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1.
PLoS One ; 13(6): e0198256, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29870545

RESUMO

Enzastaurin is a Protein Kinase C-ß selective inhibitor that was developed to treat cancers. Protein Kinase C-ß is an important enzyme for a variety of neuronal functions; in particular, previous rodent studies have reported deficits in spatial and fear-conditioned learning and memory with lower levels of Protein Kinase C-ß. Due to Enzastaurin's mechanism of action, the present study investigated the consequences of Enzastaurin exposure on learning and memory in 12-month-old Fischer-344 male rats. Rats were treated daily with subcutaneous injections of either vehicle or Enzastaurin, and behaviorally tested using the spatial reference memory Morris Water Maze. Rats treated with Enzastaurin exhibited decreased overnight retention and poorer performance on the latter testing day, indicating a mild, but significant, memory impairment. There were no differences during the probe trial indicating that all animals were able to spatially localize the platform to the proper quadrant by the end of testing. RNA isolated from the hippocampus was analyzed using Next Generation Sequencing (Illumina). No statistically significant transcriptional differences were noted. Our findings suggest that acute Enzastaurin treatment can impair hippocampal-based learning and memory performance, with no effects on transcription in the hippocampus. We propose that care should be taken in future clinical trials that utilize Protein Kinase C-ß inhibitors, to monitor for possible cognitive effects, future research should examine if these effects are fully reversible.

2.
Hippocampus ; 27(7): 784-793, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28380666

RESUMO

The rs17070145-T variant of the WWC1 gene, coding for the KIBRA protein, has been associated with both increased episodic memory performance and lowered risk for late onset Alzheimer's disease, although the mechanism behind this protective effect has not been completely elucidated. To achieve a better understanding of the pathways modulated by rs17070145 and its associated functional variant(s), we used laser capture microdissection (LCM) and RNA-sequencing to investigate the effect of rs17070145 genotypes on whole transcriptome expression in the human hippocampus (HP) of 22 neuropathologically normal individuals, with a specific focus on the dentate gyrus (DG) and at the pyramidal cells (PC) of CA1 and CA3 sub-regions. Differential expression analysis of RNA-seq data within the HP based on the rs17070145 genotype revealed an overexpression of genes involved in the MAPK signaling pathway, potentially driven by the T/T genotype. The most important contribution comes from genes dysregulated within the DG region. Other genes significantly dysregulated, and not involved in the MAPK pathway (Adj P < 0.01 and Fold Change > |1.00|) were: RSPO4 (HP); ARC, DUSP5, DNAJB5, EGR4, PPP1R15A, WBP11P1, EGR1, GADD45B (DG); CH25H, HSPA1A, HSPA1B, TNFSF9, and NPAS4 (PC). Several evidences suggested that the MAPK signaling pathway is linked with memory and learning processes. In non-neuronal cells, the KIBRA protein is phosphorylated by ERK1/2 (involved in the MAPK signaling) in cells as well as in vitro. Several of the other dysregulated genes are involved in memory and learning processes, as well as in Alzheimer's Disease. In conclusion, our results suggest that the effect of the WWC1 rs17070145 polymorphism on memory performance and Alzheimer's disease might be due to a differential regulation of the MAPK signaling, a key pathway involved in memory and learning processes.


Assuntos
Hipocampo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Sistema de Sinalização das MAP Quinases/genética , Fosfoproteínas/genética , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Feminino , Perfilação da Expressão Gênica , Genótipo , Humanos , Masculino , Polimorfismo de Nucleotídeo Único
3.
J Neurosci ; 36(42): 10750-10758, 2016 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-27798130

