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1.
BMC Genomics ; 22(1): 872, 2021 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-34863095

RESUMO

BACKGROUND: Pediatric cancers typically have a distinct genomic landscape when compared to adult cancers and frequently carry somatic gene fusion events that alter gene expression and drive tumorigenesis. Sensitive and specific detection of gene fusions through the analysis of next-generation-based RNA sequencing (RNA-Seq) data is computationally challenging and may be confounded by low tumor cellularity or underlying genomic complexity. Furthermore, numerous computational tools are available to identify fusions from supporting RNA-Seq reads, yet each algorithm demonstrates unique variability in sensitivity and precision, and no clearly superior approach currently exists. To overcome these challenges, we have developed an ensemble fusion calling approach to increase the accuracy of identifying fusions. RESULTS: Our Ensemble Fusion (EnFusion) approach utilizes seven fusion calling algorithms: Arriba, CICERO, FusionMap, FusionCatcher, JAFFA, MapSplice, and STAR-Fusion, which are packaged as a fully automated pipeline using Docker and Amazon Web Services (AWS) serverless technology. This method uses paired end RNA-Seq sequence reads as input, and the output from each algorithm is examined to identify fusions detected by a consensus of at least three algorithms. These consensus fusion results are filtered by comparison to an internal database to remove likely artifactual fusions occurring at high frequencies in our internal cohort, while a "known fusion list" prevents failure to report known pathogenic events. We have employed the EnFusion pipeline on RNA-Seq data from 229 patients with pediatric cancer or blood disorders studied under an IRB-approved protocol. The samples consist of 138 central nervous system tumors, 73 solid tumors, and 18 hematologic malignancies or disorders. The combination of an ensemble fusion-calling pipeline and a knowledge-based filtering strategy identified 67 clinically relevant fusions among our cohort (diagnostic yield of 29.3%), including RBPMS-MET, BCAN-NTRK1, and TRIM22-BRAF fusions. Following clinical confirmation and reporting in the patient's medical record, both known and novel fusions provided medically meaningful information. CONCLUSIONS: The EnFusion pipeline offers a streamlined approach to discover fusions in cancer, at higher levels of sensitivity and accuracy than single algorithm methods. Furthermore, this method accurately identifies driver fusions in pediatric cancer, providing clinical impact by contributing evidence to diagnosis and, when appropriate, indicating targeted therapies.


Assuntos
Genoma , Neoplasias , Criança , Genômica , Humanos , Neoplasias/genética , Análise de Sequência de DNA , Análise de Sequência de RNA
2.
New Phytol ; 216(4): 1247-1255, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28906557

RESUMO

The Leavenworthia self-incompatibility locus (S locus) consists of paralogs (Lal2, SCRL) of the canonical Brassicaceae S locus genes (SRK, SCR), and is situated in a genomic position that differs from the ancestral one in the Brassicaceae. Unexpectedly, in a small number of Leavenworthia alabamica plants examined, sequences closely resembling exon 1 of SRK have been found, but the function of these has remained unclear. BAC cloning and expression analyses were employed to characterize these SRK-like sequences. An SRK-positive Bacterial Artificial Chromosome clone was found to contain complete SRK and SCR sequences located close by one another in the derived genomic position of the Leavenworthia S locus, and in place of the more typical Lal2 and SCRL sequences. These sequences are expressed in stigmas and anthers, respectively, and crossing data show that the SRK/SCR haplotype is functional in self-incompatibility. Population surveys indicate that < 5% of Leavenworthia S loci possess such alleles. An ancestral translocation or recombination event involving SRK/SCR and Lal2/SCRL likely occurred, together with neofunctionalization of Lal2/SCRL, and both haplotype groups now function as Leavenworthia S locus alleles. These findings suggest that S locus alleles can have distinctly different evolutionary origins.


Assuntos
Brassicaceae/genética , Autoincompatibilidade em Angiospermas/genética , Brassicaceae/metabolismo , Flores/metabolismo , Genoma de Planta , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Homologia de Sequência do Ácido Nucleico
3.
PLoS Biol ; 11(5): e1001560, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23690750

