RESUMO
Phenylalanine hydroxylase deficient phenylketonuria (PKU) is the paradigm for a treatable inborn error of metabolism where maintaining plasma phenylalanine (Phe) in the therapeutic range relates to improved clinical outcomes. While Phe is the presumed intoxicating analyte causal in neurologic damage, the mechanism(s) of Phe toxicity has remained elusive. Altered DNA methylation is a recognized response associated with exposure to numerous small molecule toxic agents. Paralleling this effect, we hypothesized that chronic Phe over-exposure in the brain would lead to aberrant DNA methylation with secondary influence upon gene regulation that would ultimately contribute to PKU neuropathology. The PAH(enu2) mouse models human PKU with intrinsic hyperphenylalaninemia, abnormal response to Phe challenge, and neurologic deficit. To examine this hypothesis, we assessed DNA methylation patterns in brain tissues using methylated DNA immunoprecipitation and paired end sequencing in adult PAH(enu2) animals maintained under either continuous dietary Phe restriction or chronic hyperphenylalaninemia. Heterozygous PAH(enu2/WT) litter mates served as controls for normal Phe exposure. Extensive repatterning of DNA methylation was observed in brain tissue of hyperphenylalaninemic animals while Phe restricted animals displayed an attenuated pattern of aberrant DNA methylation. Affected gene coding regions displayed aberrant hypermethylation and hypomethylation. Gene body methylation of noncoding RNA genes was observed and among these microRNA genes were prominent. Of particular note, observed only in hyperphenylalaninemic animals, was hypomethylation of miRNA genes within the imprinted Dlk1-Dio3 locus on chromosome 12. Aberrant methylation of microRNA genes influenced their expression which has secondary effects upon the expression of targeted protein coding genes. Differential hypermethylation of gene promoters was exclusive to hyperphenylalaninemic PAH(enu2) animals. Genes with synaptic involvement were targets of promoter hypermethylation that resulted in down-regulation of their expression. Gene dysregulation secondary to abnormal DNA methylation may be contributing to PKU neuropathology. These results suggest drugs that prevent or correct aberrant DNA methylation may offer a novel therapeutic option to management of neurological symptoms in PKU patients.
Assuntos
Metilação de DNA/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Iodeto Peroxidase/genética , Fenilalanina Hidroxilase/genética , Fenilcetonúrias/genética , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Proteínas de Ligação ao Cálcio , Modelos Animais de Doenças , Regulação da Expressão Gênica , Impressão Genômica , Humanos , Fígado/metabolismo , Fígado/patologia , Camundongos , Fenilalanina/genética , Fenilalanina/metabolismo , Fenilalanina Hidroxilase/deficiência , Regiões Promotoras Genéticas , RNA não Traduzido/genéticaRESUMO
Maternal PKU Syndrome (MPKU) is an embryopathy resulting from in utero phenylalanine (PHE) toxicity secondary to maternal phenylalanine hydroxylase deficient phenylketonuria (PKU). Clinical phenotypes in MPKU include mental retardation, microcephaly, in utero growth restriction, and congenital heart defects. Numerous in utero toxic exposures alter DNA methylation in the fetus. The PAH(enu2) mouse is a model of classical PKU while offspring born of hyperphenylalaninemic dams model MPKU. We investigated offspring of PAH(enu2) dams to determine if altered patterns of DNA methylation occurred in response to in utero PHE exposure. As neurologic deficit is the most prominent MPKU phenotype, methylome patterns were assessed in brain tissue using methylated DNA immunoprecipitation and paired-end sequencing. Brain tissues were assessed in E18.5-19 fetuses of PHE unrestricted PAH(enu2) dams, PHE restricted PAH(enu2) dams, and heterozygous(wt/enu2) control dams. Extensive methylome repatterning was observed in offspring of hyperphenylalaninemic dams while the offspring of PHE restricted dams displayed attenuated methylome repatterning. Methylation within coding regions was dominated by noncoding RNA genes. Differential methylation of promoters targeted protein coding genes. To assess the impact of methylome repatterning on gene expression, brain tissue in experimental and control animals were queried with microarrays assessing expression of microRNAs and protein coding genes. Altered expression of methylome-modified microRNAs and protein coding genes was extensive in offspring of hyperphenylalaninemic dams while minimal changes were observed in offspring of PHE restricted dams. Several genes displaying significantly reduced expression have roles in neurological function or genetic disease with neurological phenotypes. These data indicate in utero PHE toxicity alters DNA methylation in the brain which has downstream impact upon gene expression. Altered gene expression may contribute to pathophysiology of neurologic presentation in MPKU.