RESUMO

Ventral regions of the medulla oblongata of the brainstem are populated by astrocytes sensitive to physiological changes in PCO2/[H+]. These astrocytes respond to decreases in pH with elevations in intracellular Ca2+ and facilitated exocytosis of ATP-containing vesicles. Released ATP propagates Ca2+ excitation among neighboring astrocytes and activates neurons of the brainstem respiratory network triggering adaptive increases in breathing. The mechanisms linking increases in extracellular and/or intracellular PCO2/[H+] with Ca2+ responses in chemosensitive astrocytes remain unknown. Fluorescent imaging of changes in [Na+]i and/or [Ca2+]i in individual astrocytes was performed in organotypic brainstem slice cultures and acute brainstem slices of adult rats. It was found that astroglial [Ca2+]i responses triggered by decreases in pH are preceded by Na+ entry, markedly reduced by inhibition of Na+/HCO3- cotransport (NBC) or Na+/Ca2+ exchange (NCX), and abolished in Na+-free medium or by combined NBC/NCX blockade. Acidification-induced [Ca2+]i responses were also dramatically reduced in brainstem astrocytes of mice deficient in the electrogenic Na+/HCO3- cotransporter NBCe1. Sensitivity of astrocytes to changes in pH was not affected by inhibition of Na+/H+ exchange or blockade of phospholipase C. These results suggest that in pH-sensitive astrocytes, acidification activates NBCe1, which brings Na+ inside the cell. Raising [Na+]i activates NCX to operate in a reverse mode, leading to Ca2+ entry followed by activation of downstream signaling pathways. Coupled NBC and NCX activities are, therefore, suggested to be responsible for functional CO2/H+ sensitivity of astrocytes that contribute to homeostatic regulation of brain parenchymal pH and control of breathing. SIGNIFICANCE STATEMENT: Brainstem astrocytes detect physiological changes in pH, activate neurons of the neighboring respiratory network, and contribute to the development of adaptive respiratory responses to the increases in the level of blood and brain PCO2/[H+]. The mechanisms underlying astroglial pH sensitivity remained unknown and here we show that in brainstem astrocytes acidification activates Na+/HCO3- cotransport, which brings Na+ inside the cell. Raising [Na+]i activates the Na+/Ca2+ exchanger to operate in a reverse mode leading to Ca2+ entry. This identifies a plausible mechanism of functional CO2/H+ sensitivity of brainstem astrocytes, which play an important role in homeostatic regulation of brain pH and control of breathing.


Assuntos
Astrócitos/efeitos dos fármacos , Dióxido de Carbono/farmacologia , Hidrogênio/farmacologia , Trifosfato de Adenosina/metabolismo , Animais , Astrócitos/metabolismo , Bicarbonatos/metabolismo , Sinalização do Cálcio , Exocitose , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Neuroglia/efeitos dos fármacos , Neuroglia/metabolismo , Ratos , Respiração , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Sódio/metabolismo , Simportadores de Sódio-Bicarbonato/antagonistas & inibidores , Simportadores de Sódio-Bicarbonato/genética , Simportadores de Sódio-Bicarbonato/metabolismo , Trocador de Sódio e Cálcio/antagonistas & inibidores , Trocador de Sódio e Cálcio/metabolismo
4.
Cold Spring Harb Mol Case Stud ; 2(5): a000851, 2016 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-27626064

RESUMO

Recently, mutations in the zinc finger MYND-type containing 11 (ZMYND11) gene were identified in patients with autism spectrum disorders, intellectual disability, aggression, and complex neuropsychiatric features, supporting that this gene is implicated in 10p15.3 microdeletion syndrome. We report a novel de novo variant in the ZMYND11 gene (p.Ser421Asn) in a patient with a complex neurodevelopmental phenotype. The patient is a 24-yr-old Caucasian/Filipino female with seizures, global developmental delay, sensorineural hearing loss, hypotonia, dysmorphic features, and other features including a happy disposition and ataxic gait similar to Angelman syndrome. In addition, this patient had uncommon features including eosinophilic esophagitis and multiple, severe allergies not described in similar ZMYND11 cases. This new case further supports the association of ZMYND11 with autistic-like phenotypes and suggests that ZMYND11 should be included in the list of potentially causative candidate genes in cases with complex neurodevelopmental phenotypes.

5.
Neurol Genet ; 2(3): e75, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-27275011

RESUMO

OBJECTIVE: We sought to determine the underlying cortical gene expression changes associated with Parkinson dementia using a next-generation RNA sequencing approach. METHODS: In this study, we used RNA sequencing to evaluate differential gene expression and alternative splicing in the posterior cingulate cortex from neurologically normal control patients, patients with Parkinson disease, and patients with Parkinson disease with dementia. RESULTS: Genes overexpressed in both disease states were involved with an immune response, whereas shared underexpressed genes functioned in signal transduction or as components of the cytoskeleton. Alternative splicing analysis produced a pattern of immune and RNA-processing disturbances. CONCLUSIONS: Genes with the greatest degree of differential expression did not overlap with genes exhibiting significant alternative splicing activity. Such variation indicates the importance of broadening expression studies to include exon-level changes because there can be significant differential splicing activity with potential structural consequences, a subtlety that is not detected when examining differential gene expression alone, or is underrepresented with probe-limited array technology.

