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1.
Hum Mutat ; 2019 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-31471937

RESUMO

Microsatellite instability (MSI) testing of colorectal cancers (CRCs) is used to screen for Lynch syndrome (LS), a hereditary cancer-predisposition, and can be used to predict response to immunotherapy. Here, we present a single-molecule molecular inversion probe and sequencing-based MSI assay and demonstrate its clinical validity according to existing guidelines. We amplified 24 microsatellites in multiplex and trained a classifier using 98 CRCs, which accommodates marker specific sensitivities to MSI. Sample classification achieved 100% concordance with the MSI Analysis System v1.2 (Promega) in three independent cohorts, totaling 220 CRCs. Backward-forward stepwise selection was used to identify a 6-marker subset of equal accuracy to the 24-marker panel. Assessment of assay detection limits showed that the 24-marker panel is marginally more robust to sample variables than the 6-marker subset, detecting as little as 3% high levels of MSI DNA in sample mixtures, and requiring a minimum of 10 template molecules to be sequenced per marker for >95% accuracy. BRAF c.1799 mutation analysis was also included to streamline LS testing, with all c.1799T>A variants being correctly identified. The assay, therefore, provides a cheap, robust, automatable, and scalable MSI test with internal quality controls, suitable for clinical cancer diagnostics.

2.
Hum Mutat ; 40(5): 649-655, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30740824

RESUMO

Constitutional mismatch repair deficiency (CMMRD) is caused by germline pathogenic variants in both alleles of a mismatch repair gene. Patients have an exceptionally high risk of numerous pediatric malignancies and benefit from surveillance and adjusted treatment. The diversity of its manifestation, and ambiguous genotyping results, particularly from PMS2, can complicate diagnosis and preclude timely patient management. Assessment of low-level microsatellite instability in nonneoplastic tissues can detect CMMRD, but current techniques are laborious or of limited sensitivity. Here, we present a simple, scalable CMMRD diagnostic assay. It uses sequencing and molecular barcodes to detect low-frequency microsatellite variants in peripheral blood leukocytes and classifies samples using variant frequencies. We tested 30 samples from 26 genetically-confirmed CMMRD patients, and samples from 94 controls and 40 Lynch syndrome patients. All samples were correctly classified, except one from a CMMRD patient recovering from aplasia. However, additional samples from this same patient tested positive for CMMRD. The assay also confirmed CMMRD in six suspected patients. The assay is suitable for both rapid CMMRD diagnosis within clinical decision windows and scalable screening of at-risk populations. Its deployment will improve patient care, and better define the prevalence and phenotype of this likely underreported cancer syndrome.

3.
Development ; 146(2)2019 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-30696714

RESUMO

The scarcity of embryonic/foetal material as a resource for direct study means that there is still limited understanding of human retina development. Here, we present an integrated transcriptome analysis combined with immunohistochemistry in human eye and retinal samples from 4 to 19 post-conception weeks. This analysis reveals three developmental windows with specific gene expression patterns that informed the sequential emergence of retinal cell types and enabled identification of stage-specific cellular and biological processes, and transcriptional regulators. Each stage is characterised by a specific set of alternatively spliced transcripts that code for proteins involved in the formation of the photoreceptor connecting cilium, pre-mRNA splicing and epigenetic modifiers. Importantly, our data show that the transition from foetal to adult retina is characterised by a large increase in the percentage of mutually exclusive exons that code for proteins involved in photoreceptor maintenance. The circular RNA population is also defined and shown to increase during retinal development. Collectively, these data increase our understanding of human retinal development and the pre-mRNA splicing process, and help to identify new candidate disease genes.


Assuntos
Perfilação da Expressão Gênica , Retina/embriologia , Retina/metabolismo , Processamento Alternativo/genética , Animais , Biomarcadores/metabolismo , Cílios/metabolismo , Feto/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Organogênese/genética , Células Fotorreceptoras de Vertebrados/citologia , Células Fotorreceptoras de Vertebrados/metabolismo , Análise de Componente Principal , RNA/genética , RNA/metabolismo , Precursores de RNA/genética , Precursores de RNA/metabolismo , Retina/citologia , Retina/ultraestrutura , Transcriptoma/genética
4.
PLoS One ; 13(8): e0203052, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30157243

