RESUMO
De novo variants represent a significant cause of neurodevelopmental delay and intellectual disability. A genetic basis can be identified in only half of individuals who have neurodevelopmental disorders (NDDs); this indicates that additional causes need to be elucidated. We compared the frequency of de novo variants in patient-parent trios with (n = 2,030) versus without (n = 2,755) NDDs. We identified de novo variants in TAOK1 (thousand and one [TAO] amino acid kinase 1), which encodes the serine/threonine-protein kinase TAO1, in three individuals with NDDs but not in persons who did not have NDDs. Through further screening and the use of GeneMatcher, five additional individuals with NDDs were found to have de novo variants. All eight variants were absent from gnomAD (Genome Aggregation Database). The variant carriers shared a non-specific phenotype of developmental delay, and six individuals had additional muscular hypotonia. We established a fibroblast line of one mutation carrier, and we demonstrated that reduced mRNA levels of TAOK1 could be increased upon cycloheximide treatment. These results indicate nonsense-mediated mRNA decay. Further, there was neither detectable phosphorylated TAO1 kinase nor phosphorylated tau in these cells, and mitochondrial morphology was altered. Knockdown of the ortholog gene Tao1 (Tao, CG14217) in Drosophila resulted in delayed early development. The majority of the Tao1-knockdown flies did not survive beyond the third instar larval stage. When compared to control flies, Tao1 knockdown flies revealed changed morphology of the ventral nerve cord and the neuromuscular junctions as well as a decreased number of endings (boutons). Furthermore, mitochondria in mutant flies showed altered distribution and decreased size in axons of motor neurons. Thus, we provide compelling evidence that de novo variants in TAOK1 cause NDDs.
Assuntos
Drosophila melanogaster/crescimento & desenvolvimento , Exoma/genética , Mutação , Transtornos do Neurodesenvolvimento/etiologia , Proteínas Serina-Treonina Quinases/genética , Animais , Criança , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Feminino , Heterozigoto , Humanos , Masculino , Transtornos do Neurodesenvolvimento/patologia , Fenótipo , Sequenciamento do ExomaRESUMO
PURPOSE: Next-generation sequencing (NGS) is rapidly replacing Sanger sequencing in genetic diagnostics. Sensitivity and specificity of NGS approaches are not well-defined, but can be estimated from applying NGS and Sanger sequencing in parallel. Utilizing this strategy, we aimed at optimizing exome sequencing (ES)-based diagnostics of a clinically diverse patient population. METHODS: Consecutive DNA samples from unrelated patients with suspected genetic disease were exome-sequenced; comparatively nonstringent criteria were applied in variant calling. One thousand forty-eight variants in genes compatible with the clinical diagnosis were followed up by Sanger sequencing. Based on a set of variant-specific features, predictors for true positives and true negatives were developed. RESULTS: Sanger sequencing confirmed 81.9% of ES-derived variants. Calls from the lower end of stringency accounted for the majority of the false positives, but also contained ~5% of the true positives. A predictor incorporating three variant-specific features classified 91.7% of variants with 100% specificity and 99.75% sensitivity. Confirmation status of the remaining variants (8.3%) was not predictable. CONCLUSIONS: Criteria for variant calling in ES-based diagnostics impact on specificity and sensitivity. Confirmatory sequencing for a proportion of variants, therefore, remains a necessity. Our study exemplifies how these variants can be defined on an empirical basis.
Assuntos
Sequenciamento do Exoma , Exoma/genética , Doenças Genéticas Inatas/genética , Doenças Genéticas Inatas/diagnóstico , Doenças Genéticas Inatas/patologia , Variação Genética/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Análise de Sequência de DNARESUMO
Differential gene expression is a prerequisite for the formation of multiple cell types from the fertilized egg during embryogenesis. Understanding the gene regulatory networks controlling cellular differentiation requires the identification of crucial differentially expressed control genes and, ideally, the determination of the complete transcriptomes of each individual cell type. Here, we have analyzed the transcriptomes of six major tissues dissected from mid-gestational (TS12) mouse embryos. Approximately one billion reads derived by RNA-seq analysis provided extended transcript lengths, novel first exons and alternative transcripts of known genes. We have identified 1375 genes showing tissue-specific expression, providing gene signatures for each of the six tissues. In addition, we have identified 1403 novel putative long noncoding RNA gene loci, 439 of which show differential expression. Our analysis provides the first complete transcriptome data for the mouse embryo. It offers a rich data source for the analysis of individual genes and gene regulatory networks controlling mid-gestational development.
