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1.
Artigo em Inglês | MEDLINE | ID: mdl-38521860

RESUMO

Meta-analysis of genome-wide association study data has implicated PDE4B in the pathogenesis of Alzheimer's disease (AD), the leading cause of senile dementia. PDE4B encodes one of four subtypes of cyclic adenosine monophosphate (cAMP)-specific phosphodiesterase-4 (PDE4A-D). To interrogate the involvement of PDE4B in the manifestation of AD-related phenotypes, the effects of a hypomorphic mutation (Pde4bY358C) that decreases PDE4B's cAMP hydrolytic activity were evaluated in the AppNL-G-F knock-in mouse model of AD using the Barnes maze test of spatial memory, 14C-2-deoxyglucose autoradiography, thioflavin-S staining of ß-amyloid (Aß) plaques, and inflammatory marker assay and transcriptomic analysis (RNA sequencing) of cerebral cortical tissue. At 12 months of age, AppNL-G-F mice exhibited spatial memory and brain metabolism deficits, which were prevented by the hypomorphic PDE4B in AppNL-G-F/Pde4bY358C mice, without a decrease in Aß plaque burden. RNA sequencing revealed that, among the 531 transcripts differentially expressed in AppNL-G-F versus wild-type mice, only 13 transcripts from four genes - Ide, Btaf1, Padi2, and C1qb - were differentially expressed in AppNL-G-F/Pde4bY358C versus AppNL-G-F mice, identifying their potential involvement in the protective effect of hypomorphic PDE4B. Our data demonstrate that spatial memory and cerebral glucose metabolism deficits exhibited by 12-month-old AppNL-G-F mice are prevented by targeted inhibition of PDE4B. To our knowledge, this is the first demonstration of a protective effect of PDE4B subtype-specific inhibition in a preclinical model of AD. It thus identifies PDE4B as a key regulator of disease manifestation in the AppNL-G-F model and a promising therapeutic target for AD.

2.
Am J Hum Genet ; 111(1): 119-132, 2024 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-38141607

RESUMO

Cyclin D2 (CCND2) stabilization underpins a range of macrocephaly-associated disorders through mutation of CCND2 or activating mutations in upstream genes encoding PI3K-AKT pathway components. Here, we describe three individuals with overlapping macrocephaly-associated phenotypes who carry the same recurrent de novo c.179G>A (p.Arg60Gln) variant in Myc-associated factor X (MAX). The mutation, located in the b-HLH-LZ domain, causes increased intracellular CCND2 through increased transcription but it does not cause stabilization of CCND2. We show that the purified b-HLH-LZ domain of MAXArg60Gln (Max∗Arg60Gln) binds its target E-box sequence with a lower apparent affinity. This leads to a more efficient heterodimerization with c-Myc resulting in an increase in transcriptional activity of c-Myc in individuals carrying this mutation. The recent development of Omomyc-CPP, a cell-penetrating b-HLH-LZ-domain c-Myc inhibitor, provides a possible therapeutic option for MAXArg60Gln individuals, and others carrying similar germline mutations resulting in dysregulated transcriptional c-Myc activity.


Assuntos
Megalencefalia , Proteínas Proto-Oncogênicas c-myc , Humanos , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Dimerização , Megalencefalia/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo
3.
Br J Cancer ; 125(7): 983-993, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34253873

RESUMO

BACKGROUND: Breast cancer stem cells (BCSCs) are drivers of therapy-resistance, therefore are responsible for poor survival. Molecular signatures of BCSCs from primary cancers remain undefined. Here, we identify the consistent transcriptome of primary BCSCs shared across breast cancer subtypes, and we examine the clinical relevance of ITGA7, one of the genes differentially expressed in BCSCs. METHODS: Primary BCSCs were assessed using immunohistochemistry and fluorescently labelled using Aldefluor (n = 17). Transcriptomes of fluorescently sorted BCSCs and matched non-stem cancer cells were determined using RNA-seq (n = 6). ITGA7 expression was examined in breast cancers using immunohistochemistry (n = 305), and its functional role was tested using siRNA in breast cancer cells. RESULTS: Proportions of BCSCs varied from 0 to 9.4%. 38 genes were significantly differentially expressed in BCSCs; genes were enriched for functions in vessel morphogenesis, motility, and metabolism. ITGA7 was found to be significantly downregulated in BCSCs, and low expression significantly correlated with reduced survival in patients treated with chemotherapy, and with chemoresistance in breast cancer cells in vitro. CONCLUSIONS: This study is the first to define the molecular profile of BCSCs from a range of primary breast cancers. ITGA7 acts as a predictive marker for chemotherapy response, in accordance with its downregulation in BCSCs.


