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1.
BMC Plant Biol ; 19(1): 307, 2019 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-31299897

RESUMO

BACKGROUND: DNA methylation is a crucial epigenetic modification, which is involved in many biological processes, including gene expression regulation, embryonic development, cell differentiation and genomic imprinting etc. And it also involves many key regulatory genes in eukaryotes. By tracing the evolutionary history of methylation-related genes, we can understand the origin and expansion time of these genes, which helps to understand the evolutionary history of plants, and we can also understand the changes of DNA methylation patterns in different species. However, most studies on the evolution of methylation-related genes failed to be carried out for the whole DNA methylation pathway. RESULTS: In this study, we conducted a comprehensive identification of 33 methylation-related genes in 77 species, and investigated gene origin and evolution throughout the plant kingdom. We found that the origin of genes responsible for methylation maintenance and demethylation evolved early, while most de novo methylation-related genes appeared late. The methylation-related genes were expanded by whole genome duplication and tandem replication, but were also accompanied by a large number of gene absence events in different species. The gene length and intron length varied a lot in different species, but exon structure and functional domains were relatively conserved. The phylogenetic relationships of methylation-related genes were traced to reveal the evolution history of DNA methylation in different species. The expression patterns of methylation-related genes have changed during the evolution of species, and the expression patterns of these genes in different species can be clustered into four categories. CONCLUSIONS: The study describes a global characterization of DNA methylation-related genes in the plant kingdom. The similarities and differences in origin time, gene structure and phylogenetic relationship of these genes lead us to understand the evolutionary conservation and dynamics of DNA methylation in plants.


Assuntos
Metilação de DNA , Epigênese Genética , Impressão Genômica , Plantas/genética , Evolução Molecular , Éxons/genética , Íntrons/genética , Filogenia
2.
Psychiatr Danub ; 31(2): 263-268, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31291235

RESUMO

BACKGROUND: Posttraumatic stress disorder (PTSD) is a complex stress related disorder, that follows a severe traumatic experience, characterized with an intense sense of terror, fear, and helplessness. The aim of this study is to identify associations of genetic variations within candidate genes DRD2 and DRD4 with various PTSD related phenotypes. PTSD lifetime and PTSD current subjects were analyzed separately, each of them were analyzed in a Case/Control design, as well as regarding BSI and CAPS within cases only. SUBJECTS AND METHODS: 719 (487 male, 232 female) participants who had experienced war-related trauma between 1991 and 1999 in Bosnia and Hercegovina, Kosovo and Croatia were included in the study. Sociodemographic questionnaire, Clinician Administered PTSD Scale (CAPS) and the Brief Symptom Inventory (BSI) were used to collect clinical data. RESULTS: The DRD2 rs1800497 variant and a variable number tandem repeat (VNTR) located in exon three of DRD4 were investigated for association with PTSD. In case control analyses we did not identify any significant associations. Within the PTSD current patients, we identified an association of DRD2 rs1800497 with BSI in the genotypic and the recessive model with the T allele as the risk allele. CONCLUSION: Our findings suggest that rs1800497 of DRD2 gene is involved in pathogenesis of PTSD.


Assuntos
Repetições Minissatélites , Polimorfismo Genético , Receptores de Dopamina D2/genética , Receptores de Dopamina D4/genética , Transtornos de Estresse Pós-Traumáticos/genética , Conflitos Armados/psicologia , Bósnia e Herzegóvina , Croácia , Éxons/genética , Feminino , Humanos , Kosovo , Masculino , Pessoa de Meia-Idade
3.
BMC Bioinformatics ; 20(1): 363, 2019 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-31253089

RESUMO

BACKGROUND: Missense mutations in the first five exons of F9, which encodes factor FIX, represent 40% of all mutations that cause hemophilia B. To address the ongoing debate regarding in silico identification of disease-causing mutations at these exons, we analyzed 215 missense mutations from www.factorix.org using six in silico prediction tools, which are the most common used programs for analysis prediction of impact of mutations on the protein structure and function, with further advantage of using similar approaches. We developed different algorithms to integrate multiple predictions from such tools. In order to approach a structural analysis on FIX we performed a modeling of five selected pathogenic mutations. RESULTS: SIFT, PolyPhen-2 HumDiv, SNAP2, and MutationAssessor were the most successful in identifying true non-causative and causative mutations. A proposed function integrating these algorithms (wgP4) was the most sensitive (90.1%), specific (22.6%), and accurate (87%) than similar functions, and identified 187 variants as deleterious. Clinical phenotype was significantly associated with predicted causative mutations at all five exons. However, PolyPhen-2 HumDiv was more successful in linking clinical severity to specific exons, while functions that integrate 4-6 predictions were more successful in linking phenotype to genotypes at the light chain (exons 3-5). The most important value of integrating multiple predictions is the inclusion of scores derived from different approaches. Modeling of protein structure showed the effects of pathogenic nsSNPs on structure and function of FIX. CONCLUSIONS: A simple function that integrates information from different in silico programs yields the best prediction of mutated phenotypes. However, the specificity, sensitivity, and accuracy of genotype-phenotype predictions depend on specific characteristics of the protein domain and the disease of interest as we validated by the structural analysis of selected pathogenic F9 mutations. The proposed function integrating algorithm (wgP4) might be useful for the analysis of nsSNPs impact on other genes.


