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1.
APMIS ; 128(1): 3-9, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31628675

RESUMO

Biliary tract cancers (BTC) are a rare heterogeneous disease group with a dismal prognosis and limited treatment options. The mutational landscape consists of genetic aberrations both shared by and characteristic for anatomical location. Here, we present exome sequencing data on 22 genes from a phase 2 trial using a clinically validated panel used in patients with colorectal cancer. A total of 56 patients were included in a one-armed phase 2 trial investigating the treatment combination of capecitabine, gemcitabine, oxaliplatin, and cetuximab. Tissue DNA yield and quality allowed analysis of 30 patients on our panel including 22 genes. ARID1A (33%) and TP53 (33%) were found to be most frequently mutated followed by KRAS mutations found in 20% of the patients. Mutational aberrations in ARID1A were found more prevalent than expected, whereas TP53 and KRAS were in concordance with earlier reported data. Mutation in CTNNB1 was significantly associated with poor prognosis. Our panel is clinically validated and suitable for a high volume of samples to detect mutations in patients with BTC. However, it is reasonable to assume that the clinical utility could be optimized in this patient group by extending the panel to include BTC specific mutations with potential therapeutic consequences such as IDH1/2, FGFR fusions, ERBB3, and BRCA1/2.


Assuntos
Neoplasias dos Ductos Biliares/genética , Exoma , Mutação , Adulto , Idoso , Antineoplásicos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias dos Ductos Biliares/tratamento farmacológico , Estudos de Coortes , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas Nucleares/genética , Inclusão em Parafina , Prognóstico , Proteínas Proto-Oncogênicas p21(ras)/genética , Análise de Sequência de DNA , Fatores de Transcrição/genética , Proteína Supressora de Tumor p53/genética , beta Catenina/genética
2.
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
3.
N Engl J Med ; 381(15): 1422-1433, 2019 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-31509666

RESUMO

BACKGROUND: Identifying mechanisms of diseases with complex inheritance patterns, such as macular telangiectasia type 2, is challenging. A link between macular telangiectasia type 2 and altered serine metabolism has been established previously. METHODS: Through exome sequence analysis of a patient with macular telangiectasia type 2 and his family members, we identified a variant in SPTLC1 encoding a subunit of serine palmitoyltransferase (SPT). Because mutations affecting SPT are known to cause hereditary sensory and autonomic neuropathy type 1 (HSAN1), we examined 10 additional persons with HSAN1 for ophthalmologic disease. We assayed serum amino acid and sphingoid base levels, including levels of deoxysphingolipids, in patients who had macular telangiectasia type 2 but did not have HSAN1 or pathogenic variants affecting SPT. We characterized mice with low serine levels and tested the effects of deoxysphingolipids on human retinal organoids. RESULTS: Two variants known to cause HSAN1 were identified as causal for macular telangiectasia type 2: of 11 patients with HSAN1, 9 also had macular telangiectasia type 2. Circulating deoxysphingolipid levels were 84.2% higher among 125 patients with macular telangiectasia type 2 who did not have pathogenic variants affecting SPT than among 94 unaffected controls. Deoxysphingolipid levels were negatively correlated with serine levels, which were 20.6% lower than among controls. Reduction of serine levels in mice led to increases in levels of retinal deoxysphingolipids and compromised visual function. Deoxysphingolipids caused photoreceptor-cell death in retinal organoids, but not in the presence of regulators of lipid metabolism. CONCLUSIONS: Elevated levels of atypical deoxysphingolipids, caused by variant SPTLC1 or SPTLC2 or by low serine levels, were risk factors for macular telangiectasia type 2, as well as for peripheral neuropathy. (Funded by the Lowy Medical Research Institute and others.).


Assuntos
Neuropatias Hereditárias Sensoriais e Autônomas/genética , Mutação , Telangiectasia Retiniana/genética , Serina C-Palmitoiltransferase/genética , Serina/metabolismo , Esfingolipídeos/metabolismo , Adulto , Idoso , Animais , Análise Mutacional de DNA , Modelos Animais de Doenças , Exoma/genética , Feminino , Neuropatias Hereditárias Sensoriais e Autônomas/complicações , Neuropatias Hereditárias Sensoriais e Autônomas/metabolismo , Humanos , Metabolismo dos Lipídeos , Macula Lutea/patologia , Masculino , Camundongos , Pessoa de Meia-Idade , Linhagem , Telangiectasia Retiniana/complicações , Telangiectasia Retiniana/metabolismo , Fatores de Risco , Serina/sangue , Esfingosina/análogos & derivados , Esfingosina/análise , Adulto Jovem
4.
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
5.
Nat Commun ; 10(1): 2985, 2019 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-31278258

RESUMO

Mosaic genetic variants can have major clinical impact. We systematically analyse trio exome sequence data from 4,293 probands from the DDD Study with severe developmental disorders for pathogenic postzygotic mosaicism (PZM) in the child or a clinically-unaffected parent, and use ultrahigh-depth sequencing to validate candidate mosaic variants. We observe that levels of mosaicism for small genetic variants are usually equivalent in both saliva and blood and ~3% of causative de novo mutations exhibit PZM; this is an important observation, as the sibling recurrence risk is extremely low. We identify parental PZM in 21 trios (0.5% of trios), resulting in a substantially increased sibling recurrence risk in future pregnancies. Together, these forms of mosaicism account for 40 (1%) diagnoses in our cohort. Likely child-PZM mutations occur equally on both parental haplotypes, and the penetrance of detectable mosaic pathogenic variants overall is likely to be less than half that of constitutive variants.