Assuntos
Antígenos CD/genética , Neoplasias da Mama/genética , Regulação para Baixo , Resistencia a Medicamentos Antineoplásicos , Cadeias alfa de Integrinas/genética , Células-Tronco Neoplásicas/metabolismo , Antígenos CD/metabolismo , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Feminino , Perfilação da Expressão Gênica/métodos , Humanos , Cadeias alfa de Integrinas/metabolismo , Células MCF-7 , Análise de Sequência de RNA , Análise de Sobrevida
4.
Lab Invest ; 101(4): 442-449, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-32989232

RESUMO

Short-read next generation sequencing (NGS) has become the predominant first-line technique used to diagnose patients with rare genetic conditions. Inherent limitations of short-read technology, notably for the detection and characterization of complex insertion-containing variants, are offset by the ability to concurrently screen many disease genes. "Third-generation" long-read sequencers are increasingly being deployed as an orthogonal adjunct technology, but their full potential for molecular genetic diagnosis has yet to be exploited. Here, we describe three diagnostic cases in which pathogenic mobile element insertions were refractory to characterization by short-read sequencing. To validate the accuracy of the long-read technology, we first used Sanger sequencing to confirm the integration sites and derive curated benchmark sequences of the variant-containing alleles. Long-read nanopore sequencing was then performed on locus-specific amplicons. Pairwise comparison between these data and the previously determined benchmark alleles revealed 100% identity of the variant-containing sequences. We demonstrate a number of technical advantages over existing wet-laboratory approaches, including in silico size selection of a mixed pool of amplification products, and the relative ease with which an automated informatics workflow can be established. Our findings add to a growing body of literature describing the diagnostic utility of long-read sequencing.


Assuntos
Análise Mutacional de DNA/métodos , Sequências Repetitivas Dispersas/genética , Mutagênese Insercional/genética , Sequenciamento por Nanoporos/métodos , DNA/análise , DNA/genética , Bases de Dados Genéticas , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Neoplasias/genética
5.
Free Radic Biol Med ; 162: 191-201, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33091574

RESUMO

BACKGROUND: Uric acid, a metabolic product of purine degradation in humans, is a risk factor for developing gout and type 2 diabetes, and supplementation with quercetin lowers plasma uric acid in mildly hyperuricemic men. Here we examined the mechanism of inhibition of enzymes involved in uric acid metabolism by quercetin, conjugates and microbial catabolites, and measured the effect of lowered circulating uric acid on endothelial cell gene expression. METHODS: Inhibition of adenosine deaminase (ADA), purine nucleoside phosphorylase (PNP) and xanthine oxidoreductase (XOR) activity by quercetin and metabolites was determined by HPLC. Human umbilical vein endothelial cells (HUVECs) were cultured under conditions mimicking blood flow, treated with uric acid (0, 300 or 500 µmol/L), and changes in gene expression measured using transcriptomics and quantitative droplet digital PCR. RESULTS: In human plasma, no inhibition of PNP activity was observed, and only quercetin weakly inhibited ADA. XOR was not present at sufficient amount in human plasma to use for testing, but quercetin, quercetin-3'-sulfate and the gut microbial metabolite 3',4'-dihydroxyphenylacetic acid inhibited bovine milk XOR. Several changes were observed in gene expression in HUVECs under flow compared to static conditions, but after uric acid treatment, only very few changes were detected. CONCLUSIONS: We propose that the main mechanism by which quercetin, as quercetin-3'-sulfate, lowers uric acid in vivo is through inhibition of XOR, and not ADA nor PNP. The pertinent shift in uric acid concentration was not sufficient to produce significant changes in endothelial gene expression in a cell model.