Assuntos
Biologia Computacional/métodos , Simulação por Computador , Éxons/genética , Fator IX/genética , Hemofilia B/genética , Mutação de Sentido Incorreto , Algoritmos , Genótipo , Humanos , Fenótipo
4.
BMC Bioinformatics ; 20(1): 324, 2019 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-31195961

RESUMO

BACKGROUND: As DNA sequencing technologies are improving and getting cheaper, genomic data can be utilized for diagnosis of many diseases such as cancer. Human raw genome data is huge in size for computational systems. Therefore, there is a need for a compact and accurate representation of the valuable information in DNA. The occurrence of complex genetic disorders often results from multiple gene mutations. The effect of each mutation is not equal for the development of a disease. Inspired from the field of information retrieval, we propose using the term frequency (tf) and BM25 term weighting measures with the inverse document frequency (idf) and relevance frequency (rf) measures to weight genes based on their mutations. The underlying assumption is that the more mutations a gene has in patients with a certain disease and the less mutations it has in other patients, the more discriminative that gene is. RESULTS: We evaluated the proposed representations on the task of cancer type classification. We applied various machine learning techniques using the tf-idf and tf-rf schemes and their BM25 versions. Our results show that the BM25-tf-rf representation leads to improved classification accuracy and f-score values compared to the other representations. The highest accuracy (76.44%) and f-score (76.95%) are achieved with the BM25-tf-rf based data representation. CONCLUSIONS: As a result of our experiments, the BM25-tf-rf scheme and the proposed neural network model is shown to be the best performing classification system for our case study of cancer type classification. This system is further utilized for causal gene analysis. Examples from the most effective genes that are used for decision making are found to be in the literature as target or causal genes.


Assuntos
Genômica/métodos , Modelos Genéticos , Modelos Estatísticos , Mutação/genética , Bases de Dados Genéticas , Éxons/genética , Humanos , Íntrons/genética , Aprendizado de Máquina , Neoplasias/genética , Redes Neurais (Computação)
5.
BMC Bioinformatics ; 20(Suppl 8): 283, 2019 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-31182012

RESUMO

BACKGROUND: Numerous essential algorithms and methods, including entropy-based quantitative methods, have been developed to analyze complex DNA sequences since the last decade. Exons and introns are the most notable components of DNA and their identification and prediction are always the focus of state-of-the-art research. RESULTS: In this study, we designed an integrated entropy-based analysis approach, which involves modified topological entropy calculation, genomic signal processing (GSP) method and singular value decomposition (SVD), to investigate exons and introns in DNA sequences. We optimized and implemented the topological entropy and the generalized topological entropy to calculate the complexity of DNA sequences, highlighting the characteristics of repetition sequences. By comparing digitalizing entropy values of exons and introns, we observed that they are significantly different. After we converted DNA data to numerical topological entropy value, we applied SVD method to effectively investigate exon and intron regions on a single gene sequence. Additionally, several genes across five species are used for exon predictions. CONCLUSIONS: Our approach not only helps to explore the complexity of DNA sequence and its functional elements, but also provides an entropy-based GSP method to analyze exon and intron regions. Our work is feasible across different species and extendable to analyze other components in both coding and noncoding region of DNA sequences.


Assuntos
Entropia , Éxons/genética , Íntrons/genética , Algoritmos , Sequência de Bases , Cromossomos Humanos/genética , DNA/genética , Genoma Humano , Humanos , Regiões Promotoras Genéticas/genética , Curva ROC , Análise de Sequência de DNA/métodos , Processamento de Sinais Assistido por Computador
6.
An Acad Bras Cienc ; 91(2): e20180945, 2019 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-31241704

