15 research outputs found
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Use of whole genome sequencing to determine genetic basis of suspected mitochondrial disorders: cohort study
Funder: University of Cambridge; FundRef: http://dx.doi.org/10.13039/501100000735Funder: Alzheimer's Society; FundRef: http://dx.doi.org/10.13039/501100000320Funder: Leverhulme Trust; FundRef: http://dx.doi.org/10.13039/501100000275Funder: National Institute for Health Research; FundRef: http://dx.doi.org/10.13039/501100000272Funder: Department of Health; FundRef: http://dx.doi.org/10.13039/501100000276Funder: Evelyn Trust; FundRef: http://dx.doi.org/10.13039/501100004282Funder: Wellcome Trust; FundRef: http://dx.doi.org/10.13039/100004440Funder: Medical Research Council; FundRef: http://dx.doi.org/10.13039/501100000265Abstract: Objective: To determine whether whole genome sequencing can be used to define the molecular basis of suspected mitochondrial disease. Design: Cohort study. Setting: National Health Service, England, including secondary and tertiary care. Participants: 345 patients with suspected mitochondrial disorders recruited to the 100 000 Genomes Project in England between 2015 and 2018. Intervention: Short read whole genome sequencing was performed. Nuclear variants were prioritised on the basis of gene panels chosen according to phenotypes, ClinVar pathogenic/likely pathogenic variants, and the top 10 prioritised variants from Exomiser. Mitochondrial DNA variants were called using an in-house pipeline and compared with a list of pathogenic variants. Copy number variants and short tandem repeats for 13 neurological disorders were also analysed. American College of Medical Genetics guidelines were followed for classification of variants. Main outcome measure: Definite or probable genetic diagnosis. Results: A definite or probable genetic diagnosis was identified in 98/319 (31%) families, with an additional 6 (2%) possible diagnoses. Fourteen of the diagnoses (4% of the 319 families) explained only part of the clinical features. A total of 95 different genes were implicated. Of 104 families given a diagnosis, 39 (38%) had a mitochondrial diagnosis and 65 (63%) had a non-mitochondrial diagnosis. Conclusion: Whole genome sequencing is a useful diagnostic test in patients with suspected mitochondrial disorders, yielding a diagnosis in a further 31% after exclusion of common causes. Most diagnoses were non-mitochondrial disorders and included developmental disorders with intellectual disability, epileptic encephalopathies, other metabolic disorders, cardiomyopathies, and leukodystrophies. These would have been missed if a targeted approach was taken, and some have specific treatments
Widespread genomic influences on phenotype in Dravet syndrome, a ‘monogenic’ condition
Dravet syndrome is an archetypal rare severe epilepsy, considered “monogenic”, typically caused by loss-of-function SCN1A variants. Despite a recognisable core phenotype, its marked phenotypic heterogeneity is incompletely explained by differences in the causal SCN1A variant or clinical factors. In 34 adults with SCN1A-related Dravet syndrome, we show additional genomic variation beyond SCN1A contributes to phenotype and its diversity, with an excess of rare variants in epilepsy-related genes as a set and examples of blended phenotypes, including one individual with an ultra-rare DEPDC5 variant and focal cortical dysplasia. Polygenic risk scores for intelligence are lower, and for longevity, higher, in Dravet syndrome than in epilepsy controls. The causal, major-effect, SCN1A variant may need to act against a broadly compromised genomic background to generate the full Dravet syndrome phenotype, whilst genomic resilience may help to ameliorate the risk of premature mortality in adult Dravet syndrome survivors
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Spectrum of mutational signatures in T-cell lymphoma reveals a key role for UV radiation in cutaneous T-cell lymphoma