Assuntos
Diabetes Mellitus Tipo 2 , Ácido Úrico , Animais , Bovinos , Células Endoteliais , Endotélio , Expressão Gênica , Humanos , Masculino , Quercetina/farmacologia
6.
Nat Commun ; 11(1): 6149, 2020 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-33262343

RESUMO

Autoimmune connective tissue diseases arise in a stepwise fashion from asymptomatic preclinical autoimmunity. Type I interferons have a crucial role in the progression to established autoimmune diseases. The cellular source and regulation in disease initiation of these cytokines is not clear, but plasmacytoid dendritic cells have been thought to contribute to excessive type I interferon production. Here, we show that in preclinical autoimmunity and established systemic lupus erythematosus, plasmacytoid dendritic cells are not effector cells, have lost capacity for Toll-like-receptor-mediated cytokine production and do not induce T cell activation, independent of disease activity and the blood interferon signature. In addition, plasmacytoid dendritic cells have a transcriptional signature indicative of cellular stress and senescence accompanied by increased telomere erosion. In preclinical autoimmunity, we show a marked enrichment of an interferon signature in the skin without infiltrating immune cells, but with interferon-κ production by keratinocytes. In conclusion, non-hematopoietic cellular sources, rather than plasmacytoid dendritic cells, are responsible for interferon production prior to clinical autoimmunity.


Assuntos
Autoimunidade , Células Dendríticas/imunologia , Interferon Tipo I/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Citocinas/genética , Citocinas/imunologia , Humanos , Interferon Tipo I/genética , Lúpus Eritematoso Sistêmico/genética , Ativação Linfocitária , Linfócitos T/imunologia , Receptores Toll-Like/genética , Receptores Toll-Like/imunologia
7.
Cancers (Basel) ; 12(10)2020 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-33007872

RESUMO

Cholangiocarcinoma (CCA) is a rare disease with poor outcomes and limited research efforts into novel treatment options. A systematic review of CCA biomarkers was undertaken to identify promising biomarkers that may be used for theranosis (therapy and diagnosis). MEDLINE/EMBASE databases (1996-2019) were systematically searched using two strategies to identify biomarker studies of CCA. The PANTHER Go-Slim classification system and STRING network version 11.0 were used to interrogate the identified biomarkers. The TArget Selection Criteria for Theranosis (TASC-T) score was used to rank identified proteins as potential targetable biomarkers for theranosis. The following proteins scored the highest, CA9, CLDN18, TNC, MMP9, and EGFR, and they were evaluated in detail. None of these biomarkers had high sensitivity or specificity for CCA but have potential for theranosis. This review is unique in that it describes the process of selecting suitable markers for theranosis, which is also applicable to other diseases. This has highlighted existing validated markers of CCA that can be used for active tumor targeting for the future development of targeted theranostic delivery systems. It also emphasizes the relevance of bioinformatics in aiding the search for validated biomarkers that could be repurposed for theranosis.

8.
Lab Invest ; 100(1): 135-146, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31273287

RESUMO

The widespread use of genome-wide diagnostic screening methods has greatly increased the frequency with which incidental (but possibly pathogenic) copy number changes affecting single genes are detected. These findings require validation to allow appropriate clinical management. Deletion variants can usually be readily validated using a range of short-read next-generation sequencing (NGS) strategies, but the characterization of duplication variants at nucleotide resolution remains challenging. This presents diagnostic problems, since pathogenicity cannot generally be assessed without knowing the structure of the variant. We have used a novel Cas9 enrichment strategy, in combination with long-read single-molecule nanopore sequencing, to address this need. We describe the nucleotide-level resolution of two problematic cases, both of whom presented with neurodevelopmental problems and were initially investigated by array CGH. In the first case, an incidental 1.7-kb imbalance involving a partial duplication of VHL exon 3 was detected. This variant was inherited from the patient's father, who had a history of renal cancer at 38 years. In the second case, an incidental ~200-kb de novo duplication that included DMD exons 30-44 was resolved. In both cases, the long-read data yielded sufficient information to enable Sanger sequencing to define the rearrangement breakpoints, and creation of breakpoint-spanning PCR assays suitable for testing of relatives. Our Cas9 enrichment and nanopore sequencing approach can be readily adopted by molecular diagnostic laboratories for cost-effective and rapid characterization of challenging duplication-containing alleles. We also anticipate that in future this method may prove useful for characterizing acquired translocations in tumor cells, and for precisely identifying transgene integration sites in mouse models.