RESUMO

DNA methylation is essential for spatiotemporally-regulated gene expression in embryonic development. TBX22 (Chr X: 107667964-107688978) functioning as a transcriptional repressor affects DNA binding, sumoylation, and transcriptional repression associated with X-linked cleft palate. This study aimed to explore the relationship and potential mechanism between TBX22 exon 5 methylation and palatal shelf fusion induced by all-trans retinoic acid (ATRA). We performed DNA methylation profiling, using MethylRAD-seq, after high throughput sequencing of mouse embryos from control (n=9) and ATRA-treated (to induce cleft palate, n=9) C57BL/6J mice at embryonic gestation days(E) 13.5, 14.5 and 16.5. TBX22 exon 5 was hyper-methylated at the CpG site at E13.5 (P=0.025, log2FC=1.5) and E14.5 (P=0.011, log2FC:1.5) in ATRA-treated, whereas methylation TBX22 exon 5 at the CpG site was not significantly different at E16.5 (P=0.808, log2FC=-0.2) between control and ATRA-treated. MSP results showed a similar trend consistent with the MethylRAD-seq results. qPCR showed the change in TBX22 exon 5 expression level negatively correlated with its TBX22 exon 5 methylation level. These results indicate that changes in TBX22 exon 5 methylation might play an important regulatory role during palatal shelf fusion, and may enlighten the development of novel epigenetic biomarkers in the treatment of CP in the future.


Assuntos
Fissura Palatina/embriologia , Desenvolvimento Embrionário/genética , Éxons/genética , Doenças Genéticas Ligadas ao Cromossomo X/embriologia , Proteínas com Domínio T/genética , Animais , Fissura Palatina/genética , Modelos Animais de Doenças , Feminino , Doenças Genéticas Ligadas ao Cromossomo X/genética , Metilação , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo , Gravidez
7.
Nat Commun ; 10(1): 2673, 2019 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-31209208

RESUMO

Alternative splicing performs a central role in expanding genomic coding capacity and proteomic diversity. However, programming of splicing patterns in engineered biological systems remains underused. Synthetic approaches thus far have predominantly focused on controlling expression of a single protein through alternative splicing. Here, we describe a modular and extensible platform for regulating four programmable exons that undergo a mutually exclusive alternative splicing event to generate multiple functionally-distinct proteins. We present an intron framework that enforces the mutual exclusivity of two internal exons and demonstrate a graded series of consensus sequence elements of varying strengths that set the ratio of two mutually exclusive isoforms. We apply this framework to program the DNA-binding domains of modular transcription factors to differentially control downstream gene activation. This splicing platform advances an approach for generating diverse isoforms and can ultimately be applied to program modular proteins and increase coding capacity of synthetic biological systems.


Assuntos
Processamento Alternativo/genética , Regulação da Expressão Gênica/genética , Engenharia Genética/métodos , RNA/genética , Fatores de Transcrição/genética , Motivos de Aminoácidos/genética , Animais , Linhagem Celular , Biologia Computacional , Sequência Consenso/genética , Éxons/genética , Biblioteca Gênica , Genes Reporter/genética , Humanos , Íntrons/genética , Mutagênese Sítio-Dirigida/métodos , Domínios Proteicos/genética , Isoformas de Proteínas/genética , RNA/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Genética
8.
Mol Biol (Mosk) ; 53(3): 411-420, 2019.
Artigo em Russo | MEDLINE | ID: mdl-31184606

RESUMO

Antithrombin III (AT3) belongs to the superfamily of serine protease inhibitors (serpins) and is a major anticoagulant in physiological conditions. Based on SERPINC1 gene, a minigene coding for human AT3, which is valuable for medicine and biotechnology, was constructed by minimizing the size of lengthy introns and preserving the splicing site-flanking sequences. An analysis of the minigene splicing pattern identified one correct AT3 transcript and two alternatively spliced transcripts, which formed either due to minigene exons 2 and 3 skipping or an aberrant exon insertion via splicing at cryptic splicing sites in intron 1 of the minigene. Site-directed mutagenesis of the cryptic splicing sites successfully optimized the splicing pattern of the AT3 minigene to completely prevent the generation of the alternative transcripts. The presence of the cryptic splicing sites in intron 1 of the minigene was confirmed with Human Splicing Finder v. 3.1 software, thus demonstrating that putative alternative splicing sites are possible to identify in minimized or hybrid introns of minigenes and to eliminate via mutagenesis before experimentally testing the minigene splicing patterns. The approach to the design of minigenes together with the bioinformatical analysis of the nucleotide sequences of minigene introns can be used to construct minigenes in order to generate transgenic animals producing economically valuable proteins in the milk.