Assuntos
Deficiências do Desenvolvimento/genética , Exoma/genética , Mosaicismo , Sequenciamento Completo do Exoma/métodos , Criança , Estudos de Coortes , Deficiências do Desenvolvimento/diagnóstico , Feminino , Testes Genéticos/métodos , Variação Genética , Haplótipos/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Herança Materna/genética , Pais , Herança Paterna/genética
6.
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
7.
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
8.
Immunogenetics ; 71(8-9): 531-544, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31321455

RESUMO

Indian rhesus macaque major histocompatibility complex (MHC) variation can influence the outcomes of transplantation and infectious disease studies. Frequently, rhesus macaques are MHC genotyped to identify variants that could account for unexpected results. Since the MHC is only one region in the genome where variation could impact experimental outcomes, strategies for simultaneously profiling variation in the macaque MHC and the remainder of the protein coding genome would be useful. Here we determine MHC class I and class II genotypes using target-capture probes enriched for MHC sequences, a method we term macaque exome sequence (MES) genotyping. For a cohort of 27 Indian rhesus macaques, we describe two methods for obtaining MHC genotypes from MES data and demonstrate that the MHC class I and class II genotyping results obtained with these methods are 98.1% and 98.7% concordant, respectively, with expected MHC genotypes. In contrast, conventional MHC genotyping results obtained by deep sequencing of short multiplex PCR amplicons were only 92.6% concordant with expectations for this cohort.


Assuntos
Exoma/genética , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe I/genética , Macaca mulatta/genética , Polimorfismo Genético , Animais , Haplótipos , Sequenciamento Completo do Exoma
9.
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
11.
PLoS Genet ; 15(6): e1008180, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31170158

RESUMO

Multiple sclerosis (MS) is an inflammatory disease of the central nervous system characterized by myelin loss and neuronal dysfunction. Although the majority of patients do not present familial aggregation, Mendelian forms have been described. We performed whole-exome sequencing analysis in 132 patients from 34 multi-incident families, which nominated likely pathogenic variants for MS in 12 genes of the innate immune system that regulate the transcription and activation of inflammatory mediators. Rare missense or nonsense variants were identified in genes of the fibrinolysis and complement pathways (PLAU, MASP1, C2), inflammasome assembly (NLRP12), Wnt signaling (UBR2, CTNNA3, NFATC2, RNF213), nuclear receptor complexes (NCOA3), and cation channels and exchangers (KCNG4, SLC24A6, SLC8B1). These genes suggest a disruption of interconnected immunological and pro-inflammatory pathways as the initial event in the pathophysiology of familial MS, and provide the molecular and biological rationale for the chronic inflammation, demyelination and neurodegeneration observed in MS patients.


Assuntos
Predisposição Genética para Doença , Inflamação/genética , Esclerose Múltipla/genética , Transcriptoma/genética , Adulto , Códon sem Sentido , Doenças Desmielinizantes/genética , Doenças Desmielinizantes/patologia , Exoma/genética , Feminino , Humanos , Inflamação/metabolismo , Inflamação/patologia , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla/metabolismo , Esclerose Múltipla/patologia , Bainha de Mielina/genética , Bainha de Mielina/patologia , Degeneração Neural/genética , Degeneração Neural/patologia , Neurônios/metabolismo , Neurônios/patologia , Linhagem , Sequenciamento Completo do Exoma , Adulto Jovem
12.
J Clin Neurosci ; 67: 139-144, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31182267

RESUMO

Spinocerebellar ataxia with axonal neuropathy type 1 (SCAN1; OMIM #607250), an exceedingly rare disorder having been documented in only a single family from Saudi Arabia, is the result of an unusual mutation in the tyrosyl DNA phosphodiesterase 1 gene (TDP1). We performed high-throughput sequencing (whole exome and ataxia gene panel) in two apparently unrelated Omani families segregating sensorimotor neuropathy and ataxia in an autosomal recessive fashion. Following validation by Sanger sequencing, all affected subjects (n = 4) were confirmed to carry the known SCAN1 pathogenic homozygous variant in the TDP1 gene, NM_001008744.1:c.1478A > G (p.His493Arg). In keeping with the initial description, our patients demonstrated progressive ataxia, cerebellar atrophy and disabling axonal sensori-motor neuropathy (n = 4), hypercholesterolemia (n = 2) and elevated serum alpha fetoprotein (n = 3). In addition, our patients also had mild cognitive deficits in multiple domains (n = 3), a feature not previously reported. Our findings independently revalidate the phenotype of TDP1 mutation and expand the clinical spectrum to include mild cognitive deficits. Haplotype sharing, as determined by DNA microarray (CytoScan HD), attests to a possible common founder mutation in the Arab population.


Assuntos
Ataxias Espinocerebelares/genética , Adolescente , Adulto , Exoma , Feminino , Humanos , Masculino , Mutação , Doenças do Sistema Nervoso Periférico/genética , Diester Fosfórico Hidrolases , Ataxias Espinocerebelares/diagnóstico por imagem , Ataxias Espinocerebelares/psicologia , Adulto Jovem
18.
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
19.
Biol Psychiatry ; 85(11): 889-890, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-31122339
20.
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
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