Assuntos
Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento , RNA não Traduzido/genética , Transcriptoma , Processamento Alternativo , Animais , Embrião de Mamíferos , Éxons , Feminino , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Sequenciamento de Nucleotídeos em Larga Escala , Camundongos , Camundongos Endogâmicos C57BL , RNA não Traduzido/metabolismo , Análise de Sequência de RNA , Distribuição TecidualRESUMO
The t complex responder (Tcr) encoded by the mouse t haplotype is able to cause phenotypic differences between t and + sperm derived from t/+ males, leading to non-Mendelian inheritance. This capability of Tcr contradicts the concept of phenotypic equivalence proposed for sperm cells, which develop in a syncytium and actively share gene products. By analyzing a Tcr minigene in hemizygous transgenic mice, we show that Tcr gene products are post-meiotically expressed and are retained in the haploid sperm cells. The wild-type allele of Tcr, sperm motility kinase-1 (Smok1), behaves in the same manner, suggesting that Tcr/Smok reveal a common mechanism prone to evolve non-Mendelian inheritance in mammals.
Assuntos
Regulação da Expressão Gênica , Padrões de Herança/genética , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Espermátides/metabolismo , Animais , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Dados de Sequência Molecular , Fenótipo , Espermátides/ultraestruturaAssuntos
Processamento Alternativo/genética , Éxons/genética , Mutação da Fase de Leitura/genética , Hidrocefalia/complicações , Hidrocefalia/genética , Nefropatias/complicações , Nefropatias/genética , Proteínas de Membrana/genética , Proteínas Supressoras de Tumor/genética , Feminino , Homozigoto , Humanos , Hidrocefalia/diagnóstico por imagem , Lactente , Nefropatias/diagnóstico por imagem , Imageamento por Ressonância Magnética , Masculino , LinhagemRESUMO
Despite clear technical superiority of genome sequencing (GS) over other diagnostic methods such as exome sequencing (ES), few studies are available regarding the advantages of its clinical application. We analyzed 1007 consecutive index cases for whom GS was performed in a diagnostic setting over a 2-year period. We reported pathogenic and likely pathogenic (P/LP) variants that explain the patients' phenotype in 212 of the 1007 cases (21.1%). In 245 additional cases (24.3%), a variant of unknown significance (VUS) related to the phenotype was reported. We especially investigated patients which had had ES with no genetic diagnosis (n = 358). For this group, GS diagnostic yield was 14.5% (52 patients with P/LP out of 358). GS should be especially indicated for ES-negative cases since up to 29.6% of them could benefit from GS testing (14.5% with P/LP, n = 52 and 15.1% with VUS, n = 54). Genetic diagnoses in most of the ES-negative/GS-positive cases were determined by technical superiority of GS, i.e., access to noncoding regions and more uniform coverage. Importantly, we reported 79 noncoding variants, of which, 41 variants were classified as P/LP. Interpretation of noncoding variants remains challenging, and in many cases, complementary methods based on direct enzyme assessment, biomarker testing and RNA analysis are needed for variant classification and diagnosis. We present the largest cohort of patients with GS performed in a clinical setting to date. The results of this study should direct the decision for GS as standard second-line, or even first-line stand-alone test.