Assuntos
Processamento Alternativo/genética , Antitrombina III/genética , Sítios de Splice de RNA/genética , Éxons/genética , Humanos , Íntrons/genética
9.
BMC Bioinformatics ; 20(1): 231, 2019 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-31068132

RESUMO

BACKGROUND: In eukaryotes, most genes code for multiple transcript isoforms that are generated through the complex and tightly regulated process of RNA splicing. Despite arising from identical precursor transcripts, alternatively spliced RNAs can have dramatically different functions. Transcriptome complexity is elevated further by the production of circular RNAs (circRNAs), another class of mature RNA that results from the splicing of a downstream splice donor to an upstream splice acceptor. While there has been a rapid expansion of circRNA catalogs in the last few years through the utilization of next generation sequencing approaches, our understanding of the mechanisms and regulation of circular RNA biogenesis, the impact that circRNA generation has on parental transcript processing, and the functions carried out by circular RNAs remains limited. RESULTS: Here, we present a visualization and analysis tool, SpliceV, that rapidly plots all relevant forward- and back-splice data, with exon and single nucleotide level coverage information from RNA-seq experiments in a publication quality format. SpliceV also integrates analysis features that assist investigations into splicing regulation and transcript functions through the display of predicted RNA binding protein sites and the configuration of repetitive elements along the primary transcript. CONCLUSIONS: SpliceV is an easy-to-use splicing visualization tool, compatible with both Python 2.7 and 3+, and distributed under the GNU Public License. The source code is freely available for download at https://github.com/flemingtonlab/SpliceV and can be installed from PyPI using pip.


Assuntos
Processamento Alternativo/genética , Éxons/genética , Processamento de RNA/genética , Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , RNA/genética
10.
Zhongguo Fei Ai Za Zhi ; 22(5): 299-305, 2019 May 20.
Artigo em Chinês | MEDLINE | ID: mdl-31109439

RESUMO

BACKGROUND: Adenocarcinoma is the most common type of lung cancer. It has been clinically evaluated that therapiestargeting against the epidermal growth factor receptor (EGFR) as the clinical standard first-line treatment. The response and outcome of EGFR-tyrosine kinase inhibitors (TKIs) in patients harboring common mutations in EGFR kinase domain (deletion in exon19 and L858R in exon 21) has been well demonstrated, but not in rare or complex mutations. METHODS: A total of 150 patients that harbored rare or complex mutations in EGFR diagnosed by histopathology were included in this retrospective study. The clinical-pathological characteristics of all 150 patients as well as the response and progression-free survival (PFS) in 48 patients that received EGFR-TKIs in first/second/third line treatments weredescribed and analyzed. RESULTS: Patients were divided into four groups based on the mutation types: single G719X point mutation in exon 18 (n=46, 30.7%), single L861Q point mutation in exon 21 (n=45, 30.0%), other single rare mutation (n=14, 9.3%) and complex mutations (n=45, 30.0%). The result indicated thatthere was no correlation of EGFR mutation typeswith other parameters such as gender, age, clinical stage, pathology and smoking history. For the 48 patients that received EGFR-TKIs treatment, there were no significant differencesamong 4 groups in terms of objective response rate (ORR) and disease control rate (DCR) (54.5% vs 30.0% vs 0.0% vs 35.7%, χ²=3.200, P=0.34; 90.9% vs 85.0% vs 66.7% vs 92.9%, χ²=2.162, P=0.59). The median progress-free survival (mPFS) was 11.0 months (95%CI: 4.4-17.6), and in each group of different EGFR mutation types are 15.8 months (95%CI: 9.5-22.2), 8.0 months (95%CI: 5.1-11.0), 4.9 months (95%CI: 1.4-8.4) and 23.1 months (95%CI: 15.8-30.4)(χ²=7.876, P=0.049). CONCLUSIONS: The efficiency of targeting EGFR-TKIs on different types of rare or complex mutations was heterogeneous. The PFS may be better in patients that harbored complex mutations than those with single rare mutations. Further studies with larger sample size are necessary. Moreover, to discover novel therapeutic targets and develop new drugs are imminentfor those patientswith no response to the existing treatments.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Mutação , Inibidores de Proteínas Quinases/farmacologia , Intervalo Livre de Doença , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Éxons/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Inibidores de Proteínas Quinases/uso terapêutico , Estudos Retrospectivos , Resultado do Tratamento
11.
Nat Commun ; 10(1): 2037, 2019 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-31048690

RESUMO

Genome-wide analysis of genomic signatures might reveal novel mechanisms for gastric cancer (GC) tumorigenesis. Here, we analysis structural variations (SVs) and mutational signatures via whole-genome sequencing of 168 GCs. Our data demonstrates diverse models of complex SVs operative in GC, which lead to high-level amplification of oncogenes. We find varying proportion of tandem-duplications (TDs) among individuals and identify 24 TD hotspots involving well-established cancer genes such as CCND1, ERBB2 and MYC. Specifically, we nominate a novel hotspot involving the super-enhancer of ZFP36L2 presents in approximately 10% GCs from different cohorts, the oncogenic role of which is further confirmed by experimental data. In addition, our data reveal a mutational signature, specifically occurring in noncoding region, significantly enriched in tumors with cadherin 1 mutations, and associated with poor prognoses. Collectively, our data suggest that TDs might serve as an important mechanism for cancer gene activation and provide a novel signature for stratification.