RESUMO

Self-incompatibility (SI) is the flowering plant reproductive system in which self pollen tube growth is inhibited, thereby preventing self-fertilization. SI has evolved independently in several different flowering plant lineages. In all Brassicaceae species in which the molecular basis of SI has been investigated in detail, the product of the S-locus receptor kinase (SRK) gene functions as receptor in the initial step of the self pollen-rejection pathway, while that of the S-locus cysteine-rich (SCR) gene functions as ligand. Here we examine the hypothesis that the S locus in the Brassicaceae genus Leavenworthia is paralogous with the S locus previously characterized in other members of the family. We also test the hypothesis that self-compatibility in this group is based on disruption of the pollen ligand-producing gene. Sequence analysis of the S-locus genes in Leavenworthia, phylogeny of S alleles, gene expression patterns, and comparative genomics analyses provide support for both hypotheses. Of special interest are two genes located in a non-S locus genomic region of Arabidopsis lyrata that exhibit domain structures, sequences, and phylogenetic histories similar to those of the S-locus genes in Leavenworthia, and that also share synteny with these genes. These A. lyrata genes resemble those comprising the A. lyrata S locus, but they do not function in self-recognition. Moreover, they appear to belong to a lineage that diverged from the ancestral Brassicaceae S-locus genes before allelic diversification at the S locus. We hypothesize that there has been neo-functionalization of these S-locus-like genes in the Leavenworthia lineage, resulting in evolution of a separate ligand-receptor system of SI. Our results also provide support for theoretical models that predict that the least constrained pathway to the evolution of self-compatibility is one involving loss of pollen gene function.


Assuntos
Brassicaceae/genética , Evolução Molecular , Alelos , Sequência de Aminoácidos , Brassicaceae/classificação , Regulação da Expressão Gênica de Plantas , Humanos , Dados de Sequência Molecular , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Alinhamento de Sequência
4.
Mol Ther Methods Clin Dev ; 8: 121-130, 2018 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-29387734

RESUMO

RNAi emerged as a prospective molecular therapy nearly 15 years ago. Since then, two major RNAi platforms have been under development: oligonucleotides and gene therapy. Oligonucleotide-based approaches have seen more advancement, with some promising therapies that may soon reach market. In contrast, vector-based approaches for RNAi therapy have remained largely in the pre-clinical realm, with limited clinical safety and efficacy data to date. We are developing a gene therapy approach to treat the autosomal-dominant disorder facioscapulohumeral muscular dystrophy. Our strategy involves silencing the myotoxic gene DUX4 using adeno-associated viral vectors to deliver targeted microRNA expression cassettes (miDUX4s). We previously demonstrated proof of concept for this approach in mice, and we are now taking additional steps here to assess safety issues related to miDUX4 overexpression and sequence-specific off-target silencing. In this study, we describe improvements in vector design and expansion of our miDUX4 sequence repertoire and report differential toxicity elicited by two miDUX4 sequences, of which one was toxic and the other was not. This study provides important data to help advance our goal of translating RNAi gene therapy for facioscapulohumeral muscular dystrophy.

5.
Evolution ; 70(6): 1212-24, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27139712

RESUMO

The evolution of self-compatibility (SC) is the first step in the evolutionary transition in plants from outcrossing enforced by self-incompatibility (SI) to self-fertilization. In the Brassicaceae, SI is controlled by alleles of two tightly linked genes at the S-locus. Despite permitting inbreeding, mutations at the S-locus leading to SC may be selected if they provide reproductive assurance and/or gain a transmission advantage in a population when SC plants self- and outcross. Positive selection can leave a genomic signature in the regions physically linked to the focus of selection when selection has occurred recently. From an SC population of Leavenworthia alabamica with a known nonfunctional mutation at the S-locus, we collected sequence data from a ∼690 Kb region surrounding the S-locus, as well as from regions not linked to the S-locus. To test for recent positive selection acting at the S-locus, we examined polymorphism and the site-frequency spectra. Using forward simulations, we demonstrate that recent selection of the strength expected for SC at a locus formerly under balancing selection can generate patterns similar to those seen in our empirical data.


Assuntos
Brassicaceae/fisiologia , Polinização , Autofertilização , Alabama , Reprodução
6.
Evolution ; 66(6): 1849-61, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22671551

RESUMO

The adoption of self-fertilization from an ancestral outcrossing state is one of the most common evolutionary transitions in the flowering plants. In the mustard family, outcrossing is typically enforced by sporophytic self-incompatibility (SI), but there are also many self-compatible species. The genus Leavenworthia contains taxa that either possess or lack SI. Here, we present data showing that SI is associated with strict outcrossing and that there is widespread trans-specific sequence polymorphism at the locus involved in the recognition of self-pollen (the S-locus). This ancestral polymorphism is consistent with the presence of an outcrossing mating system in the common ancestor of Leavenworthia species, and suggests that there have been several independent losses of SI in the group. When compared with other mustard species, the bulk of Leavenworthia S-allele sequences are highly diverged from those found in other Brassicaceae and show relatively low levels of nucleotide diversity, a pattern that suggests the common ancestor of the genus likely underwent a strong population bottleneck. The hypothesis of postbottleneck S-locus rediversification is supported by tests showing stronger positive selection acting on S-alleles from Leavenworthia than those found in other Brassicaceae.


Assuntos
Alelos , Brassicaceae/genética , Evolução Molecular , Filogenia , Brassicaceae/fisiologia , Códon , Genes de Plantas , Seleção Genética
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