Assuntos
Sequenciamento do Exoma/normas , Doenças Genéticas Inatas/diagnóstico , Testes Genéticos/normas , Adolescente , Criança , Pré-Escolar , Feminino , Frequência do Gene , Doenças Genéticas Inatas/epidemiologia , Doenças Genéticas Inatas/genética , Testes Genéticos/estatística & dados numéricos , Humanos , Lactente , Recém-Nascido , Masculino , Diagnóstico Pré-Natal/normas , Diagnóstico Pré-Natal/estatística & dados numéricos , Sensibilidade e Especificidade , Sequenciamento do Exoma/estatística & dados numéricosRESUMO
Neurometabolic disorders are often inherited and complex disorders that result from abnormalities of enzymes important for development and function of the nervous system. Recently, biallelic mutations in NAXE (APOA1BP) were found in patients with an infantile, lethal, neurometabolic disease. Here, exome sequencing was performed in two affected sisters and their healthy parents. The best candidate, NAXE, was tested for replication in exome sequencing data from 4351 patients with neurodevelopmental disorders. Quantitative RT-PCR, western blot and form factor analysis were performed to assess NAXE expression, protein levels and to analyze mitochondrial morphology in fibroblasts. Vitamin B3 was administered to one patient. Compound heterozygous missense (c.757G>A: p.Gly253Ser) and splicing (c.665-1G>A) variants in NAXE were identified in both affected sisters. In contrast to the previously reported patients with biallelic NAXE variants, our patients showed a milder phenotype with disease onset in early adulthood with psychosis, cognitive impairment, seizures, cerebellar ataxia and spasticity. The symptoms fluctuated. Additional screening of NAXE identified three novel homozygous missense variants (p.Lys245Gln, p.Asp218Asn, p.Ile214Val) in three patients with overlapping phenotype (fluctuating disease course, respiratory insufficiency, movement disorder). Lastly, patients with the c.665-1G>A splicing variant showed a significant reduction of NAXE expression compared to control fibroblasts and undetectable NAXE protein levels compared to control fibroblasts. Based on the metabolic pathway, vitamin B3 and coenzyme Q treatment was introduced in one patient in addition to antiepileptic treatment. This combination and avoidance of triggers was associated with continuous motor and cognitive improvement. The NAXE variants identified in this study suggest a loss-of-function mechanism leading to an insufficient NAD(P)HX repair system. Importantly, symptoms of patients with NAXE variants may improve with vitamin B3/coenzyme Q administration.
Assuntos
Encefalopatias Metabólicas Congênitas/genética , Racemases e Epimerases/genética , Encefalopatias Metabólicas Congênitas/tratamento farmacológico , Feminino , Humanos , Masculino , Mutação de Sentido Incorreto , Transtornos do Neurodesenvolvimento/genética , Niacinamida/uso terapêutico , Linhagem , Ubiquinona/análogos & derivados , Ubiquinona/uso terapêutico , Adulto JovemRESUMO
BACKGROUND: Rare denovo variants represent a significant cause of neurodevelopmental delay and intellectual disability (ID). METHODS: Exome sequencing was performed on 4351 patients with global developmental delay, seizures, microcephaly, macrocephaly, motor delay, delayed speech and language development, or ID according to Human Phenotype Ontology (HPO) terms. All patients had previously undergone whole exome sequencing as part of diagnostic genetic testing with a focus on variants in genes implicated in neurodevelopmental disorders up to January 2017. This resulted in a genetic diagnosis in 1336 of the patients. In this study, we specifically searched for variants in 14 recently implicated novel neurodevelopmental disorder (NDD) genes. RESULTS: We identified 65 rare, protein-changing variants in 11 of these 14 novel candidate genes. Fourteen variants in CDK13, CHD4, KCNQ3, KMT5B, TCF20, and ZBTB18 were scored pathogenic or likely pathogenic. Of note, two of these patients had a previously identified cause of their disease, and thus, multiple molecular diagnoses were made including pathogenic/likely pathogenic variants in FOXG1 and CDK13 or in TMEM237 and KMT5B. CONCLUSIONS: Looking for pathogenic variants in newly identified NDD genes enabled us to provide a molecular diagnosis to 14 patients and their close relatives and caregivers. This underlines the relevance of re-evaluation of existing exome data on a regular basis to improve the diagnostic yield and serve the needs of our patients.