Assuntos
Transtorno do Espectro Autista/genética , Proteínas do Citoesqueleto/genética , Distrofina/genética , Duplicação Gênica , Chaperonas Moleculares/genética , Sequenciamento por Nanoporos/métodos , Adolescente , Proteína 9 Associada à CRISPR , Pré-Escolar , Hibridização Genômica Comparativa , Feminino , Humanos , Masculino
9.
Elife ; 82019 10 24.
Artigo em Inglês | MEDLINE | ID: mdl-31647415

RESUMO

N6-methyladenosine (m6A) is the most abundant internal RNA modification of cellular mRNAs. m6A is recognised by YTH domain-containing proteins, which selectively bind to m6A-decorated RNAs regulating their turnover and translation. Using an m6A-modified hairpin present in the Kaposi's sarcoma associated herpesvirus (KSHV) ORF50 RNA, we identified seven members from the 'Royal family' as putative m6A readers, including SND1. RIP-seq and eCLIP analysis characterised the SND1 binding profile transcriptome-wide, revealing SND1 as an m6A reader. We further demonstrate that the m6A modification of the ORF50 RNA is critical for SND1 binding, which in turn stabilises the ORF50 transcript. Importantly, SND1 depletion leads to inhibition of KSHV early gene expression showing that SND1 is essential for KSHV lytic replication. This work demonstrates that members of the 'Royal family' have m6A-reading ability, greatly increasing their epigenetic functions beyond protein methylation.


Assuntos
Adenosina/análogos & derivados , Endonucleases/metabolismo , Herpesvirus Humano 8/crescimento & desenvolvimento , Interações Hospedeiro-Patógeno , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , Replicação Viral , Adenosina/metabolismo , Biologia Computacional , Células HEK293 , Humanos , Ligação Proteica , Análise de Sequência de RNA
11.
Am J Hum Genet ; 103(5): 727-739, 2018 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-30388400

RESUMO

Primary defects in motile cilia result in dysfunction of the apparatus responsible for generating fluid flows. Defects in these mechanisms underlie disorders characterized by poor mucus clearance, resulting in susceptibility to chronic recurrent respiratory infections, often associated with infertility; laterality defects occur in about 50% of such individuals. Here we report biallelic variants in LRRC56 (known as oda8 in Chlamydomonas) identified in three unrelated families. The phenotype comprises laterality defects and chronic pulmonary infections. High-speed video microscopy of cultured epithelial cells from an affected individual showed severely dyskinetic cilia but no obvious ultra-structural abnormalities on routine transmission electron microscopy (TEM). Further investigation revealed that LRRC56 interacts with the intraflagellar transport (IFT) protein IFT88. The link with IFT was interrogated in Trypanosoma brucei. In this protist, LRRC56 is recruited to the cilium during axoneme construction, where it co-localizes with IFT trains and is required for the addition of dynein arms to the distal end of the flagellum. In T. brucei carrying LRRC56-null mutations, or a variant resulting in the p.Leu259Pro substitution corresponding to the p.Leu140Pro variant seen in one of the affected families, we observed abnormal ciliary beat patterns and an absence of outer dynein arms restricted to the distal portion of the axoneme. Together, our findings confirm that deleterious variants in LRRC56 result in a human disease and suggest that this protein has a likely role in dynein transport during cilia assembly that is evolutionarily important for cilia motility.


Assuntos
Transporte Biológico/genética , Flagelos/genética , Depuração Mucociliar/genética , Mutação/genética , Proteínas/genética , Adulto , Alelos , Axonema/genética , Linhagem Celular , Chlamydomonas/genética , Cílios/genética , Dineínas/genética , Células Epiteliais/patologia , Feminino , Células HEK293 , Humanos , Lactente , Masculino , Fenótipo , Trypanosoma brucei brucei/genética
12.
Endocrine ; 59(3): 677-684, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29327300

