factor paralogs, AFF1, AFF2, AFF3, and AFF4, are components of the transcriptional super elongation complex that regulates expression of genes involved in neurogenesis and development. We describe an autosomal dominant disorder associated with de novo missense variants in the degron of AFF3, a nine amino acid sequence important for its binding to ubiquitin ligase, or with de novo deletions of this region. The sixteen affected individuals we identified, along with two previously reported individuals, present with a recognizable pattern of anomalies, which we named KINSSHIP syndrome (KI for horseshoe kidney, NS for Nievergelt/Savarirayan type of mesomelic dysplasia, S for seizures, H for hypertrichosis, I for intellectual disability, and P for pulmonary involvement), partially overlapping the AFF4-associated CHOPS syndrome. Whereas homozygous Aff3 knockout mice display skeletal anomalies, kidney defects, brain malformations, and neurological anomalies, knockin animals modeling one of the microdeletions and the most common of the missense variants identified in affected individuals presented with lower mesomelic limb deformities like KINSSHIP-affected individuals and early lethality, respectively. Overexpression of AFF3 in zebrafish resulted in body axis anomalies, providing some support for the pathological effect of increased amount of AFF3. The only partial phenotypic overlap of AFF3- and AFF4-associated syndromes and the previously published transcriptome analyses of ALF transcription factors suggest that these factors are not redundant and each contributes uniquely to proper development.
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Encefalopatias/genética , Epilepsia/genética , Rim Fundido/genética , Deficiência Intelectual/genética , Mutação de Sentido Incorreto , Proteínas Nucleares/genética , Osteocondrodisplasias/genética , Adolescente , Sequência de Aminoácidos , Animais , Encefalopatias/etiologia , Criança , Pré-Escolar , Epilepsia/complicações , Evolução Molecular , Feminino , Frequência do Gene , Humanos , Lactente , Masculino , Camundongos , Modelos Moleculares , Proteínas Nucleares/química , Proteínas Nucleares/deficiência , Fenótipo , Estabilidade Proteica , Síndrome , Fatores de Elongação da Transcrição/química , Fatores de Elongação da Transcrição/genética , Adulto Jovem , Peixe-Zebra/genéticaRESUMO
We conducted genome sequencing to search for rare variation contributing to early-onset Alzheimer's disease (EOAD) and frontotemporal dementia (FTD). Discovery analysis was conducted on 435 cases and 671 controls of European ancestry. Burden testing for rare variation associated with disease was conducted using filters based on variant rarity (less than one in 10,000 or private), computational prediction of deleteriousness (CADD) (10 or 15 thresholds), and molecular function (protein loss-of-function [LoF] only, coding alteration only, or coding plus non-coding variants in experimentally predicted regulatory regions). Replication analysis was conducted on 16,434 independent cases and 15,587 independent controls. Rare variants in TET2 were enriched in the discovery combined EOAD and FTD cohort (p = 4.6 × 10-8, genome-wide corrected p = 0.0026). Most of these variants were canonical LoF or non-coding in predicted regulatory regions. This enrichment replicated across several cohorts of Alzheimer's disease (AD) and FTD (replication only p = 0.0029). The combined analysis odds ratio was 2.3 (95% confidence interval [CI] 1.6-3.4) for AD and FTD. The odds ratio for qualifying non-coding variants considered independently from coding variants was 3.7 (95% CI 1.7-9.4). For LoF variants, the combined odds ratio (for AD, FTD, and amyotrophic lateral sclerosis, which shares clinicopathological overlap with FTD) was 3.1 (95% CI 1.9-5.2). TET2 catalyzes DNA demethylation. Given well-defined changes in DNA methylation that occur during aging, rare variation in TET2 may confer risk for neurodegeneration by altering the homeostasis of key aging-related processes. Additionally, our study emphasizes the relevance of non-coding variation in genetic studies of complex disease.