RESUMO

Somatic mutations in mononucleotide repeats are commonly used to assess the mismatch repair status of tumours. Current tests focus on repeats with a length above 15bp, which tend to be somatically more unstable than shorter ones. These longer repeats also have a substantially higher PCR error rate, and tests that use capillary electrophoresis for fragment size analysis often require expert interpretation. In this communication, we present a panel of 17 short repeats (length 7-12bp) for sequence-based microsatellite instability (MSI) testing. Using a simple scoring procedure that incorporates the allelic distribution of the mutant repeats, and analysis of two cohort of tumours totalling 209 samples, we show that this panel is able to discriminate between MMR proficient and deficient tumours, even when constitutional DNA is not available. In the training cohort, the method achieved 100% concordance with fragment analysis, while in the testing cohort, 4 discordant samples were observed (corresponding to 97% concordance). Of these, 2 showed discrepancies between fragment analysis and immunohistochemistry and one was reclassified after re-testing using fragment analysis. These results indicate that our approach offers the option of a reliable, scalable routine test for MSI.

5.
BMC Genomics ; 19(1): 276, 2018 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-29678151

RESUMO

BACKGROUND: Circular RNAs (circRNAs) are predominantly derived from protein coding genes, and some can act as microRNA sponges or transcriptional regulators. Changes in circRNA levels have been identified during human development which may be functionally important, but lineage-specific analyses are currently lacking. To address this, we performed RNAseq analysis of human embryonic stem (ES) cells differentiated for 90 days towards 3D laminated retina. RESULTS: A transcriptome-wide increase in circRNA expression, size, and exon count was observed, with circRNA levels reaching a plateau by day 45. Parallel statistical analyses, controlling for sample and locus specific effects, identified 239 circRNAs with expression changes distinct from the transcriptome-wide pattern, but these all also increased in abundance over time. Surprisingly, circRNAs derived from long non-coding RNAs (lncRNAs) were found to account for a significantly larger proportion of transcripts from their loci of origin than circRNAs from coding genes. The most abundant, circRMST:E12-E6, showed a > 100X increase during differentiation accompanied by an isoform switch, and accounts for > 99% of RMST transcripts in many adult tissues. The second most abundant, circFIRRE:E10-E5, accounts for > 98% of FIRRE transcripts in differentiating human ES cells, and is one of 39 FIRRE circRNAs, many of which include multiple unannotated exons. CONCLUSIONS: Our results suggest that during human ES cell differentiation, changes in circRNA levels are primarily globally controlled. They also suggest that RMST and FIRRE, genes with established roles in neurogenesis and topological organisation of chromosomal domains respectively, are processed as circular lncRNAs with only minor linear species.

6.
PLoS One ; 13(2): e0192223, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29425227

RESUMO

Regular aspirin use is associated with reduced risk of colorectal cancer (CRC). Variation in aspirin's chemoprevention efficacy has been attributed to the presence of single nucleotide polymorphisms (SNPs). We conducted a meta-analysis using two large population-based case-control datasets, the UK-Leeds Colorectal Cancer Study Group and the NIH-Colon Cancer Family Registry, having a combined total of 3325 cases and 2262 controls. The aim was to assess 42 candidate SNPs in 15 genes whose association with colorectal cancer risk was putatively modified by aspirin use, in the literature. Log odds ratios (ORs) and standard errors were estimated for each dataset separately using logistic regression adjusting for age, sex and study site, and dataset-specific results were combined using random effects meta-analysis. Meta-analysis showed association between SNPs rs6983267, rs11694911 and rs2302615 with CRC risk reduction (All P<0.05). Association for SNP rs6983267 in the CCAT2 gene only was noteworthy after multiple test correction (P = 0.001). Site-specific analysis showed association between SNPs rs1799853 and rs2302615 with reduced colon cancer risk only (P = 0.01 and P = 0.004, respectively), however neither reached significance threshold following multiple test correction. Meta-analysis of SNPs rs2070959 and rs1105879 in UGT1A6 gene showed interaction between aspirin use and CRC risk (Pinteraction = 0.01 and 0.02, respectively); stratification by aspirin use showed an association for decreased CRC risk for aspirin users having a wild-type genotype (rs2070959 OR = 0.77, 95% CI = 0.68-0.86; rs1105879 OR = 0.77 95% CI = 0.69-0.86) compared to variant allele cariers. The direction of the interaction however is in contrast to that published in studies on colorectal adenomas. Both SNPs showed potential site-specific interaction with aspirin use and colon cancer risk only (Pinteraction = 0.006 and 0.008, respectively), with the direction of association similar to that observed for CRC. Additionally, they showed interaction between any non-steroidal anti-inflammatory drugs (including aspirin) use and CRC risk (Pinteraction = 0.01 for both). All gene x environment (GxE) interactions however were not significant after multiple test correction. Candidate gene investigation indicated no evidence of GxE interaction between genetic variants in genes involved in aspirin pathways, regular aspirin use and colorectal cancer risk.