RESUMO

CONTEXT: Pseudoacromegaly describes conditions with an acromegaly related physical appearance without abnormalities in the growth hormone (GH) axis. Acromegaloid facies, together with hypertrichosis, are typical manifestations of Cantú syndrome. CASE DESCRIPTION: We present a three-generation family with 5 affected members, with marked acromegaloid facies and prominent hypertrichosis, due to a novel missense variant in the ABCC9 gene. The proband, a 2-year-old girl, was referred due to marked hypertrichosis, noticed soon after birth, associated with coarsening of her facial appearance. Her endocrine assessment, including of the GH axis, was normal. The proband's father, paternal aunt, and half-sibling were referred to the Endocrine department for exclusion of acromegaly. Although the GH axis was normal in all, two subjects had clinically non-functioning pituitary macroadenomas, a feature which has not previously been associated with Cantú syndrome. CONCLUSIONS: Activating mutations in the ABCC9 and, less commonly, KCNJ8 genes-representing the two subunits of the ATP-sensitive potassium channel-have been linked with Cantú syndrome. Interestingly, minoxidil, a well-known ATP-sensitive potassium channel agonist, can cause a similar phenotype. There is no clear explanation why activating this channel would lead to acromegaloid features or hypertrichosis. This report raises awareness for this complex condition, especially for adult or pediatric endocrinologists who might see these patients referred for evaluation of acromegaloid features or hirsutism. The link between Cantú syndrome and pituitary adenomas is currently unclear.


Assuntos
Adenoma/complicações , Cardiomegalia/complicações , Hipertricose/complicações , Osteocondrodisplasias/complicações , Neoplasias Hipofisárias/complicações , Adenoma/diagnóstico por imagem , Adenoma/genética , Adulto , Cardiomegalia/diagnóstico por imagem , Cardiomegalia/genética , Pré-Escolar , Feminino , Humanos , Hipertricose/diagnóstico por imagem , Hipertricose/genética , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Mutação de Sentido Incorreto , Osteocondrodisplasias/diagnóstico por imagem , Osteocondrodisplasias/genética , Neoplasias Hipofisárias/diagnóstico por imagem , Neoplasias Hipofisárias/genética , Receptores de Sulfonilureias/genética , Adulto Jovem
13.
Mol Diagn Ther ; 21(6): 685-692, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28986857

RESUMO

BACKGROUND: Diagnostic genetic testing programmes based on next-generation DNA sequencing have resulted in the accrual of large datasets of targeted raw sequence data. Most diagnostic laboratories process these data through an automated variant-calling pipeline. Validation of the chosen analytical methods typically depends on confirming the detection of known sequence variants. Despite improvements in short-read alignment methods, current pipelines are known to be comparatively poor at detecting large insertion/deletion mutations. METHODS: We performed clinical validation of a local reassembly tool, ABRA (assembly-based realigner), through retrospective reanalysis of a cohort of more than 2000 hereditary cancer cases. RESULTS: ABRA enabled detection of a 96-bp deletion, 4-bp insertion mutation in PMS2 that had been initially identified using a comparative read-depth approach. We applied an updated pipeline incorporating ABRA to the entire cohort of 2000 cases and identified one previously undetected pathogenic variant, a 23-bp duplication in PTEN. We demonstrate the effect of read length on the ability to detect insertion/deletion variants by comparing HiSeq2500 (2 × 101-bp) and NextSeq500 (2 × 151-bp) sequence data for a range of variants and thereby show that the limitations of shorter read lengths can be mitigated using appropriate informatics tools. CONCLUSIONS: This work highlights the need for ongoing development of diagnostic pipelines to maximize test sensitivity. We also draw attention to the large differences in computational infrastructure required to perform day-to-day versus large-scale reprocessing tasks.


Assuntos
Biologia Computacional/métodos , Análise Mutacional de DNA/métodos , Testes Genéticos/métodos , Neoplasias/genética , Inibidor p16 de Quinase Dependente de Ciclina , Inibidor de Quinase Dependente de Ciclina p18/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Endonuclease PMS2 de Reparo de Erro de Pareamento/genética , PTEN Fosfo-Hidrolase/genética , Sensibilidade e Especificidade , Análise de Sequência de DNA/métodos
14.
J Mol Diagn ; 19(6): 933-940, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28867604