Assuntos
Metilação de DNA , Fenilcetonúria Materna/genética , Animais , Encéfalo/metabolismo , Modelos Animais de Doenças , Feminino , Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/fisiologia , Gravidez , Regiões Promotoras Genéticas , Interferência de RNA , Análise de Sequência de DNARESUMO
BACKGROUND: Preeclampsia is a pregnancy-specific disorder that remains a leading cause of maternal, fetal and neonatal morbidity and mortality, and is associated with risk for future cardiovascular disease. There are no reliable predictors, specific preventative measures or treatments other than delivery. A widely held view is that the antecedents of preeclampsia lie with impaired placentation in early pregnancy. Accordingly, we hypothesized dysregulation of global gene expression in first trimester placentas of women who later manifested preeclampsia. METHODS: Surplus chorionic villus sampling (CVS) tissues were collected at 10-12 weeks gestation in 160 patients with singleton fetuses. Four patients developed preeclampsia, and their banked CVS specimens were matched to 8 control samples from patients with unaffected pregnancies. Affymetrix HG-U133 Plus 2.0 GeneChips were utilized for microarray analysis. Naïve Bayes prediction modeling and pathway analysis were conducted. qRT-PCR examined three of the dysregulated genes. RESULTS: Thirty-six differentially expressed genes were identified in the preeclampsia placentas. qRT-PCR verified the microarray analysis. Thirty-one genes were down-regulated. Many were related to inflammation/immunoregulation and cell motility. Decidual gene dysregulation was prominent. No evidence was found for alterations in hypoxia and oxidative stress regulated genes. CONCLUSIONS: To our knowledge, this is the first study to show dysregulation of gene expression in the early placentas of women approximately 6 months before developing preeclampsia, thereby reinforcing a placental origin of the disorder. We hypothesize that placentation in preeclampsia is compromised in the first trimester by maternal and fetal immune dysregulation, abnormal decidualization, or both, thereby impairing trophoblast invasion. Several of the genes provide potential targets for the development of clinical biomarkers in maternal blood during the first trimester. Supplementary materials are available for this article via the publisher's online edition.
Assuntos
Vilosidades Coriônicas/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Predisposição Genética para Doença/genética , Estudo de Associação Genômica Ampla , Pré-Eclâmpsia/genética , Adulto , Biomarcadores/metabolismo , Regulação para Baixo , Feminino , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Pré-Eclâmpsia/metabolismo , Gravidez , Primeiro Trimestre da GravidezRESUMO
Most eubacteria, and all eukaryotes examined thus far, encode homologs of the DNA mismatch repair protein MutS. Although eubacteria encode only one or two MutS-like proteins, eukaryotes encode at least six distinct MutS homolog (MSH) proteins, corresponding to conserved (orthologous) gene families. This suggests evolution of individual gene family lines of descent by several duplication/specialization events. Using quantitative phylogenetic analyses (RASA, or relative apparent synapomorphy analysis), we demonstrate that comparison of complete MutS protein sequences, rather than highly conserved C-terminal domains only, maximizes information about evolutionary relationships. We identify a novel, highly conserved middle domain, as well as clearly delineate an N-terminal domain, previously implicated in mismatch recognition, that shows family-specific patterns of aromatic and charged amino acids. Our final analysis, in contrast to previous analyses of MutS-like sequences, yields a stable phylogenetic tree consistent with the known biochemical functions of MutS/MSH proteins, that now assigns all known eukaryotic MSH proteins to a monophyletic group, whose branches correspond to the respective specialized gene families. The rooted phylogenetic tree suggests their derivation from a mitochondrial MSH1-like protein, itself the descendent of the MutS of a symbiont in a primitive eukaryotic precursor.