Assuntos
Aspirina/metabolismo , Neoplasias Colorretais/epidemiologia , Grupo com Ancestrais do Continente Europeu , Polimorfismo de Nucleotídeo Único , Aspirina/administração & dosagem , Austrália/epidemiologia , Canadá/epidemiologia , Estudos de Casos e Controles , Neoplasias Colorretais/prevenção & controle , Humanos , Fatores de Risco , Estados Unidos/epidemiologia
8.
BMC Bioinformatics ; 17: 31, 2016 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-26758031

RESUMO

BACKGROUND: Transcripts, which have been subject to Post-transcriptional exon shuffling (PTES), have an exon order inconsistent with the underlying genomic sequence. These have been identified in a wide variety of tissues and cell types from many eukaryotes, and are now known to be mostly circular, cytoplasmic, and non-coding. Although there is no uniformly ascribed function, several have been shown to be involved in gene regulation. Accurate identification of these transcripts can, however, be difficult due to artefacts from a wide variety of sources. RESULTS: Here, we present a computational method, PTESFinder, to identify these transcripts from high throughput RNAseq data. Uniquely, it systematically excludes potential artefacts emanating from pseudogenes, segmental duplications, and template switching, and outputs both PTES and canonical exon junction counts to facilitate comparative analyses. In comparison with four existing methods, PTESFinder achieves highest specificity and comparable sensitivity at a variety of read depths. PTESFinder also identifies between 13 % and 41.6 % more structures, compared to publicly available methods recently used to identify human circular RNAs. CONCLUSIONS: With high sensitivity and specificity, user-adjustable filters that target known sources of false positives, and tailored output to facilitate comparison of transcript levels, PTESFinder will facilitate the discovery and analysis of these poorly understood transcripts.


Assuntos
Processamento Alternativo , Biologia Computacional/métodos , Regulação da Expressão Gênica , Genômica/métodos , RNA , Éxons , Genoma , Humanos , Software
9.
Blood ; 127(9): e1-e11, 2016 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-26660425

RESUMO

In platelets, splicing and translation occur in the absence of a nucleus. However, the integrity and stability of mRNAs derived from megakaryocyte progenitor cells remain poorly quantified on a transcriptome-wide level. As circular RNAs (circRNAs) are resistant to degradation by exonucleases, their abundance relative to linear RNAs can be used as a surrogate marker for mRNA stability in the absence of transcription. Here we show that circRNAs are enriched in human platelets 17- to 188-fold relative to nucleated tissues and 14- to 26-fold relative to samples digested with RNAse R to selectively remove linear RNA. We compare RNAseq read depths inside and outside circRNAs to provide in silico evidence of transcript circularity, show that exons within circRNAs are enriched on average 12.7 times in platelets relative to nucleated tissues and identify 3162 genes significantly enriched for circRNAs, including some where all RNAseq reads appear to be derived from circular molecules. We also confirm that this is a feature of other anucleate cells through transcriptome sequencing of mature erythrocytes, demonstrate that circRNAs are not enriched in cultured megakaryocytes, and demonstrate that linear RNAs decay more rapidly than circRNAs in platelet preparations. Collectively, these results suggest that circulating platelets have lost >90% of their progenitor mRNAs and that translation in platelets occurs against the backdrop of a highly degraded transcriptome. Finally, we find that transcripts previously classified as products of reverse transcriptase template switching are both enriched in platelets and resistant to decay, countering the recent suggestion that up to 50% of rearranged RNAs are artifacts.


