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1.
Gene ; 590(1): 1-4, 2016 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-27259663

RESUMO

Amelogenesis imperfecta (AI) is a clinically and genetically heterogeneous disorder of tooth development which is due to aberrant deposition or composition of enamel. Both syndromic and isolated forms exist; they may be inherited in an X-linked, autosomal recessive, or autosomal dominant manner. WDR72 is one of ten currently known genes for recessive isolated AI; nine WDR72 mutations affecting single nucleotides have been described to date. Based on whole exome sequencing in a large consanguineous AI pedigree, we obtained evidence for presence of a multi-exonic WDR72 deletion. A home-made multiplex ligation-dependent probe amplification assay was used to confirm the aberration, to narrow its extent, and to identify heterozygous carriers. Our study extends the mutational spectrum for WDR72 to include large deletions, and supports a relevance of the previously proposed loss-of-function mechanism. It also introduces an easy-to-use and highly sensitive tool for detecting WDR72 copy number alterations.


Assuntos
Amelogênese Imperfeita/genética , Sequência de Bases , Éxons , Dosagem de Genes , Proteínas/genética , Deleção de Sequência , Amelogênese Imperfeita/metabolismo , Amelogênese Imperfeita/patologia , Consanguinidade , Esmalte Dentário/metabolismo , Esmalte Dentário/patologia , Exoma , Feminino , Expressão Gênica , Heterozigoto , Humanos , Masculino , Reação em Cadeia da Polimerase Multiplex , Linhagem , Análise de Sequência de DNA
2.
Nat Genet ; 47(6): 647-53, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25961942

RESUMO

Cardiovascular disease is the most common cause of death worldwide, and hypertension is the major risk factor. Mendelian hypertension elucidates mechanisms of blood pressure regulation. Here we report six missense mutations in PDE3A (encoding phosphodiesterase 3A) in six unrelated families with mendelian hypertension and brachydactyly type E (HTNB). The syndrome features brachydactyly type E (BDE), severe salt-independent but age-dependent hypertension, an increased fibroblast growth rate, neurovascular contact at the rostral-ventrolateral medulla, altered baroreflex blood pressure regulation and death from stroke before age 50 years when untreated. In vitro analyses of mesenchymal stem cell-derived vascular smooth muscle cells (VSMCs) and chondrocytes provided insights into molecular pathogenesis. The mutations increased protein kinase A-mediated PDE3A phosphorylation and resulted in gain of function, with increased cAMP-hydrolytic activity and enhanced cell proliferation. Levels of phosphorylated VASP were diminished, and PTHrP levels were dysregulated. We suggest that the identified PDE3A mutations cause the syndrome. VSMC-expressed PDE3A deserves scrutiny as a therapeutic target for the treatment of hypertension.


Assuntos
Braquidactilia/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/genética , Hipertensão/congênito , Adolescente , Adulto , Sequência de Aminoácidos , Animais , Sequência de Bases , Estudos de Casos e Controles , Diferenciação Celular , Criança , Feminino , Estudos de Associação Genética , Células HeLa , Humanos , Hipertensão/genética , Cinética , Masculino , Células-Tronco Mesenquimais/fisiologia , Camundongos , Pessoa de Meia-Idade , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Miócitos de Músculo Liso/fisiologia , Linhagem
3.
Mol Genet Genomic Med ; 2(5): 393-401, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25333064

RESUMO

Usher syndrome is an autosomal recessive disorder characterized both by deafness and blindness. For the three clinical subtypes of Usher syndrome causal mutations in altogether 12 genes and a modifier gene have been identified. Due to the genetic heterogeneity of Usher syndrome, the molecular analysis is predestined for a comprehensive and parallelized analysis of all known genes by next-generation sequencing (NGS) approaches. We describe here the targeted enrichment and deep sequencing for exons of Usher genes and compare the costs and workload of this approach compared to Sanger sequencing. We also present a bioinformatics analysis pipeline that allows us to detect single-nucleotide variants, short insertions and deletions, as well as copy number variations of one or more exons on the same sequence data. Additionally, we present a flexible in silico gene panel for the analysis of sequence variants, in which newly identified genes can easily be included. We applied this approach to a cohort of 44 Usher patients and detected biallelic pathogenic mutations in 35 individuals and monoallelic mutations in eight individuals of our cohort. Thirty-nine of the sequence variants, including two heterozygous deletions comprising several exons of USH2A, have not been reported so far. Our NGS-based approach allowed us to assess single-nucleotide variants, small indels, and whole exon deletions in a single test. The described diagnostic approach is fast and cost-effective with a high molecular diagnostic yield.