Assuntos
Sequenciamento do Exoma , Testes Genéticos , Transtornos do Neurodesenvolvimento/diagnóstico , Transtornos do Neurodesenvolvimento/genética , Adolescente , Ontologias Biológicas , Criança , Pré-Escolar , Feminino , Humanos , Masculino , FenótipoRESUMO
We report our results of 1000 diagnostic WES cases based on 2819 sequenced samples from 54 countries with a wide phenotypic spectrum. Clinical information given by the requesting physicians was translated to HPO terms. WES processes were performed according to standardized settings. We identified the underlying pathogenic or likely pathogenic variants in 307 families (30.7%). In further 253 families (25.3%) a variant of unknown significance, possibly explaining the clinical symptoms of the index patient was identified. WES enabled timely diagnosing of genetic diseases, validation of causality of specific genetic disorders of PTPN23, KCTD3, SCN3A, PPOX, FRMPD4, and SCN1B, and setting dual diagnoses by detecting two causative variants in distinct genes in the same patient. We observed a better diagnostic yield in consanguineous families, in severe and in syndromic phenotypes. Our results suggest that WES has a better yield in patients that present with several symptoms, rather than an isolated abnormality. We also validate the clinical benefit of WES as an effective diagnostic tool, particularly in nonspecific or heterogeneous phenotypes. We recommend WES as a first-line diagnostic in all cases without a clear differential diagnosis, to facilitate personal medical care.
Assuntos
Exoma , Testes Genéticos/métodos , Técnicas de Genotipagem/métodos , Análise de Sequência de DNA/métodos , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Flavoproteínas/genética , Testes Genéticos/normas , Técnicas de Genotipagem/normas , Humanos , Lactente , Recém-Nascido , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Pessoa de Meia-Idade , Proteínas Mitocondriais/genética , Canal de Sódio Disparado por Voltagem NAV1.3/genética , Núcleo Familiar , Fenótipo , Canais de Potássio/genética , Proteínas Tirosina Fosfatases não Receptoras/genética , Protoporfirinogênio Oxidase/genética , Análise de Sequência de DNA/normas , Canais de Sódio/genética , Subunidade beta-1 do Canal de Sódio Disparado por Voltagem/genéticaRESUMO
BACKGROUND: While the song of all songbirds is controlled by the same neural circuit, the hormone dependence of singing behavior varies greatly between species. For this reason, songbirds are ideal organisms to study ultimate and proximate mechanisms of hormone-dependent behavior and neuronal plasticity. RESULTS: We present the high quality assembly and annotation of a female 1.2-Gbp canary genome. Whole genome alignments between the canary and 13 genomes throughout the bird taxa show a much-conserved synteny, whereas at the single-base resolution there are considerable species differences. These differences impact small sequence motifs like transcription factor binding sites such as estrogen response elements and androgen response elements. To relate these species-specific response elements to the hormone-sensitivity of the canary singing behavior, we identify seasonal testosterone-sensitive transcriptomes of major song-related brain regions, HVC and RA, and find the seasonal gene networks related to neuronal differentiation only in the HVC. Testosterone-sensitive up-regulated gene networks of HVC of singing males concerned neuronal differentiation. Among the testosterone-regulated genes of canary HVC, 20% lack estrogen response elements and 4 to 8% lack androgen response elements in orthologous promoters in the zebra finch. CONCLUSIONS: The canary genome sequence and complementary expression analysis reveal intra-regional evolutionary changes in a multi-regional neural circuit controlling seasonal singing behavior and identify gene evolution related to the hormone-sensitivity of this seasonal singing behavior. Such genes that are testosterone- and estrogen-sensitive specifically in the canary and that are involved in rewiring of neurons might be crucial for seasonal re-differentiation of HVC underlying seasonal song patterning.