Assuntos
Plaquetas/metabolismo , Estabilidade de RNA/genética , RNA/genética , Transcriptoma/genética , Éxons/genética , Exorribonucleases/metabolismo , Humanos , Megacariócitos/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Reprodutibilidade dos Testes
10.
Acta Neuropathol Commun ; 1: 35, 2013 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-24252690

RESUMO

BACKGROUND: Medulloblastomas, the most frequent malignant brain tumours affecting children, comprise at least 4 distinct clinicogenetic subgroups. Aberrant sonic hedgehog (SHH) signalling is observed in approximately 25% of tumours and defines one subgroup. Although alterations in SHH pathway genes (e.g. PTCH1, SUFU) are observed in many of these tumours, high throughput genomic analyses have identified few other recurring mutations. Here, we have mutagenised the Ptch+/- murine tumour model using the Sleeping Beauty transposon system to identify additional genes and pathways involved in SHH subgroup medulloblastoma development. RESULTS: Mutagenesis significantly increased medulloblastoma frequency and identified 17 candidate cancer genes, including orthologs of genes somatically mutated (PTEN, CREBBP) or associated with poor outcome (PTEN, MYT1L) in the human disease. Strikingly, these candidate genes were enriched for transcription factors (p=2x10-5), the majority of which (6/7; Crebbp, Myt1L, Nfia, Nfib, Tead1 and Tgif2) were linked within a single regulatory network enriched for genes associated with a differentiated neuronal phenotype. Furthermore, activity of this network varied significantly between the human subgroups, was associated with metastatic disease, and predicted poor survival specifically within the SHH subgroup of tumours. Igf2, previously implicated in medulloblastoma, was the most differentially expressed gene in murine tumours with network perturbation, and network activity in both mouse and human tumours was characterised by enrichment for multiple gene-sets indicating increased cell proliferation, IGF signalling, MYC target upregulation, and decreased neuronal differentiation. CONCLUSIONS: Collectively, our data support a model of medulloblastoma development in SB-mutagenised Ptch+/- mice which involves disruption of a novel transcription factor network leading to Igf2 upregulation, proliferation of GNPs, and tumour formation. Moreover, our results identify rational therapeutic targets for SHH subgroup tumours, alongside prognostic biomarkers for the identification of poor-risk SHH patients.


Assuntos
Meduloblastoma/genética , Meduloblastoma/metabolismo , Neurônios/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Animais , Neoplasias Encefálicas/diagnóstico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Cerebelo/metabolismo , Cerebelo/patologia , Progressão da Doença , Expressão Gênica , Humanos , Fator de Crescimento Insulin-Like II/genética , Fator de Crescimento Insulin-Like II/metabolismo , Masculino , Meduloblastoma/diagnóstico , Meduloblastoma/patologia , Camundongos , Camundongos Endogâmicos CBA , Camundongos Transgênicos , Receptores Patched , Receptor Patched-1 , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo
11.
PLoS Genet ; 7(12): e1002390, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22194695

RESUMO

Alternative splicing amplifies the information content of the genome, creating multiple mRNA isoforms from single genes. The evolutionarily conserved splicing activator Tra2ß (Sfrs10) is essential for mouse embryogenesis and implicated in spermatogenesis. Here we find that Tra2ß is up-regulated as the mitotic stem cell containing population of male germ cells differentiate into meiotic and post-meiotic cells. Using CLIP coupled to deep sequencing, we found that Tra2ß binds a high frequency of exons and identified specific G/A rich motifs as frequent targets. Significantly, for the first time we have analysed the splicing effect of Sfrs10 depletion in vivo by generating a conditional neuronal-specific Sfrs10 knock-out mouse (Sfrs10(fl/fl); Nestin-Cre(tg/+)). This mouse has defects in brain development and allowed correlation of genuine physiologically Tra2ß regulated exons. These belonged to a novel class which were longer than average size and importantly needed multiple cooperative Tra2ß binding sites for efficient splicing activation, thus explaining the observed splicing defects in the knockout mice. Regulated exons included a cassette exon which produces a meiotic isoform of the Nasp histone chaperone that helps monitor DNA double-strand breaks. We also found a previously uncharacterised poison exon identifying a new pathway of feedback control between vertebrate Tra2 proteins. Both Nasp-T and the Tra2a poison exon are evolutionarily conserved, suggesting they might control fundamental developmental processes. Tra2ß protein isoforms lacking the RRM were able to activate specific target exons indicating an additional functional role as a splicing co-activator. Significantly the N-terminal RS1 domain conserved between flies and humans was essential for the splicing activator function of Tra2ß. Versions of Tra2ß lacking this N-terminal RS1 domain potently repressed the same target exons activated by full-length Tra2ß protein.


