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BACKGROUND - The K3326X variant in BRCA2 (BRCA2*c.9976A>T; p.Lys3326*; rs11571833) has been found to be associated with small increased risks of breast cancer. However, it is not clear to what extent linkage disequilibrium with fully pathogenic mutations might account for this association. There is scant information about the effect of K3326X in other hormone-related cancers.
METHODS - Using weighted logistic regression, we analyzed data from the large iCOGS study including 76 637 cancer case patients and 83 796 control patients to estimate odds ratios (ORw) and 95% confidence intervals (CIs) for K3326X variant carriers in relation to breast, ovarian, and prostate cancer risks, with weights defined as probability of not having a pathogenic BRCA2 variant. Using Cox proportional hazards modeling, we also examined the associations of K3326X with breast and ovarian cancer risks among 7183 BRCA1 variant carriers. All statistical tests were two-sided.
RESULTS - The K3326X variant was associated with breast (ORw = 1.28, 95% CI = 1.17 to 1.40, P = 5.9x10(-) (6)) and invasive ovarian cancer (ORw = 1.26, 95% CI = 1.10 to 1.43, P = 3.8x10(-3)). These associations were stronger for serous ovarian cancer and for estrogen receptor-negative breast cancer (ORw = 1.46, 95% CI = 1.2 to 1.70, P = 3.4x10(-5) and ORw = 1.50, 95% CI = 1.28 to 1.76, P = 4.1x10(-5), respectively). For BRCA1 mutation carriers, there was a statistically significant inverse association of the K3326X variant with risk of ovarian cancer (HR = 0.43, 95% CI = 0.22 to 0.84, P = .013) but no association with breast cancer. No association with prostate cancer was observed.
CONCLUSIONS - Our study provides evidence that the K3326X variant is associated with risk of developing breast and ovarian cancers independent of other pathogenic variants in BRCA2. Further studies are needed to determine the biological mechanism of action responsible for these associations.
© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: firstname.lastname@example.org.
OBJECTIVE - Animal studies and in vitro human studies suggest that certain opioid analgesics impair crucial immune functions. This study was undertaken to determine whether opioid use is associated with increased risk of serious infection in patients with rheumatoid arthritis (RA).
METHODS - We conducted a self-controlled case series analysis on a retrospective cohort of 13,796 patients with RA enrolled in Tennessee Medicaid in 1995-2009. Within-person comparisons of the risk of hospitalization for serious infection during periods of opioid use versus non-use were performed using conditional Poisson regression. Fixed confounders were accounted for by design; time-varying confounders included age and use of disease-modifying antirheumatic drugs, glucocorticoids, and proton-pump inhibitors. In additional analyses, risks associated with new opioid use, use of opioids known to have immunosuppressive properties, use of long-acting opioids, and different opioid dosages were assessed. Sensitivity analyses were performed to account for potential protopathic bias and confounding by indication.
RESULTS - Among 1,790 patients with RA who had at least 1 hospitalization for serious infection, the adjusted incidence rate of serious infection was higher during periods of current opioid use compared to non-use, with an incidence rate ratio (IRR) of 1.39 (95% confidence interval [95% CI] 1.19-1.62). The incidence rate was also higher during periods of long-acting opioid use, immunosuppressive opioid use, and new opioid use compared to non-use (IRR 2.01 [95% CI 1.52-2.66], IRR 1.72 [95% CI 1.33-2.23], and IRR 2.38 [95% CI 1.65-3.42], respectively). Results of sensitivity analyses were consistent with the main findings.
CONCLUSION - In within-person comparisons of patients with RA, opioid use was associated with an increased risk of hospitalization for serious infection.
© 2016, American College of Rheumatology.
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by the selective death of motor neurons. Causative mutations in the global RNA-processing proteins TDP-43 and FUS among others, as well as their aggregation in ALS patients, have identified defects in RNA metabolism as an important feature in this disease. Lethal congenital contracture syndrome 1 and lethal arthrogryposis with anterior horn cell disease are autosomal recessive fetal motor neuron diseases that are caused by mutations in another global RNA-processing protein, hGle1. In this study, we carried out the first screening of GLE1 in ALS patients (173 familial and 760 sporadic) and identified 2 deleterious mutations (1 splice site and 1 nonsense mutation) and 1 missense mutation. Functional analysis of the deleterious mutants revealed them to be unable to rescue motor neuron pathology in zebrafish morphants lacking Gle1. Furthermore, in HeLa cells, both mutations caused a depletion of hGle1 at the nuclear pore where it carries out an essential role in nuclear export of mRNA. These results suggest a haploinsufficiency mechanism and point to a causative role for GLE1 mutations in ALS patients. This further supports the involvement of global defects in RNA metabolism in ALS.
© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: email@example.com.
AIMS - Positional cloning and candidate gene approaches have shown that atrial fibrillation (AF) is a complex disease with familial aggregation. Here, we employed whole-exome sequencing (WES) in AF kindreds to identify variants associated with familial AF.