Assuntos
Evolução Biológica , Canários/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Genoma , Hormônios/farmacologia , Estações do Ano , Vocalização Animal/efeitos dos fármacos , Animais , Cromossomos/genética , Ilhas de CpG/genética , Feminino , Perfilação da Expressão Gênica , Ontologia Genética , Redes Reguladoras de Genes/efeitos dos fármacos , Hibridização In Situ , Cariotipagem , Masculino , Anotação de Sequência Molecular , Especificidade de Órgãos/efeitos dos fármacos , Especificidade de Órgãos/genética , Regiões Promotoras Genéticas/genética , Proteoma/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sequências Repetitivas de Ácido Nucleico/genética , Análise de Sequência de DNA , Análise de Sequência de RNA , Testosterona/farmacologia , Transcriptoma/genéticaRESUMO
Genetic testing for cystic fibrosis and CFTR-related disorders mostly relies on laborious molecular tools that use Sanger sequencing to scan for mutations in the CFTR gene. We have explored a more efficient genetic screening strategy based on next-generation sequencing (NGS) of the CFTR gene. We validated this approach in a cohort of 177 patients with previously known CFTR mutations and polymorphisms. Genomic DNA was amplified using the Ion AmpliSeq™ CFTR panel. The DNA libraries were pooled, barcoded, and sequenced using an Ion Torrent PGM sequencer. The combination of different robust bioinformatics tools allowed us to detect previously known pathogenic mutations and polymorphisms in the 177 samples, without detecting spurious pathogenic calls. In summary, the assay achieves a sensitivity of 94.45% (95% CI: 92% to 96.9%), with a specificity of detecting nonvariant sites from the CFTR reference sequence of 100% (95% CI: 100% to 100%), a positive predictive value of 100% (95% CI: 100% to 100%), and a negative predictive value of 99.99% (95% CI: 99.99% to 100%). In addition, we describe the observed allelic frequencies of 94 unique definitely and likely pathogenic, uncertain, and neutral CFTR variants, some of them not previously annotated in the public databases. Strikingly, a seven exon spanning deletion as well as several more technically challenging variants such as pathogenic poly-thymidine-guanine and poly-thymidine (poly-TG-T) tracts were also detected. Targeted NGS is ready to substitute classical molecular methods to perform genetic testing on the CFTR gene.
RESUMO
Mesoderm formation in the mouse embryo initiates around E6.5 at the primitive streak and continues until the end of axis extension at E12.5. It requires the process of epithelial-to-mesenchymal transition (EMT), wherein cells detach from the epithelium, adopt mesenchymal cell morphology, and gain competence to migrate. It was shown previously that, prior to mesoderm formation, the transcription factor SRF (Serum Response Factor) is essential for the formation of the primitive streak. To elucidate the role of murine Srf in mesoderm formation during axis extension we conditionally inactivated Srf in nascent mesoderm using the T(s)::Cre driver mouse. Defects in mutant embryos became apparent at E8.75 in the heart and in the allantois. From E9.0 onwards body axis elongation was arrested. Using genome-wide expression analysis, combined with SRF occupancy data from ChIP-seq analysis, we identified a set of direct SRF target genes acting in posterior nascent mesoderm which are enriched for transcripts associated with migratory function. We further show that cell migration is impaired in Srf mutant embryos. Thus, the primary role for SRF in the nascent mesoderm during elongation of the embryonic body axis is the activation of a migratory program, which is a prerequisite for axis extension.
Assuntos
Mesoderma/embriologia , Mesoderma/metabolismo , Fator de Resposta Sérica/metabolismo , Animais , Padronização Corporal/genética , Padronização Corporal/fisiologia , Caderinas/metabolismo , Movimento Celular/genética , Movimento Celular/fisiologia , Transição Epitelial-Mesenquimal/fisiologia , Proteínas Fetais/deficiência , Proteínas Fetais/genética , Proteínas Fetais/metabolismo , Adesões Focais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Mesoderma/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Fator de Resposta Sérica/deficiência , Fator de Resposta Sérica/genética , Fibras de Estresse/metabolismo , Proteínas com Domínio T/deficiência , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Vimentina/metabolismoRESUMO
The histone-modifying complexes PRC2 and TrxG/MLL play pivotal roles in determining the activation state of genes controlling pluripotency, lineage commitment, and cell differentiation. Long noncoding RNAs (lncRNAs) can bind to either complex, and some have been shown to act as modulators of PRC2 or TrxG/MLL activity. Here we show that the lateral mesoderm-specific lncRNA Fendrr is essential for proper heart and body wall development in the mouse. Embryos lacking Fendrr displayed upregulation of several transcription factors controlling lateral plate or cardiac mesoderm differentiation, accompanied by a drastic reduction in PRC2 occupancy along with decreased H3K27 trimethylation and/or an increase in H3K4 trimethylation at their promoters. Fendrr binds to both the PRC2 and TrxG/MLL complexes, suggesting that it acts as modulator of chromatin signatures that define gene activity. Thus, we identified an lncRNA that plays an essential role in the regulatory networks controlling the fate of lateral mesoderm derivatives.