Assuntos
Desenvolvimento Embrionário/genética , Éxons/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Processamento Alternativo/genética , Animais , Autoantígenos/genética , Autoantígenos/metabolismo , Sequência de Bases , Sítios de Ligação , Encéfalo/anormalidades , Diferenciação Celular , Quebras de DNA de Cadeia Dupla , Evolução Molecular , Células Germinativas/citologia , Masculino , Meiose/genética , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Fatores de Processamento de Serina-Arginina , Espermatogênese/genética
12.
Genome Res ; 21(11): 1788-99, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21948523

RESUMO

In silico analyses have established that transcripts from some genes can be processed into RNAs with rearranged exon order relative to genomic structure (post-transcriptional exon shuffling, or PTES). Although known to contribute to transcriptome diversity in some species, to date the structure, distribution, abundance, and functional significance of human PTES transcripts remains largely unknown. Here, using high-throughput transcriptome sequencing, we identify 205 putative human PTES products from 176 genes. We validate 72 out of 112 products analyzed using RT-PCR, and identify additional PTES products structurally related to 61% of validated targets. Sequencing of these additional products reveals GT-AG dinucleotides at >95% of the splice junctions, confirming that they are processed by the spliceosome. We show that most PTES transcripts are expressed in a wide variety of human tissues, that they can be polyadenylated, and that some are conserved in mouse. We also show that they can extend into 5' and 3' UTRs, consistent with formation via trans-splicing of independent pre-mRNA molecules. Finally, we use real-time PCR to compare the abundance of PTES exon junctions relative to canonical exon junctions within the transcripts from seven genes. PTES exon junctions are present at <0.01% to >90% of the levels of canonical junctions, with transcripts from MAN1A2, PHC3, TLE4, and CDK13 exhibiting the highest levels. This is the first systematic experimental analysis of PTES in human, and it suggests both that the phenomenon is much more widespread than previously thought and that some PTES transcripts could be functional.


Assuntos
Evolução Molecular , Éxons , Processamento Pós-Transcricional do RNA , Animais , Sequência de Bases , Embaralhamento de DNA , Regulação da Expressão Gênica , Rearranjo Gênico , Humanos , Camundongos , Dados de Sequência Molecular , Estrutura Molecular , Precursores de RNA/metabolismo , Sítios de Splice de RNA , Processamento de RNA , RNA Mensageiro/metabolismo , Transcriptoma , Regiões não Traduzidas/genética
13.
Eur J Cancer ; 43(9): 1467-75, 2007 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17449239

RESUMO

Overexpression of the human MYCN oncogene driven by a tyrosine hydroxylase promoter causes tumours in transgenic mice that recapitulate the childhood cancer neuroblastoma. To establish an in vitro model to study this process, a series of isogenic cell lines were developed from these MYCN-driven murine tumours. Lines were established from tumours arising in homozygous and hemizygous MYCN transgenic mice. Hemizygous tumours gave rise to cell lines growing only in suspension. Homozygous tumours gave rise to similar suspension lines as well as morphologically distinct substrate-adherent lines characteristic of human S-type neuroblastoma cells. FISH analysis demonstrated selective MYCN transgene amplification in cell lines derived from hemizygous mice. Comparative genomic hybridisation (CGH) and fluorescence in situ hybridisation (FISH) analysis confirmed a range of neuroblastoma-associated genetic changes in the various lines, in particular, gain of regions syntenic with human 17q. These isogenic lines together with the transgenic mice thus represent valuable models for investigating the biological characteristics of aggressive neuroblastoma.


Assuntos
Neuroblastoma/genética , Proteínas Nucleares/genética , Proteínas Oncogênicas/genética , Animais , Linhagem Celular Tumoral , Aberrações Cromossômicas , Expressão Gênica , Heterozigoto , Homozigoto , Humanos , Cariotipagem , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Camundongos Transgênicos , Proteína Proto-Oncogênica N-Myc , Transplante de Neoplasias , Ploidias
14.
PLoS Med ; 3(10): e431, 2006 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17076561

