RESUMO
BACKGROUND: Clinical interpretation of the large number of rare variants identified by high throughput sequencing (HTS) technologies is challenging. The aim of this study was to explore the clinical implications of a HTS strategy for patients with hypertrophic cardiomyopathy (HCM) using a targeted HTS methodology and workflow developed for patients with a range of inherited cardiovascular diseases. By comparing the sequencing results with published findings and with sequence data from a large-scale exome sequencing screen of UK individuals, we sought to quantify the strength of the evidence supporting causality for detected candidate variants. METHODS AND RESULTS: 223 unrelated patients with HCM (46±15 years at diagnosis, 74% males) were studied. In order to analyse coding, intronic and regulatory regions of 41 cardiovascular genes, we used solution-based sequence capture followed by massive parallel resequencing on Illumina GAIIx. Average read-depth in the 2.1 Mb target region was 120. Rare (frequency<0.5%) non-synonymous, loss-of-function and splice-site variants were defined as candidates. Excluding titin, we identified 152 distinct candidate variants in sarcomeric or associated genes (89 novel) in 143 patients (64%). Four sarcomeric genes (MYH7, MYBPC3, TNNI3, TNNT2) showed an excess of rare single non-synonymous single-nucleotide polymorphisms (nsSNPs) in cases compared to controls. The estimated probability that a nsSNP in these genes is pathogenic varied between 57% and near certainty depending on the location. We detected an additional 94 candidate variants (73 novel) in desmosomal, and ion-channel genes in 96 patients (43%). CONCLUSIONS: This study provides the first large-scale quantitative analysis of the prevalence of sarcomere protein gene variants in patients with HCM using HTS technology. Inclusion of other genes implicated in inherited cardiac disease identifies a large number of non-synonymous rare variants of unknown clinical significance.
Assuntos
Cardiomiopatia Hipertrófica/genética , Sequenciamento de Nucleotídeos em Larga Escala , Sarcômeros/genética , Adulto , Substituição de Aminoácidos/genética , Cardiomiopatia Hipertrófica/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Linhagem , Polimorfismo de Nucleotídeo Único , Sarcômeros/metabolismoRESUMO
Oral rotavirus vaccines have consistently proven to be less immunogenic among infants in developing countries. Discrepancies in the intestinal microbiota, including a greater burden of enteropathogens and an altered commensal community composition, may contribute to this trend by inhibiting the replication of vaccine viruses. To test this possibility, we performed a nested case-control study in Vellore, India, in which we compared the intestinal microbiota of infants who responded serologically or not after two doses of Rotarix delivered at 6 and 10â¯weeks of age as part of a clinical trial (CTRI/2012/05/002677). The prevalence of 40 bacterial, viral, and eukaryotic pathogen targets was assessed in pre-vaccination stool samples from 325 infants using singleplex real-time PCR on a Taqman array card (TAC). In a subset of 170 infants, we assessed bacterial microbiota composition by sequencing the 16S rRNA gene V4 region. Contrary to expectations, responders were more likely than non-responders to harbor ≥1 bacterial enteropathogen at dose 1 (26% [40/156] vs 13% [21/157] of infants with TAC results who completed the study per protocol; χ2, Pâ¯=â¯.006), although this was not apparent at dose 2 (24% [38/158] vs 23% [36/158]; Pâ¯=â¯.790). Rotavirus shedding after dose 1 was negatively correlated with the replication of co-administered oral poliovirus vaccine (OPV). We observed no consistent differences in composition or diversity of the 16S bacterial microbiota according to serological response, although rotavirus shedding was associated with slightly more bacterial taxa pre-vaccination. Overall, our findings demonstrate an inhibitory effect of co-administered OPV on the first dose of Rotarix, consistent with previous studies, but in the context of OPV co-administration we did not find a strong association between other components of the intestinal microbiota at the time of vaccination and Rotarix immunogenicity.
Assuntos
Anticorpos Antivirais/sangue , Microbioma Gastrointestinal , Microbiota , Infecções por Rotavirus/prevenção & controle , Vacinas contra Rotavirus/administração & dosagem , Vacinas contra Rotavirus/imunologia , Animais , Bactérias/classificação , Bactérias/isolamento & purificação , Estudos de Casos e Controles , Feminino , Humanos , Índia , Lactente , Masculino , Parasitos/classificação , Parasitos/isolamento & purificação , Vacina Antipólio Oral/administração & dosagem , Reação em Cadeia da Polimerase em Tempo Real , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/imunologia , Eliminação de Partículas Virais , Vírus/classificação , Vírus/isolamento & purificaçãoRESUMO
Carboxypeptidase E is a peptide processing enzyme, involved in cleaving numerous peptide precursors, including neuropeptides and hormones involved in appetite control and glucose metabolism. Exome sequencing of a morbidly obese female from a consanguineous family revealed homozygosity for a truncating mutation of the CPE gene (c.76_98del; p.E26RfsX68). Analysis detected no CPE expression in whole blood-derived RNA from the proband, consistent with nonsense-mediated decay. The morbid obesity, intellectual disability, abnormal glucose homeostasis and hypogonadotrophic hypogonadism seen in this individual recapitulates phenotypes in the previously described fat/fat and Cpe knockout mouse models, evidencing the importance of this peptide/hormone-processing enzyme in regulating body weight, metabolism, and brain and reproductive function in humans.