Assuntos
Oncogenes/genética , Neoplasias Gástricas/genética , Fatores de Transcrição/genética , Transcriptoma/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos CD/genética , Caderinas/genética , Carcinogênese/genética , Linhagem Celular Tumoral , Elementos Facilitadores Genéticos/genética , Éxons/genética , Feminino , Duplicação Gênica/genética , Variação Estrutural do Genoma , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Estômago/patologia , Neoplasias Gástricas/mortalidade , Neoplasias Gástricas/patologia , Análise de Sobrevida , Sequenciamento Completo do Genoma
12.
Mol Med Rep ; 19(6): 4711-4718, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31059048

RESUMO

Autosomal recessive cornea plana is a very rare hereditary ocular disease, characterized by a flattened corneal curvature, marked hyperopia due to low refractive power and frequently consequent accommodative esotropia. Other features include various cornea anterior segment abnormalities, without systemic problems. The purpose of the present study was to investigate the clinical and molecular alterations in a Chinese family with cornea plana. Full ophthalmic examinations of the patients were performed, including slit­lamp examination, fundus examination and ocular ultrasound. Whole­exome sequencing data were screened for pathological variants in the proband, which were confirmed by Sanger sequencing. One novel missense mutation, c.242A>G (p.N81S) and another novel 7 base­pair deletion mutation, c.772­779del (p.G258Cfs*30), were detected in the keratocan (KERA) gene; two affected siblings inherited these variations in a compound heterozygous state, which were derived from the clinically unaffected heterozygous father (c.772_779del) and mother (c.242A>G), respectively. Neither mutation was observed in unrelated healthy controls (n=200). Multiple computer software predictions supported the pathogenicity of the two variants. Furthermore, protein modeling prediction was performed to better understand the molecular basis of cornea plana, particularly the importance of the leucine­rich repeat domain. This study presents the 14th pathogenic KERA mutations identified worldwide and the first in East Asia so far, to the best of our knowledge. These findings guided prenatal diagnosis for the family in question and expand on the variant spectrum of KERA, therefore facilitating genetic counseling.


Assuntos
Doenças da Córnea/genética , Genes Recessivos/genética , Proteoglicanas/genética , Grupo com Ancestrais do Continente Asiático , Sequência de Bases , China , Córnea/anormalidades , Córnea/patologia , Doenças da Córnea/diagnóstico , Doenças da Córnea/patologia , Distrofias Hereditárias da Córnea/diagnóstico , Distrofias Hereditárias da Córnea/genética , Análise Mutacional de DNA , Éxons/genética , Anormalidades do Olho/genética , Anormalidades do Olho/patologia , Feminino , Humanos , Mutação de Sentido Incorreto , Linhagem , Análise de Sequência , Deleção de Sequência , Sequenciamento Completo do Exoma
13.
Int J Mol Sci ; 20(9)2019 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-31086020

RESUMO

Bombyx mori doublesex (Bmdsx) functions as a double-switch gene in the final step of the sex-determination cascade in the silkworm Bombyx mori. The P-element somatic inhibitor (PSI) protein in B. mori interacts with Bmdsx pre-mRNA in CE1 as an exonic splicing silencer to promote male-specific splicing of Bmdsx. However, the character of the interaction between BmPSI and Bmdsx pre-mRNA remains unclear. Electrophoretic mobility shift assay (EMSA) results showed that the four KH_1 motifs in BmPSI are all essential for the binding, especially the former two KH_1 motifs. Three active sites (I116, L127, and IGGI) in the KH_1 motif were found to be necessary for the binding through EMSA, circular dichroism (CD) spectroscopy, and isothermal titration calorimetry (ITC). The PSI homologous protein in S. litura (SlPSI) was purified and the binding of SlPSI and CE1 was verified. Compared with BmPSI, the mutant SlPSI proteins of I116 and IGGI lost their ability to bind to CE1. In conclusion, the binding of PSI and dsx pre-mRNA are generally conserved in both B. mori and S. litura. These findings provide clues for sex determination in Lepidoptera.


