Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 95
Filtrar
Mais filtros

Base de dados
País/Região como assunto
Tipo de documento
Intervalo de ano de publicação
1.
Ann Neurol ; 94(4): 696-712, 2023 10.
Artigo em Inglês | MEDLINE | ID: mdl-37255483

RESUMO

OBJECTIVE: Primary mitochondrial diseases (PMDs) are heterogeneous disorders caused by inherited mitochondrial dysfunction. Classically defined neuropathologically as subacute necrotizing encephalomyelopathy, Leigh syndrome spectrum (LSS) is the most frequent manifestation of PMD in children, but may also present in adults. A major challenge for accurate diagnosis of LSS in the genomic medicine era is establishing gene-disease relationships (GDRs) for this syndrome with >100 monogenic causes across both nuclear and mitochondrial genomes. METHODS: The Clinical Genome Resource (ClinGen) Mitochondrial Disease Gene Curation Expert Panel (GCEP), comprising 40 international PMD experts, met monthly for 4 years to review GDRs for LSS. The GCEP standardized gene curation for LSS by refining the phenotypic definition, modifying the ClinGen Gene-Disease Clinical Validity Curation Framework to improve interpretation for LSS, and establishing a scoring rubric for LSS. RESULTS: The GDR with LSS across the nuclear and mitochondrial genomes was classified as definitive for 31 of 114 GDRs curated (27%), moderate for 38 (33%), limited for 43 (38%), and disputed for 2 (2%). Ninety genes were associated with autosomal recessive inheritance, 16 were maternally inherited, 5 were autosomal dominant, and 3 were X-linked. INTERPRETATION: GDRs for LSS were established for genes across both nuclear and mitochondrial genomes. Establishing these GDRs will allow accurate variant interpretation, expedite genetic diagnosis of LSS, and facilitate precision medicine, multisystem organ surveillance, recurrence risk counseling, reproductive choice, natural history studies, and determination of eligibility for interventional clinical trials. ANN NEUROL 2023;94:696-712.


Assuntos
Doença de Leigh , Doenças Mitocondriais , Criança , Humanos , Doença de Leigh/diagnóstico , Doença de Leigh/genética , Mitocôndrias
2.
Int J Mol Sci ; 23(11)2022 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-35682905

RESUMO

Tumor biopsy can identify prognostic biomarkers for metastatic uveal melanoma (UM), however aqueous humor (AH) liquid biopsy may serve as an adjunct. This study investigated whether the AH of UM eyes has sufficient circulating tumor DNA (ctDNA) to perform genetic analysis. This is a case series of 37 AH samples, taken before or after radiation, and one tumor wash sample, from 12 choroidal and 8 ciliary body (CB) melanoma eyes. AH was analyzed for nucleic acid concentrations. AH DNA and one tumor wash sample underwent shallow whole-genome sequencing followed by Illumina sequencing to detect somatic copy number alterations (SCNAs). Four post-radiation AH underwent targeted sequencing of BAP1 and GNAQ genes. Post-radiation AH had significantly higher DNA and miRNA concentrations than paired pre-radiation samples. Highly recurrent UM SCNAs were identified in 0/11 post-radiation choroidal and 6/8 post-radiation CB AH. SCNAs were highly concordant in a CB post-radiation AH with its matched tumor (r = 0.978). BAP1 or GNAQ variants were detected in 3/4 post-radiation AH samples. AH is a source of ctDNA in UM eyes, particularly in post-radiation CB eyes. For the first time, UM SCNAs and mutations were identified in AH-derived ctDNA. Suggesting that AH can serve as a liquid biopsy for UM.


Assuntos
Melanoma , Neoplasias Uveais , Humor Aquoso , Humanos , Biópsia Líquida , Melanoma/diagnóstico , Melanoma/genética , Melanoma/patologia , Mutação , Recidiva Local de Neoplasia , Neoplasias Uveais/diagnóstico , Neoplasias Uveais/genética , Neoplasias Uveais/patologia
3.
Bioinformatics ; 36(15): 4353-4356, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32484858