Funder: Galderma; doi: http://dx.doi.org/10.13039/501100009754Funder: NIHR-BRC Cambridge core grantFunder: National Institute for Health Research; doi: http://dx.doi.org/10.13039/501100000272Funder: NHS EnglandAbstract: T-cell non-Hodgkin’s lymphomas develop following transformation of tissue resident T-cells. We performed a meta-analysis of whole exome sequencing data from 403 patients with eight subtypes of T-cell non-Hodgkin’s lymphoma to identify mutational signatures and associated recurrent gene mutations. Signature 1, indicative of age-related deamination, was prevalent across all T-cell lymphomas, reflecting the derivation of these malignancies from memory T-cells. Adult T-cell leukemia-lymphoma was specifically associated with signature 17, which was found to correlate with the IRF4 K59R mutation that is exclusive to Adult T-cell leukemia-lymphoma. Signature 7, implicating UV exposure was uniquely identified in cutaneous T-cell lymphoma (CTCL), contributing 52% of the mutational burden in mycosis fungoides and 23% in Sezary syndrome. Importantly this UV signature was observed in CD4 + T-cells isolated from the blood of Sezary syndrome patients suggesting extensive re-circulation of these T-cells through skin and blood. Analysis of non-Hodgkin’s T-cell lymphoma cases submitted to the national 100,000 WGS project confirmed that signature 7 was only identified in CTCL strongly implicating UV radiation in the pathogenesis of cutaneous T-cell lymphoma
GWAS meta-analysis of intrahepatic cholestasis of pregnancy implicates multiple hepatic genes and regulatory elements
Intrahepatic cholestasis of pregnancy (ICP) is a pregnancy-specific liver disorder affecting 0.5–2% of pregnancies. The majority of cases present in the third trimester with pruritus, elevated serum bile acids and abnormal serum liver tests. ICP is associated with an increased risk of adverse outcomes, including spontaneous preterm birth and stillbirth. Whilst rare mutations affecting hepatobiliary transporters contribute to the aetiology of ICP, the role of common genetic variation in ICP has not been systematically characterised to date. Here, we perform genome-wide association studies (GWAS) and meta-analyses for ICP across three studies including 1138 cases and 153,642 controls. Eleven loci achieve genome-wide significance and have been further investigated and fine-mapped using functional genomics approaches. Our results pinpoint common sequence variation in liver-enriched genes and liver-specific cis-regulatory elements as contributing mechanisms to ICP susceptibility
Androgen receptor and androgen-responsive gene FKBP5 are independent prognostic indicators for esophageal adenocarcinoma
Background: esophageal adenocarcinoma is a male-dominant disease, but the role of androgens is unclear.Aims: to examine the expression and clinical correlates of the androgen receptor (AR) and the androgen-responsive gene FK506-binding protein 5 (FKBP5) in esophageal adenocarcinoma.Methods: expression of AR and FKBP5 was determined by immunohistochemistry. The effect of the AR ligand 5?-dihydrotestosterone (DHT) on the expression of a panel of androgen-responsive genes was measured in AR-positive and AR-negative esophageal adenocarcinoma cell lines. Correlations in expression between androgen-responsive genes were analyzed in an independent cohort of esophageal adenocarcinoma tissues.Results: there was AR staining in 75 of 77 cases (97 %), and FKBP5 staining in 49 (64 %), all of which had nuclear AR. Nuclear AR with FKBP5 expression was associated with decreased median survival (451 vs. 2800 days) and was an independent prognostic indicator (HR 2.894, 95 % CI 1.396–6.002, p = 0.0043) in multivariable Cox proportional hazards models. DHT induced a significant increase in expression of the androgen-responsive genes FKBP5, HMOX1, FBXO32, VEGFA, WNT5A, and KLK3 only in AR-positive cells in vitro. Significant correlations in expression were observed between these androgen-responsive genes in an independent cohort of esophageal adenocarcinoma tissues.Conclusion: nuclear AR and expression of FKBP5 is associated with decreased survival in esophageal adenocarcinoma<br/