Assuntos
Desenvolvimento Embrionário , Coração/embriologia , Proteína de Leucina Linfoide-Mieloide/metabolismo , Complexo Repressor Polycomb 2/metabolismo , RNA Longo não Codificante/metabolismo , Animais , Diferenciação Celular/genética , Metilação de DNA , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Histona-Lisina N-Metiltransferase , Histonas/metabolismo , Proteínas de Homeodomínio/metabolismo , Camundongos , Dados de Sequência Molecular , Regiões Promotoras Genéticas , RNA Longo não Codificante/genética , Fatores de Transcrição/metabolismo , Proteína Homeobox PITX2RESUMO
Saccharomyces cerevisiae strain W303 is a widely used model organism. However, little is known about its genetic origins, as it was created in the 1970s from crossing yeast strains of uncertain genealogy. To obtain insights into its ancestry and physiology, we sequenced the genome of its variant W303-K6001, a yeast model of ageing research. The combination of two next-generation sequencing (NGS) technologies (Illumina and Roche/454 sequencing) yielded an 11.8 Mb genome assembly at an N50 contig length of 262 kb. Although sequencing was substantially more precise and sensitive than whole-genome tiling arrays, both NGS platforms produced a number of false positives. At a 378× average coverage, only 74 per cent of called differences to the S288c reference genome were confirmed by both techniques. The consensus W303-K6001 genome differs in 8133 positions from S288c, predicting altered amino acid sequence in 799 proteins, including factors of ageing and stress resistance. The W303-K6001 (85.4%) genome is virtually identical (less than equal to 0.5 variations per kb) to S288c, and thus originates in the same ancestor. Non-S288c regions distribute unequally over the genome, with chromosome XVI the most (99.6%) and chromosome XI the least (54.5%) S288c-like. Several of these clusters are shared with Σ1278B, another widely used S288c-related model, indicating that these strains share a second ancestor. Thus, the W303-K6001 genome pictures details of complex genetic relationships between the model strains that date back to the early days of experimental yeast genetics. Moreover, this study underlines the necessity of combining multiple NGS and genome-assembling techniques for achieving accurate variant calling in genomic studies.
Assuntos
Genoma Fúngico , Genômica/métodos , Saccharomyces cerevisiae/genética , Análise de Sequência de DNA/métodos , Sequência de Bases , Mapeamento Cromossômico , Cromossomos Fúngicos/genética , DNA Fúngico/química , DNA Fúngico/genética , Genes Fúngicos/genética , Dados de Sequência Molecular , Filogenia , Polimorfismo de Nucleotídeo Único , Saccharomyces cerevisiae/classificação , Proteínas de Saccharomyces cerevisiae/genética , Homologia de Sequência do Ácido Nucleico , Especificidade da EspécieRESUMO
The linkage of disease gene mapping with DNA sequencing is an essential strategy for defining the genetic basis of a disease. New massively parallel sequencing procedures will greatly facilitate this process, although enrichment for the target region before sequencing remains necessary. For this step, various DNA capture approaches have been described that rely on sequence-defined probe sets. To avoid making assumptions on the sequences present in the targeted region, we accessed specific cytogenetic regions in preparation for next-generation sequencing. We directly microdissected the target region in metaphase chromosomes, amplified it by degenerate oligonucleotide-primed PCR, and obtained sufficient material of high quality for high-throughput sequencing. Sequence reads could be obtained from as few as six chromosomal fragments. The power of cytogenetic enrichment followed by next-generation sequencing is that it does not depend on earlier knowledge of sequences in the region being studied. Accordingly, this method is uniquely suited for situations in which the sequence of a reference region of the genome is not available, including population-specific or tumor rearrangements, as well as previously unsequenced genomic regions such as centromeres.