RESUMO

BACKGROUND: Sequence analysis of the regulators of complement activation (RCA) cluster of genes at chromosome position 1q32 shows evidence of several large genomic duplications. These duplications have resulted in a high degree of sequence identity between the gene for factor H (CFH) and the genes for the five factor H-related proteins (CFHL1-5; aliases CFHR1-5). CFH mutations have been described in association with atypical haemolytic uraemic syndrome (aHUS). The majority of the mutations are missense changes that cluster in the C-terminal region and impair the ability of factor H to regulate surface-bound C3b. Some have arisen as a result of gene conversion between CFH and CFHL1. In this study we tested the hypothesis that nonallelic homologous recombination between low-copy repeats in the RCA cluster could result in the formation of a hybrid CFH/CFHL1 gene that predisposes to the development of aHUS. METHODS AND FINDINGS: In a family with many cases of aHUS that segregate with the RCA cluster we used cDNA analysis, gene sequencing, and Southern blotting to show that affected individuals carry a heterozygous CFH/CFHL1 hybrid gene in which exons 1-21 are derived from CFH and exons 22/23 from CFHL1. This hybrid encodes a protein product identical to a functionally significant CFH mutant (c.3572C>T, S1191L and c.3590T>C, V1197A) that has been previously described in association with aHUS. CONCLUSIONS: CFH mutation screening is recommended in all aHUS patients prior to renal transplantation because of the high risk of disease recurrence post-transplant in those known to have a CFH mutation. Because of our finding it will be necessary to implement additional screening strategies that will detect a hybrid CFH/CFHL1 gene.


Assuntos
Síndrome Hemolítico-Urêmica/genética , Proteínas Mutantes Quiméricas/genética , Adulto , Idoso de 80 Anos ou mais , Southern Blotting , Proteínas Inativadoras do Complemento C3b/genética , Fator H do Complemento/genética , Proteínas do Sistema Complemento/genética , Quebras de DNA , Análise Mutacional de DNA , Feminino , Dosagem de Genes , Predisposição Genética para Doença , Humanos , Masculino , Pessoa de Meia-Idade , Técnicas de Amplificação de Ácido Nucleico/métodos , Reação em Cadeia da Polimerase/métodos , Análise de Sequência de DNA
15.
Hum Mutat ; 27(3): 292-3, 2006 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-16470555

RESUMO

Many of the complement regulatory genes within the RCA cluster (1q32) have arisen through genomic duplication and the resulting high degree of sequence identity is likely to predispose to gene conversion events. The highest degree of identity is between the genes for factor H (CFH) and five factor H-related proteins--CFHL1, CFHL2, CFHL3, CFHL4, and CFHL5. CFH mutations are associated with atypical hemolytic uremic syndrome (aHUS). In the Newcastle cohort of 157 aHUS patients we have identified CFH mutations in 25 families or individuals. Eleven of these 25 independent mutations are either c.3226C>G,Q1076E; c.3572C>T,S1191L; c.3590T>C,V1197A or combined c.3572C>T,S1191L/c.3590T>C,V1197A. Sequence analysis shows that all four of these changes could have arisen as a result of gene conversion between CFH and CFHL1. Analysis of parental samples in two patients with S1191L/V1197A has shown that the changes are de novo thus providing conclusive evidence that gene conversion is the mutational mechanism in these two cases. To confirm that S1191L and V1197A are disease predisposing we examined their functional significance in three ways - analysis of the C3b/C3d binding characteristics of recombinant mutant S1191L/V1197A protein, heparin affinity chromatography and haemolytic assays of serum samples from aHUS patients carrying these changes. The results showed that these changes resulted in impaired C3b binding and a defective capacity to control complement activation on cellular surfaces. We, therefore, provide conclusive evidence that gene conversion is responsible for functionally significant CFH mutations in aHUS.


Assuntos
Cromossomos Humanos Par 1 , Fator H do Complemento/genética , Síndrome Hemolítico-Urêmica/genética , Família Multigênica , Mutação , Animais , Sequência de Bases , Estudos de Coortes , Eritrócitos/citologia , Conversão Gênica , Humanos , Modelos Genéticos , Dados de Sequência Molecular , Homologia de Sequência do Ácido Nucleico , Ovinos
16.
Am J Hum Genet ; 77(5): 824-40, 2005 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-16252241

RESUMO

Approximately 5% of the human genome consists of segmental duplications that can cause genomic mutations and may play a role in gene innovation. Reticulate evolutionary processes, such as unequal crossing-over and gene conversion, are known to occur within specific duplicon families, but the broader contribution of these processes to the evolution of human duplications remains poorly characterized. Here, we use phylogenetic profiling to analyze multiple alignments of 24 human duplicon families that span >8 Mb of DNA. Our results indicate that none of them are evolving independently, with all alignments showing sharp discontinuities in phylogenetic signal consistent with reticulation. To analyze these results in more detail, we have developed a quartet method that estimates the relative contribution of nucleotide substitution and reticulate processes to sequence evolution. Our data indicate that most of the duplications show a highly significant excess of sites consistent with reticulate evolution, compared with the number expected by nucleotide substitution alone, with 15 of 30 alignments showing a >20-fold excess over that expected. Using permutation tests, we also show that at least 5% of the total sequence shares 100% sequence identity because of reticulation, a figure that includes 74 independent tracts of perfect identity >2 kb in length. Furthermore, analysis of a subset of alignments indicates that the density of reticulation events is as high as 1 every 4 kb. These results indicate that phylogenetic relationships within recently duplicated human DNA can be rapidly disrupted by reticulate evolution. This finding has important implications for efforts to finish the human genome sequence, complicates comparative sequence analysis of duplicon families, and could profoundly influence the tempo of gene-family evolution.