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Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Mutação com Perda de Função/genética , Doenças Neurodegenerativas/genética , Proteínas Proto-Oncogênicas/deficiência , Proteínas Proto-Oncogênicas/genética , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/genética , Animais , Cognição , Dioxigenases , Feminino , Demência Frontotemporal/genética , Humanos , Masculino , CamundongosRESUMO
TBR1, a T-box transcription factor expressed in the cerebral cortex, regulates the expression of several candidate genes for autism spectrum disorders (ASD). Although TBR1 has been reported as a high-confidence risk gene for ASD and intellectual disability (ID) in functional and clinical reports since 2011, TBR1 has only recently been recorded as a human disease gene in the OMIM database. Currently, the neurodevelopmental disorders and structural brain anomalies associated with TBR1 variants are not well characterized. Through international data sharing, we collected data from 25 unreported individuals and compared them with data from the literature. We evaluated structural brain anomalies in seven individuals by analysis of MRI images, and compared these with anomalies observed in TBR1 mutant mice. The phenotype included ID in all individuals, associated to autistic traits in 76% of them. No recognizable facial phenotype could be identified. MRI analysis revealed a reduction of the anterior commissure and suggested new features including dysplastic hippocampus and subtle neocortical dysgenesis. This report supports the role of TBR1 in ID associated with autistic traits and suggests new structural brain malformations in humans. We hope this work will help geneticists to interpret TBR1 variants and diagnose ASD probands.
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Transtorno Autístico/genética , Anormalidades Craniofaciais/genética , Deficiência Intelectual/genética , Fenótipo , Proteínas com Domínio T/genética , Adolescente , Adulto , Animais , Transtorno Autístico/patologia , Criança , Pré-Escolar , Cognição , Anormalidades Craniofaciais/patologia , Feminino , Hipocampo/diagnóstico por imagem , Hipocampo/patologia , Humanos , Deficiência Intelectual/patologia , Masculino , Camundongos , Mutação , Neocórtex/diagnóstico por imagem , Neocórtex/patologia , Síndrome , Proteínas com Domínio T/metabolismoRESUMO
Tissue-engineered graft substitutes have shown great potential to treat large bone defects. While we usually assume that therapeutic approaches developed for appendicular bone healing could be similarly translated for application in craniofacial reconstruction and vice versa, this is not necessarily accurate. In addition to those more well-known healing-associated factors, such as age, lifestyle (e.g., nutrition and smoking), preexisting disease (e.g., diabetes), medication, and poor blood supply, the developmental origins and surrounding tissue of the wound sites can largely affect the fracture healing outcome as well as designed treatments. Therefore, the strategies developed for long bone fracture repair might not be suitable or directly applicable to skull bone repair. In this review, we discuss aspects of development, healing process, structure, and tissue engineering considerations between calvarial and long bones to assist in designing the tailored bone repair strategies. Impact Statement We summarized, in this review, the existing body of knowledge between long bone and calvarial bone with regard to their development and healing, tissue structure, and consideration of current tissue engineering strategies. By highlighting their similarities and differences, we propose that tailored tissue engineering strategies, such as scaffold features, growth factor usage, and the source of cells for tissue or region-specific bone repair, are necessary to ensure an optimized healing outcome.
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Doenças Ósseas/terapia , Osso e Ossos/citologia , Consolidação da Fratura , Osteogênese , Crânio/citologia , Engenharia Tecidual/métodos , Animais , HumanosRESUMO
BACKGROUND: Large calvarial defects represent a major reconstructive challenge, as they do not heal spontaneously. Infection causes inflammation and scarring, further reducing the healing capacity of the calvaria. Bone morphogenetic protein-2 (BMP2) has been shown to stimulate osteogenesis but has significant side effects in high doses. BMP2 has not been tested in combination with antiinflammatory cytokines such as interleukin-10. METHODS: Sixteen New Zealand White rabbits underwent 15 × 15-mm flap calvarectomies. The flap was incubated in Staphylococcus aureus and replaced, and infection and scarring were allowed to develop. The flap was subsequently removed and the wound débrided. A 15 × 15-mm square of acellular dermal matrix biopatterned with low-dose BMP2, interleukin-10, or a combination was implanted. Computed tomographic scans were taken over 42 days. Rabbits were then killed and histology was performed. RESULTS: Defects treated with BMP2 showed significantly (p < 0.05) greater osseous regeneration than untreated controls. Interleukin-10 did not significantly augment the healing achieved with BMP2, and interleukin-10 alone did not significantly increase healing compared with controls. Histology showed evidence of bone formation in defects treated with BMP2. Untreated controls and defects treated with interleukin-10 alone showed only fibrous tissue in the defect site. CONCLUSIONS: Low-dose BMP2 delivered directly to the scarred calvarial defect augments bony healing. Interleukin-10 at the dose applied did not significantly augment healing alone or in combination with BMP2. Healing had not finished at 42 days and analysis at later time points or the use of higher doses of BMP2 may yield greater healing.