4.
Naturwissenschaften ; 101(11): 939-54, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25228346

RESUMO

How mutations accumulate in genomes is the central question of molecular evolution theories. However, our understanding of this process is far from complete. Drake's rule is a notoriously universal property of genomes from microbes to mammals-the number of (functional) mutations per-genome per-generation is approximately constant within a phylum, despite the orders of magnitude differences in genome sizes and diverse populations' properties. So far, there is no concise explanation for this phenomenon. A formal model for the storage of genetic information suggests that a genome of any species operates near its maximum informational storage capacity, and the mutation rate per-genome per-generation is near its upper limit, providing a simple explanation for the rule with minimal assumptions.


Assuntos
Simulação por Computador , Evolução Molecular , Modelos Genéticos , Mutação
5.
PLoS One ; 8(8): e70151, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23940540

RESUMO

The identification of disease-causing mutations in next-generation sequencing (NGS) data requires efficient filtering techniques. In patients with rare recessive diseases, compound heterozygosity of pathogenic mutations is the most likely inheritance model if the parents are non-consanguineous. We developed a web-based compound heterozygous filter that is suited for data from NGS projects and that is easy to use for non-bioinformaticians. We analyzed the power of compound heterozygous mutation filtering by deriving background distributions for healthy individuals from different ethnicities and studied the effectiveness in trios as well as more complex pedigree structures. While usually more then 30 genes harbor potential compound heterozygotes in single exomes, this number can be markedly reduced with every additional member of the pedigree that is included in the analysis. In a real data set with exomes of four family members, two sisters affected by Mabry syndrome and their healthy parents, the disease-causing gene PIGO, which harbors the pathogenic compound heterozygous variants, could be readily identified. Compound heterozygous filtering is an efficient means to reduce the number of candidate mutations in studies aiming at identifying recessive disease genes in non-consanguineous families. A web-server is provided to make this filtering strategy available at www.gene-talk.de.


Assuntos
Biologia Computacional/métodos , Heterozigoto , Sequenciamento de Nucleotídeos em Larga Escala , Exoma/genética , Humanos , Mutação , Linhagem
6.
Genome Med ; 5(7): 69, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23902830

RESUMO

With exome sequencing becoming a tool for mutation detection in routine diagnostics there is an increasing need for platform-independent methods of quality control. We present a genotype-weighted metric that allows comparison of all the variant calls of an exome to a high-quality reference dataset of an ethnically matched population. The exome-wide genotyping accuracy is estimated from the distance to this reference set, and does not require any further knowledge about data generation or the bioinformatics involved. The distances of our metric are visualized by non-metric multidimensional scaling and serve as an intuitive, standardizable score for the quality assessment of exome data.

7.
Blood ; 122(7): 1312-5, 2013 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-23733340

RESUMO

To ascertain the genetic basis of a paroxysmal nocturnal hemoglobinuria (PNH) case without somatic mutations in PIGA, we performed deep next-generation sequencing on all exons of known genes of the glycosylphosphatidylinositol (GPI) anchor synthesis pathway. We identified a heterozygous germline splice site mutation in PIGT and a somatic 8-MB deletion in granulocytes affecting the other copy of PIGT. PIGA is essential for GPI anchor synthesis, whereas PIGT is essential for attachment of the preassembled GPI anchor to proteins. Although a single mutation event in the X-chromosomal gene PIGA is known to cause GPI-anchored protein deficiency, 2 such hits are required in the autosomal gene PIGT. Our data indicate that PNH can occur even in the presence of fully assembled GPI if its transfer to proteins is defective in hematopoietic stem cells.


Assuntos
Aciltransferases/genética , Mutação em Linhagem Germinativa/genética , Hemoglobinúria Paroxística/genética , Mutação/genética , Adulto , Processamento Alternativo/genética , Animais , Células CHO , Estudos de Casos e Controles , Hibridização Genômica Comparativa , Cricetulus , Éxons/genética , Feminino , Citometria de Fluxo , Genes Ligados ao Cromossomo X , Humanos , Hibridização in Situ Fluorescente , Análise de Sequência de DNA , Deleção de Sequência
8.
Cancer Cell ; 23(2): 159-70, 2013 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-23410972

RESUMO

Early-onset prostate cancer (EO-PCA) represents the earliest clinical manifestation of prostate cancer. To compare the genomic alteration landscapes of EO-PCA with "classical" (elderly-onset) PCA, we performed deep sequencing-based genomics analyses in 11 tumors diagnosed at young age, and pursued comparative assessments with seven elderly-onset PCA genomes. Remarkable age-related differences in structural rearrangement (SR) formation became evident, suggesting distinct disease pathomechanisms. Whereas EO-PCAs harbored a prevalence of balanced SRs, with a specific abundance of androgen-regulated ETS gene fusions including TMPRSS2:ERG, elderly-onset PCAs displayed primarily non-androgen-associated SRs. Data from a validation cohort of > 10,000 patients showed age-dependent androgen receptor levels and a prevalence of SRs affecting androgen-regulated genes, further substantiating the activity of a characteristic "androgen-type" pathomechanism in EO-PCA.