RESUMO

SUMMARY: A number of methods have been devised to address the need for targeted genomic resequencing. One of these methods, region-specific extraction (RSE) is characterized by the capture of long DNA fragments (15-20 kb) by magnetic beads, after enzymatic extension of oligonucleotides hybridized to selected genomic regions. Facilitating the selection of the most appropriate capture oligos for targeting a region of interest, satisfying the properties of temperature (Tm) and entropy (ΔG), while minimizing the formation of primer-dimers in a pooled experiment, is therefore necessary. Manual design and selection of oligos becomes very challenging, complicated by factors such as length of the target region and number of targeted regions. Here we describe, AnthOligo, a web-based application developed to optimally automate the process of generation of oligo sequences used to target and capture the continuum of large and complex genomic regions. Apart from generating oligos for RSE, this program may have wider applications in the design of customizable internal oligos to be used as baits for gene panel analysis or even probes for large-scale comparative genomic hybridization array processes. AnthOligo was tested by capturing the Major Histocompatibility Complex (MHC) of a random sample.The application provides users with a simple interface to upload an input file in BED format and customize parameters for each task. The task of probe design in AnthOligo commences when a user uploads an input file and concludes with the generation of a result-set containing an optimal set of region-specific oligos. AnthOligo is currently available as a public web application with URL: http://antholigo.chop.edu. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.


Assuntos
Genoma , Genômica , Hibridização Genômica Comparativa , Complexo Principal de Histocompatibilidade , Oligonucleotídeos/genética
4.
Genes Chromosomes Cancer ; 59(10): 584-590, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32390242

RESUMO

Retinoblastoma (RB) is a childhood intraocular cancer initiated by biallelic inactivation of the RB tumor suppressor gene (RB1-/- ). RB can be hereditary (germline RB1 pathogenic allele is present) or non-hereditary. Somatic copy number alterations (SCNAs) contribute to subsequent tumorigenesis. Previous studies of only enucleated RB eyes have reported associations between heritability status and the prevalence of SCNAs. Herein, we use an aqueous humor (AH) liquid biopsy to investigate RB genomic profiles in the context of germline RB1 status, age, and International Intraocular Retinoblastoma Classification (IIRC) clinical grouping for both enucleated and salvaged eyes. Between 2014 and 2019, AH was sampled from a total of 54 eyes of 50 patients. Germline RB1 status was determined from clinical blood testing, and cell-free DNA from AH was analyzed for SCNAs. Of the 50 patients, 23 (46.0%; 27 eyes) had hereditary RB, and 27 (54.0%, 27 eyes) had non-hereditary RB. Median age at diagnosis was comparable between hereditary (13 ± 10 months) and non-hereditary (13 ± 8 months) eyes (P = 0.818). There was no significant difference in the prevalence or number of SCNAs based on (1) hereditary status (P > 0.56) or (2) IIRC grouping (P > 0.47). There was, however, a significant correlation between patient age at diagnosis, and (1) number of total SCNAs (r[52] = 0.672, P < 0.00001) and (2) number of highly-recurrent RB SCNAs (r[52] = 0.616, P < 0.00001). This evidence does not support the theory that specific molecular or genomic subtypes exist between hereditary and non-hereditary RB; rather, the prevalence of genomic alterations in RB eyes is strongly related to patient age at diagnosis.


Assuntos
Instabilidade Genômica , Neoplasias da Retina/genética , Retinoblastoma/genética , Fatores Etários , Criança , Pré-Escolar , Variações do Número de Cópias de DNA , Testes Genéticos/estatística & dados numéricos , Células Germinativas/metabolismo , Humanos , Lactente , Prevalência , Retina/metabolismo , Neoplasias da Retina/diagnóstico , Neoplasias da Retina/epidemiologia , Retinoblastoma/diagnóstico , Retinoblastoma/epidemiologia , Proteínas de Ligação a Retinoblastoma/genética , Ubiquitina-Proteína Ligases/genética
5.
Hum Mutat ; 41(12): 2028-2057, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32906214

RESUMO

Mitochondrial DNA (mtDNA) variant pathogenicity interpretation has special considerations given unique features of the mtDNA genome, including maternal inheritance, variant heteroplasmy, threshold effect, absence of splicing, and contextual effects of haplogroups. Currently, there are insufficient standardized criteria for mtDNA variant assessment, which leads to inconsistencies in clinical variant pathogenicity reporting. An international working group of mtDNA experts was assembled within the Mitochondrial Disease Sequence Data Resource Consortium and obtained Expert Panel status from ClinGen. This group reviewed the 2015 American College of Medical Genetics and Association of Molecular Pathology standards and guidelines that are widely used for clinical interpretation of DNA sequence variants and provided further specifications for additional and specific guidance related to mtDNA variant classification. These Expert Panel consensus specifications allow for consistent consideration of the unique aspects of the mtDNA genome that directly influence variant assessment, including addressing mtDNA genome composition and structure, haplogroups and phylogeny, maternal inheritance, heteroplasmy, and functional analyses unique to mtDNA, as well as specifications for utilization of mtDNA genomic databases and computational algorithms.