RESUMO

Like many clinical diagnostic laboratories, the Yorkshire Regional Genetics Service undertakes routine investigation of cancer-predisposed individuals by high-throughput sequencing of patient DNA that has been target-enriched for genes associated with hereditary cancer. Accurate diagnosis using such reagents requires alertness regarding rare nonpathogenic variants that may interfere with variant calling. In a cohort of 2042 such cases, we identified 5 that initially appeared to be carriers of a 95-bp deletion of SMAD4 intron 6. More detailed analysis indicated that these individuals all carried one copy of a SMAD4 processed gene. Because of its interference with diagnostic analysis, we characterized this processed gene in detail. Whole-genome sequencing and confirmatory Sanger sequencing of junction PCR products were used to show that in each of the 5 cases, the SMAD4 processed gene was integrated at the same position on chromosome 9, located within the last intron of the SCAI gene. This rare polymorphic processed gene therefore reflects the occurrence of a single ancestral retrotransposition event. Compared to the reference SMAD4 mRNA sequence NM_005359.5 (https://www.ncbi.nlm.nih.gov/nucleotide), the 5' and 3' untranslated regions of the processed gene are both truncated, but its open reading frame is unaltered. Our experience leads us to advocate the use of an RNA-seq aligner as part of diagnostic assay quality assurance, since this allows recognition of processed pseudogenes in a comparatively facile automated fashion.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Neoplasias/genética , Proteína Smad4/genética , Fatores de Transcrição/genética , Mapeamento Cromossômico , Cromossomos Humanos Par 9/genética , Genômica , Heterozigoto , Humanos , Íntrons/genética , Neoplasias/diagnóstico , Neoplasias/patologia , Patologia Molecular/métodos , Pseudogenes/genética , Sequenciamento Completo do Genoma
15.
Sci Rep ; 7(1): 6074, 2017 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-28729694

RESUMO

Genetic deletion or pharmacological inhibition of cyclooxygenase (COX)-2 abrogates intestinal adenoma development at early stages of colorectal carcinogenesis. COX-2 is localised to stromal cells (predominantly macrophages) in human and mouse intestinal adenomas. Therefore, we tested the hypothesis that paracrine Cox-2-mediated signalling from macrophages drives adenoma growth and progression in vivo in the Apc Min/+ mouse model of intestinal tumorigenesis. Using a transgenic C57Bl/6 mouse model of Cox-2 over-expression driven by the chicken lysozyme locus (cLys-Cox-2), which directs integration site-independent, copy number-dependent transgene expression restricted to macrophages, we demonstrated that stromal macrophage Cox-2 in colorectal (but not small intestinal) adenomas from cLys-Cox-2 x Apc Min/+ mice was associated with significantly increased tumour size (P = 0.025) and multiplicity (P = 0.025), compared with control Apc Min/+ mice. Transgenic macrophage Cox-2 expression was associated with increased dysplasia, epithelial cell Cox-2 expression and submucosal tumour invasion, as well as increased nuclear ß-catenin translocation in dysplastic epithelial cells. In vitro studies confirmed that paracrine macrophage Cox-2 signalling drives catenin-related transcription in intestinal epithelial cells. Paracrine macrophage Cox-2 activity drives growth and progression of Apc Min/+ mouse colonic adenomas, linked to increased epithelial cell ß-catenin dysregulation. Stromal cell (macrophage) gene regulation and signalling represent valid targets for chemoprevention of colorectal cancer.


Assuntos
Adenoma/metabolismo , Transformação Celular Neoplásica/metabolismo , Neoplasias Colorretais/metabolismo , Ciclo-Oxigenase 2/metabolismo , Macrófagos/metabolismo , Comunicação Parácrina , Adenoma/genética , Adenoma/patologia , Animais , Transformação Celular Neoplásica/genética , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Ciclo-Oxigenase 2/genética , Modelos Animais de Doenças , Progressão da Doença , Expressão Gênica , Genes APC , Loci Gênicos , Imuno-Histoquímica , Camundongos , Camundongos Transgênicos , Especificidade de Órgãos
16.
RNA ; 23(10): 1493-1501, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28724534

RESUMO

Recent methods for transcriptome-wide N6-methyladenosine (m6A) profiling have facilitated investigations into the RNA methylome and established m6A as a dynamic modification that has critical regulatory roles in gene expression and may play a role in human disease. However, bioinformatics resources available for the analysis of m6A sequencing data are still limited. Here, we describe m6aViewer-a cross-platform application for analysis and visualization of m6A peaks from sequencing data. m6aViewer implements a novel m6A peak-calling algorithm that identifies high-confidence methylated residues with more precision than previously described approaches. The application enables data analysis through a graphical user interface, and thus, in contrast to other currently available tools, does not require the user to be skilled in computer programming. m6aViewer and test data can be downloaded here: http://dna2.leeds.ac.uk/m6a.