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Proteína Morfogenética Óssea 2/farmacologia , Regeneração Óssea/efeitos dos fármacos , Interleucina-10/farmacologia , Crânio/fisiologia , Animais , Proteína Morfogenética Óssea 2/administração & dosagem , Cicatriz/tratamento farmacológico , Combinação de Medicamentos , Interleucina-10/administração & dosagem , Masculino , Coelhos , Crânio/efeitos dos fármacos , Crânio/cirurgia , Infecções Estafilocócicas/fisiopatologia , Staphylococcus aureus , Retalhos Cirúrgicos , Tomografia Computadorizada por Raios XRESUMO
Bone morphogenetic protein 2 (BMP2) bioprinted on biological matrix induces osseous regeneration in large calvarial defects in rabbits, both uncomplicated and scarred. Healing in unfavorable defects scarred from previous infection is decreased due in part to the lack of vascularity. This impedes the access of mesenchymal stem cells, key to osseous regeneration and the efficacy of BMP2, to the wound bed. The authors hypothesized that bioprinted vascular endothelial growth factor (VEGF) would augment the osseous regeneration achieved with low dose biopatterned BMP2 alone. Thirteen New Zealand white rabbits underwent subtotal calvariectomy using a dental cutting burr. Care was taken to preserve the underlying dura. A 15 mm × 15âmm flap of bone was cut away and incubated in a 1 × 108 cfu/mL planktonic solution of S aureus before reimplantation. After 2 weeks of subsequent infection the flap was removed and the surgical wound debrided followed by 10 days of antibiotic treatment. On postoperative day 42 the calvarial defects were treated with acellular dermal matrix bioprinted with nothing (control), VEGF, BMP2, BMP2/VEGF combined. Bone growth was analyzed with serial CT and postmortem histology. Defects treated with BMP2 (BMP2 alone and BMP2/VEGF combination) showed significantly greater healing than control and VEGF treated defect (Pâ<â0.5). Vascular endothelial growth factor treated defect demonstrated less healing than control and VEGF/BMP2 combination treatments achieved less healing than BMP2 alone though these differences were nonsignificant. Low dose BMP2-patterned acellular dermal matrix improves healing of scarred calvarial defects. Vascular endothelial growth factor at the doses applied in this study failed to increase healing.
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Proteína Morfogenética Óssea 2/farmacologia , Regeneração Óssea/efeitos dos fármacos , Procedimentos de Cirurgia Plástica/métodos , Crânio/cirurgia , Fator de Crescimento Transformador beta/farmacologia , Fator A de Crescimento do Endotélio Vascular/farmacologia , Cicatrização/efeitos dos fármacos , Animais , Modelos Animais de Doenças , Coelhos , Proteínas Recombinantes/farmacologiaRESUMO
Diamond-Blackfan Anemia (DBA) is a rare polygenic disorder defined by congenital hypoplastic anemia with marked decrease or absence of bone marrow erythroid precursors. Identifying the specific genetic etiology is important for counseling and clinical management. A 6-yr-old boy with a clinical diagnosis of DBA has been followed by our pediatric hematology team since birth. His clinical course includes transfusion-dependent hypoplastic anemia and progressive autoimmune cytopenias. Genetic testing failed to identify a causative mutation in any of the classical DBA-associated genes. He and his parents underwent trio whole-exome sequencing (WES) with no genetic etiology identified initially. Clinical persistence and suspicion led to testing for adenosine deaminase 2 (ADA2) activity and whole-genome sequencing (WGS) that identified compound heterozygous pathogenic mutations in the ADA2-encoding CECR1 gene, a recently appreciated etiology for congenital hypoplastic anemia. This case illustrates current challenges in genetic testing and how they can be overcome by multidisciplinary expertise in clinical medicine and genomics.