Assuntos
DNA Mitocondrial/genética , Variação Genética , Guias como Assunto , Sociedades Científicas , Bases de Dados Genéticas , Árvores de Decisões , Haplótipos/genética , Humanos , Fenótipo , Padrões de Referência
6.
Hum Mol Genet ; 27(19): 3305-3312, 2018 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-29917077

RESUMO

Leigh syndrome is a frequent, heterogeneous pediatric presentation of mitochondrial oxidative phosphorylation (OXPHOS) disease, manifesting with psychomotor retardation and necrotizing lesions in brain deep gray matter. OXPHOS occurs at the inner mitochondrial membrane through the integrated activity of five protein complexes, of which complex V (CV) functions in a dimeric form to directly generate adenosine triphosphate (ATP). Mutations in several different structural CV subunits cause Leigh syndrome; however, dimerization defects have not been associated with human disease. We report four Leigh syndrome subjects from three unrelated Ashkenazi Jewish families harboring a homozygous splice-site mutation (c.87 + 1G>C) in a novel CV subunit disease gene, USMG5. The Ashkenazi population allele frequency is 0.57%. This mutation produces two USMG5 transcripts, wild-type and lacking exon 3. Fibroblasts from two Leigh syndrome probands had reduced wild-type USMG5 mRNA expression and undetectable protein. The mutation did not alter monomeric CV expression, but reduced both CV dimer expression and ATP synthesis rate. Rescue with wild-type USMG5 cDNA in proband fibroblasts restored USMG5 protein, increased CV dimerization and enhanced ATP production rate. These data demonstrate that a recurrent USMG5 splice-site founder mutation in the Ashkenazi Jewish population causes autosomal recessive Leigh syndrome by reduction of CV dimerization and ATP synthesis.


Assuntos
Doença de Leigh/genética , Mitocôndrias/genética , Doenças Mitocondriais/genética , ATPases Mitocondriais Próton-Translocadoras/genética , Trifosfato de Adenosina/biossíntese , Criança , Pré-Escolar , Dimerização , Éxons/genética , Efeito Fundador , Frequência do Gene , Haplótipos , Humanos , Lactente , Recém-Nascido , Judeus/genética , Doença de Leigh/metabolismo , Doença de Leigh/patologia , Masculino , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Doenças Mitocondriais/metabolismo , Doenças Mitocondriais/patologia , Mutação , Fosforilação Oxidativa , Sítios de Splice de RNA/genética , Sequenciamento do Exoma
7.
Hum Mutat ; 39(6): 806-810, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29539190

RESUMO

Accurate mitochondrial DNA (mtDNA) variant annotation is essential for the clinical diagnosis of diverse human diseases. Substantial challenges to this process include the inconsistency in mtDNA nomenclatures, the existence of multiple reference genomes, and a lack of reference population frequency data. Clinicians need a simple bioinformatics tool that is user-friendly, and bioinformaticians need a powerful informatics resource for programmatic usage. Here, we report the development and functionality of the MSeqDR mtDNA Variant Tool set (mvTool), a one-stop mtDNA variant annotation and analysis Web service. mvTool is built upon the MSeqDR infrastructure (https://mseqdr.org), with contributions of expert curated data from MITOMAP (https://www.mitomap.org) and HmtDB (https://www.hmtdb.uniba.it/hmdb). mvTool supports all mtDNA nomenclatures, converts variants to standard rCRS- and HGVS-based nomenclatures, and annotates novel mtDNA variants. Besides generic annotations from dbNSFP and Variant Effect Predictor (VEP), mvTool provides allele frequencies in more than 47,000 germline mitogenomes, and disease and pathogenicity classifications from MSeqDR, Mitomap, HmtDB and ClinVar (Landrum et al., 2013). mvTools also provides mtDNA somatic variants annotations. "mvTool API" is implemented for programmatic access using inputs in VCF, HGVS, or classical mtDNA variant nomenclatures. The results are reported as hyperlinked html tables, JSON, Excel, and VCF formats. MSeqDR mvTool is freely accessible at https://mseqdr.org/mvtool.php.