Assuntos
Adenosina/análogos & derivados , Biologia Computacional/métodos , Análise de Sequência de RNA/métodos , Software , Adenosina/análise , Interface Usuário-Computador
17.
Am J Hum Genet ; 98(4): 735-43, 2016 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-27058446

RESUMO

Deficits in the basal ganglia pathways modulating cortical motor activity underlie both Parkinson disease (PD) and Huntington disease (HD). Phosphodiesterase 10A (PDE10A) is enriched in the striatum, and animal data suggest that it is a key regulator of this circuitry. Here, we report on germline PDE10A mutations in eight individuals from two families affected by a hyperkinetic movement disorder due to homozygous mutations c.320A>G (p.Tyr107Cys) and c.346G>C (p.Ala116Pro). Both mutations lead to a reduction in PDE10A levels in recombinant cellular systems, and critically, positron-emission-tomography (PET) studies with a specific PDE10A ligand confirmed that the p.Tyr107Cys variant also reduced striatal PDE10A levels in one of the affected individuals. A knock-in mouse model carrying the homologous p.Tyr97Cys variant had decreased striatal PDE10A and also displayed motor abnormalities. Striatal preparations from this animal had an impaired capacity to degrade cyclic adenosine monophosphate (cAMP) and a blunted pharmacological response to PDE10A inhibitors. These observations highlight the critical role of PDE10A in motor control across species.


Assuntos
Corpo Estriado/patologia , Hipercinese/genética , Mutação , Diester Fosfórico Hidrolases/genética , Alelos , Sequência de Aminoácidos , Animais , Modelos Animais de Doenças , Regulação da Expressão Gênica , Variação Genética , Células HEK293 , Humanos , Hipercinese/diagnóstico , Hipercinese/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Linhagem , Inibidores de Fosfodiesterase/metabolismo , Alinhamento de Sequência
18.
J Clin Res Pediatr Endocrinol ; 8(2): 125-34, 2016 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-27086651

RESUMO

OBJECTIVE: What initiates the pubertal process in humans and other mammals is still unknown. We hypothesized that gene(s) taking roles in triggering human puberty may be identified by studying a cohort of idiopathic hypogonadotropic hypogonadism (IHH). METHODS: A cohort of IHH cases was studied based on autozygosity mapping coupled with whole exome sequencing. RESULTS: Our studies revealed three independent families in which IHH/delayed puberty is associated with inactivating SRA1 variants. SRA1 was the first gene to be identified to function through its protein as well as noncoding functional ribonucleic acid products. These products act as co-regulators of nuclear receptors including sex steroid receptors as well as SF-1 and LRH-1, the master regulators of steroidogenesis. Functional studies with a mutant SRA1 construct showed a reduced co-activation of ligand-dependent activity of the estrogen receptor alpha, as assessed by luciferase reporter assay in HeLa cells. CONCLUSION: Our findings strongly suggest that SRA1 gene function is required for initiation of puberty in humans. Furthermore, SRA1 with its alternative products and functionality may provide a potential explanation for the versatility and complexity of the pubertal process.


Assuntos
Proteínas de Transporte/genética , Hipogonadismo/genética , Mutação , Puberdade Tardia/genética , Maturidade Sexual/genética , Adolescente , Adulto , Western Blotting , Estudos de Coortes , Análise Mutacional de DNA , Feminino , Genótipo , Humanos , Masculino , Microscopia de Fluorescência , Análise de Sequência com Séries de Oligonucleotídeos , Linhagem , Polimorfismo de Nucleotídeo Único , Adulto Jovem
19.
BMC Med Genet ; 17: 1, 2016 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-26729329