Assuntos
Adenosina Trifosfatases , Proteínas de Bactérias/genética , Pareamento Incorreto de Bases , Reparo do DNA/genética , Proteínas de Ligação a DNA , Proteínas de Escherichia coli , Evolução Molecular , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/química , Duplicação Gênica , Humanos , Dados de Sequência Molecular , Proteína MutS de Ligação de DNA com Erro de Pareamento , Filogenia , Homologia de Sequência de AminoácidosAssuntos
Íntrons/genética , Modelos Genéticos , Família Multigênica , Sequência de Bases , DNA Fúngico/genética , DNA de Plantas/genética , Eucariotos/genética , Evolução Molecular , Fungos/genética , Genes Fúngicos , Genes de Plantas , Dados de Sequência Molecular , Método de Monte Carlo , Alinhamento de Sequência , Homologia de Sequência do Ácido NucleicoRESUMO
The aim of this study was to use molecular identification methods, such as 16S RNA gene sequence and reverse-capture checkerboard hybridization, for identification of the bacteria associated with dental caries and with dental health in a subset of 204 twins aged 1.5 to 7 years old. A total of 448 plaque samples (118 collected from caries-free subjects and 330 from caries-active subjects) were used for analysis. We compared the bacteria found in biofilms of children exhibiting severe dental caries, with different degrees of lesion severity, with those found in biofilms of caries-free children. A panel of 82 bacterial species was selected, and a PCR-based reverse-capture checkerboard method was used for detection. A simple univariate test was used to determine the overabundance and underabundance of bacterial species in the diseased and in the healthy groups. Features identified with this univariate test were used to construct a probabilistic disease prediction model. Furthermore, a method for the analysis of global patterns of gene expression was performed to permit simultaneous analysis of the abundance of significant species by allowing cross-bacterial comparisons of abundance profiles between caries-active and caries-free subjects. Our results suggested that global patterns of microbial abundance in this population are very distinctive. The top bacterial species found to be overabundant in the caries-active group were Actinomyces sp. strain B19SC, Streptococcus mutans, and Lactobacillus spp., which exhibited an inverse relationship to beneficial bacterial species, such as Streptococcus parasanguinis, Abiotrophia defectiva, Streptococcus mitis, Streptococcus oralis, and Streptococcus sanguinis.
Assuntos
Cárie Dentária/microbiologia , Bactérias Gram-Positivas/isolamento & purificação , Criança , Pré-Escolar , Bactérias Gram-Positivas/genética , Humanos , Lactente , Reação em Cadeia da Polimerase , Polissorbatos , RNA Bacteriano/análise , RNA Bacteriano/genética , RNA Ribossômico 16S/análise , RNA Ribossômico 16S/genética , Especificidade da EspécieRESUMO
Long branches in a true phylogeny tend to disrupt hierarchical character covariation (phylogenetic signal) in the distribution of traits among organisms. The distortion of hierarchical structure in character-state matrices can lead to errors in the estimation of phylogenetic relationships and inconsistency of methods of phylogenetic inference. Examination of trees distorted by long-branch attraction will not reveal the identities of problematic taxa, in part because the distortion can mask long branches by reducing inferred branch lengths and through errors in branching order. Here we present a simple method for the detection of taxa whose placement in evolutionary trees is made difficult by the effects of long-branch attraction. The method is an extension of a tree-independent conceptual framework of phylogenetic data exploration (RASA). Taxa that are likely to attract are revealed because long branches leave distinct footprints in the distribution of character states among taxa, and these traces can be directly observed in the error structure of the RASA regression. Problematic taxa are identified using a new diagnostic plot called the taxon variance plot, in which the apparent cladistic and phenetic variances contributed by individual taxa are compared. The procedure for identifying long edges employs algorithms solved in polynomial time and can be applied to morphological, molecular, and mixed characters. The efficacy of the method is demonstrated using simulated evolution and empirical evidence of long branches in a set of recently published sequences. We show that the accuracy of evolutionary trees can be improved by detecting and combating the potentially misleading influences of long-branch taxa.