Assuntos
DNA Mitocondrial/genética , Doenças Genéticas Inatas/genética , Genoma Mitocondrial/genética , Mitocôndrias/genética , Biologia Computacional , Bases de Dados Genéticas , Doenças Genéticas Inatas/patologia , Humanos , Anotação de Sequência Molecular , Software
8.
Am J Hum Genet ; 96(5): 753-64, 2015 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-25892112

RESUMO

The 22q11.2 deletion syndrome (22q11DS; velocardiofacial/DiGeorge syndrome; VCFS/DGS) is the most common microdeletion syndrome and the phenotypic presentation is highly variable. Approximately 65% of individuals with 22q11DS have a congenital heart defect (CHD), mostly of the conotruncal type, and/or an aortic arch defect. The etiology of this phenotypic variability is not currently known. We hypothesized that copy-number variants (CNVs) outside the 22q11.2 deleted region might increase the risk of being born with a CHD in this sensitized population. Genotyping with Affymetrix SNP Array 6.0 was performed on two groups of subjects with 22q11DS separated by time of ascertainment and processing. CNV analysis was completed on a total of 949 subjects (cohort 1, n = 562; cohort 2, n = 387), 603 with CHDs (cohort 1, n = 363; cohort 2, n = 240) and 346 with normal cardiac anatomy (cohort 1, n = 199; cohort 2, n = 147). Our analysis revealed that a duplication of SLC2A3 was the most frequent CNV identified in the first cohort. It was present in 18 subjects with CHDs and 1 subject without (p = 3.12 × 10(-3), two-tailed Fisher's exact test). In the second cohort, the SLC2A3 duplication was also significantly enriched in subjects with CHDs (p = 3.30 × 10(-2), two-tailed Fisher's exact test). The SLC2A3 duplication was the most frequent CNV detected and the only significant finding in our combined analysis (p = 2.68 × 10(-4), two-tailed Fisher's exact test), indicating that the SLC2A3 duplication might serve as a genetic modifier of CHDs and/or aortic arch anomalies in individuals with 22q11DS.


Assuntos
Variações do Número de Cópias de DNA/genética , Síndrome de DiGeorge/genética , Transportador de Glucose Tipo 3/genética , Cardiopatias Congênitas/genética , Adulto , Aorta Torácica/fisiopatologia , Síndrome de DiGeorge/fisiopatologia , Feminino , Genótipo , Cardiopatias Congênitas/fisiopatologia , Humanos , Masculino , Polimorfismo de Nucleotídeo Único
9.
Hum Mol Genet ; 24(1): 230-42, 2015 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-25168386

RESUMO

Primary cilia are sensory organelles present on most mammalian cells. The assembly and maintenance of primary cilia are facilitated by intraflagellar transport (IFT), a bidirectional protein trafficking along the cilium. Mutations in genes coding for IFT components have been associated with a group of diseases called ciliopathies. These genetic disorders can affect a variety of organs including the retina. Using whole exome sequencing in three families, we identified mutations in Intraflagellar Transport 172 Homolog [IFT172 (Chlamydomonas)] that underlie an isolated retinal degeneration and Bardet-Biedl syndrome. Extensive functional analyses of the identified mutations in cell culture, rat retina and in zebrafish demonstrated their hypomorphic or null nature. It has recently been reported that mutations in IFT172 cause a severe ciliopathy syndrome involving skeletal, renal, hepatic and retinal abnormalities (Jeune and Mainzer-Saldino syndromes). Here, we report for the first time that mutations in this gene can also lead to an isolated form of retinal degeneration. The functional data for the mutations can partially explain milder phenotypes; however, the involvement of modifying alleles in the IFT172-associated phenotypes cannot be excluded. These findings expand the spectrum of disease associated with mutations in IFT172 and suggest that mutations in genes originally reported to be associated with syndromic ciliopathies should also be considered in subjects with non-syndromic retinal dystrophy.


Assuntos
Síndrome de Bardet-Biedl/genética , Síndrome de Bardet-Biedl/patologia , Proteínas de Transporte/genética , Retina/patologia , Retinose Pigmentar/genética , Retinose Pigmentar/patologia , Proteínas Adaptadoras de Transdução de Sinal , Adolescente , Adulto , Animais , Células Cultivadas , Proteínas do Citoesqueleto , Exoma , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Mutação , Linhagem , Ratos , Retina/metabolismo , Análise de Sequência de DNA , Adulto Jovem , Peixe-Zebra
11.
Genet Med ; 19(6): 643-651, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-27735924