METHODS AND RESULTS - WES was performed on 18 individuals in six modestly sized familial AF kindreds. After filtering very rare variants by multiple metrics, we identified 39 very rare and potentially pathogenic variants [minor allele frequency (MAF) ≤0.04%] in genes not previously associated with AF. Despite stringent filtering >1 very rare variants in the 5/6 of the kindreds were identified, whereas no plausible variants contributing to familial AF were found in 1/6 of the kindreds. Two candidate AF variants in the calcium channel subunit genes (CACNB2 and CACNA2D4) were identified in two separate families using expression data and predicted function.
CONCLUSION - By coupling family data with exome sequencing, we identified multiple very rare potentially pathogenic variants in five of six families, suggestive of a complex disease mechanism, whereas none were identified in the remaining AF pedigree. This study highlights some important limitations and challenges associated with performing WES in AF including the importance of having large well-curated multi-generational pedigrees, the issue of potential AF misclassification, and limitations of WES technology when applied to a complex disease.
Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2014. For permissions please email: firstname.lastname@example.org.
PURPOSE - Mutations in receptor tyrosine kinase (RTK) genes can confer resistance to receptor-targeted therapies. A T798M mutation in the HER2 oncogene has been shown to confer resistance to the tyrosine kinase inhibitor (TKI) lapatinib. We studied the mechanisms of HER2-T798M-induced resistance to identify potential strategies to overcome that resistance.
EXPERIMENTAL DESIGN - HER2-T798M was stably expressed in BT474 and MCF10A cells. Mutant cells and xenografts were evaluated for effects of the mutation on proliferation, signaling, and tumor growth after treatment with combinations of inhibitors targeting the EGFR/HER2/HER3/PI3K axis.
RESULTS - A low 3% allelic frequency of the T798M mutant shifted 10-fold the IC50 of lapatinib. In mutant-expressing cells, lapatinib did not block basal phosphorylation of HER2, HER3, AKT, and ERK1/2. In vitro kinase assays showed increased autocatalytic activity of HER2-T798M. HER3 association with PI3K p85 was increased in mutant-expressing cells. BT474-T798M cells were also resistant to the HER2 antibody trastuzumab. These cells were sensitive to the pan-PI3K inhibitors BKM120 and XL147 and the irreversible HER2/EGFR TKI afatinib but not the MEK1/2 inhibitor CI-1040, suggesting continued dependence of the mutant cells on ErbB receptors and downstream PI3K signaling. BT474-T798M cells showed increased expression of the EGFR ligands EGF, TGFα, amphiregulin, and HB-EGF. Addition of the EGFR neutralizing antibody cetuximab or lapatinib restored trastuzumab sensitivity of BT474-T798M cells and xenografts, suggesting that increased EGFR ligand production was causally associated with drug resistance.
CONCLUSIONS - Simultaneous blockade of HER2 and EGFR should be an effective treatment strategy against HER2 gene-amplified breast cancer cells harboring T798M mutant alleles.
OBJECTIVE - Genetic epilepsies and many other human genetic diseases display phenotypic heterogeneity, often for unknown reasons. Disease severity associated with nonsense mutations is dependent partially on mutation gene location and resulting efficiency of nonsense-mediated mRNA decay (NMD) to eliminate potentially toxic proteins. Nonsense mutations in the last exon do not activate NMD, thus producing truncated proteins. We compared the protein metabolism and the impact on channel biogenesis, function, and cellular homeostasis of truncated γ2 subunits produced by GABRG2 nonsense mutations associated with epilepsy of different severities and by a nonsense mutation in the last exon unassociated with epilepsy.
METHODS - γ-Aminobutyric acid type A receptor subunits were coexpressed in non-neuronal cells and neurons. NMD was studied using minigenes that support NMD. Protein degradation rates were determined using (35) S radiolabeling pulse chase. Channel function was determined by whole cell recordings, and subunits trafficking and cellular toxicity were determined using flow cytometry, immunoblotting, and immunohistochemistry.
RESULTS - Although all GABRG2 nonsense mutations resulted in loss of γ2 subunit surface expression, the truncated subunits had different degradation rates and stabilities, suppression of wild-type subunit biogenesis and function, amounts of conjugation with polyubiquitin, and endoplasmic reticulum stress levels.
INTERPRETATION - We compared molecular phenotypes of GABRG2 nonsense mutations. The findings suggest that despite the common loss of mutant allele function, each mutation produced different intracellular levels of trafficking-deficient subunits. The concentration-dependent suppression of wild-type channel function and cellular disturbance resulting from differences in mutant subunit metabolism may contribute to associated epilepsy severities and by implication to phenotypic heterogeneity in many inherited human diseases.
© 2013 American Neurological Association.