6.
Hum Mutat ; 37(8): 812-9, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27068579

RESUMO

Although there are nearly 100 different causative genes identified for nonsyndromic hearing loss (NSHL), Sanger sequencing-based DNA diagnostics usually only analyses three, namely, GJB2, SLC26A4, and OTOF. As this is seen as inadequate, there is a need for high-throughput diagnostic methods to detect disease-causing variations, including single-nucleotide variations (SNVs), insertions/deletions (Indels), and copy-number variations (CNVs). In this study, a targeted resequencing panel for hearing loss was developed including 79 genes for NSHL and selected forms of syndromic hearing loss. One-hundred thirty one presumed autosomal-recessive NSHL (arNSHL) patients of Western-European ethnicity were analyzed for SNVs, Indels, and CNVs. In addition, we established a straightforward variant classification system to deal with the large number of variants encountered. We estimate that combining prescreening of GJB2 with our panel leads to a diagnosis in 25%-30% of patients. Our data show that after GJB2, the most commonly mutated genes in a Western-European population are TMC1, MYO15A, and MYO7A (3.1%). CNV analysis resulted in the identification of causative variants in two patients in OTOA and STRC. One of the major challenges for diagnostic gene panels is assigning pathogenicity for variants. A collaborative database collecting all identified variants from multiple centers could be a valuable resource for hearing loss diagnostics.


Assuntos
Predisposição Genética para Doença , Perda Auditiva Neurossensorial/diagnóstico , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Mutação , Análise de Sequência de DNA/métodos , Conexinas/genética , Variações do Número de Cópias de DNA , Exoma , Proteínas Ligadas por GPI/genética , Perda Auditiva Neurossensorial/genética , Humanos , Mutação INDEL , Proteínas de Membrana/genética , Miosinas/genética , Polimorfismo de Nucleotídeo Único
7.
Hear Res ; 333: 266-274, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26341477

RESUMO

The mammalian inner ear consists of the cochlea and the vestibular labyrinth (utricle, saccule, and semicircular canals), which participate in both hearing and balance. Proper development and life-long function of these structures involves a highly complex coordinated system of spatial and temporal gene expression. The characterization of the inner ear transcriptome is likely important for the functional study of auditory and vestibular components, yet, primarily due to tissue unavailability, detailed expression catalogues of the human inner ear remain largely incomplete. We report here, for the first time, comprehensive transcriptome characterization of the adult human cochlea, ampulla, saccule and utricle of the vestibule obtained from patients without hearing abnormalities. Using RNA-Seq, we measured the expression of >50,000 predicted genes corresponding to approximately 200,000 transcripts, in the adult inner ear and compared it to 32 other human tissues. First, we identified genes preferentially expressed in the inner ear, and unique either to the vestibule or cochlea. Next, we examined expression levels of specific groups of potentially interesting RNAs, such as genes implicated in hearing loss, long non-coding RNAs, pseudogenes and transcripts subject to nonsense mediated decay (NMD). We uncover the spatial specificity of expression of these RNAs in the hearing/balance system, and reveal evidence of tissue specific NMD. Lastly, we investigated the non-syndromic deafness loci to which no gene has been mapped, and narrow the list of potential candidates for each locus. These data represent the first high-resolution transcriptome catalogue of the adult human inner ear. A comprehensive identification of coding and non-coding RNAs in the inner ear will enable pathways of auditory and vestibular function to be further defined in the study of hearing and balance. Expression data are freely accessible at https://www.tgen.org/home/research/research-divisions/neurogenomics/supplementary-data/inner-ear-transcriptome.aspx.


Assuntos
Orelha Interna/química , Perfilação da Expressão Gênica/métodos , RNA Mensageiro/genética , RNA não Traduzido/genética , Transcriptoma , Animais , Biologia Computacional , Bases de Dados Genéticas , Surdez/genética , Surdez/fisiopatologia , Predisposição Genética para Doença , Audição/genética , Humanos , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Análise de Sequência de RNA
8.
Neurosurgery ; 78(6): 835-43, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-26606670

RESUMO

BACKGROUND: Stress-induced cardiomyopathy (SIC) is a poorly understood condition associated with periods of emotional and physical stress. The clinical approaches for management of SIC are supportive and reactive to patient symptoms. OBJECTIVE: To utilize next-generation exome sequencing to define genetic variation associated with, and potentially responsible for, this disease. METHODS: We performed exome sequencing of 7 white female patients with SIC. Filtering of the identified variants was performed to limit our investigation to those sequences that passed quality control criteria, were rare or novel, were determined algorithmically to have high impact on the associated protein, and were within regions of high species conservation. All variants were verified by using Sanger sequencing. RESULTS: Exome-sequencing analysis revealed that each patient carried predicted deleterious variants affecting known cardiomyopathy genes. In each case, the identified variant was either not previously found in public human genome data or was previously annotated in a database of clinical variants associated with cardiac dysfunction. CONCLUSION: Patients with SIC harbor deleterious mutations in established cardiomyopathy genes at a level higher than healthy controls. We hypothesize that patients at highest risk for SIC likely live in a compensated state of cardiac dysfunction that manifests clinically only after the myocardium is stressed. In short, we propose that SIC is another example of an occult cardiomyopathy with a distinct physiological trigger and suggest that alternative clinical approaches to these patients may be warranted. ABBREVIATIONS: CADD, Combined Annotation Dependent DepletionFPKM, fragments per kilobase pair of exon per million fragments mappedNHLBI GO ESP, National Heart, Lung, and Blood Institute Grand Opportunity Exome Sequencing ProjectPCR, polymerase chain reactionSIC, stress-induced cardiomyopathy.


Assuntos
Cardiomiopatias/genética , Predisposição Genética para Doença/genética , Estresse Fisiológico/genética , Estresse Psicológico/genética , Adulto , Grupo com Ancestrais do Continente Europeu , Exoma , Feminino , Variação Genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino
9.
PLoS One ; 10(10): e0135076, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26474411

RESUMO

We have previously hypothesized a biological pathway of activity-dependent synaptic plasticity proteins that addresses the dual genetic and environmental contributions to schizophrenia. Accordingly, variations in the immediate early gene EGR3, and its target ARC, should influence schizophrenia susceptibility. We used a pooled Next-Generation Sequencing approach to identify variants across these genes in U.S. populations of European (EU) and African (AA) descent. Three EGR3 and one ARC SNP were selected and genotyped for validation, and three SNPs were tested for association in a replication cohort. In the EU group of 386 schizophrenia cases and 150 controls EGR3 SNP rs1877670 and ARC SNP rs35900184 showed significant associations (p = 0.0078 and p = 0.0275, respectively). In the AA group of 185 cases and 50 controls, only the ARC SNP revealed significant association (p = 0.0448). The ARC SNP did not show association in the Han Chinese (CH) population. However, combining the EU, AA, and CH groups revealed a highly significant association of ARC SNP rs35900184 (p = 2.353 x 10(-7); OR [95% CI] = 1.54 [1.310-1.820]). These findings support previously reported associations between EGR3 and schizophrenia. Moreover, this is the first report associating an ARC SNP with schizophrenia and supports recent large-scale GWAS findings implicating the ARC complex in schizophrenia risk. These results support the need for further investigation of the proposed pathway of environmentally responsive, synaptic plasticity-related, schizophrenia genes.


Assuntos
Proteínas do Citoesqueleto/genética , Proteína 3 de Resposta de Crescimento Precoce/genética , Proteínas do Tecido Nervoso/genética , Polimorfismo de Nucleotídeo Único , Esquizofrenia/genética , Grupo com Ancestrais do Continente Asiático , China/etnologia , Feminino , Técnicas de Genotipagem , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Fatores de Risco , Esquizofrenia/etnologia
10.
Genome Res ; 25(11): 1646-55, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26232412

RESUMO

Canine transmissible venereal tumor (CTVT) is a parasitic cancer clone that has propagated for thousands of years via sexual transfer of malignant cells. Little is understood about the mechanisms that converted an ancient tumor into the world's oldest known continuously propagating somatic cell lineage. We created the largest existing catalog of canine genome-wide variation and compared it against two CTVT genome sequences, thereby separating alleles derived from the founder's genome from somatic mutations that must drive clonal transmissibility. We show that CTVT has undergone continuous adaptation to its transmissible allograft niche, with overlapping mutations at every step of immunosurveillance, particularly self-antigen presentation and apoptosis. We also identified chronologically early somatic mutations in oncogenesis- and immune-related genes that may represent key initiators of clonal transmissibility. Thus, we provide the first insights into the specific genomic aberrations that underlie CTVT's dogged perseverance in canids around the world.


Assuntos
Doenças do Cão/genética , Cães/genética , Estudos de Associação Genética , Tumores Venéreos Veterinários/genética , Animais , Apoptose , Autoantígenos/genética , Proteínas Adaptadoras de Sinalização CARD/genética , Moléculas de Adesão Celular/genética , Linhagem Celular Tumoral , Linhagem da Célula/genética , Colágeno Tipo XI/genética , Proteínas de Ligação a DNA/genética , Doenças do Cão/diagnóstico , Variação Genética , Genoma , Fatores de Troca do Nucleotídeo Guanina/genética , Proteoglicanas de Heparan Sulfato/genética , Proteínas dos Microfilamentos/genética , Mutação , Miotonina Proteína Quinase/genética , Filogenia , Análise de Componente Principal , Análise de Sequência de DNA , Tumores Venéreos Veterinários/diagnóstico
11.
Mol Genet Genomic Med ; 3(4): 283-301, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-26247046

RESUMO

Neuromuscular diseases (NMD) account for a significant proportion of infant and childhood mortality and devastating chronic disease. Determining the specific diagnosis of NMD is challenging due to thousands of unique or rare genetic variants that result in overlapping phenotypes. We present four unique childhood myopathy cases characterized by relatively mild muscle weakness, slowly progressing course, mildly elevated creatine phosphokinase (CPK), and contractures. We also present two additional cases characterized by severe prenatal/neonatal myopathy. Prior extensive genetic testing and histology of these cases did not reveal the genetic etiology of disease. Here, we applied whole exome sequencing (WES) and bioinformatics to identify likely causal pathogenic variants in each pedigree. In two cases, we identified novel pathogenic variants in COL6A3. In a third case, we identified novel likely pathogenic variants in COL6A6 and COL6A3. We identified a novel splice variant in EMD in a fourth case. Finally, we classify two cases as calcium channelopathies with identification of novel pathogenic variants in RYR1 and CACNA1S. These are the first cases of myopathies reported to be caused by variants in COL6A6 and CACNA1S. Our results demonstrate the utility and genetic diagnostic value of WES in the broad class of NMD phenotypes.

12.
PLoS One ; 10(7): e0131797, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26176221

RESUMO

A 3-year-old female patient presenting with an unknown syndrome of a neonatal progeroid appearance, lipodystrophy, pulmonary hypertension, cutis marmorata, feeding disorder and failure to thrive was investigated by whole-genome sequencing. This revealed a de novo, heterozygous, frame-shift mutation in the Caveolin1 gene (CAV1) (p.Phe160X). Mutations in CAV1, encoding the main component of the caveolae in plasma membranes, cause Berardinelli-Seip congenital lipodystrophy type 3 (BSCL). Although BSCL is recessive, heterozygous carriers either show a reduced phenotype of partial lipodystrophy, pulmonary hypertension, or no phenotype. To investigate the pathogenic mechanisms underlying this syndrome in more depth, we performed next generation RNA sequencing of peripheral blood, which showed several dysregulated pathways in the patient that might be related to the phenotypic progeroid features (apoptosis, DNA repair/replication, mitochondrial). Secondly, we found a significant down-regulation of known Cav1 interaction partners, verifying the dysfunction of CAV1. Other known progeroid genes and lipodystrophy genes were also dysregulated. Next, western blotting of lysates of cultured fibroblasts showed that the patient shows a significantly decreased expression of wild-type CAV1 protein, demonstrating a loss-of-function mutation, though her phenotype is more severe that other heterozygotes with similar mutations. This phenotypic variety could be explained by differences in genetic background. Indications for this are supported by additional rare variants we found in AGPAT2 and LPIN1 lipodystrophy genes. CAV1, AGPAT2 and LPIN1 all play an important role in triacylglycerol (TAG) biosynthesis in adipose tissue, and the defective function in different parts of this pathway, though not all to the same extend, could contribute to a more severe lipoatrophic phenotype in this patient. In conclusion, we report, for the first time, an association of CAV1 dysfunction with a syndrome of severe premature aging and lipodystrophy. This may contribute to a better understanding of the aging process and pathogenic mechanisms that contribute to premature aging.


Assuntos
Caveolina 1/genética , Retardo do Crescimento Fetal/genética , Lipodistrofia Generalizada Congênita/genética , Progéria/genética , Aciltransferases/genética , Pré-Escolar , Códon sem Sentido , Feminino , Retardo do Crescimento Fetal/patologia , Mutação da Fase de Leitura , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Lipodistrofia Generalizada Congênita/patologia , Fenótipo , Fosfatidato Fosfatase/genética , Progéria/patologia , Análise de Sequência de RNA , Índice de Gravidade de Doença
13.
Invest Ophthalmol Vis Sci ; 56(6): 3896-904, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26091538

RESUMO

PURPOSE: Aicardi syndrome (AIC) is a congenital neurodevelopmental disorder characterized by infantile spasms, agenesis of the corpus callosum, and chorioretinal lacunae. Variation in phenotype and disease severity is well documented, but chorioretinal lacunae represent the most constant pathological feature. Aicardi syndrome is believed to be an X-linked-dominant disorder occurring almost exclusively in females, although 46, XY males with AIC have been described. The purpose of this study is to identify genetic factors and pathways involved in AIC. METHODS: We performed exome/genome sequencing of 10 children diagnosed with AIC and their parents and performed RNA sequencing on blood samples from nine cases, their parents, and unrelated controls. RESULTS: We identified a de novo mutation in autosomal gene TEAD1, expressed in the retina and brain, in a patient with AIC. Mutations in TEAD1 have previously been associated with Sveinsson's chorioretinal atrophy, characterized by chorioretinal degeneration. This demonstrates that TEAD1 mutations can lead to different chorioretinal complications. In addition, we found that altered expression of genes associated with synaptic plasticity, neuronal development, retinal development, and cell cycle control/apoptosis is an important underlying potential pathogenic mechanism shared among cases. Last, we found a case with skewed X inactivation, supporting the idea that nonrandom X inactivation might be important in AIC. CONCLUSIONS: We expand the phenotype of TEAD1 mutations, demonstrate its importance in chorioretinal complications, and propose the first putative pathogenic mechanisms underlying AIC. Our data suggest that AIC is a genetically heterogeneous disease and is not restricted to the X chromosome, and that TEAD1 mutations may be present in male patients.


Assuntos
Síndrome de Aicardi/genética , Proteínas de Ligação a DNA/genética , Predisposição Genética para Doença/genética , Mutação , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Adulto , Criança , Pré-Escolar , Análise Mutacional de DNA/métodos , Feminino , Perfilação da Expressão Gênica , Humanos , Masculino , Análise de Sequência de DNA , Análise de Sequência de RNA
14.
Ann Neurol ; 77(3): 547-52, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25559091

RESUMO

We used whole-exome sequencing to identify variants other than APOE associated with the rate of hippocampal atrophy in amnestic mild cognitive impairment. An in-silico predicted missense variant in REST (rs3796529) was found exclusively in subjects with slow hippocampal volume loss and validated using unbiased whole-brain analysis and meta-analysis across 5 independent cohorts. REST is a master regulator of neurogenesis and neuronal differentiation that has not been previously implicated in Alzheimer's disease. These findings nominate REST and its functional pathways as protective and illustrate the potential of combining next-generation sequencing with neuroimaging to discover novel disease mechanisms and potential therapeutic targets.


Assuntos
Amnésia/genética , Disfunção Cognitiva/genética , Progressão da Doença , Exoma/genética , Hipocampo/patologia , Proteínas Repressoras/genética , Idoso , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Doença de Alzheimer/fisiopatologia , Amnésia/patologia , Amnésia/fisiopatologia , Atrofia/genética , Atrofia/patologia , Disfunção Cognitiva/patologia , Disfunção Cognitiva/fisiopatologia , Hipocampo/fisiopatologia , Humanos , Masculino , Mutação de Sentido Incorreto , Fatores de Proteção , Análise de Sequência de DNA/métodos
15.
Pac Symp Biocomput ; : 56-67, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25592568

RESUMO

The ability to rapidly sequence the tumor and germline DNA of an individual holds the eventual promise of revolutionizing our ability to match targeted therapies to tumors harboring the associated genetic biomarkers. Analyzing high throughput genomic data consisting of millions of base pairs and discovering alterations in clinically actionable genes in a structured and real time manner is at the crux of personalized testing. This requires a computational architecture that can monitor and track a system within a regulated environment as terabytes of data are reduced to a small number of therapeutically relevant variants, delivered as a diagnostic laboratory developed test. These high complexity assays require data structures that enable real-time and retrospective ad-hoc analysis, with a capability of updating to keep up with the rapidly changing genomic and therapeutic options, all under a regulated environment that is relevant under both CMS and FDA depending on application. We describe a flexible computational framework that uses a paired tumor/normal sample allowing for complete analysis and reporting in approximately 24 hours, providing identification of single nucleotide changes, small insertions and deletions, chromosomal rearrangements, gene fusions and gene expression with positive predictive values over 90%. In this paper we present the challenges in integrating clinical, genomic and annotation databases to provide interpreted draft reports which we utilize within ongoing clinical research protocols. We demonstrate the need to retire from existing performance measurements of accuracy and specificity and measure metrics that are meaningful to a genomic diagnostic environment. This paper presents a three-tier infrastructure that is currently being used to analyze an individual genome and provide available therapeutic options via a clinical report. Our framework utilizes a non-relational variant-centric database that is scaleable to a large amount of data and addresses the challenges and limitations of a relational database system. Our system is continuously monitored via multiple trackers each catering differently to the diversity of users involved in this process. These trackers designed in analytics web-app framework provide status updates for an individual sample accurate to a few minutes. In this paper, we also present our outcome delivery process that is designed and delivered adhering to the standards defined by various regulation agencies involved in clinical genomic testing.


Assuntos
Biomarcadores Tumorais/genética , Neoplasias/genética , Medicina de Precisão/métodos , Ensaios Clínicos como Assunto , Biologia Computacional , Bases de Dados Genéticas , Perfilação da Expressão Gênica , Variação Genética , Genômica , Humanos , Terapia de Alvo Molecular , Neoplasias/terapia
16.
Eur J Hum Genet ; 23(1): 110-5, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24939585

RESUMO

We performed a genome-wide association study (GWAS) to identify the genes responsible for age-related hearing impairment (ARHI), the most common form of hearing impairment in the elderly. Analysis of common variants, with and without adjustment for stratification and environmental covariates, rare variants and interactions, as well as gene-set enrichment analysis, showed no variants with genome-wide significance. No evidence for replication of any previously reported genes was found. A study of the genetic architecture indicates for the first time that ARHI is highly polygenic in nature, with probably no major genes involved. The phenotype depends on the aggregated effect of a large number of SNPs, of which the individual effects are undetectable in a modestly powered GWAS. We estimated that 22% of the variance in our data set can be explained by the collective effect of all genotyped SNPs. A score analysis showed a modest enrichment in causative SNPs among the SNPs with a P-value below 0.01.


Assuntos
Estudo de Associação Genômica Ampla , Perda Auditiva/etiologia , Herança Multifatorial , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Biologia Computacional , Bases de Dados Genéticas , Meio Ambiente , Epistasia Genética , Feminino , Predisposição Genética para Doença , Genótipo , Perda Auditiva/metabolismo , Humanos , Masculino , Fenótipo , Polimorfismo de Nucleotídeo Único , Característica Quantitativa Herdável , Fatores de Risco , Transdução de Sinais
17.
PLoS One ; 9(12): e113036, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25503791

RESUMO

In females, X chromosome inactivation (XCI) is an epigenetic, gene dosage compensatory mechanism by inactivation of one copy of X in cells. Random XCI of one of the parental chromosomes results in an approximately equal proportion of cells expressing alleles from either the maternally or paternally inherited active X, and is defined by the XCI ratio. Skewed XCI ratio is suggestive of non-random inactivation, which can play an important role in X-linked genetic conditions. Current methods rely on indirect, semi-quantitative DNA methylation-based assay to estimate XCI ratio. Here we report a direct approach to estimate XCI ratio by integrated, family-trio based whole-exome and mRNA sequencing using phase-by-transmission of alleles coupled with allele-specific expression analysis. We applied this method to in silico data and to a clinical patient with mild cognitive impairment but no clear diagnosis or understanding molecular mechanism underlying the phenotype. Simulation showed that phased and unphased heterozygous allele expression can be used to estimate XCI ratio. Segregation analysis of the patient's exome uncovered a de novo, interstitial, 1.7 Mb deletion on Xp22.31 that originated on the paternally inherited X and previously been associated with heterogeneous, neurological phenotype. Phased, allelic expression data suggested an 83∶20 moderately skewed XCI that favored the expression of the maternally inherited, cytogenetically normal X and suggested that the deleterious affect of the de novo event on the paternal copy may be offset by skewed XCI that favors expression of the wild-type X. This study shows the utility of integrated sequencing approach in XCI ratio estimation.


Assuntos
Cromossomos Humanos X/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Doenças do Sistema Nervoso/genética , RNA Mensageiro/análise , Análise de Sequência de RNA/métodos , Inativação do Cromossomo X , Adolescente , Desequilíbrio Alélico , Criança , Simulação por Computador , Exoma , Feminino , Humanos , Deleção de Sequência , Adulto Jovem
18.
PLoS One ; 9(11): e112755, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25393235

RESUMO

Necrotizing meningoencephalitis (NME) affects toy and small breed dogs causing progressive, often fatal, inflammation and necrosis in the brain. Genetic risk loci for NME previously were identified in pug dogs, particularly associated with the dog leukocyte antigen (DLA) class II complex on chromosome 12, but have not been investigated in other susceptible breeds. We sought to evaluate Maltese and Chihuahua dogs, in addition to pug dogs, to identify novel or shared genetic risk factors for NME development. Genome-wide association testing of single nucleotide polymorphisms (SNPs) in Maltese dogs with NME identified 2 regions of genome-wide significance on chromosomes 4 (chr4:74522353T>A, p = 8.1×10-7) and 15 (chr15:53338796A>G, p = 1.5×10-7). Haplotype analysis and fine-mapping suggests that ILR7 and FBXW7, respectively, both important for regulation of immune system function, could be the underlying associated genes. Further evaluation of these regions and the previously identified DLA II locus across all three breeds, revealed an enrichment of nominal significant SNPs associated with chromosome 15 in pug dogs and DLA II in Maltese and Chihuahua dogs. Meta-analysis confirmed effect sizes the same direction in all three breeds for both the chromosome 15 and DLA II loci (p = 8.6×10-11 and p = 2.5×10-7, respectively). This suggests a shared genetic background exists between all breeds and confers susceptibility to NME, but effect sizes might be different among breeds. In conclusion, we identified the first genetic risk factors for NME development in the Maltese, chromosome 4 and chromosome 15, and provide evidence for a shared genetic risk between breeds associated with chromosome 15 and DLA II. Last, DLA II and IL7R both have been implicated in human inflammatory diseases of the central nervous system such as multiple sclerosis, suggesting that similar pharmacotherapeutic targets across species should be investigated.


Assuntos
Doenças do Cão/genética , Proteínas F-Box/genética , Antígenos de Histocompatibilidade Classe II/genética , Meningoencefalite/genética , Meningoencefalite/veterinária , Receptores de Interleucina-7/genética , Animais , Cruzamento , Cromossomos de Mamíferos , Doenças do Cão/imunologia , Doenças do Cão/patologia , Cães , Proteínas F-Box/imunologia , Feminino , Loci Gênicos , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Haplótipos , Antígenos de Histocompatibilidade Classe II/imunologia , Masculino , Meningoencefalite/imunologia , Meningoencefalite/patologia , Polimorfismo de Nucleotídeo Único , Receptores de Interleucina-7/imunologia , Fatores de Risco
19.
PLoS Genet ; 10(9): e1004606, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25188341

RESUMO

Alzheimer's disease (AD) and related dementias are a major public health challenge and present a therapeutic imperative for which we need additional insight into molecular pathogenesis. We performed a genome-wide association study and analysis of known genetic risk loci for AD dementia using neuropathologic data from 4,914 brain autopsies. Neuropathologic data were used to define clinico-pathologic AD dementia or controls, assess core neuropathologic features of AD (neuritic plaques, NPs; neurofibrillary tangles, NFTs), and evaluate commonly co-morbid neuropathologic changes: cerebral amyloid angiopathy (CAA), Lewy body disease (LBD), hippocampal sclerosis of the elderly (HS), and vascular brain injury (VBI). Genome-wide significance was observed for clinico-pathologic AD dementia, NPs, NFTs, CAA, and LBD with a number of variants in and around the apolipoprotein E gene (APOE). GalNAc transferase 7 (GALNT7), ATP-Binding Cassette, Sub-Family G (WHITE), Member 1 (ABCG1), and an intergenic region on chromosome 9 were associated with NP score; and Potassium Large Conductance Calcium-Activated Channel, Subfamily M, Beta Member 2 (KCNMB2) was strongly associated with HS. Twelve of the 21 non-APOE genetic risk loci for clinically-defined AD dementia were confirmed in our clinico-pathologic sample: CR1, BIN1, CLU, MS4A6A, PICALM, ABCA7, CD33, PTK2B, SORL1, MEF2C, ZCWPW1, and CASS4 with 9 of these 12 loci showing larger odds ratio in the clinico-pathologic sample. Correlation of effect sizes for risk of AD dementia with effect size for NFTs or NPs showed positive correlation, while those for risk of VBI showed a moderate negative correlation. The other co-morbid neuropathologic features showed only nominal association with the known AD loci. Our results discovered new genetic associations with specific neuropathologic features and aligned known genetic risk for AD dementia with specific neuropathologic changes in the largest brain autopsy study of AD and related dementias.


Assuntos
Doença de Alzheimer/diagnóstico , Doença de Alzheimer/genética , Demência/diagnóstico , Demência/etiologia , Estudo de Associação Genômica Ampla , Membro 1 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Encéfalo/patologia , Estudos de Casos e Controles , Mapeamento Cromossômico , Cromossomos Humanos Par 18 , Cromossomos Humanos Par 9 , Predisposição Genética para Doença , Hipocampo/metabolismo , Hipocampo/patologia , Humanos , N-Acetilgalactosaminiltransferases/genética , Razão de Chances , Fenótipo , Placa Amiloide , Locos de Características Quantitativas
20.
PLoS One ; 9(8): e105004, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25140675

RESUMO

Lizards, which are amniote vertebrates like humans, are able to lose and regenerate a functional tail. Understanding the molecular basis of this process would advance regenerative approaches in amniotes, including humans. We have carried out the first transcriptomic analysis of tail regeneration in a lizard, the green anole Anolis carolinensis, which revealed 326 differentially expressed genes activating multiple developmental and repair mechanisms. Specifically, genes involved in wound response, hormonal regulation, musculoskeletal development, and the Wnt and MAPK/FGF pathways were differentially expressed along the regenerating tail axis. Furthermore, we identified 2 microRNA precursor families, 22 unclassified non-coding RNAs, and 3 novel protein-coding genes significantly enriched in the regenerating tail. However, high levels of progenitor/stem cell markers were not observed in any region of the regenerating tail. Furthermore, we observed multiple tissue-type specific clusters of proliferating cells along the regenerating tail, not localized to the tail tip. These findings predict a different mechanism of regeneration in the lizard than the blastema model described in the salamander and the zebrafish, which are anamniote vertebrates. Thus, lizard tail regrowth involves the activation of conserved developmental and wound response pathways, which are potential targets for regenerative medical therapies.


Assuntos
Lagartos/fisiologia , Regeneração/genética , Cauda/fisiologia , Cicatrização/genética , Animais , Lagartos/genética , Transcriptoma
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