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1.
Exp Suppl ; 111: 33-52, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31588527

RESUMO

Molecular genetic methods have become an organic part of everyday clinical practice. In the past, molecular diagnostic tests were carried out for genetic diagnosis of a particular monogenic disease. In these situations the tests itself were used for identification of one particular genetic alteration (e.g., point mutation or deletion) of the gene of interest. Later, parallel with the development of the technology, the focus has shifted by allowing investigating at once targeted gene panels and even the whole exome/genome behind a suspected genetic disorder. Historically for these purposes, array-based methods (oligonucleotide arrays) and then next-generation sequencing-based methods have been used. High-throughput methods have been fundamentally transforming the everyday, routine genetic diagnostics, but older molecular techniques still have a role in clinical genetics. Here, we summarize the most important molecular genetic methods and shed light to the advantages and disadvantages of their application in routine diagnostics. We mainly focus on methods used for detection of germline alterations.


Assuntos
Testes Genéticos , Sequenciamento de Nucleotídeos em Larga Escala , Técnicas de Diagnóstico Molecular , Reação em Cadeia da Polimerase em Tempo Real , Exoma , Humanos , Mutação
2.
Stud Health Technol Inform ; 264: 1845-1846, 2019 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-31438372

RESUMO

Next-generation sequencing has prompted the development of numerous -omics applications. Along with experimental procedures, various computational pipelines became available to address the inherent complexities concerning the volume and quality of data. These pipelines are effective and routinely applied; however, interpreting their outcomes into actionable evidence is still poorly addressed. In this context, this work proposes a method for translating patient genomic profiles to drug response aberrations by integrating pharmacogenomic data into sequencing data analysis pipelines.


Assuntos
Biologia Computacional , Exoma , Relação Dose-Resposta a Droga , Genômica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos
3.
Zhongguo Dang Dai Er Ke Za Zhi ; 21(7): 640-643, 2019 Jul.
Artigo em Chinês | MEDLINE | ID: mdl-31315761

RESUMO

OBJECTIVE: To explore the value and significance of the clinical application of whole exome sequencing (WES) in monogenic hereditary disorders in critically ill newborns. METHODS: The critically ill newborns in the neonatal intensive care unit with suspected hereditary diseases or unclear clinical diagnosis from June 2016 to December 2018 were enrolled. The whole blood samples from both newborns and parents were collected for WES. The detected genetic mutations were classified, the mutations associated with clinical phenotypes were searched for, and Sanger sequencing was performed to verify the mutations. RESULTS: A total of 45 newborns were enrolled, including 22 males and 23 females, and the median age of onset was 2.0 days. Of the 45 newborns, 12 (27%) were confirmed with monogenic hereditary disorders by molecular diagnostics, and the median age at diagnosis was 31.5 days. Of the 12 newborns with monogenic hereditary disorders, 5 (42%) were partially associated with clinical phenotypes but confirmed with monogenic hereditary disorders by additional information supplement and analysis. The improvement rate of newborns with monogenic hereditary disorders was 67% (8/12) after treatment. CONCLUSIONS: WES technology is a powerful tool for finding genetic mutations in monogenic hereditary disorders in critically ill newborns and can play a crucial role in clinical decision-making. However, a comprehensive interpretation of sequence data requires physicians to take the clinical phenotypes and the results of WES into consideration simultaneously.


Assuntos
Estado Terminal , Exoma , Feminino , Humanos , Recém-Nascido , Masculino , Mutação , Fenótipo , Sequenciamento Completo do Exoma
4.
BMC Evol Biol ; 19(1): 144, 2019 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-31311498

RESUMO

BACKGROUND: Rapid accumulation of vertebrate genome sequences render comparative genomics a powerful approach to study macro-evolutionary events. The assessment of phylogenic relationships between species routinely depends on the analysis of sequence homology at the nucleotide or protein level. RESULTS: We analyzed mRNA GC content, codon usage and divergence of orthologous proteins in 55 vertebrate genomes. Data were visualized in genome-wide landscapes using a sliding window approach. Landscapes of GC content reveal both evolutionary conservation of clustered genes, and lineage-specific changes, so that it was possible to construct a phylogenetic tree that closely matched the classic "tree of life". Landscapes of GC content also strongly correlated to landscapes of amino acid usage: positive correlation with glycine, alanine, arginine and proline and negative correlation with phenylalanine, tyrosine, methionine, isoleucine, asparagine and lysine. Peaks of GC content correlated strongly with increased protein divergence. CONCLUSIONS: Landscapes of base- and amino acid composition of the coding genome opens a new approach in comparative genomics, allowing identification of discrete regions in which protein evolution accelerated over deep evolutionary time. Insight in the evolution of genome structure may spur novel studies assessing the evolutionary benefit of genes in particular genomic regions.