A Novel Androgen Receptor Amino Terminal Region Reveals Two Classes of Amino/Carboxyl Interaction-Deficient Variants with Divergent Capacity to Activate Responsive Sites in Chromatin
The androgen receptor (AR) is an important signaling molecule in multiple tissues, yet its mode of action and cell-specific activities remain enigmatic. AR function has been best studied in the prostate, in which it is essential for growth and homeostasis of the normal organ as well as each stage of cancer development. Investigation of mechanisms responsible for continued AR action that evolve during prostate cancer progression or after hormonal management of the disease have been instructive in defining AR signaling pathways. In the current paper, we use sequence similarity and the collocation of somatic mutations in prostate cancer to define residues 501–535 of the AR amino-terminal domain as an important mediator of receptor function. Specifically, the 501–535 region is required for optimal interaction of the amino-terminal domain with both the p160 coactivator, nuclear receptor coactivator-2, and the AR-ligand binding domain in the amino/carboxyl (N/C) interaction. The N/C interaction is decreased by deletion of the 501–535 region but is distinct from deletion of the 23FQNLF27 peptide in that it does not affect the capacity of the AR to activate transcription from a chromatin integrated reporter or recruitment of the receptor to androgen-responsive loci in vivo. Collectively, we have been able to outline two classes of N/C-deficient AR variant that are divergent in their capacity to act in a chromatin context, thereby further defining the interplay between N/C interaction and coregulator recruitment via multiple receptor domains. These mechanisms are likely to be key determinants of the cell and promoter specific activities of the AR
Molecular and structural basis of androgen receptor responses to dihydrotestosterone, medroxyprogesterone acetate and Δ4-tibolone
Medroxyprogesterone acetate (MPA) has widely been used in hormone replacement therapy (HRT), and is associated with an increased risk of breast cancer, possibly due to disruption of androgen receptor (AR) signaling. In contrast, the synthetic HRT Tibolone does not increase breast density, and is rapidly metabolized to estrogenic 3α-OH-tibolone and 3β-OH-tibolone, and a delta-4 isomer (Δ4-TIB) that has both androgenic and progestagenic properties. Here, we show that 5α-dihydrotestosterone (DHT) and Δ4-TIB, but not MPA, stabilize AR protein levels, initiate specific AR intramolecular interactions critical for AR transcriptional regulation, and increase proliferation of AR positive MDA-MB-453 breast cancer cells. Structural modeling and molecular dynamic simulation indicate that Δ4-TIB induces a more stable AR structure than does DHT, and MPA a less stable one. Microarray expression analyses confirms that the molecular actions of Δ4-TIB more closely resembles DHT in breast cancer cells than either ligand does to MPA
Diamide Linked γ‑Cyclodextrin Dimers as Molecular-Scale Delivery Systems for the Medicinal Pigment Curcumin to Prostate Cancer Cells
Diamide
linked γ-cyclodextrin (γ-CD) dimers are proposed
as molecular-scale delivery agents for the anticancer agent curcumin. <i>N,N</i>′-Bis(6<sup>A</sup>-deoxy-γ-cyclodextrin-6<sup>A</sup>-yl)succinamide (66γCD<sub>2</sub>su) and <i>N,N</i>′-bis(6<sup>A</sup>-deoxy-γ-cyclodextrin-6<sup>A</sup>-yl)urea (66γCD<sub>2</sub>ur) markedly suppress the degradation
of curcumin by forming a strong 1:1 cooperative binding complexes.