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Adenosina Desaminase/genética , Anemia de Diamond-Blackfan/genética , Anemia Hipoplástica Congênita/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Anemia de Diamond-Blackfan/diagnóstico , Anemia Hipoplástica Congênita/diagnóstico , Medula Óssea/fisiopatologia , Criança , Testes Genéticos/métodos , Humanos , Masculino , Mutação , Pais , Proteínas Ribossômicas , Sequenciamento do ExomaRESUMO
Mutations that alter signaling of RAS/MAPK-family proteins give rise to a group of Mendelian diseases known as RASopathies. However, among RASopathies, the matrix of genotype-phenotype relationships is still incomplete, in part because there are many RAS-related proteins and in part because the phenotypic consequences may be variable and/or pleiotropic. Here, we describe a cohort of ten cases, drawn from six clinical sites and over 16,000 sequenced probands, with de novo protein-altering variation in RALA, a RAS-like small GTPase. All probands present with speech and motor delays, and most have intellectual disability, low weight, short stature, and facial dysmorphism. The observed rate of de novo RALA variants in affected probands is significantly higher (p = 4.93 x 10(-11)) than expected from the estimated random mutation rate. Further, all de novo variants described here affect residues within the GTP/GDP-binding region of RALA; in fact, six alleles arose at only two codons, Val25 and Lys128. The affected residues are highly conserved across both RAL- and RAS-family genes, are devoid of variation in large human population datasets, and several are homologous to positions at which disease-associated variants have been observed in other GTPase genes. We directly assayed GTP hydrolysis and RALA effector-protein binding of the observed variants, and found that all but one tested variant significantly reduced both activities compared to wild-type. The one exception, S157A, reduced GTP hydrolysis but significantly increased RALA-effector binding, an observation similar to that seen for oncogenic RAS variants. These results show the power of data sharing for the interpretation and analysis of rare variation, expand the spectrum of molecular causes of developmental disability to include RALA, and provide additional insight into the pathogenesis of human disease caused by mutations in small GTPases.
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Deficiências do Desenvolvimento/genética , Deficiência Intelectual/genética , Proteínas Mitocondriais/genética , Mutação , Domínios e Motivos de Interação entre Proteínas/genética , Proteínas ral de Ligação ao GTP/genética , Proteínas ras/genética , Fácies , Genótipo , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Proteínas Mitocondriais/química , Modelos Moleculares , Mutação de Sentido Incorreto , Fenótipo , Conformação Proteica , Proteínas ral de Ligação ao GTP/química , Proteínas ras/químicaRESUMO
Corpus callosum malformations are associated with a broad range of neurodevelopmental diseases. We report that de novo mutations in MAST1 cause mega-corpus-callosum syndrome with cerebellar hypoplasia and cortical malformations (MCC-CH-CM) in the absence of megalencephaly. We show that MAST1 is a microtubule-associated protein that is predominantly expressed in post-mitotic neurons and is present in both dendritic and axonal compartments. We further show that Mast1 null animals are phenotypically normal, whereas the deletion of a single amino acid (L278del) recapitulates the distinct neurological phenotype observed in patients. In animals harboring Mast1 microdeletions, we find that the PI3K/AKT3/mTOR pathway is unperturbed, whereas Mast2 and Mast3 levels are diminished, indicative of a dominant-negative mode of action. Finally, we report that de novo MAST1 substitutions are present in patients with autism and microcephaly, raising the prospect that mutations in this gene give rise to a spectrum of neurodevelopmental diseases.