Assuntos
Simulação por Computador , Evolução Molecular , Duplicação Gênica , Genoma Humano , Biologia Computacional , Rearranjo Gênico , Humanos , Análise de Sequência de DNA
17.
Genes Chromosomes Cancer ; 40(2): 158-63, 2004 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15101050

RESUMO

Gain of chromosome arm 17q is the most frequent chromosomal change in human neuroblastoma and is a powerful predictor of adverse outcome of disease. This suggests that the region of gain includes a gene or genes critical for tumor pathogenesis. Analyses of breakpoint positions have revealed that the shortest region of gain (SRG) extends from MPO (17q23.1) to 17qter. Because this encompasses >300 genes, it precludes the identification of candidate genes from human breakpoint data alone. However, mouse chromosome 11, which is syntenic to human chromosome 17, is gained in up to 30% of neuroblastoma tumors developed in a murine MYCN transgenic model of this disease. To confirm that this key genetic change indicates the involvement of a molecular pathway conserved between mouse and man and is not occurring coincidentally in the transgenic model, we used fluorescence in situ hybridization to analyze sporadic cases of both mouse and rat neuroblastoma. Our results confirmed the presence of chromosome 11 gain in all three of the mouse cell lines we analyzed, with the SRG extending from Stat5b (101.6 Mb) to tel. In addition, the rat neuroblastoma cell line harbors an extra copy of distal chromosome 10, extending from 92.8 to 109.3 Mb, which is also syntenic to human 17q. Comparison of the regions gained in all three species has excluded 4.2 Mb from the previously defined region of 17q gain in humans as a likely location of the candidate gene or genes, and strongly suggests that the molecular etiology of neuroblastoma is similar in all three species.


Assuntos
Cromossomos Humanos Par 17/genética , Neuroblastoma/genética , Sintenia/genética , Animais , Linhagem Celular Tumoral , Deleção Cromossômica , Cromossomos/genética , Mapeamento de Sequências Contíguas , Modelos Animais de Doenças , Amplificação de Genes/genética , Marcadores Genéticos/genética , Predisposição Genética para Doença/genética , Humanos , Hibridização in Situ Fluorescente , Camundongos , Camundongos Transgênicos , Neuroblastoma/etiologia , Neuroblastoma/patologia , Ratos
18.
Genome Res ; 13(9): 2059-68, 2003 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-12915487

RESUMO

The existence of latent centromeres has been proposed as a possible explanation for the ectopic emergence of neocentromeres in humans. This hypothesis predicts an association between the position of neocentromeres and the position of ancient centromeres inactivated during karyotypic evolution. Human chromosomal region 15q24-26 is one of several hotspots where multiple cases of neocentromere emergence have been reported, and it harbors a high density of chromosome-specific duplicons, rearrangements of which have been implicated as a susceptibility factor for panic and phobic disorders with joint laxity. We investigated the evolutionary history of this region in primates and found that it contains the site of an ancestral centromere which became inactivated about 25 million years ago, after great apes/Old World monkeys diverged. This inactivation has followed a noncentromeric chromosomal fission of an ancestral chromosome which gave rise to phylogenetic chromosomes XIV and XV in human and great apes. Detailed mapping of the ancient centromere and two neocentromeres in 15q24-26 has established that the neocentromere domains map approximately 8 Mb proximal and 1.5 Mb distal of the ancestral centromeric region, but that all three map within 500 kb of duplicons, copies of which flank the centromere in Old World Monkey species. This suggests that the association between neocentromere and ancestral centromere position on this chromosome may be due to the persistence of recombinogenic duplications accrued within the ancient pericentromere, rather than the retention of "centromere-competent" sequences per se. The high frequency of neocentromere emergence in the 15q24-26 region and the high density of clinically important duplicons are, therefore, understandable in the light of the evolutionary history of this region.