RESUMO

PURPOSE: Despite substantial progress in sequencing, current strategies can genetically solve only approximately 55-60% of inherited retinal degeneration (IRD) cases. This can be partially attributed to elusive mutations in the known IRD genes, which are not easily identified by the targeted next-generation sequencing (NGS) or Sanger sequencing approaches. We hypothesized that copy-number variations (CNVs) are a major contributor to the elusive genetic causality of IRDs. METHODS: Twenty-eight cases previously unsolved with a targeted NGS were investigated with whole-genome single-nucleotide polymorphism (SNP) and comparative genomic hybridization (CGH) arrays. RESULTS: Deletions in the IRD genes were detected in 5 of 28 families, including a de novo deletion. We suggest that the de novo deletion occurred through nonallelic homologous recombination (NAHR) and we constructed a genomic map of NAHR-prone regions with overlapping IRD genes. In this article, we also report an unusual case of recessive retinitis pigmentosa due to compound heterozygous mutations in SNRNP200, a gene that is typically associated with the dominant form of this disease. CONCLUSIONS: CNV mapping substantially increased the genetic diagnostic rate of IRDs, detecting genetic causality in 18% of previously unsolved cases. Extending the search to other structural variations will probably demonstrate an even higher contribution to genetic causality of IRDs.Genet Med advance online publication 13 October 2016.


Assuntos
Variações do Número de Cópias de DNA , Degeneração Retiniana/genética , Adolescente , Criança , Mapeamento Cromossômico , Estudos de Coortes , Hibridização Genômica Comparativa , Saúde da Família , Feminino , Deleção de Genes , Predisposição Genética para Doença , Genoma , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único
12.
Am J Med Genet A ; 173(5): 1390-1395, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28371217

RESUMO

We report a family in which two brothers had an undiagnosed genetic disorder comprised of dysmorphic features, microcephaly, severe intellectual disability (non-verbal), mild anemia, and cryptorchidism. Both developed osteosarcoma. Trio exome sequencing (using blood samples from the younger brother and both parents) was performed and a nonsense NM_000489.4:c.7156C>T (p.Arg2386*) mutation in the ATRX gene was identified in the proband (hemizygous) and in the mother's peripheral blood DNA (heterozygous). The mother is healthy, does not exhibit any clinical manifestations of ATR-X syndrome and there was no family history of cancer. The same hemizygous pathogenic variant was confirmed in the affected older brother's skin tissue by subsequent Sanger sequencing. Chromosomal microarray studies of both brothers' osteosarcomas revealed complex copy number alterations consistent with the clinical diagnosis of osteosarcoma. Recently, somatic mutations in the ATRX gene have been observed as recurrent alterations in both osteosarcoma and brain tumors. However, it is unclear if there is any association between osteosarcoma and germline ATRX mutations, specifically in patients with constitutional ATR-X syndrome. This is the first report of osteosarcoma diagnosed in two males with ATR-X syndrome, suggesting a potential increased risk for cancer in patients with this disorder.


Assuntos
DNA Helicases/genética , Deficiência Intelectual/genética , Deficiência Intelectual Ligada ao Cromossomo X/genética , Proteínas Nucleares/genética , Osteossarcoma/genética , Talassemia alfa/genética , Adolescente , Adulto , Sequência de Bases , Exoma/genética , Feminino , Mutação em Linhagem Germinativa , Heterozigoto , Humanos , Deficiência Intelectual/complicações , Deficiência Intelectual/fisiopatologia , Masculino , Deficiência Intelectual Ligada ao Cromossomo X/complicações , Deficiência Intelectual Ligada ao Cromossomo X/fisiopatologia , Osteossarcoma/complicações , Osteossarcoma/fisiopatologia , Linhagem , Irmãos , Proteína Nuclear Ligada ao X , Talassemia alfa/complicações , Talassemia alfa/fisiopatologia
13.
Hum Mutat ; 37(6): 540-548, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-26919060

RESUMO

MSeqDR is the Mitochondrial Disease Sequence Data Resource, a centralized and comprehensive genome and phenome bioinformatics resource built by the mitochondrial disease community to facilitate clinical diagnosis and research investigations of individual patient phenotypes, genomes, genes, and variants. A central Web portal (https://mseqdr.org) integrates community knowledge from expert-curated databases with genomic and phenotype data shared by clinicians and researchers. MSeqDR also functions as a centralized application server for Web-based tools to analyze data across both mitochondrial and nuclear DNA, including investigator-driven whole exome or genome dataset analyses through MSeqDR-Genesis. MSeqDR-GBrowse genome browser supports interactive genomic data exploration and visualization with custom tracks relevant to mtDNA variation and mitochondrial disease. MSeqDR-LSDB is a locus-specific database that currently manages 178 mitochondrial diseases, 1,363 genes associated with mitochondrial biology or disease, and 3,711 pathogenic variants in those genes. MSeqDR Disease Portal allows hierarchical tree-style disease exploration to evaluate their unique descriptions, phenotypes, and causative variants. Automated genomic data submission tools are provided that capture ClinVar compliant variant annotations. PhenoTips will be used for phenotypic data submission on deidentified patients using human phenotype ontology terminology. The development of a dynamic informed patient consent process to guide data access is underway to realize the full potential of these resources.