Assuntos
Composição de Bases/genética , Evolução Molecular , Exoma/genética , Proteínas/genética , Vertebrados/genética , Animais , Códon/genética , Genoma , Humanos , Mamíferos/genética , Filogenia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Répteis/genética
5.
BMC Bioinformatics ; 20(1): 342, 2019 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-31208315

RESUMO

BACKGROUND: Whole exome sequencing (WES) is a cost-effective method that identifies clinical variants but it demands accurate variant caller tools. Currently available tools have variable accuracy in predicting specific clinical variants. But it may be possible to find the best combination of aligner-variant caller tools for detecting accurate single nucleotide variants (SNVs) and small insertion and deletion (InDels) separately. Moreover, many important aspects of InDel detection are overlooked while comparing the performance of tools, particularly its base pair length. RESULTS: We assessed the performance of variant calling pipelines using the combinations of four variant callers and five aligners on human NA12878 and simulated exome data. We used high confidence variant calls from Genome in a Bottle (GiaB) consortium for validation, and GRCh37 and GRCh38 as the human reference genome. Based on the performance metrics, both BWA and Novoalign aligners performed better with DeepVariant and SAMtools callers for detecting SNVs, and with DeepVariant and GATK for InDels. Furthermore, we obtained similar results on human NA24385 and NA24631 exome data from GiaB. CONCLUSION: In this study, DeepVariant with BWA and Novoalign performed best for detecting accurate SNVs and InDels. The accuracy of variant calling was improved by merging the top performing pipelines. The results of our study provide useful recommendations for analysis of WES data in clinical genomics.


Assuntos
Simulação por Computador , Polimorfismo de Nucleotídeo Único/genética , Sequenciamento Completo do Exoma , Pareamento de Bases/genética , Exoma/genética , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Mutação INDEL/genética , Curva ROC
7.
BMC Bioinformatics ; 20(1): 254, 2019 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-31096927

RESUMO

BACKGROUND: Next-generation sequencing is revolutionising diagnosis and treatment of rare diseases, however its application to understanding common disease aetiology is limited. Rare disease applications binarily attribute genetic change(s) at a single locus to a specific phenotype. In common diseases, where multiple genetic variants within and across genes contribute to disease, binary modelling cannot capture the burden of pathogenicity harboured by an individual across a given gene/pathway. We present GenePy, a novel gene-level scoring system for integration and analysis of next-generation sequencing data on a per-individual basis that transforms NGS data interpretation from variant-level to gene-level. This simple and flexible scoring system is intuitive and amenable to integration for machine learning, network and topological approaches, facilitating the investigation of complex phenotypes. RESULTS: Whole-exome sequencing data from 508 individuals were used to generate GenePy scores. For each variant a score is calculated incorporating: i) population allele frequency estimates; ii) individual zygosity, determined through standard variant calling pipelines and; iii) any user defined deleteriousness metric to inform on functional impact. GenePy then combines scores generated for all variants observed into a single gene score for each individual. We generated a matrix of ~ 14,000 GenePy scores for all individuals for each of sixteen popular deleteriousness metrics. All per-gene scores are corrected for gene length. The majority of genes generate GenePy scores < 0.01 although individuals harbouring multiple rare highly deleterious mutations can accumulate extremely high GenePy scores. In the absence of a comparator metric, we examine GenePy performance in discriminating genes known to be associated with three common, complex diseases. A Mann-Whitney U test conducted on GenePy scores for this positive control gene in cases versus controls demonstrates markedly more significant results (p = 1.37 × 10- 4) compared to the most commonly applied association tool that combines common and rare variation (p = 0.003). CONCLUSIONS: Per-gene per-individual GenePy scores are intuitive when assessing genetic variation in individual patients or comparing scores between groups. GenePy outperforms the currently accepted best practice tools for combining common and rare variation. GenePy scores are suitable for downstream data integration with transcriptomic and proteomic data that also report at the gene level.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Software , Virulência/genética , Alelos , Estudos de Coortes , Bases de Dados Genéticas , Exoma , Frequência do Gene/genética , Humanos , Fenótipo , Polimorfismo de Nucleotídeo Único/genética , Sequenciamento Completo do Exoma , Zigoto/metabolismo
13.
Hum Genet ; 138(7): 703-713, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31139930