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Agenesia do Corpo Caloso/genética , Cerebelo/anormalidades , Regulação da Expressão Gênica no Desenvolvimento/genética , Malformações do Desenvolvimento Cortical/genética , Proteínas Associadas aos Microtúbulos/genética , Mutação/genética , Malformações do Sistema Nervoso/genética , Agenesia do Corpo Caloso/complicações , Agenesia do Corpo Caloso/diagnóstico por imagem , Agenesia do Corpo Caloso/patologia , Animais , Animais Recém-Nascidos , Apoptose/genética , Encéfalo/metabolismo , Encéfalo/patologia , Células Cultivadas , Cerebelo/diagnóstico por imagem , Criança , Deficiências do Desenvolvimento/complicações , Deficiências do Desenvolvimento/diagnóstico por imagem , Deficiências do Desenvolvimento/genética , Modelos Animais de Doenças , Embrião de Mamíferos , Feminino , Humanos , Masculino , Malformações do Desenvolvimento Cortical/complicações , Malformações do Desenvolvimento Cortical/diagnóstico por imagem , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/deficiência , Proteínas do Tecido Nervoso/metabolismo , Malformações do Sistema Nervoso/complicações , Malformações do Sistema Nervoso/diagnóstico por imagem , Fator de Transcrição PAX6/metabolismoRESUMO
The Clinical Sequencing Evidence-Generating Research (CSER) consortium, now in its second funding cycle, is investigating the effectiveness of integrating genomic (exome or genome) sequencing into the clinical care of diverse and medically underserved individuals in a variety of healthcare settings and disease states. The consortium comprises a coordinating center, six funded extramural clinical projects, and an ongoing National Human Genome Research Institute (NHGRI) intramural project. Collectively, these projects aim to enroll and sequence over 6,100 participants in four years. At least 60% of participants will be of non-European ancestry or from underserved settings, with the goal of diversifying the populations that are providing an evidence base for genomic medicine. Five of the six clinical projects are enrolling pediatric patients with various phenotypes. One of these five projects is also enrolling couples whose fetus has a structural anomaly, and the sixth project is enrolling adults at risk for hereditary cancer. The ongoing NHGRI intramural project has enrolled primarily healthy adults. Goals of the consortium include assessing the clinical utility of genomic sequencing, exploring medical follow up and cascade testing of relatives, and evaluating patient-provider-laboratory level interactions that influence the use of this technology. The findings from the CSER consortium will offer patients, healthcare systems, and policymakers a clearer understanding of the opportunities and challenges of providing genomic medicine in diverse populations and settings, and contribute evidence toward developing best practices for the delivery of clinically useful and cost-effective genomic sequencing in diverse healthcare settings.
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Genoma Humano/genética , Adulto , Análise Custo-Benefício/métodos , Atenção à Saúde/métodos , Europa (Continente) , Exoma/genética , Genômica/métodos , Humanos , National Human Genome Research Institute (U.S.) , Fenótipo , Estados Unidos , Sequenciamento Completo do Genoma/métodosRESUMO
Purpose: Colorectal cancer is the third most common cancer worldwide, causing approximately 700,000 deaths each year. The majority of colorectal cancers begin as adenomas. Definitive screening for colorectal adenomas is currently accomplished through colonoscopy but, owing largely to costs and invasiveness, is typically limited to patient groups at higher risk by virtue of age or family history. We sought to determine if blood-based small RNA markers could detect colorectal adenoma.Experimental Design: We applied high-depth small RNA sequencing to plasma from a large (n = 189) cohort of patients, balanced for age, sex, and ancestry. Our analytical methodology allowed for the detection of both microRNAs and other small RNA species. We replicated sequencing results by qPCR on plasma samples from an independent cohort (n = 140).Results: We found several small RNA species with significant associations to colorectal adenoma, including both microRNAs and non-microRNA small RNAs. These associations were robust to correction for patient covariates, including age. Among the adenoma-associated small RNAs, two, a miR-335-5p isoform and an un-annotated small RNA, were validated by qPCR in an independent cohort. A classifier trained on measures of these two RNAs in the discovery cohort yields an AUC of 0.755 (0.775 with age) for adenoma detection in the independent cohort. This classifier accurately detects adenomas in patients under 50 and is robust to sex or ancestry.Conclusions: Circulating small RNAs (including but not limited to miRNAs) discovered by sequencing and validated by qPCR identify patients with colorectal adenomas effectively. Clin Cancer Res; 24(9); 2092-9. ©2018 AACR.
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Adenoma/sangue , Adenoma/genética , Biomarcadores Tumorais , Ácidos Nucleicos Livres , Neoplasias Colorretais/sangue , Neoplasias Colorretais/genética , Pequeno RNA não Traduzido/sangue , Pequeno RNA não Traduzido/genética , Adenoma/diagnóstico , Neoplasias Colorretais/diagnóstico , Biologia Computacional/métodos , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Curva ROC , Reprodutibilidade dos TestesRESUMO
OBJECTIVE: Craniosynostosis (CS) involves the premature fusion of one or more cranial sutures. The etiology of CS is complex and mutations in more than 50 distinct genes have been causally linked to the disorder. Many of the genes that have been associated with CS in humans play an essential role in tissue patterning and early craniofacial development. Among these genes are members of the Hedgehog (HH) and Notch signal transduction pathways, including the GLI family member Gli3, Indian Hedgehog ( Ihh), the RAS oncogene family member Rab23, and the Notch ligand JAGGED1 ( Jag1). We have previously described a colony of rabbits with a heritable pattern of coronal suture synostosis, although the genetic basis for synostosis within this model remains unknown. The present study was performed to determine if coding errors in Gli3, Ihh, Rab23, or Jag1 could be causally linked to craniosynostosis in this unique animal model. DESIGN: Sequencing of cDNA templates was performed using samples obtained from wild-type and craniosynostotic rabbits. RESULTS: Several nucleotide polymorphisms were identified in Gli3, Ihh, and Rab23, although these variants failed to segregate by phenotype. No nucleotide polymorphisms were identified in Jag1. CONCLUSIONS: These data indicate that the causal locus for heritable craniosynostosis in this rabbit model is not located within the protein coding regions of Gli3, Ihh, Rab23, or Jag1.
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Craniossinostoses/genética , Polimorfismo de Nucleotídeo Único , Animais , Western Blotting , Modelos Animais de Doenças , Regulação da Expressão Gênica no Desenvolvimento , Genótipo , Proteínas Hedgehog/genética , Proteína Jagged-1/genética , Fenótipo , Reação em Cadeia da Polimerase , Coelhos , Transdução de Sinais , Proteína Gli3 com Dedos de Zinco/genética , Proteínas rab de Ligação ao GTP/genéticaRESUMO
OBJECTIVES: An overexpression of Tgf-ß2 leads to calvarial hyperostosis and suture fusion in individuals with craniosynostosis. Inhibition of Tgf-ß2 may help rescue fusing sutures and restore normal growth. The present study was designed to test this hypothesis. DESIGN: Twenty-eight New Zealand White rabbits with delayed-onset coronal synostosis had radiopaque markers placed on either side of the coronal sutures at 10 days of age. The rabbits were randomly assigned to: (1) sham control rabbits (n = 10), (2) rabbits with control IgG (100 µg/suture) delivered in a collagen vehicle (n = 9), and (3) rabbits with Tgf-ß2 neutralizing antibody (100 µg/suture) delivered in a collagen vehicle (n = 9). Longitudinal growth data were collected at 10, 25, 42, and 84 days of age. Sutures were harvested at 84 days of age for histomorphometry. RESULTS: Radiographic analysis showed significantly greater ( P < .05) coronal suture marker separation, craniofacial length, cranial vault length, height, shape indices, cranial base length, and more lordotic cranial base angles in rabbits treated with anti-Tgf-ß2 antibody than in controls at 42 and 84 days of age. Histologically, rabbits treated with anti-Tgf-ß2 antibody at 84 days of age had patent and significantly ( P < .05) wider coronal sutures and greater sutural area compared to controls. CONCLUSIONS: These data support our hypothesis that antagonism of Tgf-ß2 may rescue fusing coronal sutures and facilitate craniofacial growth in this rabbit model. These findings also suggest that cytokine therapy may have clinical significance in infants with progressive postgestational craniosynostosis.
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Suturas Cranianas , Craniossinostoses , Fator de Crescimento Transformador beta2 , Animais , Coelhos , Animais Recém-Nascidos , Suturas Cranianas/diagnóstico por imagem , Suturas Cranianas/efeitos dos fármacos , Suturas Cranianas/crescimento & desenvolvimento , Craniossinostoses/diagnóstico por imagem , Craniossinostoses/prevenção & controle , Modelos Animais de Doenças , Distribuição Aleatória , Fator de Crescimento Transformador beta2/antagonistas & inibidoresRESUMO
PURPOSE: As massively parallel sequencing is increasingly being used for clinical decision making, it has become critical to understand parameters that affect sequencing quality and to establish methods for measuring and reporting clinical sequencing standards. In this report, we propose a definition for reduced coverage regions and describe a set of standards for variant calling in clinical sequencing applications. METHODS: To enable sequencing centers to assess the regions of poor sequencing quality in their own data, we optimized and used a tool (ExCID) to identify reduced coverage loci within genes or regions of particular interest. We used this framework to examine sequencing data from 500 patients generated in 10 projects at sequencing centers in the National Human Genome Research Institute/National Cancer Institute Clinical Sequencing Exploratory Research Consortium. RESULTS: This approach identified reduced coverage regions in clinically relevant genes, including known clinically relevant loci that were uniquely missed at individual centers, in multiple centers, and in all centers. CONCLUSION: This report provides a process road map for clinical sequencing centers looking to perform similar analyses on their data.
Assuntos
Sequenciamento do Exoma/métodos , Análise de Sequência de DNA/métodos , Sequenciamento Completo do Genoma/métodos , Sequência de Bases , Mapeamento Cromossômico , Exoma , Genoma Humano , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Análise de Sequência de DNA/normas , SoftwareRESUMO
BACKGROUND: Toll-like receptor 4 (TLR4) has been implicated in inflammation-induced bone destruction in various chronic bone diseases; however, its direct influence on bone healing is not well understood. The authors' previous study showed accelerated bone healing with higher osteoclastogenesis gene expression in toll-like receptor 4 knockout mice (TLR4). This study aimed to further elucidate the underlying cellular mechanisms during fracture healing by generating a myeloid cell-specific toll-like receptor 4 knockout model (Lyz-TLR4 mice). METHODS: Calvarial defects, 1.8 mm in diameter, were created in wild-type, TLR4, and Lyz-TLR4 mice. Bone healing was investigated using micro-computed tomography and histologic, histomorphometric, and immunohistochemistry analyses. Primary bone marrow-derived cells were also isolated from wild-type, TLR4, and Lyz-TLR4 mice to measure their osteoclast differentiation and resorption properties. RESULTS: A similar faster bone healing response, with active intramembranous bone formation, intense osteopontin staining, and more osteoblast infiltration, was observed in TLR4 and Lyz-TLR4 mice. Tartrate-resistant acid phosphatase staining showed more osteoclast infiltration in Lyz-TLR4 mice than in wild-type mice at day 7. Primary bone marrow-derived cells isolated from TLR4 and Lyz-TLR4 mice presented enhanced osteoclastogenesis and resorption activity compared with those from wild-type mice. Comparable M0, M1, and M2 macrophage infiltration was found among all groups at days 1, 4, and 7. CONCLUSIONS: This study revealed that inactivation of toll-like receptor 4 in myeloid cells enhanced osteoclastogenesis and accelerated healing response during skull repair. Together with the role of toll-like receptor 4 in inflammation-mediated bone destruction, it suggests that toll-like receptor 4 might regulate inflammation-induced osteoclastogenesis under different clinical settings.
Assuntos
Consolidação da Fratura/fisiologia , Células Mieloides , Crânio/lesões , Receptor 4 Toll-Like/fisiologia , Animais , Feminino , Camundongos , Modelos AnimaisRESUMO
BACKGROUND: The limitations of autologous and alloplastic reconstruction for craniofacial bone defects have created a clinical need for viable tissue-engineering strategies. Recombinant human bone morphogenetic protein-2 (rhBMP-2) has shown promise in this setting. The aim of this study was to determine the long-term biomechanical properties of rhBMP-2-mediated calvarial reconstruction. METHODS: Twelve-week-old New Zealand White rabbits underwent subtotal calvarectomy. Defects were repaired in one of several groups: immediate reconstruction with autologous graft, immediate reconstruction with cryopreserved bone graft, immediate reconstruction with rhBMP-2 (favorable), and delayed reconstruction with rhBMP-2 following infection and subsequent débridement (unfavorable). Cryopreserved reconstructions were measured at 6 weeks; autologous reconstructions were measured at 6 weeks and 6 months; and both favorable and unfavorable rhBMP-2 reconstructions were assessed at 6 weeks, 6 months, and 1 year after reconstruction. Healing was assessed with computed tomography. An unconfined compression test was performed for biomechanical analysis. Stress at 20 percent strain, percentage relaxation, tangent modulus, and final strain at 1800 N were compared between groups. RESULTS: Nearly complete radiographic coverage was achieved by 6 months for autologous reconstruction and by 6 weeks for rhBMP-2 reconstruction. Favorable rhBMP-2 reconstruction demonstrated a larger final strain at 1800 N through 1 year compared with native bone. Bone in unfavorable rhBMP-2 reconstruction was more compressible than native bone, with a larger final strain at 1800 N at 1 year. There were no significant differences between favorable and unfavorable groups. CONCLUSIONS: Despite providing radiographic coverage, the biomechanical properties of rhBMP-2 bone differ from those of native bone. Further studies are warranted to determine how these properties affect overall strength and structural integrity.
Assuntos
Proteína Morfogenética Óssea 2/uso terapêutico , Transplante Ósseo , Procedimentos de Cirurgia Plástica/métodos , Crânio/cirurgia , Fator de Crescimento Transformador beta/uso terapêutico , Animais , Fenômenos Biomecânicos , Doenças Ósseas/microbiologia , Doenças Ósseas/cirurgia , Infecções/cirurgia , Coelhos , Proteínas Recombinantes/uso terapêutico , Crânio/anatomia & histologia , Crânio/fisiologiaRESUMO
BACKGROUND: Inflammation is integral to the injury response. The inflammatory response is essential to the host defense against infection and also to tissue regeneration and repair. Toll-like receptors (TLRs) are critical activators of the innate immune response and present attractive therapeutic targets for inflammation-modulated tissue regeneration. The authors' previous study showed that depletion of TLR4 resulted in accelerated skull bone healing concurrent with increased expression of osteoclastogenic genes. As such, in the present study, the authors used various knockout mouse models for TLR4 and its associated signaling mediators as tools to further understand the role of Toll-like receptor-mediated inflammation in calvarial bone healing. METHODS: Calvarial defects (1.8-mm diameter) were created in wild-type, TLR4 knockout (TLR4), TLR2, MyD88, TRIF, TLR4 knockout in myeloid cell (Lyz-TLR4), and TLR4 knockout in dendritic-lineage cell (CD11c-TLR4) mice. Bone healing was examined using micro-computed tomographic, histologic, and histomorphometric analyses. RESULTS: Micro-computed tomographic and histomorphometric analyses revealed that TLR4-deficient mice (TLR4, Lyz-TLR4, and CD11c-TLR4) exhibited a faster intramembraneous healing response at postoperative day 7, whereas MyD88 and CD11c-TLR4 mice showed enhanced bone healing at day 28. CONCLUSIONS: The authors' data suggest a detrimental role for TLR4 in CD11c cells, mediated by Myd88 signaling, during calvarial bone healing. The authors have demonstrated that Toll-like receptor signaling components affect calvarial bone healing, establishing a link between the skeletal and immune systems during craniofacial bone healing. Toll-like receptor signaling components might be used to initiate enhanced healing in bone defects to improve clinical outcomes.
Assuntos
Antígeno CD11c/fisiologia , Fator 88 de Diferenciação Mieloide/fisiologia , Crânio/lesões , Receptor 4 Toll-Like/fisiologia , Cicatrização/fisiologia , Animais , Antígeno CD11c/genética , Feminino , Camundongos , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/genética , Transdução de Sinais , Receptor 4 Toll-Like/genéticaRESUMO
OBJECTIVE: To assess the prevalence of somatic MTOR mutations in focal cortical dysplasia (FCD) and of germline MTOR mutations in a broad range of epilepsies. METHODS: We collected 20 blood-brain paired samples from patients with FCD and searched for somatic variants using deep-targeted gene panel sequencing. Germline mutations in MTOR were assessed in a French research cohort of 93 probands with focal epilepsies and in a diagnostic Danish cohort of 245 patients with a broad range of epilepsies. Data sharing among collaborators allowed us to ascertain additional germline variants in MTOR. RESULTS: We detected recurrent somatic variants (p.Ser2215Phe, p.Ser2215Tyr, and p.Leu1460Pro) in the MTOR gene in 37% of participants with FCD II and showed histologic evidence for activation of the mTORC1 signaling cascade in brain tissue. We further identified 5 novel de novo germline missense MTOR variants in 6 individuals with a variable phenotype from focal, and less frequently generalized, epilepsies without brain malformations, to macrocephaly, with or without moderate intellectual disability. In addition, an inherited variant was found in a mother-daughter pair with nonlesional autosomal dominant nocturnal frontal lobe epilepsy. CONCLUSIONS: Our data illustrate the increasingly important role of somatic mutations of the MTOR gene in FCD and germline mutations in the pathogenesis of focal epilepsy syndromes with and without brain malformation or macrocephaly.