Assuntos
Biologia Computacional/métodos , Bases de Dados Genéticas , Doenças Mitocondriais/genética , Variação Genética , Genoma Mitocondrial , Genômica , Humanos , Disseminação de Informação , Interface Usuário-Computador , Navegador
14.
BMC Genomics ; 17: 486, 2016 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-27393338

RESUMO

BACKGROUND: The ability to capture and sequence large contiguous DNA fragments represents a significant advancement towards the comprehensive characterization of complex genomic regions. While emerging sequencing platforms are capable of producing several kilobases-long reads, the fragment sizes generated by current DNA target enrichment technologies remain a limiting factor, producing DNA fragments generally shorter than 1 kbp. The DNA enrichment methodology described herein, Region-Specific Extraction (RSE), produces DNA segments in excess of 20 kbp in length. Coupling this enrichment method to appropriate sequencing platforms will significantly enhance the ability to generate complete and accurate sequence characterization of any genomic region without the need for reference-based assembly. RESULTS: RSE is a long-range DNA target capture methodology that relies on the specific hybridization of short (20-25 base) oligonucleotide primers to selected sequence motifs within the DNA target region. These capture primers are then enzymatically extended on the 3'-end, incorporating biotinylated nucleotides into the DNA. Streptavidin-coated beads are subsequently used to pull-down the original, long DNA template molecules via the newly synthesized, biotinylated DNA that is bound to them. We demonstrate the accuracy, simplicity and utility of the RSE method by capturing and sequencing a 4 Mbp stretch of the major histocompatibility complex (MHC). Our results show an average depth of coverage of 164X for the entire MHC. This depth of coverage contributes significantly to a 99.94 % total coverage of the targeted region and to an accuracy that is over 99.99 %. CONCLUSIONS: RSE represents a cost-effective target enrichment method capable of producing sequencing templates in excess of 20 kbp in length. The utility of our method has been proven to generate superior coverage across the MHC as compared to other commercially available methodologies, with the added advantage of producing longer sequencing templates amenable to DNA sequencing on recently developed platforms. Although our demonstration of the method does not utilize these DNA sequencing platforms directly, our results indicate that the capture of long DNA fragments produce superior coverage of the targeted region.


Assuntos
Variação Genética , Genoma Humano , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Hibridização Genômica Comparativa/métodos , Primers do DNA , Bases de Dados Genéticas , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Complexo Principal de Histocompatibilidade/genética , Hibridização de Ácido Nucleico , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de DNA
15.
Am J Hum Genet ; 93(3): 482-95, 2013 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-23993194

RESUMO

Whole-exome sequencing and autozygosity mapping studies, independently performed in subjects with defective combined mitochondrial OXPHOS-enzyme deficiencies, identified a total of nine disease-segregating FBXL4 mutations in seven unrelated mitochondrial disease families, composed of six singletons and three siblings. All subjects manifested early-onset lactic acidemia, hypotonia, and developmental delay caused by severe encephalomyopathy consistently associated with progressive cerebral atrophy and variable involvement of the white matter, deep gray nuclei, and brainstem structures. A wide range of other multisystem features were variably seen, including dysmorphism, skeletal abnormalities, poor growth, gastrointestinal dysmotility, renal tubular acidosis, seizures, and episodic metabolic failure. Mitochondrial respiratory chain deficiency was present in muscle or fibroblasts of all tested individuals, together with markedly reduced oxygen consumption rate and hyperfragmentation of the mitochondrial network in cultured cells. In muscle and fibroblasts from several subjects, substantially decreased mtDNA content was observed. FBXL4 is a member of the F-box family of proteins, some of which are involved in phosphorylation-dependent ubiquitination and/or G protein receptor coupling. We also demonstrate that FBXL4 is targeted to mitochondria and localizes in the intermembrane space, where it participates in an approximately 400 kDa protein complex. These data strongly support a role for FBXL4 in controlling bioenergetic homeostasis and mtDNA maintenance. FBXL4 mutations are a recurrent cause of mitochondrial encephalomyopathy onset in early infancy.