Circadian rhythms are oscillations in biological processes that function as a key adaptation to the daily rhythms of most environments. In the model cyanobacterial circadian clock system, the core oscillator proteins are encoded by the gene cluster kaiABC. Genes with high expression and functional importance, such as the kai genes, are usually encoded by optimal codons, yet the codon-usage bias of the kaiBC genes is not optimized for translational efficiency. We discovered a relationship between codon usage and a general property of circadian rhythms called conditionality; namely, that endogenous rhythmicity is robustly expressed under some environmental conditions but not others. Despite the generality of circadian conditionality, however, its molecular basis is unknown for any system. Here we show that in the cyanobacterium Synechococcus elongate, non-optimal codon usage was selected as a post-transcriptional mechanism to switch between circadian and non-circadian regulation of gene expression as an adaptive response to environmental conditions. When the kaiBC sequence was experimentally optimized to enhance expression of the KaiB and KaiC proteins, intrinsic rhythmicity was enhanced at cool temperatures that are experienced by this organism in its natural habitat. However, fitness at those temperatures was highest in cells in which the endogenous rhythms were suppressed at cool temperatures as compared with cells exhibiting high-amplitude rhythmicity. These results indicate natural selection against circadian systems in cyanobacteria that are intrinsically robust at cool temperatures. Modulation of circadian amplitude is therefore crucial to its adaptive significance. Moreover, these results show the direct effects of codon usage on a complex phenotype and organismal fitness. Our work also challenges the long-standing view of directional selection towards optimal codons, and provides a key example of natural selection against optimal codons to achieve adaptive responses to environmental changes.
Previous studies from our and other groups have demonstrated that the majority of γ-aminobutyric acid (GABA)(A) receptor subunit mutations produce mutant subunits with impaired biogenesis and trafficking. These GABA(A) receptor mutations include missense, nonsense, deletion, or insertion mutations that result in a frameshift with premature translation-termination codons (PTCs) and splice-site mutations. Frameshift or splice-site mutations produce mutant proteins with PTCs, thus generating nonfunctional truncated proteins. All of these mutant GABA(A) receptor subunits are subject to cellular quality control at the messenger RNA (mRNA) or protein level. These quality-control checkpoints shape the cell's response to the presence of the mutant subunits and attempt to reduce the impact of the mutant subunit on GABA(A) receptor expression and function. The check points prevent nonfunctioning or malfunctioning GABA(A) receptor subunits from trafficking to the cell surface or to synapses, and help to ensure that the receptor channels trafficked to the membrane and synapses are indeed functional. However, if and how these quality control or check points impact the posttranslational modifications of functional GABA(A) receptor channels such as receptor phosphorylation and ubiquitination and their involvement in mediating GABAergic inhibitory synaptic strength needs to be investigated in the near future.
Wiley Periodicals, Inc. © 2012 International League Against Epilepsy.
To complement the recent genomic sequencing of Chinese hamster ovary (CHO) cells, proteomic analysis was performed on CHO cells including the cellular proteome, secretome, and glycoproteome using tandem mass spectrometry (MS/MS) of multiple fractions obtained from gel electrophoresis, multidimensional liquid chromatography, and solid phase extraction of glycopeptides (SPEG). From the 120 different mass spectrometry analyses generating 682,097 MS/MS spectra, 93,548 unique peptide sequences were identified with at most 0.02 false discovery rate (FDR). A total of 6164 grouped proteins were identified from both glycoproteome and proteome analysis, representing an 8-fold increase in the number of proteins currently identified in the CHO proteome. Furthermore, this is the first proteomic study done using the CHO genome exclusively, which provides for more accurate identification of proteins. From this analysis, the CHO codon frequency was determined and found to be distinct from humans, which will facilitate expression of human proteins in CHO cells. Analysis of the combined proteomic and mRNA data sets indicated the enrichment of a number of pathways including protein processing and apoptosis but depletion of proteins involved in steroid hormone and glycosphingolipid metabolism. Five-hundred four of the detected proteins included N-acetylation modifications, and 1292 different proteins were observed to be N-glycosylated. This first large-scale proteomic analysis will enhance the knowledge base about CHO capabilities for recombinant expression and provide information useful in cell engineering efforts aimed at modifying CHO cellular functions.
BACKGROUND - To facilitate the use of automated databases for studies of sudden cardiac death, we previously developed a computerized case definition that had a positive predictive value between 86% and 88%. However, the definition has not been specifically validated for prescription opioid users, for whom out-of-hospital overdose deaths may be difficult to distinguish from sudden cardiac death.
FINDINGS - We assembled a cohort of persons 30-74 years of age prescribed propoxyphene or hydrocodone who had no life-threatening non-cardiovascular illness, diagnosed drug abuse, residence in a nursing home in the past year, or hospital stay within the past 30 days. Medical records were sought for a sample of 140 cohort deaths within 30 days of a prescription fill meeting the computer case definition. Of the 140 sampled deaths, 81 were adjudicated; 73 (90%) were sudden cardiac deaths. Two deaths had possible opioid overdose; after removing these two the positive predictive value was 88%.
CONCLUSIONS - These findings are consistent with our previous validation studies and suggest the computer case definition of sudden cardiac death is a useful tool for pharmacoepidemiologic studies of opioid analgesics.