Assuntos
Bombyx/genética , Proteínas de Insetos/genética , Processamento de RNA/genética , Spodoptera/genética , Processamento Alternativo/genética , Animais , Varredura Diferencial de Calorimetria , Dicroísmo Circular , Ensaio de Desvio de Mobilidade Eletroforética , Éxons/genética , Feminino , Masculino , Ligação Proteica
14.
Gene ; 706: 6-12, 2019 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-31022435

RESUMO

Factor XII (FXII) is a coagulation protein that initiates surface-activation of the coagulation cascade in vitro. The protein's in vivo role, however, remains poorly defined. Factor XII deficiency, or Hageman trait, is a rare hereditary disorder that is not associated with bleeding, and wide variations in FXII activity (FXII:C) exist among healthy people. While FXII-deficient knockout mice appear to be resistant to arterial thrombosis, human F12 polymorphisms that influence FXII:C have not been associated with thrombotic risk in population surveys. Factor XII deficiency is a naturally occurring hereditary trait in domestic cats. We undertook phenotypic and genotypic analyses of FXII-deficient cats for comparative studies with the human disease counterpart. A retrospective review of feline submissions to our laboratory revealed that FXII deficiency is common in domestic cats, and also present in many different breeds. The trait has a geographic bias toward the Midwestern United States. Clinical history, coagulation assays, and samples for F12 sequencing were obtained from 26 FXII deficient cats. None of the cats had experienced abnormal bleeding and their residual FXII:C was related to F12 mutation number and mutation-type. We found 2 high frequency F12 mutations: an exon 13 missense mutation (c.1631G > C) and an exon 11 deletion mutation (c.1321delC), and additional sequence variants throughout the gene. Factor XII deficiency in pet cat populations provides an animal model system to help clarify the biologic actions and clinical relevance of FXII protein.


Assuntos
Gatos/genética , Deficiência do Fator XII/genética , Fator XII/genética , Animais , Éxons/genética , Fator XII/fisiologia , Deficiência do Fator XII/veterinária , Mutação , Polimorfismo Genético/genética , Estudos Retrospectivos , Deleção de Sequência , Estados Unidos
15.
Science ; 364(6435): 74-78, 2019 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-30948549

RESUMO

A core question in evolutionary biology is whether convergent phenotypic evolution is driven by convergent molecular changes in proteins or regulatory regions. We combined phylogenomic, developmental, and epigenomic analysis of 11 new genomes of paleognathous birds, including an extinct moa, to show that convergent evolution of regulatory regions, more so than protein-coding genes, is prevalent among developmental pathways associated with independent losses of flight. A Bayesian analysis of 284,001 conserved noncoding elements, 60,665 of which are corroborated as enhancers by open chromatin states during development, identified 2355 independent accelerations along lineages of flightless paleognaths, with functional consequences for driving gene expression in the developing forelimb. Our results suggest that the genomic landscape associated with morphological convergence in ratites has a substantial shared regulatory component.


Assuntos
Evolução Biológica , Epigênese Genética , Evolução Molecular , Voo Animal , Paleógnatas/anatomia & histologia , Paleógnatas/genética , Animais , Teorema de Bayes , Cromatina/metabolismo , Sequência Conservada , Elementos Facilitadores Genéticos , Epigenômica , Éxons/genética , Extinção Biológica , Membro Anterior/anatomia & histologia , Paleógnatas/fisiologia , Fenótipo , Filogenia
16.
Nat Commun ; 10(1): 1634, 2019 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-30967552

RESUMO

Gene correction in human long-term hematopoietic stem cells (LT-HSCs) could be an effective therapy for monogenic diseases of the blood and immune system. Here we describe an approach for X-linked sSevere cCombined iImmunodeficiency (SCID-X1) using targeted integration of a cDNA into the endogenous start codon to functionally correct disease-causing mutations throughout the gene. Using a CRISPR-Cas9/AAV6 based strategy, we achieve up to 20% targeted integration frequencies in LT-HSCs. As measures of the lack of toxicity we observe no evidence of abnormal hematopoiesis following transplantation and no evidence of off-target mutations using a high-fidelity Cas9 as a ribonucleoprotein complex. We achieve high levels of targeting frequencies (median 45%) in CD34+ HSPCs from six SCID-X1 patients and demonstrate rescue of lymphopoietic defect in a patient derived HSPC population in vitro and in vivo. In sum, our study provides specificity, toxicity and efficacy data supportive of clinical development of genome editing to treat SCID-Xl.