Assuntos
Predisposição Genética para Doença , Encefalomiopatias Mitocondriais/genética , Proteínas Mitocondriais/genética , Mutação/genética , Idade de Início , Criança , Pré-Escolar , Cromossomos Humanos Par 6/genética , DNA Complementar/genética , Proteínas F-Box/química , Proteínas F-Box/genética , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Genes Recessivos/genética , Células HEK293 , Humanos , Lactente , Recém-Nascido , Masculino , Mitocôndrias/metabolismo , Encefalomiopatias Mitocondriais/epidemiologia , Músculo Esquelético/patologia , Proteínas Mutantes/metabolismo , Fosforilação Oxidativa , Linhagem , Transporte Proteico , Frações Subcelulares/metabolismo , Síndrome , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genética
16.
Gastroenterology ; 149(7): 1872-1883.e9, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26344056

RESUMO

BACKGROUND & AIMS: DNA structural lesions are prevalent in sporadic colorectal cancer. Therefore, we proposed that gene variants that predispose to DNA double-strand breaks (DSBs) would be found in patients with familial colorectal carcinomas of an undefined genetic basis (UFCRC). METHODS: We collected primary T cells from 25 patients with UFCRC and matched patients without colorectal cancer (controls) and assayed for DSBs. We performed exome sequence analyses of germline DNA from 20 patients with UFCRC and 5 undiagnosed patients with polyposis. The prevalence of identified variants in genes linked to DNA integrity was compared with that of individuals without a family history of cancer. The effects of representative variants found to be associated with UFCRC was confirmed in functional assays with HCT116 cells. RESULTS: Primary T cells from most patients with UFCRC had increased levels of the DSB marker γ(phosphorylated)histone2AX (γH2AX) after treatment with DNA damaging agents, compared with T cells from controls (P < .001). Exome sequence analysis identified a mean 1.4 rare variants per patient that were predicted to disrupt functions of genes relevant to DSBs. Controls (from public databases) had a much lower frequency of variants in the same genes (P < .001). Knockdown of representative variant genes in HCT116 CRC cells increased γH2AX. A detailed analysis of immortalized patient-derived B cells that contained variants in the Werner syndrome, RecQ helicase-like gene (WRN, encoding T705I), and excision repair cross-complementation group 6 (ERCC6, encoding N180Y) showed reduced levels of these proteins and increased DSBs, compared with B cells from controls. This phenotype was rescued by exogenous expression of WRN or ERCC6. Direct analysis of the recombinant variant proteins confirmed defective enzymatic activities. CONCLUSIONS: These results provide evidence that defects in suppression of DSBs underlie some cases of UFCRC; these can be identified by assays of circulating lymphocytes. We specifically associated UFCRC with variants in WRN and ERCC6 that reduce the capacity for repair of DNA DSBs. These observations could lead to a simple screening strategy for UFCRC, and provide insight into the pathogenic mechanisms of colorectal carcinogenesis.


Assuntos
Biomarcadores Tumorais/genética , Neoplasias Colorretais/genética , Quebras de DNA de Cadeia Dupla , Variação Genética , Linfócitos T/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/metabolismo , Estudos de Casos e Controles , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Biologia Computacional , DNA Helicases/genética , DNA Helicases/metabolismo , Reparo do DNA , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Bases de Dados Genéticas , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Exoma , Feminino , Frequência do Gene , Técnicas de Silenciamento de Genes , Predisposição Genética para Doença , Instabilidade Genômica , Células HCT116 , Hereditariedade , Histonas/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Mutagênicos/farmacologia , Fenótipo , Fosforilação , Proteínas de Ligação a Poli-ADP-Ribose , RecQ Helicases/genética , RecQ Helicases/metabolismo , Análise de Sequência de DNA , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/metabolismo , Transfecção , Regulação para Cima , Helicase da Síndrome de Werner
17.
Bioinformatics ; 31(8): 1310-2, 2015 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-25505086

RESUMO

MOTIVATION: All current mitochondrial haplogroup classification tools require variants to be detected from an alignment with the reference sequence and to be properly named according to the canonical nomenclature standards for describing mitochondrial variants, before they can be compared with the haplogroup determining polymorphisms. With the emergence of high-throughput sequencing technologies and hence greater availability of mitochondrial genome sequences, there is a strong need for an automated haplogroup classification tool that is alignment-free and agnostic to reference sequence. RESULTS: We have developed a novel mitochondrial genome haplogroup-defining algorithm using a k-mer approach namely Phy-Mer. Phy-Mer performs equally well as the leading haplogroup classifier, HaploGrep, while avoiding the errors that may occur when preparing variants to required formats and notations. We have further expanded Phy-Mer functionality such that next-generation sequencing data can be used directly as input. AVAILABILITY AND IMPLEMENTATION: Phy-Mer is publicly available under the GNU Affero General Public License v3.0 on GitHub (https://github.com/danielnavarrogomez/phy-mer). CONTACT: Xiaowu_Gai@meei.harvard.edu SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.