RESUMO

Neural tube defects (NTD) result from complex mechanisms between genes, nutrition and environment. The identification of genetic predictors by genome exome sequencing and their influence on genome methylation need further consideration. Gene variants related to 1-CM metabolism (1-CM) could influence the methylation of genes involved in neural tube embryogenesis through impaired synthesis of S-adenosyl methionine. We performed exome sequencing of 6116 genes referenced in OMIM and NTD risk and genome-wide methylation in 23 NTD cases. We replicated the most significant associations in 81 other cases. The analysis of exome sequencing identified one gene of 1-CM, LRP2, and one gene of Sonic Hedgehog (SHH), GLI3, in the 23 NTD cases. The analysis restricted to genes of 1-CM and neural tube embryogenesis identified five gene predictors of 1-CM (LRP2, rs137983840; MMAA, rs148142853; TCN2, rs35838082; FPGS, rs41306702; BHMT, rs763726268) and two of SHH (GLI3, rs35364414; MKS1, rs151023718). We replicated the association of TCN2, BHMT and GLI3 with NTD risk in the 81 cases. We found a significant hemimethylation of CFAP46 that may influence SHH activation in one case, who carried risk alleles in BHMT, LRP2, MMAA and GLI3. In conclusion, we identified new candidate genes and rare variants that highlight an interacting influence of genes involved in SHH and 1-CM in the puzzle of genetic components of NTD risk.


Assuntos
Biomarcadores/metabolismo , Carbono/metabolismo , Exoma , Proteínas Hedgehog/genética , Defeitos do Tubo Neural/genética , Vitamina B 12/metabolismo , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Estudos de Associação Genética , Humanos , Masculino , Defeitos do Tubo Neural/metabolismo , Defeitos do Tubo Neural/patologia , Transdução de Sinais , Sequenciamento Completo do Exoma , Adulto Jovem
14.
Eur J Med Genet ; 62(7): 103662, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31071488

RESUMO

Limb-girdle muscular dystrophies (LGMDs) are a group of genetically heterogeneous muscular diseases that predominantly affect the proximal muscles. Pathogenic variants in TNPO3 have been associated with a rare, autosomal dominant limb-girdle muscular dystrophy 1F (LGMD1F) in a large Italian-Spanish family and an isolated LGMD1F case. Here we present two individuals from a Hungarian family with an early-onset, slowly progressive muscular dystrophy. Both the female proband and her affected son had delayed early motor milestones including first walking at 14 months and 18 months, respectively. Both present with progressive weakness of facial, bulbar, axial, and distal muscles especially of the lower extremities. Electromyography indicated myogenic damage and muscle biopsy from the proband showed myopathic alterations with sarcoplasmic masses and signs of mitochondrial dysfunction. Exome sequencing of the female proband identified a novel c.2767delC p.(Arg923AspfsTer17) variant in TNPO3. Sanger sequencing confirmed the presence of the TNPO3 variant in the affected son; the unaffected son did not have the variant. The identification of the c.2767delC variant further supports the clinical significance of TNPO3 and expands the clinical spectrum of TNPO3-associated LGMD1F.


Assuntos
Distrofia Muscular do Cíngulo dos Membros/genética , beta Carioferinas/genética , Adulto , Exoma , Feminino , Humanos , Lactente , Masculino , Distrofia Muscular do Cíngulo dos Membros/patologia , Mutação , Linhagem
15.
Nature ; 570(7759): 71-76, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31118516

RESUMO

Protein-coding genetic variants that strongly affect disease risk can yield relevant clues to disease pathogenesis. Here we report exome-sequencing analyses of 20,791 individuals with type 2 diabetes (T2D) and 24,440 non-diabetic control participants from 5 ancestries. We identify gene-level associations of rare variants (with minor allele frequencies of less than 0.5%) in 4 genes at exome-wide significance, including a series of more than 30 SLC30A8 alleles that conveys protection against T2D, and in 12 gene sets, including those corresponding to T2D drug targets (P = 6.1 × 10-3) and candidate genes from knockout mice (P = 5.2 × 10-3). Within our study, the strongest T2D gene-level signals for rare variants explain at most 25% of the heritability of the strongest common single-variant signals, and the gene-level effect sizes of the rare variants that we observed in established T2D drug targets will require 75,000-185,000 sequenced cases to achieve exome-wide significance. We propose a method to interpret these modest rare-variant associations and to incorporate these associations into future target or gene prioritization efforts.


Assuntos
Diabetes Mellitus Tipo 2/genética , Exoma/genética , Sequenciamento Completo do Exoma , Animais , Estudos de Casos e Controles , Técnicas de Apoio para a Decisão , Feminino , Frequência do Gene , Estudo de Associação Genômica Ampla , Humanos , Masculino , Camundongos , Camundongos Knockout
16.
Nat Med ; 25(5): 792-804, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31068711

RESUMO

Precision health relies on the ability to assess disease risk at an individual level, detect early preclinical conditions and initiate preventive strategies. Recent technological advances in omics and wearable monitoring enable deep molecular and physiological profiling and may provide important tools for precision health. We explored the ability of deep longitudinal profiling to make health-related discoveries, identify clinically relevant molecular pathways and affect behavior in a prospective longitudinal cohort (n = 109) enriched for risk of type 2 diabetes mellitus. The cohort underwent integrative personalized omics profiling from samples collected quarterly for up to 8 years (median, 2.8 years) using clinical measures and emerging technologies including genome, immunome, transcriptome, proteome, metabolome, microbiome and wearable monitoring. We discovered more than 67 clinically actionable health discoveries and identified multiple molecular pathways associated with metabolic, cardiovascular and oncologic pathophysiology. We developed prediction models for insulin resistance by using omics measurements, illustrating their potential to replace burdensome tests. Finally, study participation led the majority of participants to implement diet and exercise changes. Altogether, we conclude that deep longitudinal profiling can lead to actionable health discoveries and provide relevant information for precision health.


Assuntos
Big Data , Diabetes Mellitus Tipo 2/etiologia , Medicina de Precisão/estatística & dados numéricos , Adulto , Idoso , Doenças Cardiovasculares/etiologia , Estudos de Coortes , Exoma , Feminino , Microbioma Gastrointestinal , Humanos , Resistência à Insulina , Estudos Longitudinais , Masculino , Metaboloma , Pessoa de Meia-Idade , Modelos Biológicos , Fatores de Risco , Transcriptoma
18.
PLoS Comput Biol ; 15(4): e1006953, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30986244

RESUMO

Determining the cancer type and molecular subtype has important clinical implications. The primary site is however unknown for some malignancies discovered in the metastatic stage. Moreover liquid biopsies may be used to screen for tumoral DNA, which upon detection needs to be assigned to a site-of-origin. Classifiers based on genomic features are a promising approach to prioritize the tumor anatomical site, type and subtype. We examined the predictive ability of causal (driver) somatic mutations in this task, comparing it against global patterns of non-selected (passenger) mutations, including features based on regional mutation density (RMD). In the task of distinguishing 18 cancer types, the driver mutations-mutated oncogenes or tumor suppressors, pathways and hotspots-classified 36% of the patients to the correct cancer type. In contrast, the features based on passenger mutations did so at 92% accuracy, with similar contribution from the RMD and the trinucleotide mutation spectra. The RMD and the spectra covered distinct sets of patients with predictions. In particular, introducing the RMD features into a combined classification model increased the fraction of diagnosed patients by 50 percentage points (at 20% FDR). Furthermore, RMD was able to discriminate molecular subtypes and/or anatomical site of six major cancers. The advantage of passenger mutations was upheld under high rates of false negative mutation calls and with exome sequencing, even though overall accuracy decreased. We suggest whole genome sequencing is valuable for classifying tumors because it captures global patterns emanating from mutational processes, which are informative of the underlying tumor biology.


Assuntos
Biologia Computacional/métodos , Neoplasias/classificação , Neoplasias/genética , Algoritmos , DNA de Neoplasias/classificação , DNA de Neoplasias/genética , Exoma/genética , Genômica , Humanos , Aprendizado de Máquina , Mutação/genética , Software , Sequenciamento Completo do Exoma/métodos , Sequenciamento Completo do Genoma/métodos
19.
N Engl J Med ; 380(15): 1421-1432, 2019 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-30970187