Assuntos
Rearranjo Gênico , Genômica , Proteínas de Fusão Oncogênica/genética , Neoplasias da Próstata/genética , Receptores Androgênicos/genética , Serina Endopeptidases/genética , Transativadores/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Biologia Computacional , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Pessoa de Meia-Idade , Regulador Transcricional ERG
9.
Nucleic Acids Res ; 37(18): e123, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19620212

RESUMO

High-throughput complementary DNA sequencing (RNA-Seq) is a powerful tool for whole-transcriptome analysis, supplying information about a transcript's expression level and structure. However, it is difficult to determine the polarity of transcripts, and therefore identify which strand is transcribed. Here, we present a simple cDNA sequencing protocol that preserves information about a transcript's direction. Using Saccharomyces cerevisiae and mouse brain transcriptomes as models, we demonstrate that knowing the transcript's orientation allows more accurate determination of the structure and expression of genes. It also helps to identify new genes and enables studying promoter-associated and antisense transcription. The transcriptional landscapes we obtained are available online.


Assuntos
DNA Complementar/química , Perfilação da Expressão Gênica , Análise de Sequência de DNA/métodos , Animais , Nucleotídeos de Desoxiuracil/metabolismo , Genes Fúngicos , Camundongos , Regiões Promotoras Genéticas , RNA Antissenso/biossíntese , Reprodutibilidade dos Testes , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transcrição Gênica
10.
PLoS One ; 4(5): e5548, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19440246

RESUMO

BACKGROUND: Cancer re-sequencing programs rely on DNA isolated from fresh snap frozen tissues, the preparation of which is combined with additional preservation efforts. Tissue samples at pathology departments are routinely stored as formalin-fixed and paraffin-embedded (FFPE) samples and their use would open up access to a variety of clinical trials. However, FFPE preparation is incompatible with many down-stream molecular biology techniques such as PCR based amplification methods and gene expression studies. METHODOLOGY/PRINCIPAL FINDINGS: Here we investigated the sample quality requirements of FFPE tissues for massively parallel short-read sequencing approaches. We evaluated key variables of pre-fixation, fixation related and post-fixation processes that occur in routine medical service (e.g. degree of autolysis, duration of fixation and of storage). We also investigated the influence of tissue storage time on sequencing quality by using material that was up to 18 years old. Finally, we analyzed normal and tumor breast tissues using the Sequencing by Synthesis technique (Illumina Genome Analyzer, Solexa) to simultaneously localize genome-wide copy number alterations and to detect genomic variations such as substitutions and point-deletions and/or insertions in FFPE tissue samples. CONCLUSIONS/SIGNIFICANCE: The application of second generation sequencing techniques on small amounts of FFPE material opens up the possibility to analyze tissue samples which have been collected during routine clinical work as well as in the context of clinical trials. This is in particular important since FFPE samples are amply available from surgical tumor resections and histopathological diagnosis, and comprise tissue from precursor lesions, primary tumors, lymphogenic and/or hematogenic metastases. Large-scale studies using this tissue material will result in a better prediction of the prognosis of cancer patients and the early identification of patients which will respond to therapy.


Assuntos
Formaldeído/química , Neoplasias/metabolismo , Inclusão em Parafina/métodos , Análise de Sequência de DNA/métodos , Fixação de Tecidos/métodos , Adolescente , Biologia Computacional , Análise Mutacional de DNA/métodos , Humanos , Técnicas In Vitro
11.
Science ; 321(5891): 956-60, 2008 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-18599741

RESUMO

The functional complexity of the human transcriptome is not yet fully elucidated. We report a high-throughput sequence of the human transcriptome from a human embryonic kidney and a B cell line. We used shotgun sequencing of transcripts to generate randomly distributed reads. Of these, 50% mapped to unique genomic locations, of which 80% corresponded to known exons. We found that 66% of the polyadenylated transcriptome mapped to known genes and 34% to nonannotated genomic regions. On the basis of known transcripts, RNA-Seq can detect 25% more genes than can microarrays. A global survey of messenger RNA splicing events identified 94,241 splice junctions (4096 of which were previously unidentified) and showed that exon skipping is the most prevalent form of alternative splicing.