The results presented in this study describe the potential efficacy
of 66γCD<sub>2</sub>su and 66γCD<sub>2</sub>ur for intracellular
curcumin delivery to cancer cells. Cellular viability assays demonstrated
a dose-dependent antiproliferative effect of curcumin in human prostate
cancer (PC-3) cells that was preserved by the curcumin-66γCD<sub>2</sub>su complex. In contrast, delivery of curcumin by 66γCD<sub>2</sub>ur significantly delayed the antiproliferative effect. We
observed similar patterns of gene regulation in PC-3 cells for curcumin
complexed with either 66γCD<sub>2</sub>su or 66γCD<sub>2</sub>ur in comparison to curcumin alone, although curcumin delivered
by either 66γCD<sub>2</sub>su or 66γCD<sub>2</sub>ur induces
a slightly higher up-regulation of heme oxygenase-1. Highlighting
their nontoxic nature, neither 66γCD<sub>2</sub>su nor 66γCD<sub>2</sub>ur carriers alone had any measurable effect on cell proliferation
or candidate gene expression in PC-3 cells. Finally, confocal fluorescence
imaging and uptake studies were used to demonstrate the intracellular
delivery of curcumin by 66γCD<sub>2</sub>su and 66γCD<sub>2</sub>ur. Overall, these results demonstrate effective intracellular
delivery and action of curcumin when complexed with 66γCD<sub>2</sub>su and 66γCD<sub>2</sub>ur, providing further evidence
of their potential applications to deliver curcumin effectively in
cancer and other treatment settings
Research resource: interplay between the genomic and transcriptional networks of androgen receptor and estrogen receptor alpha in luminal breast cancer cells
The cellular response to circulating sex steroids is more than the sum of individual hormone actions, instead representing an interplay between activities of the evolutionarily related steroid hormone receptors. An example of this interaction is in breast cancer, where the risk of dying from estrogen receptor-_ (ER_)-positive disease decreases approximately 4-fold when androgen receptor (AR) expression is high. In this study, we used chromatin immunoprecipitation sequencing (ChIP-seq) and microarray expression profiling to investigate the genomic and transcriptional cross talk between AR and ER_ signaling in a luminal breast cancer cell line model, ZR-75–1. Expression profiling demonstrated reciprocal interference between 5_-dihydrotestosterone (DHT)- and 17_-estradiol (E2)-induced transcriptional programs. Specifically, regulation of 26% of E2 and 15% of DHT target genes was significantly affected by cotreatment with the other hormone, in the majority of cases (78–83%) antagonistically. Pathway analysis suggested that DHT cotreatment, for example, depleted E2-regulated pathways in cell survival and proliferation. ChIP-seq identified substantial overlap between the steroid receptor cistromes in ZR-75–1 cells, with 10–13% of AR- and ER_-binding sites located within 10 kb of the other receptor. Enrichment of androgen response elements in ER_-binding sites and vice versa was revealed by motif analysis, and AR-binding sites were enriched about E2-responsive genes affected by DHT cotreatment. Targeted ChIP and expression analysis revealed locus-specific outcomes when AR and ER_ bind to the same DNA region. This work provides the first cistrome data for two steroid receptors in the same cell, insight into the antagonistic interplay between estrogens and androgens in luminal breast cancer, and an important resource for future work aimed at evaluating interrelated steroid receptors in different cellular systems.Eleanor F. Need, Luke A. Selth, Tiffany J. Harris, Stephen N. Birrell, Wayne D. Tilley and Grant Buchana
Long terminal repeats act as androgen-responsive enhancers for the PSA-kallikrein locus
Free to read on publisher website The androgen receptor (AR) signaling pathway is a common therapeutic target for prostate cancer, because it is critical for the survival of both hormone-responsive and castrate-resistant tumor cells. Most of the detailed understanding that we have of AR transcriptional activation has been gained by studying classical target genes. For more than two decades, Kallikrein 3 (KLK3) (prostate-specific antigen) has been used as a prototypical AR target gene, because it is highly androgen responsive in prostate cancer cells. Three regions upstream of the KLK3 gene, including the distal enhancer, are known to contain consensus androgen-responsive elements required for AR-mediated transcriptional activation. Here, we show that KLK3 is one of a specific cluster of androgen-regulated genes at the centromeric end of the kallikrein locus with enhancers that evolved from the long terminal repeat (LTR) (LTR40a) of an endogenous retrovirus. Ligand-dependent recruitment of the AR to individual LTR-derived enhancers results in concurrent up-regulation of endogenous KLK2, KLK3, and KLKP1 expression in LNCaP prostate cancer cells. At the molecular level, a kallikrein-specific duplication within the LTR is required for maximal androgen responsiveness. Therefore, KLK3 represents a subset of target genes regulated by repetitive elements but is not typical of the whole spectrum of androgen-responsive transcripts. These data provide a novel and more detailed understanding of AR transcriptional activation and emphasize the importance of repetitive elements as functional regulatory unit