RESUMO

BACKGROUND: Hirschsprung's disease, or congenital aganglionosis, is a developmental disorder of the enteric nervous system and is the most common cause of intestinal obstruction in neonates and infants. The disease has more than 80% heritability, including significant associations with rare and common sequence variants in genes related to the enteric nervous system, as well as with monogenic and chromosomal syndromes. METHODS: We genotyped and exome-sequenced samples from 190 patients with Hirschsprung's disease to quantify the genetic burden in patients with this condition. DNA sequence variants, large copy-number variants, and karyotype variants in probands were considered to be pathogenic when they were significantly associated with Hirschsprung's disease or another neurodevelopmental disorder. Novel genes were confirmed by functional studies in the mouse and human embryonic gut and in zebrafish embryos. RESULTS: The presence of five or more variants in four noncoding elements defined a widespread risk of Hirschsprung's disease (48.4% of patients and 17.1% of controls; odds ratio, 4.54; 95% confidence interval [CI], 3.19 to 6.46). Rare coding variants in 24 genes that play roles in enteric neural-crest cell fate, 7 of which were novel, were also common (34.7% of patients and 5.0% of controls) and conferred a much greater risk than noncoding variants (odds ratio, 10.02; 95% CI, 6.45 to 15.58). Large copy-number variants, which were present in fewer patients (11.4%, as compared with 0.2% of controls), conferred the highest risk (odds ratio, 63.07; 95% CI, 36.75 to 108.25). At least one identifiable genetic risk factor was found in 72.1% of the patients, and at least 48.4% of patients had a structural or regulatory deficiency in the gene encoding receptor tyrosine kinase (RET). For individual patients, the estimated risk of Hirschsprung's disease ranged from 5.33 cases per 100,000 live births (approximately 1 per 18,800) to 8.38 per 1000 live births (approximately 1 per 120). CONCLUSIONS: Among the patients in our study, Hirschsprung's disease arose from common noncoding variants, rare coding variants, and copy-number variants affecting genes involved in enteric neural-crest cell fate that exacerbate the widespread genetic susceptibility associated with RET. For individual patients, the genotype-specific odds ratios varied by a factor of approximately 67, which provides a basis for risk stratification and genetic counseling. (Funded by the National Institutes of Health.).


Assuntos
Variação Genética , Genótipo , Doença de Hirschsprung/genética , Exoma , Feminino , Predisposição Genética para Doença , Humanos , Masculino , Mutação , Razão de Chances , Penetrância , Análise de Sequência de DNA , Sequenciamento Completo do Exoma
20.
BMC Cancer ; 19(1): 313, 2019 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-30947698

RESUMO

BACKGROUND: In the majority of familial breast cancer (BC) families, the etiology of the disease remains unresolved. To identify missing BC heritability resulting from relatively rare variants (minor allele frequency ≤ 1%), we have performed whole exome sequencing followed by variant analysis in a virtual panel of 492 cancer-associated genes on BC patients from BRCA1 and BRCA2 negative families with elevated BC risk. METHODS: BC patients from 54 BRCA1 and BRCA2-negative families with elevated BC risk and 120 matched controls were considered for germline DNA whole exome sequencing. Rare variants identified in the exome and in a virtual panel of cancer-associated genes [492 genes associated with different types of (hereditary) cancer] were compared between BC patients and controls. Nonsense, frame-shift indels and splice-site variants (strong protein-damaging variants, called PDAVs later on) observed in BC patients within the genes of the panel, which we estimated to possess the highest probability to predispose to BC, were further validated using an alternative sequencing procedure. RESULTS: Exome- and cancer-associated gene panel-wide variant analysis show that there is no significant difference in the average number of rare variants found in BC patients compared to controls. However, the genes in the cancer-associated gene panel with nonsense variants were more than two-fold over-represented in women with BC and commonly involved in the DNA double-strand break repair process. Approximately 44% (24 of 54) of BC patients harbored 31 PDAVs, of which 11 were novel. These variants were found in genes associated with known or suspected BC predisposition (PALB2, BARD1, CHEK2, RAD51C and FANCA) or in predisposing genes linked to other cancer types but not well-studied in the context of familial BC (EXO1, RECQL4, CCNH, MUS81, TDP1, DCLRE1A, DCLRE1C, PDE11A and RINT1) and genes associated with different hereditary syndromes but not yet clearly associated with familial cancer syndromes (ABCC11, BBS10, CD96, CYP1A1, DHCR7, DNAH11, ESCO2, FLT4, HPS6, MYH8, NME8 and TTC8). Exome-wide, only a few genes appeared to be enriched for PDAVs in the familial BC patients compared to controls. CONCLUSIONS: We have identified a series of novel candidate BC predisposition variants/genes. These variants/genes should be further investigated in larger cohorts/case-control studies. Other studies including co-segregation analyses in affected families, locus-specific loss of heterozygosity and functional studies should shed further light on their relevance for BC risk.


Assuntos
Neoplasias da Mama/genética , Exoma/genética , Predisposição Genética para Doença , Adulto , Idoso , Proteína BRCA1/genética , Proteína BRCA2/genética , Estudos de Casos e Controles , Feminino , Humanos , Pessoa de Meia-Idade , Mutação , Sequenciamento Completo do Exoma
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