Assuntos
DNA Complementar/genética , Edição de Genes/métodos , Transplante de Células-Tronco Hematopoéticas , Subunidade gama Comum de Receptores de Interleucina/genética , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/terapia , Animais , Antígenos CD34/metabolismo , Sistemas CRISPR-Cas/genética , Linhagem Celular , Códon de Iniciação/genética , Éxons/genética , Sangue Fetal/citologia , Vetores Genéticos/genética , Voluntários Saudáveis , Células-Tronco Hematopoéticas/metabolismo , Humanos , Masculino , Camundongos , Mutação , Parvovirinae/genética , Cultura Primária de Células , Fatores de Tempo , Transdução Genética/métodos , Quimeras de Transplante/genética , Transplante Heterólogo/métodos , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/genética
17.
PLoS Comput Biol ; 15(4): e1006937, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30973878

RESUMO

Gestational alcohol exposure causes fetal alcohol spectrum disorder (FASD) and is a prominent cause of neurodevelopmental disability. Whole transcriptome sequencing (RNA-Seq) offer insights into mechanisms underlying FASD, but gene-level analysis provides limited information regarding complex transcriptional processes such as alternative splicing and non-coding RNAs. Moreover, traditional analytical approaches that use multiple hypothesis testing with a false discovery rate adjustment prioritize genes based on an adjusted p-value, which is not always biologically relevant. We address these limitations with a novel approach and implemented an unsupervised machine learning model, which we applied to an exon-level analysis to reduce data complexity to the most likely functionally relevant exons, without loss of novel information. This was performed on an RNA-Seq paired-end dataset derived from alcohol-exposed neural fold-stage chick crania, wherein alcohol causes facial deficits recapitulating those of FASD. A principal component analysis along with k-means clustering was utilized to extract exons that deviated from baseline expression. This identified 6857 differentially expressed exons representing 1251 geneIDs; 391 of these genes were identified in a prior gene-level analysis of this dataset. It also identified exons encoding 23 microRNAs (miRNAs) having significantly differential expression profiles in response to alcohol. We developed an RDAVID pipeline to identify KEGG pathways represented by these exons, and separately identified predicted KEGG pathways targeted by these miRNAs. Several of these (ribosome biogenesis, oxidative phosphorylation) were identified in our prior gene-level analysis. Other pathways are crucial to facial morphogenesis and represent both novel (focal adhesion, FoxO signaling, insulin signaling) and known (Wnt signaling) alcohol targets. Importantly, there was substantial overlap between the exomes themselves and the predicted miRNA targets, suggesting these miRNAs contribute to the gene-level expression changes. Our novel application of unsupervised machine learning in conjunction with statistical analyses facilitated the discovery of signaling pathways and miRNAs that inform mechanisms underlying FASD.


Assuntos
Éxons/genética , Transtornos do Espectro Alcoólico Fetal/genética , MicroRNAs/genética , Aprendizado de Máquina não Supervisionado , Animais , Big Data , Embrião de Galinha , Análise por Conglomerados , Biologia Computacional , Bases de Dados de Ácidos Nucleicos/estatística & dados numéricos , Modelos Animais de Doenças , Etanol/toxicidade , Feminino , Perfilação da Expressão Gênica/estatística & dados numéricos , Humanos , Gravidez , Análise de Componente Principal , Aprendizado de Máquina não Supervisionado/estatística & dados numéricos
18.
Genet Sel Evol ; 51(1): 12, 2019 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-30987584

RESUMO

BACKGROUND: In quail, two feather colour phenotypes i.e. fawn-2/beige and yellow are associated with the ASIP locus. The aim of our study was to characterize the structural modifications within this locus that explain the yellow mutation (large deletion) and the fawn-2/beige mutation (assumed to be caused by a different structural modification). RESULTS: For the yellow phenotype, we identified a complex mutation that involves a 141,162-bp long deletion. For the fawn-2/beige phenotype, we identified a 71-kb tandem duplication that comprises one unchanged copy of ASIP and one copy present in the ITCH-ASIP fusion gene, which leads to a transcript coding for a normal ASIP protein. Although this agrees with previous reports that reported an increased level of ASIP transcripts in the skin of mutant animals, we show that in the skin from fawn-2/beige embryos, this level is higher than expected with a simple duplication of the ASIP gene. Thus, we hypothesize that the 5' region of the ITCH-ASIP fusion gene leads to a higher transcription level than the 5' region of the ASIP gene. CONCLUSIONS: We were able to conclude that the fawn-2 and beige phenotypes are caused by the same allele at the ASIP locus. Both of the associated mutations fawn-2/beige and yellow lead to the formation of a fusion gene, which encodes a transcript for the ASIP protein. In both cases, transcription of ASIP depends on the promoter of a different gene, which includes alternative up-regulating sequences. However, we cannot exclude the possibility that the loss of the 5' region of the ASIP gene itself has additional impacts, especially for the fawn-2/beige mutation. In addition, in several other species including mammals, the existence of other dominant gain-of-function structural modifications that are localized upstream of the ASIP coding sequences has been reported, which supports our hypothesis that repressors in the 5' region of ASIP are absent in the fawn-2/beige mutant.