Assuntos
Processamento Alternativo , Perfilação da Expressão Gênica , Genoma Humano , Sítios de Splice de RNA , RNA Mensageiro/genética , Análise de Sequência de RNA , Linhagem Celular , Linhagem Celular Tumoral , Biologia Computacional , DNA Complementar , DNA Intergênico , Éxons , Humanos , Íntrons , Análise de Sequência com Séries de Oligonucleotídeos , RNA Polimerase II/metabolismo
12.
DNA Cell Biol ; 24(9): 529-42, 2005 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16153154

RESUMO

Gene duplication is the main source of evolutionary novelties. However, the problem with duplicates is that the purifying selection overlooks deleterious mutations in the redundant sequence, which therefore, instead of gaining a new function, often degrades into a functionless pseudogene. This risk of functional loss instead of gain is much higher for small populations of higher organisms with a slow and complex development. We propose that it is the epigenetic tissue/stage-complementary silencing of duplicates that makes them exposable to the purifying selection, thus saving them from pseudogenization and opening the way towards new function(s). Our genome-wide analyses of gene duplicates in several eukaryotic species combined with the phylogenetic comparison of vertebrate alpha- and beta-globin gene clusters strongly support this epigenetic complementation (EC) model. The distinctive condition for a new duplicate to survive by the EC mechanism seems to be its repositioning to an ectopic site, which is accompanied by changes in the rate and direction of mutagenesis. The most distinguished in this respect is the human genome. In this review, we extend and discuss the data on the EC- and repositioning-dependent fate of gene duplicates with the special emphasis on the problem of detecting brief postduplication period of adaptive evolution driven by positive selection. Accordingly, we propose a new CpG-focused measure of selection that is insensitive to translocation-caused biases in mutagenesis.


Assuntos
Epigênese Genética/genética , Evolução Molecular , Inativação Gênica , Genes Duplicados/genética , Modelos Genéticos , Filogenia , Seleção Genética , Animais , Genômica/métodos , Globinas/genética , Humanos , Mutagênese , Translocação Genética/genética
13.
J Mol Evol ; 59(3): 372-84, 2004 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-15553091

RESUMO

It is well known that repositioning of a gene often exerts a strong impact on its own expression and whole development. Here we report the results of genome-wide analyses suggesting that repositioning may also radically change the evolutionary fate of gene duplicates. As an indicator of these changes, we used the GC content of gene pairs which originated by duplication. This indicator turned out to be duplicate-asymmetric, which means that genes in a pair differ significantly in GC content despite their apparent origin from a common ancestor. Such an asymmetry necessarily implies that after duplication two originally identical genes mutated in opposite directions-toward GC-rich and GC-poor content, respectively. In mammalian genomes, this trend is definitely associated with presumably methylated hypermutable CpG sites, and in a typical GC-asymmetric gene pair, its two member genes are embedded in GC-contrasting isochores. However, we unexpectedly found similar significant GC asymmetry in fish, fly, worm, and yeast. This means that neither methylation alone nor methylation in combination with isochores can be counted as a primary cause of the GC asymmetry; rather they represent specific realizations of some universal principle of genome evolution. Remarkably, genes from pairs with the greatest GC asymmetry tend to be on different chromosomes, suggesting that the mutational difference between gene duplicates is associated with translocation of a new gene to a different place in the genome, whereas GC symmetric pairs demonstrate the opposite tendency. A recently emerged extra gene copy is usually on the same chromosome as is its parent but quickly, by 0.05 substitution per synonymous site, either has perished or occupies a different chromosome. During this earliest posttranslocation period, the ratio of nonsynonymous/synonymous base substitutions is unusually high, suggesting a rapid adaptive evolution of novel functions. In a general context of evolution by gene duplication, our interpretation of this position-dependent GC asymmetry between duplicated genes is that evolution of redundant genes toward a new function has often been associated with their very early, postduplication repositioning in the genome, with a concomitant abrupt change in epigenetic control of tissue/stage-specific expression and an increase in the mutation rate. Of eight eukaryotic genomes studied, the most distinguished in this respect is the human genome.


Assuntos
Evolução Molecular , Ordem dos Genes/genética , Genes Duplicados/genética , Genoma , Animais , Composição de Bases/genética , Biologia Computacional/métodos , Bases de Dados de Ácidos Nucleicos , Drosophila melanogaster/genética , Genômica/métodos , Humanos , Isocoros/genética , Camundongos/genética , Mutação/genética , Plantas/genética , Ratos/genética , Saccharomyces cerevisiae/genética , Seleção Genética , Takifugu/genética , Translocação Genética/genética
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