RESUMO

BACKGROUND: The widespread adoption of high-throughput sequencing technologies by genetic diagnostic laboratories has enabled significant expansion of their testing portfolios. Rare autosomal recessive conditions have been a particular focus of many new services. Here we report a cohort of 26 patients referred for genetic analysis of Joubert (JBTS) and Meckel-Gruber (MKS) syndromes, two clinically and genetically heterogeneous neurodevelopmental conditions that define a phenotypic spectrum, with MKS at the severe end. METHODS: Exome sequencing was performed for all cases, using Agilent SureSelect v5 reagents and Illumina paired-end sequencing. For two cases medium-coverage (9×) whole genome sequencing was subsequently undertaken. RESULTS: Using a standard analysis pipeline for the detection of single nucleotide and small insertion or deletion variants, molecular diagnoses were confirmed in 12 cases (4%). Seeking to determine whether our cohort harboured pathogenic copy number variants (CNV), in JBTS- or MKS-associated genes, targeted comparative read-depth analysis was performed using FishingCNV. These analyses identified a putative intragenic AHI1 deletion that included three exons spanning at least 3.4 kb and an intergenic MPP4 to TMEM237 deletion that included exons spanning at least 21.5 kb. Whole genome sequencing enabled confirmation of the deletion-containing alleles and precise characterisation of the mutation breakpoints at nucleotide resolution. These data were validated following development of PCR-based assays that could be subsequently used for "cascade" screening and/or prenatal diagnosis. CONCLUSIONS: Our investigations expand the AHI1 and TMEM237 mutation spectrum and highlight the importance of performing CNV screening of disease-associated genes. We demonstrate a robust increasingly cost-effective CNV detection workflow that is applicable to all MKS/JBTS referrals.


Assuntos
Cerebelo/anormalidades , Mapeamento Cromossômico , Transtornos da Motilidade Ciliar/diagnóstico , Transtornos da Motilidade Ciliar/genética , Encefalocele/diagnóstico , Encefalocele/genética , Exoma , Doenças Renais Policísticas/diagnóstico , Doenças Renais Policísticas/genética , Retina/anormalidades , Anormalidades Múltiplas/diagnóstico , Anormalidades Múltiplas/genética , Alelos , Estudos de Coortes , Variações do Número de Cópias de DNA , Éxons , Anormalidades do Olho/diagnóstico , Anormalidades do Olho/genética , Testes Genéticos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Doenças Renais Císticas/diagnóstico , Doenças Renais Císticas/genética , Diagnóstico Pré-Natal , Retinose Pigmentar , Análise de Sequência de DNA , Deleção de Sequência
20.
J Med Genet ; 52(12): 797-803, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26424145

RESUMO

BACKGROUND: The genetic aetiology of neurodevelopmental defects is extremely diverse, and the lack of distinctive phenotypic features means that genetic criteria are often required for accurate diagnostic classification. We aimed to identify the causative genetic lesions in two families in which eight affected individuals displayed variable learning disability, spasticity and abnormal gait. METHODS: Autosomal recessive inheritance was suggested by consanguinity in one family and by sibling recurrences with normal parents in the second. Autozygosity mapping and exome sequencing, respectively, were used to identify the causative gene. RESULTS: In both families, biallelic loss-of-function mutations in HACE1 were identified. HACE1 is an E3 ubiquitin ligase that regulates the activity of cellular GTPases, including Rac1 and members of the Rab family. In the consanguineous family, a homozygous mutation p.R219* predicted a truncated protein entirely lacking its catalytic domain. In the other family, compound heterozygosity for nonsense mutation p.R748* and a 20-nt insertion interrupting the catalytic homologous to the E6-AP carboxyl terminus (HECT) domain was present; western blot analysis of patient cells revealed an absence of detectable HACE1 protein. CONCLUSION: HACE1 mutations underlie a new autosomal recessive neurodevelopmental disorder. Previous studies have implicated HACE1 as a tumour suppressor gene; however, since cancer predisposition was not observed either in homozygous or heterozygous mutation carriers, this concept may require re-evaluation.


Assuntos
Transtornos do Neurodesenvolvimento/genética , Ubiquitina-Proteína Ligases/deficiência , Células Cultivadas , Criança , Pré-Escolar , Análise Mutacional de DNA , Feminino , Genes Recessivos , Humanos , Lactente , Masculino , Linhagem , Polimorfismo de Nucleotídeo Único , Síndrome , Ubiquitina-Proteína Ligases/genética
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