Assuntos
Modelos Genéticos , Filogenia , ClassificaçãoRESUMO
Considerable confusion remains among theoreticians and practicioners of phylogenetic science on the use of outgroup taxa. Here, we show that, despite claims to the contrary, details of the optimal ingroup topology can be changed by switching outgroup taxa. This has serious implications for phylogenetic accuracy. We delineate between the process of outgroup selection and the various possible processes involved in using an outgroup taxon after one has been selected. Criteria are needed for the determination that particular outgroup taxa do not reduce the accuracy of evolutionary tree topologies and inferred character state transformations. We compare previous results from a sensitivity bootstrap analysis of the mitochondrial cytochrome b phylogenetic relationships among whales to the results of a Bremer support sensitivity analysis and of a recently developed application of RASA theory to the question of putative outgroup taxon plesiomorphy content.
Assuntos
Grupo dos Citocromos b/genética , Evolução Molecular , Modelos Genéticos , Filogenia , Homologia de Sequência de Aminoácidos , Animais , Grupo dos Citocromos b/química , Mamíferos , Mitocôndrias/metabolismo , Análise de SequênciaRESUMO
Branch length estimates play a central role in maximum-likelihood (ML) and minimum-evolution (ME) methods of phylogenetic inference. For various reasons, branch length estimates are not statistically independent under ML or ME. We studied the response of correlations among branch length estimates to the degree of among-branch length heterogeneity (BLH) in the model (true) tree. The frequency and magnitude of (especially negative) correlations among branch length estimates were both shown to increase as BLH increases under simulation and analytically. For ML, we used the correct model (Jukes-Cantor). For ME, we employed ordinary least-squares (OLS) branch lengths estimated under both simple p-distances and Jukes-Cantor distances, analyzed with and without an among-site rate heterogeneity parameter. The efficiency of ME and ML was also shown to decrease in response to increased BLH. We note that the shape of the true tree will in part determine BLH and represents a critical factor in the probability of recovering the correct topology. An important finding suggests that researchers cannot expect that different branches that were in fact the same length will have the same probability of being accurately reconstructed when BLH exists in the overall tree. We conclude that methods designed to minimize the interdependencies of branch length estimates (BLEs) may (1) reduce both the variance and the covariance associated with the estimates and (2) increase the efficiency of model-based optimality criteria. We speculate on possible ways to reduce the nonindependence of BLEs under OLS and ML.
Assuntos
Evolução Molecular , Modelos Biológicos , Filogenia , Funções Verossimilhança , Modelos EstatísticosRESUMO
We have developed a new approach to the measurement of phylogenetic signal in character state matrices called relative apparent synapomorphy analysis (RASA). RASA provides a deterministic, statistical measure of natural cladistic hierarchy (phylogenetic signal) in character state matrices. The method works by determining whether a measure of the rate of increase of cladistic similarity among pairs of taxa as a function of phenetic similarity is greater than a null equiprobable rate of increase. Our investigation of the utility and limitations of RASA using simulated and bacteriophage T7 data sets indicates that the method has numerous advantages over existing measures of signal. A first advantage is computational efficiency. A second advantage is that RASA employs known methods of statistical inference, providing measurable sensitivity and power. The performance of RASA is examined under various conditions of branching evolution as the number of characters, character states per character, and mutations per branch length are varied. RASA appears to provide an unbiased and reliable measure of phylogenetic signal, and the general approach promises to be useful in the development of new techniques that should increase the rigor and reliability of phylogenetic estimates.
Assuntos
Algoritmos , Modelos Genéticos , Filogenia , Bacteriófago T7/genética , Bacteriófago T7/ultraestrutura , Simulação por Computador , Evolução Molecular , Fenótipo , SoftwareRESUMO
Plant phylogenetic estimates are most likely to be reliable when congruent evidence is obtained independently from the mitochondrial, plastid, and nuclear genomes with all methods of analysis. Here, results are presented from separate and combined genomic analyses of new and previously published data, including six and nine genes (8, 911 bp and 12,010 bp, respectively) for different subsets of taxa that suggest Amborella + Nymphaeales (water lilies) are the first-branching angiosperm lineage. Before and after tree-independent noise reduction, most individual genomic compartments and methods of analysis estimated the Amborella + Nymphaeales basal topology with high support. Previous phylogenetic estimates placing Amborella alone as the first extant angiosperm branch may have been misled because of a series of specific problems with paralogy, suboptimal outgroups, long-branch taxa, and method dependence. Ancestral character state reconstructions differ between the two topologies and affect inferences about the features of early angiosperms.