Assuntos
Centrômero/genética , Cromossomos Humanos Par 15/genética , Evolução Molecular , Duplicação Gênica , Animais , Cercopithecidae/genética , Inversão Cromossômica , Cromossomos Humanos Par 14/genética , Rearranjo Gênico/genética , Marcadores Genéticos/genética , Genoma Humano , Humanos , Mapeamento Físico do Cromossomo , Recombinação Genética/genética
19.
Genome Res ; 13(2): 159-72, 2003 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-12566394

RESUMO

Contiguous finished sequence from highly duplicated pericentromeric regions of human chromosomes is needed if we are to understand the role of pericentromeric instability in disease, and in gene and karyotype evolution. Here, we have constructed a BAC contig spanning the transition from pericentromeric satellites to genes on the short arm of human chromosome 10, and used this to generate 1.4 Mb of finished genomic sequence. Combining RT-PCR, in silico gene prediction, and paralogy analysis, we can identify two domains within the sequence. The proximal 600 kb consists of satellite-rich pericentromerically duplicated DNA which is transcript poor, containing only three unspliced transcripts. In contrast, the distal 850 kb contains four known genes (ZNF248, ZNF25, ZNF33A, and ZNF37A) and up to 32 additional transcripts of unknown function. This distal region also contains seven out of the eight intrachromosomal duplications within the sequence, including the p arm copy of the approximately 250-kb duplication which gave rise to ZNF33A and ZNF33B. By sequencing orthologs of the duplicated ZNF33 genes we have established that ZNF33A has diverged significantly at residues critical for DNA binding but ZNF33B has not, indicating that ZNF33B has remained constrained by selection for ancestral gene function. These results provide further evidence of gene formation within intrachromosomal duplications, but indicate that recent interchromosomal duplications at this centromere have involved transcriptionally inert, satellite rich DNA, which is likely to be heterochromatic. This suggests that any novel gene structures formed by these interchromosomal events would require relocation to a more open chromatin environment to be expressed.


Assuntos
Centrômero/genética , Cromossomos Humanos Par 10/genética , DNA Satélite/genética , Perfilação da Expressão Gênica/métodos , Genes/genética , Sequência de Aminoácidos/genética , Animais , Callithrix/genética , Cromossomos Humanos Par 7/genética , Mapeamento de Sequências Contíguas/métodos , Golfinhos , Evolução Molecular , Duplicação Gênica , Humanos , Lorisidae , Dados de Sequência Molecular , Pseudogenes/genética , Proteínas Repressoras/genética , Especificidade da Espécie , Suínos , Telômero/genética , Dedos de Zinco/genética
20.
Genes Chromosomes Cancer ; 34(4): 428-36, 2002 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-12112532

RESUMO

Gain of chromosome arm 17q is a powerful prognostic factor in neuroblastoma, and the distribution of 17q breakpoints suggests that the dosage of one or more genes in 17q22-23 to 17qter is critical for tumor progression. To identify the smallest region of 17q gain, we used eight probes to map translocation breakpoints in 48 primary neuroblastoma tumors. We identified at least five different breakpoints, all localized within the proximal part of 17q (from D17Z1 to MPO). The shortest region of gain identified by these probes extends from MPO (17q23.1) to 17qter. Surprisingly, we found that breakpoints localized proximal to ERBB2 (17q12) were associated with significantly better patient survival than breakpoints localized distal to ERBB2. Breakpoints localized distal to ERBB2 identified patients with a particularly poor prognosis, higher mitotic karyorrhectic index, and stage 4 disease. This implies that breakpoint position on 17q is a discriminative factor within this prognostically poor group of patients. This result also suggests that the biological effect of 17q gain during neuroblastoma progression has a complex basis. We propose that this involves dosage alterations of genes localized on both sides of the 17q breakpoints, with a gene or genes mapping between 17cen and 17q12 acting to suppress progression, and a gene or genes mapping between 17q23.1 and 17qter acting to promote tumor progression.


Assuntos
Quebra Cromossômica/genética , Mapeamento Cromossômico , Cromossomos Humanos Par 17/genética , Neuroblastoma/genética , Neuroblastoma/patologia , Biomarcadores Tumorais/genética , Genes erbB-2/genética , Humanos , Invasividade Neoplásica/genética , Estadiamento de Neoplasias , Prognóstico , Análise de Sobrevida , Células Tumorais Cultivadas
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