Assuntos
Cromossomos Humanos Par 12/genética , Cromossomos Humanos Par 1/genética , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , Biologia Computacional , Humanos , Metáfase , MicrodissecçãoRESUMO
BACKGROUND: Colorectal cancer (CRC) is with approximately 1 million cases the third most common cancer worldwide. Extensive research is ongoing to decipher the underlying genetic patterns with the hope to improve early cancer diagnosis and treatment. In this direction, the recent progress in next generation sequencing technologies has revolutionized the field of cancer genomics. However, one caveat of these studies remains the large amount of genetic variations identified and their interpretation. METHODOLOGY/PRINCIPAL FINDINGS: Here we present the first work on whole exome NGS of primary colon cancers. We performed 454 whole exome pyrosequencing of tumor as well as adjacent not affected normal colonic tissue from microsatellite stable (MSS) and microsatellite instable (MSI) colon cancer patients and identified more than 50,000 small nucleotide variations for each tissue. According to predictions based on MSS and MSI pathomechanisms we identified eight times more somatic non-synonymous variations in MSI cancers than in MSS and we were able to reproduce the result in four additional CRCs. Our bioinformatics filtering approach narrowed down the rate of most significant mutations to 359 for MSI and 45 for MSS CRCs with predicted altered protein functions. In both CRCs, MSI and MSS, we found somatic mutations in the intracellular kinase domain of bone morphogenetic protein receptor 1A, BMPR1A, a gene where so far germline mutations are associated with juvenile polyposis syndrome, and show that the mutations functionally impair the protein function. CONCLUSIONS/SIGNIFICANCE: We conclude that with deep sequencing of tumor exomes one may be able to predict the microsatellite status of CRC and in addition identify potentially clinically relevant mutations.
Assuntos
Neoplasias Colorretais/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Instabilidade de Microssatélites , Repetições de Microssatélites , Adenocarcinoma/genética , Idoso , Neoplasias do Colo/genética , Biologia Computacional/métodos , Análise Mutacional de DNA , Genômica , Humanos , Masculino , Pessoa de Meia-Idade , MutaçãoRESUMO
BACKGROUND: Due to the advanced techniques in sequencing and fragment analysis, DNA sequencers and analyzers produce vast amounts of data within short time. To administrate the large data volume conveniently, efficient data management systems are used in order to process and to store sequencers' or analyzers' data outcome. The inclusion of graphical reports in such systems is necessary to achieve a comprehensive view of the integrated data. However, the resulting data of sequencing and fragment analysis runs are stored in a proprietary format, the so-called trace or fsa format, which is only readable by programs provided by the instrument's vendor operating on the machine itself or by commercial tools designed for editing the respective data. To allow for a quick conversion of the proprietary data format into a commonly used one, toolkits are required that reach this aim and can be easily integrated into workflow systems. RESULTS: We have developed the software toolkits Trace2PS and Fsa2PS which allow to convert sequence and fragment analysis raw data files to PostScript images, respectively. The toolkits are implemented as Perl modules that can be used as standalone command line applications in conjunction with a script-based analysis pipeline, or integrated in software applications for displaying the data content of trace and fsa files. The converter modules support the commonly used file formats for storage of sequencing (ABI and SCF) and fragment analysis data (FSA). CONCLUSION: The software toolkits provide useful applications to convert sequencing and fragment analysis files from a proprietary into a more common, human-readable format. Trace2PS and FSA2PS are useful and capable in data management workflow systems like SAMS, or laboratory information systems that are used for displaying trace and fragment analysis results via web-based tools over an intranet or internet connection to users that can view their results directly on the screen.