Assuntos
Carboxipeptidase H/genética , Diabetes Mellitus Tipo 2/complicações , Deficiência Intelectual/complicações , Síndrome de Klinefelter/complicações , Mutação/genética , Obesidade Mórbida/complicações , Obesidade Mórbida/genética , Carboxipeptidase H/metabolismo , Análise Mutacional de DNA , Diabetes Mellitus Tipo 2/enzimologia , Diabetes Mellitus Tipo 2/genética , Exoma/genética , Feminino , Regulação Enzimológica da Expressão Gênica , Homozigoto , Humanos , Deficiência Intelectual/genética , Síndrome de Klinefelter/enzimologia , Síndrome de Klinefelter/genética , Masculino , Obesidade Mórbida/enzimologia , Linhagem , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Adulto JovemRESUMO
The human ZFP36 zinc finger protein family consists of ZFP36, ZFP36L1, and ZFP36L2. These proteins regulate various cellular processes, including cell apoptosis, by binding to adenine uridine rich elements in the 3' untranslated regions of sets of target mRNAs to promote their degradation. The pro-apoptotic and other functions of ZFP36 family members have been implicated in the pathogenesis of lymphoid malignancies. To identify candidate mRNAs that are targeted in the pro-apoptotic response by ZFP36L1, we reverse-engineered a gene regulatory network for all three ZFP36 family members using the 'maximum information coefficient' (MIC) for target gene inference on a large microarray gene expression dataset representing cells of diverse histological origin. Of the three inferred ZFP36L1 mRNA targets that were identified, we focussed on experimental validation of mRNA for the pro-survival protein, BCL2, as a target for ZFP36L1. RNA electrophoretic mobility shift assay experiments revealed that ZFP36L1 interacted with the BCL2 adenine uridine rich element. In murine BCL1 leukemia cells stably transduced with a ZFP36L1 ShRNA lentiviral construct, BCL2 mRNA degradation was significantly delayed compared to control lentiviral expressing cells and ZFP36L1 knockdown in different cell types (BCL1, ACHN, Ramos), resulted in increased levels of BCL2 mRNA levels compared to control cells. 3' untranslated region luciferase reporter assays in HEK293T cells showed that wild type but not zinc finger mutant ZFP36L1 protein was able to downregulate a BCL2 construct containing the BCL2 adenine uridine rich element and removal of the adenine uridine rich core from the BCL2 3' untranslated region in the reporter construct significantly reduced the ability of ZFP36L1 to mediate this effect. Taken together, our data are consistent with ZFP36L1 interacting with and mediating degradation of BCL2 mRNA as an important target through which ZFP36L1 mediates its pro-apoptotic effects in malignant B-cells.
Assuntos
Regiões 3' não Traduzidas , Fator 1 de Resposta a Butirato/genética , Regulação Neoplásica da Expressão Gênica , Proteínas Nucleares/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas de Ligação a RNA/genética , Animais , Linfócitos B , Fator 1 de Resposta a Butirato/antagonistas & inibidores , Fator 1 de Resposta a Butirato/metabolismo , Linhagem Celular Tumoral , Genes Reporter , Vetores Genéticos , Células HEK293 , Humanos , Lentivirus/genética , Lentivirus/metabolismo , Luciferases/genética , Luciferases/metabolismo , Camundongos , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Estabilidade de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/metabolismo , Elementos de Resposta , Transdução de SinaisRESUMO
The ZFP36/Tis11 family of zinc-finger proteins regulate cellular processes by binding to adenine uridine rich elements in the 3' untranslated regions of various mRNAs and promoting their degradation. We show here that ZFP36L1 expression is largely extinguished during the transition from B cells to plasma cells, in a reciprocal pattern to that of ZFP36 and the plasma cell transcription factor, BLIMP1. Enforced expression of ZFP36L1 in the mouse BCL1 cell line blocked cytokine-induced differentiation while shRNA-mediated knock-down enhanced differentiation. Reconstruction of regulatory networks from microarray gene expression data using the ARACNe algorithm identified candidate mRNA targets for ZFP36L1 including BLIMP1. Genes that displayed down-regulation in plasma cells were significantly over-represented (Pâ=â<0.0001) in a set of previously validated ZFP36 targets suggesting that ZFP36L1 and ZFP36 target distinct sets of mRNAs during plasmacytoid differentiation. ShRNA-mediated knock-down of ZFP36L1 in BCL1 cells led to an increase in levels of BLIMP1 mRNA and protein, but not for mRNAs of other transcription factors that regulate plasmacytoid differentiation (xbp1, irf4, bcl6). Finally, ZFP36L1 significantly reduced the activity of a BLIMP1 3' untranslated region-driven luciferase reporter. Taken together, these findings suggest that ZFP36L1 negatively regulates plasmacytoid differentiation, at least in part, by targeting the expression of BLIMP1.