Assuntos
Proteína Agouti Sinalizadora/genética , Pigmentação/genética , Codorniz/genética , Proteína Agouti Sinalizadora/metabolismo , Alelos , Animais , Cor , Éxons/genética , Plumas/metabolismo , Genótipo , Mutação/genética , Fenótipo , Regiões não Traduzidas/genética
19.
BMC Evol Biol ; 19(1): 89, 2019 04 11.
Artigo em Inglês | MEDLINE | ID: mdl-30975078

RESUMO

BACKGROUND: Standard evolutionary theories of aging postulate that reduced extrinsic mortality leads to evolution of longevity. Clownfishes of the genus Amphiprion live in a symbiotic relationship with sea anemones that provide protection from predators. We performed a survey and identified at least two species with a lifespan of over 20 years. Given their small size and ease of captive reproduction, clownfish lend themselves as experimental models of exceptional longevity. To identify genetic correlates of exceptional longevity, we sequenced the transcriptomes of Amphiprion percula and A. clarkii and performed a scan for positively-selected genes (PSGs). RESULTS: The PSGs that we identified in the last common clownfish ancestor were compared with PSGs detected in long-lived mole rats and short-lived killifishes revealing convergent evolution in processes such as mitochondrial biogenesis. Among individual genes, the Mitochondrial Transcription Termination Factor 1 (MTERF1), was positively-selected in all three clades, whereas the Glutathione S-Transferase Kappa 1 (GSTK1) was under positive selection in two independent clades. For the latter, homology modelling strongly suggested that positive selection targeted enzymatically important residues. CONCLUSIONS: These results indicate that specific pathways were recruited in independent lineages evolving an exceptionally extended or shortened lifespan and point to mito-nuclear balance as a key factor.


Assuntos
Evolução Biológica , Longevidade/genética , Fases de Leitura Aberta/genética , Perciformes/genética , Adaptação Fisiológica/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Éxons/genética , Proteínas de Peixes/química , Proteínas de Peixes/genética , Ontologia Genética , Biogênese de Organelas , Filogenia
20.
Cancer Sci ; 110(6): 2004-2013, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30980774

RESUMO

Anti-PUF60 autoantibodies are reportedly detected in the sera of patients with dermatomyositis and Sjögren's syndrome; however, little is known regarding its existence in the sera of cancer patients. FIR, a splicing variant of the PUF60 gene, is a transcriptional repressor of c-myc. In colorectal cancer, there is an overexpression of the dominant negative form of FIR, in which exon 2 is lacking (FIRΔexon2). Previously, large-scale SEREX (serological identification of antigens by recombinant cDNA expression cloning) screenings have identified anti-FIR autoantibodies in the sera of cancer patients. In the present study, we revealed the presence and significance of anti-FIR (FIR/FIRΔexon2) Abs in the sera of patients with esophageal squamous cell carcinoma (ESCC). Our results were validated by an amplified luminescence proximity homogeneous assay using sera of patients with various cancer types. We revealed that anti-FIRΔexon2 Ab had higher sensitivity than anti-FIR Ab. Receiver operating characteristic (ROC) analysis was applied for evaluating the use of anti-FIRΔexon2 Ab as candidate markers such as anti-p53 Ab and carcinoembryonic antigen, and the highest area under the ROC curve was observed in the combination of anti-FIRΔexon2 Ab and anti-p53 Ab. In summary, our results suggest the use of anti-FIRΔexon2 Ab in combination with the anti-p53 Ab as a predictive marker for ESCC. The area under the ROC curve was further increased in the advanced stage of ESCC. The value of anti-FIRΔexon2 autoantibody as novel clinical indicator against ESCC and as a companion diagnostic tool is discussed.


Assuntos
Autoanticorpos/imunologia , Neoplasias Esofágicas/imunologia , Carcinoma de Células Escamosas do Esôfago/imunologia , Fatores de Troca do Nucleotídeo Guanina/imunologia , Fatores de Processamento de RNA/imunologia , Proteínas Repressoras/imunologia , Autoanticorpos/sangue , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/imunologia , Neoplasias Esofágicas/diagnóstico , Neoplasias Esofágicas/genética , Carcinoma de Células Escamosas do Esôfago/diagnóstico , Carcinoma de Células Escamosas do Esôfago/genética , Éxons/genética , Feminino , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Masculino , Pessoa de Meia-Idade , Processamento de RNA , Fatores de Processamento de RNA/genética , Curva ROC , Proteínas Repressoras/genética , Proteína Supressora de Tumor p53/imunologia
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