RESUMO
The vertebral column and skeletal muscles of vertebrates are derived from the paraxial mesoderm, which is laid down initially as two stripes of mesenchymal cells alongside the neural tube and subsequently segmented. Previous work has shown that the wingless-type MMTV integration site family (WNT), fibroblast growth factor- and Delta-Notch signalling pathways control presomitic mesoderm (psm) formation and segmentation. Here, we show that the expression of mesogenin 1, a basic helix-loop-helix transcription factor, which is essential for psm maturation, is regulated by synergism between WNT signalling and the T-box 6 transcription factor, involving a feed-forward control mechanism. These findings emphasize the crucial role of WNT signalling in the control of psm formation, maturation and segmentation.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Desenvolvimento Embrionário , Regulação da Expressão Gênica no Desenvolvimento , Mesoderma/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Wnt/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/análise , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Mesoderma/química , Camundongos , Camundongos Transgênicos , Regiões Promotoras Genéticas , Transdução de Sinais , Proteínas com Domínio T , Fatores de Transcrição/análise , beta-Galactosidase/análise , beta-Galactosidase/genéticaRESUMO
Post-germinative growth of oilseeds is dependent on the breakdown of the stored lipid reserves. Long-chain acyl-CoA synthetase activities (LACS) are critically involved in this process by activating the released free fatty acids and thus feeding the beta-oxidation cycle in glyoxysomes. Here we report on the identification of two LACS genes, AtLACS6 and AtLACS7 from Arabidopsis thaliana coding for peroxisomal LACS proteins. The subcellular localization was verified by co-expression studies of spectral variants of the green fluorescent protein (GFP). While AtLACS6 is targeted by a type 2 (PTS2) peroxisomal targeting sequence, for AtLACS7 a functional PTS1 as well as a PTS2 could be demonstrated. Possible explanations for this potentially redundant targeting information will be discussed. Expression studies of both genes revealed a strong induction 1 day after germination resembling the expression pattern of other genes involved in beta-oxidation. Analysis of the substrate specificities of the two LACS proteins demonstrated enzymatic activity for both enzymes with the whole spectrum of fatty acids found in stored lipid reserves. These results suggest that both LACS proteins might have overlapping functions and are able to initiate beta-oxidation in plant peroxisomes.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/enzimologia , Coenzima A Ligases/genética , Ácidos Graxos/metabolismo , Peroxissomos/metabolismo , Proteínas Repressoras , Proteínas de Saccharomyces cerevisiae , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/metabolismo , Coenzima A Ligases/metabolismo , Escherichia coli/genética , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Proteínas de Fluorescência Verde , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Dados de Sequência Molecular , Oxirredução , Filogenia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Basic helix-loop-helix (bHLH) transcription factors (TFs) belong to a family of transcriptional regulators present in three eukaryotic kingdoms. Many different functions have been identified for these proteins in animals, including the control of cell proliferation and development of specific cell lineages. Their mechanism for controlling gene transcription often involves homodimerization or heterodimerization. In plants, little is known about the bHLH family, but we have determined that there are 133 bHLH genes in Arabidopsis thaliana and have confirmed that at least 113 of them are expressed. The AtbHLH genes constitute one of the largest families of transcription factors in A. thaliana with significantly more members than are found in most animal species and about an equivalent number to those in vertebrates. Comparisons with animal sequences suggest that the majority of plant bHLH genes have evolved from the ancestral group B class of bHLH genes. By studying the AtbHLH genes collectively, twelve subfamilies have been identified. Within each of these main groups, there are conserved amino acid sequence motifs outside the DNA binding domain. Potential gene redundancy among members of smaller subgroups has been analyzed, and the resulting information is presented to provide a simplified visual interpretation of the gene family, identifying related genes that are likely to share similar functions. Based on the current characterization of a limited number of plant bHLH proteins, we predict that this family of TFs has a range of different roles in plant cell and tissue development as well as plant metabolism.