Assuntos
Algoritmos , DNA Mitocondrial/genética , Variação Genética/genética , Haplótipos/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise de Sequência de DNA/métodos , Humanos , Software
18.
RNA Biol ; 13(5): 477-85, 2016 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-26950678

RESUMO

We report a Caucasian boy with intractable epilepsy and global developmental delay. Whole-exome sequencing identified the likely genetic etiology as a novel p.K212E mutation in the X-linked gene HSD17B10 for mitochondrial short-chain dehydrogenase/reductase SDR5C1. Mutations in HSD17B10 cause the HSD10 disease, traditionally classified as a metabolic disorder due to the role of SDR5C1 in fatty and amino acid metabolism. However, SDR5C1 is also an essential subunit of human mitochondrial RNase P, the enzyme responsible for 5'-processing and methylation of purine-9 of mitochondrial tRNAs. Here we show that the p.K212E mutation impairs the SDR5C1-dependent mitochondrial RNase P activities, and suggest that the pathogenicity of p.K212E is due to a general mitochondrial dysfunction caused by reduction in SDR5C1-dependent maturation of mitochondrial tRNAs.


Assuntos
3-Hidroxiacil-CoA Desidrogenases/genética , Deficiências do Desenvolvimento/genética , Epilepsia Resistente a Medicamentos/genética , Mutação , Ribonuclease P/metabolismo , Análise de Sequência de DNA/métodos , Criança , Exoma , Genes Ligados ao Cromossomo X , Humanos , Masculino , Mitocôndrias/genética , Mitocôndrias/metabolismo , RNA de Transferência/metabolismo
19.
Int Urogynecol J ; 27(5): 723-33, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26423260

RESUMO

INTRODUCTION AND HYPOTHESIS: Many adult women have resident urinary bacteria (urinary microbiome/microbiota). In adult women affected by urinary urgency incontinence (UUI), the etiologic and/or therapeutic role of the urinary microbiome/microbiota remains unknown. We hypothesized that microbiome/microbiota characteristics would relate to clinically relevant treatment response to UUI medication per os. METHODS: Adult women initiating medication treatment orally for UUI and a comparator group of unaffected women were recruited in a tertiary care health-care system. All participants provided baseline clinical data and urine samples. Women with UUI were given 5 mg solifenacin, with potential dose escalation to 10 mg for inadequate UUI symptom control at 4 weeks. Additional data and urine samples were collected from women with UUI at 4 and 12 weeks. The samples were assessed using 16S ribosomal RNA (rRNA) gene sequencing and enhanced quantitative urine culturing. The primary outcome was treatment response as measured by the validated Patient Global Symptom Control (PGSC) questionnaire. Clinically relevant UUI symptom control was defined as a 4 or 5 score on the PGSC. RESULTS: Diversity and composition of the urinary microbiome/microbiota of women with and without UUI differed at baseline. Women with UUI had more bacteria and a more diverse microbiome/microbiota. The clinical response to solifenacin in UUI participants was related to baseline microbiome/microbiota, with responders more likely to have fewer bacteria and a less diverse community at baseline. Nonresponders had a more diverse community that often included bacteria not typically found in responders. CONCLUSIONS: Knowledge of an individual's urinary microbiome/microbiota may help refine UUI treatment. Complementary tools, DNA sequencing, and expanded urine culture provide information about bacteria that appear to be related to UUI incontinence status and treatment response in this population of adult women.


Assuntos
Bacteriúria/microbiologia , Microbiota , Antagonistas Muscarínicos/uso terapêutico , RNA Ribossômico 16S/análise , Succinato de Solifenacina/uso terapêutico , Incontinência Urinária de Urgência/tratamento farmacológico , Incontinência Urinária de Urgência/microbiologia , Sistema Urinário/microbiologia , Actinomyces/isolamento & purificação , Administração Oral , Adulto , Idoso , Estudos de Casos e Controles , Contagem de Colônia Microbiana , Corynebacterium/isolamento & purificação , Feminino , Humanos , Lactobacillus/isolamento & purificação , Pessoa de Meia-Idade , Antagonistas Muscarínicos/administração & dosagem , Estudos Prospectivos , Succinato de Solifenacina/administração & dosagem , Streptococcus/isolamento & purificação , Resultado do Tratamento
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA