The parasitoid wasp Nasonia vitripennis is an emerging genetic model for functional analysis of DNA methylation. Here, we characterize genome-wide methylation at a base-pair resolution, and compare these results to gene expression across five developmental stages and to methylation patterns reported in other insects. An accurate assessment of DNA methylation across the genome is accomplished using bisulfite sequencing of adult females from a highly inbred line. One-third of genes show extensive methylation over the gene body, yet methylated DNA is not found in non-coding regions and rarely in transposons. Methylated genes occur in small clusters across the genome. Methylation demarcates exon-intron boundaries, with elevated levels over exons, primarily in the 5′ regions of genes. It is also elevated near the sites of translational initiation and termination, with reduced levels in 5′ and 3′ UTRs. Methylated genes have higher median expression levels and lower expression variation across development stages than non-methylated genes. There is no difference in frequency of differential splicing between methylated and non-methylated genes, and as yet no established role for methylation in regulating alternative splicing in Nasonia. Phylogenetic comparisons indicate that many genes maintain methylation status across long evolutionary time scales. Nasonia methylated genes are more likely to be conserved in insects, but even those that are not conserved show broader expression across development than comparable non-methylated genes. Finally, examination of duplicated genes shows that those paralogs that have lost methylation in the Nasonia lineage following gene duplication evolve more rapidly, show decreased median expression levels, and increased specialization in expression across development. Methylation of Nasonia genes signals constitutive transcription across developmental stages, whereas non-methylated genes show more dynamic developmental expression patterns. We speculate that loss of methylation may result in increased developmental specialization in evolution and acquisition of methylation may lead to broader constitutive expression.
Insects use methylation to modulate genome function in a different manner from vertebrates. Here, we quantified the global methylation profile in a parasitic wasp species, Nasonia vitripennis, a model with some advantages over ant and honeybee for functional and genetic analyses of methylation, such as short generation time, inbred lines, and inter-fertile species. Using a highly inbred line permitted us to precisely characterize DNA methylation, which is compared to gene expression variation across developmental stages, and contrasted to other insect species. DNA methylation is almost exclusively on the 5′-most 1 kbp coding exons, and ∼1/3 of protein coding genes are methylated. Methylated genes tend to occur in small clusters in the genome. Unlike many organisms, Nasonia leaves nearly all transposable element genes non-methylated. Methylated genes exhibit more uniform expression across developmental stages for both moderately and highly expressed genes, suggesting that DNA methylation is marking the genes for constitutive expression. Among pairs of differentially methylated duplicated genes, the paralogs that lose DNA methylation after duplication in the Nasonia lineage show lower expression and greater specialization of expression. Finally, by comparative analysis, we show that methylated genes are more conserved at three different time scales during evolution.
Professional judgment is necessary to assess occupational exposure in population-based case-control studies; however, the assessments lack transparency and are time-consuming to perform. To improve transparency and efficiency, we systematically applied decision rules to the questionnaire responses to assess diesel exhaust exposure in the New England Bladder Cancer Study, a population-based case-control study.
2,631 participants reported 14,983 jobs; 2,749 jobs were administered questionnaires (‘modules’) with diesel-relevant questions. We applied decision rules to assign exposure metrics based solely on the occupational history responses (OH estimates) and based on the module responses (module estimates); we combined the separate OH and module estimates (OH/module estimates). Each job was also reviewed one at a time to assign exposure (one-by-one review estimates). We evaluated the agreement between the OH, OH/module, and one-by-one review estimates.
The proportion of exposed jobs was 20–25% for all jobs, depending on approach, and 54–60% for jobs with diesel-relevant modules. The OH/module and one-by-one review had moderately high agreement for all jobs (κw=0.68–0.81) and for jobs with diesel-relevant modules (κw=0.62–0.78) for the probability, intensity, and frequency metrics. For exposed subjects, the Spearman correlation statistic was 0.72 between the cumulative OH/module and one-by-one review estimates.
The agreement seen here may represent an upper level of agreement because the algorithm and one-by-one review estimates were not fully independent. This study shows that applying decision-based rules can reproduce a one-by-one review, increase transparency and efficiency, and provide a mechanism to replicate exposure decisions in other studies.
Ecologic studies have reported that solar ultraviolet radiation (UVR) exposure is associated with cancer, but little evidence is available from prospective studies. We aimed to assess the association between an objective measure of ambient UVR exposure and risk of total and site-specific cancer in a large, regionally diverse cohort (450,934 white, non-Hispanic subjects (50-71 years old) in the prospective NIH-AARP Diet and Health Study) after accounting for individual-level confounding risk factors. Estimated erythemal UVR exposure from satellite Total Ozone Mapping Spectrometer (TOMS) data from NASA was linked to the U.S. Census Bureau 2000 census tract (centroid) of baseline residence for each subject. We used Cox proportional hazards models adjusted for multiple potential confounders to estimate hazard ratios (HR) and 95% confidence intervals (CI) for quartiles of UVR exposure. Restricted cubic splines examined non-linear relationships. Over 9 years of follow-up, UVR exposure was inversely associated with total cancer risk (N=75,917; highest vs. lowest quartile, HR=0.97 (0.95, 0.99), p-trend<0.001). In site-specific cancer analyses, UVR exposure was associated with increased melanoma risk (highest vs. lowest quartile, HR=1.22 (1.13, 1.32), p-trend<0.001) and decreased risk of Non-Hodgkin’s lymphoma (HR=0.82 (0.74, 0.92)) and colon (HR=0.88 (0.82, 0.96)), squamous cell lung (HR=0.86 (0.75, 0.98)), pleural (HR=0.57 (0.38, 0.84)), prostate (HR=0.91 (0.88, 0.95)), kidney (HR=0.83 (0.73, 0.94)), and bladder (HR=0.88 (0.81, 0.96)) cancers (all p-trend<0.05). We also found non-linear associations for some cancer sites, including the thyroid and pancreas. Our results add to mounting evidence for the influential role of UVR exposure on cancer.
Ultraviolet radiation; cancer; vitamin D; prospective
Exploring spatial-temporal patterns of disease incidence and mortality can identify areas of significantly elevated or decreased risk, providing potential etiologic clues. Several methodological issues arise in spatial-temporal analysis of cancer, including population mobility, disease latency, and confounding, but applying modern statistical methods to case-control studies with residential histories can address these issues. As an example, we present a spatial-temporal analysis of non-Hodgkin lymphoma (NHL) risk using data from Los Angeles County, one of four centers in a population-based case-control study. Using residential histories, we fitted generalized additive models (GAMs) adjusted for known risk factors to model spatially the probability that an individual had NHL and identify areas of significantly elevated NHL risk. In previous analyses using models with single lag times, the lag time of 20 years yielded the most significant decrease in model deviance. To better assess cumulative effects of unmeasured environmental exposures over space and time, we considered models that allowed for multiple residences per subject through spatial smoothing functions of residential location at different times. We found that the model with the best goodness-of-fit included components for residential change and residential duration, although the model that included residential duration was not meaningfully better than the model that included only residential change. The estimated cumulative spatial risk surface from the model with residential change amplified the risk surface in some areas compared with the surface based on the model with a single component for the most significant time lag.
cancer; generalized additive model; spatial risk; latency; exposure
Transposable elements (TEs) are abundant in the human genome, and some are capable of generating new insertions through RNA intermediates. In cancer, the disruption of cellular mechanisms that normally suppress TE activity may facilitate mutagenic retrotranspositions. We performed single-nucleotide resolution analysis of TE insertions in 43 high-coverage whole-genome sequencing data sets from five cancer types. We identified 194 high-confidence somatic TE insertions, as well as thousands of polymorphic TE insertions in matched normal genomes. Somatic insertions were present in epithelial tumors but not in blood or brain cancers. Somatic L1 insertions tend to occur in genes that are commonly mutated in cancer, disrupt the expression of the target genes, and are biased toward regions of cancer-specific DNA hypomethylation, highlighting their potential impact in tumorigenesis.
Bacteria to eukaryote lateral gene transfers (LGT) are an important potential source of material for the evolution of novel genetic traits. The explosion in the number of newly sequenced genomes provides opportunities to identify and characterize examples of these lateral gene transfer events, and to assess their role in the evolution of new genes. In this paper, we describe an ancient lepidopteran LGT of a glycosyl hydrolase family 31 gene (GH31) from an Enterococcus bacteria. PCR amplification between the LGT and a flanking insect gene confirmed that the GH31 was integrated into the Bombyx mori genome and was not a result of an assembly error. Database searches in combination with degenerate PCR on a panel of 7 lepidopteran families confirmed that the GH31 LGT event occurred deep within the Order approximately 65–145 million years ago. The most basal species in which the LGT was found is Plutella xylostella (superfamily: Yponomeutoidea). Array data from Bombyx mori shows that GH31 is expressed, and low dN/dS ratios indicates the LGT coding sequence is under strong stabilizing selection. These findings provide further support for the proposition that bacterial LGTs are relatively common in insects and likely to be an underappreciated source of adaptive genetic material.
schizophrenia; sequencing; SNV; genetic; association; mutation; DISC1
Individual exposure to ultraviolet radiation (UVR) is challenging to measure, particularly for diseases with substantial latency periods between first exposure and diagnosis of outcome, such as cancer. To guide the choice of surrogates for long-term UVR exposure in epidemiologic studies, we assessed how well stable sun-related individual characteristics and environmental/meteorological factors predicted daily personal UVR exposure measurements.
We evaluated 123 United States Radiologic Technologists subjects who wore personal UVR dosimeters for 8 hours daily for up to 7 days (N = 837 days). Potential predictors of personal UVR derived from a self-administered questionnaire, and public databases that provided daily estimates of ambient UVR and weather conditions. Factors potentially related to personal UVR exposure were tested individually and in a model including all significant variables.
The strongest predictors of daily personal UVR exposure in the full model were ambient UVR, latitude, daily rainfall, and skin reaction to prolonged sunlight (R2 = 0.30). In a model containing only environmental and meteorological variables, ambient UVR, latitude, and daily rainfall were the strongest predictors of daily personal UVR exposure (R2 = 0.25).
In the absence of feasible measures of individual longitudinal sun exposure history, stable personal characteristics, ambient UVR, and weather parameters may help estimate long-term personal UVR exposure.
The development of a microneedle-based biosensor array for multiplexed in situ detection of exercise-induced metabolic acidosis, tumor microenvironment, and other variations in tissue chemistry is described. Simultaneous and selective amperometric detection of pH, glucose, and lactate over a range of physiologically-relevant concentrations in complex media is demonstrated. Furthermore, materials modified with a cell-resistant (Lipidure®) coating were shown to inhibit macrophage adhesion; no signs of coating delamination were noted over a 48-hour period.
microneedle biosensor; microneedle; multiplexed detection; tumor microenvironment; carbon paste
To characterize tumor growth and metastatic potential in head and neck squamous cell carcinoma (HNSCC) cell lines in an orthotopic murine model of oral tongue cancer, and to correlate TP53 mutation status with these findings.
Cells from each of 48 HNSCC cell lines were orthotopically injected into the oral tongues of nude mice. Tumor volume, cervical lymph node metastasis, and mouse survival were recorded. Direct sequencing of the TP53 gene and western blot analysis for the p53 protein after induction with 5-fluorouracil was performed. Cell lines were categorized as either mutant TP53 or wild-type TP53, and lines with TP53 mutation were further categorized on the basis of type of mutation (disruptive or non-disruptive), and level of p53 protein expression. The behavior of tumors in these different groups was compared.
The 48 HNSCC cell lines showed a wide range of behavior from highly aggressive and metastatic to no tumor formation. Mice injected with cells harboring disruptive TP53 mutations had faster tumor growth, greater incidence of cervical lymph node metastasis, and shorter survival than mice injected with cells lacking these mutations.
HNSCC cell lines display a wide spectrum of behavior in an orthotopic model of oral cancer. Cell lines with disruptive TP53 mutations are more aggressive in this system, corroborating clinical reports that have linked these mutations to poor patient outcome.
head and neck squamous cell carcinoma; TP53; disruptive TP53 mutation; cervical lymph node metastasis; orthotopic nude mouse model
Ion channel mutations are an important cause of rare Mendelian disorders affecting brain, heart, and other tissues. We performed parallel exome sequencing of 237 channel genes in a well characterized human sample, comparing variant profiles of unaffected individuals to those with the most common neuronal excitability disorder, sporadic idiopathic epilepsy. Rare missense variation in known Mendelian disease genes is prevalent in both groups at similar complexity, revealing that even deleterious ion channel mutations confer uncertain risk to an individual depending on the other variants with which they are combined. Our findings indicate that variant discovery via large scale sequencing efforts is only a first step in illuminating the complex allelic architecture underlying personal disease risk. We propose that in silico modeling of channel variation in realistic cell and network models will be crucial to future strategies assessing mutation profile pathogenicity and drug response in individuals with a broad spectrum of excitability disorders.
The Cancer Genome Atlas (TCGA) Network recently comprehensively catalogued the molecular aberrations in 487 high-grade serous ovarian cancers, with much remaining to be elucidated regarding the microRNAs (miRNAs). Here, using TCGA ovarian data, we surveyed the miRNAs, in the context of their predicted gene targets.
Methods and Results
Integration of miRNA and gene patterns yielded evidence that proximal pairs of miRNAs are processed from polycistronic primary transcripts, and that intronic miRNAs and their host gene mRNAs derive from common transcripts. Patterns of miRNA expression revealed multiple tumor subtypes and a set of 34 miRNAs predictive of overall patient survival. In a global analysis, miRNA:mRNA pairs anti-correlated in expression across tumors showed a higher frequency of in silico predicted target sites in the mRNA 3′-untranslated region (with less frequency observed for coding sequence and 5′-untranslated regions). The miR-29 family and predicted target genes were among the most strongly anti-correlated miRNA:mRNA pairs; over-expression of miR-29a in vitro repressed several anti-correlated genes (including DNMT3A and DNMT3B) and substantially decreased ovarian cancer cell viability.
This study establishes miRNAs as having a widespread impact on gene expression programs in ovarian cancer, further strengthening our understanding of miRNA biology as it applies to human cancer. As with gene transcripts, miRNAs exhibit high diversity reflecting the genomic heterogeneity within a clinically homogeneous disease population. Putative miRNA:mRNA interactions, as identified using integrative analysis, can be validated. TCGA data are a valuable resource for the identification of novel tumor suppressive miRNAs in ovarian as well as other cancers.
Aims. (1) To assess the efficacy and safety of pediatric office-based sedation for ophthalmologic procedures using a pediatric sedation service model. (2) To assess the reduction in hospital charges of this model of care delivery compared to the operating room (OR) setting for similar procedures. Background. Sedation is used to facilitate pediatric procedures and to immobilize patients for imaging and examination. We believe that the pediatric sedation service model can be used to facilitate office-based deep sedation for brief ophthalmologic procedures and examinations. Methods. After IRB approval, all children who underwent office-based ophthalmologic procedures at our institution between January 1, 2000 and July 31, 2008 were identified using the sedation service database and the electronic health record. A comparison of hospital charges between similar procedures in the operating room was performed. Results. A total of 855 procedures were reviewed. Procedure completion rate was 100% (C.I. 99.62–100). There were no serious complications or unanticipated admissions. Our analysis showed a significant reduction in hospital charges (average of $1287 per patient) as a result of absent OR and recovery unit charges. Conclusions. Pediatric ophthalmologic minor procedures can be performed using a sedation service model with significant reductions in hospital charges.
Phenotypic analysis of defects caused by RNA mediated interference (RNAi) in Caenorhabditis elegans has proven to be a powerful tool for determining gene function. In this study we investigated the effectiveness of RNAi in four non-model grassland soil nematodes, Oscheius sp FVV-2., Rhabditis sp, Mesorhabditis sp., and Acrobeloides sp. In contrast to reference experiments performed using C. elegans and Caenorhabditis briggsae, feeding bacteria expressing dsRNA and injecting dsRNA into the gonad did not produce the expected RNAi knockdown phenotypes in any of the grassland nematodes. Quantitative reverse-transcribed PCR (qRT-PCR) assays did not detect a statistically significant reduction in the mRNA levels of endogenous genes targeted by RNAi in Oscheius sp., and Mesorhabditis sp. From these studies we conclude that due to low effectiveness and inconsistent reproducibility, RNAi knockdown phenotypes in non-Caenorhabditis nematodes should be interpreted cautiously.
RNAi; Konza prairie; soil nematode; molecular biology
Personalized genomic medicine and surgery (PGMS) represents a new approach to health care that customizes patients’ medical treatment according to their own genetic information. This new approach is the result of increased knowledge of the human genome and ways this information can be applied by physicians in the medical and surgical management of their patients. A patient’s genotype can yield important information concerning disease susceptibility and the effectiveness of medications, therefore guiding specific, targeted imaging and treatment therapies. This review summarizes major achievements of human genomic studies and applications of genomics in health care. Five years ago we developed a model for the development of PGMS in which genomic profile guides choice of therapy. In this article we discussed our progress, including an updating of the model, and a future vision of PGMS.
Clinical cancer genetic susceptibility analysis typically proceeds sequentially beginning with the most likely causative gene. The process is time consuming and the yield is low particularly for families with unusual patterns of cancer. We determined the results of in parallel mutation analysis of a large cancer-associated gene panel. We performed deletion analysis and sequenced the coding regions of 45 genes (8 oncogenes and 37 tumor suppressor or DNA repair genes) in 48 childhood cancer patients who also (1) were diagnosed with a second malignancy under age 30, (2) have a sibling diagnosed with cancer under age 30 and/or (3) have a major congenital anomaly or developmental delay. Deleterious mutations were identified in 6 of 48 (13%) families, 4 of which met the sibling criteria. Mutations were identified in genes previously implicated in both dominant and recessive childhood syndromes including SMARCB1, PMS2, and TP53. No pathogenic deletions were identified. This approach has provided efficient identification of childhood cancer susceptibility mutations and will have greater utility as additional cancer susceptibility genes are identified. Integrating parallel analysis of large gene panels into clinical testing will speed results and increase diagnostic yield. The failure to detect mutations in 87% of families highlights that a number of childhood cancer susceptibility genes remain to be discovered.
Cancer susceptibility; tumor suppressor genes; oncogenes; mutation analysis; rhabdoid tumors
Generation of cAMP by G protein–coupled receptors (GPCRs) and its termination is currently thought to occur exclusively at the plasma membrane of cells. Under existing models of receptor regulation, this signal is primarily restricted by desensitizationof the receptors through their binding to β-arrestins. However, this paradigm is not consistent with recent observations that the parathyroid hormone receptor type 1 (PTHR) continues to stimulate cAMP production even after receptor internalization, as β-arrestins are known to rapidly bind and internalize activated PTHR. Here we show that β-arrestin1 binding prolongs rather than terminates cAMP generation by PTHR, and that cAMP generation correlates with the persistence of arrestin-receptor complexes on endosomes. We found that PTHR signaling is instead turned-off by the retromer complex, which regulates traffic of internalized receptor from endosomes to the Golgi apparatus. Thus, binding by the retromer complex regulates sustained cAMP generation triggered by an internalized GPCR.
Ciliary dysfunction leads to a broad range of overlapping phenotypes, termed collectively as ciliopathies. This grouping is underscored by genetic overlap, where causal genes can also contribute modifying alleles to clinically distinct disorders. Here we show that mutations in TTC21B/IFT139, encoding a retrograde intraflagellar transport (IFT) protein, cause both isolated nephronophthisis (NPHP) and syndromic Jeune Asphyxiating Thoracic Dystrophy (JATD). Moreover, although systematic medical resequencing of a large, clinically diverse ciliopathy cohort and matched controls showed a similar frequency of rare changes, in vivo and in vitro evaluations unmasked a significant enrichment of pathogenic alleles in cases, suggesting that TTC21B contributes pathogenic alleles to ∼5% of ciliopathy patients. Our data illustrate how genetic lesions can be both causally associated with diverse ciliopathies, as well as interact in trans with other disease-causing genes, and highlight how saturated resequencing followed by functional analysis of all variants informs the genetic architecture of disorders.
Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide. To explore the genetic origins of this cancer, we used whole exome sequencing and gene copy number analyses to study 32 primary tumors. Tumors from patients with a history of tobacco use had more mutations than did tumors from patients who did not use tobacco, and tumors that were negative for human papilloma virus (HPV) had more mutations than did HPV-positive tumors. Six of the genes that were mutated in multiple tumors were assessed in up to 88 additional HNSCCs. In addition to previously described mutations in TP53, CDKN2A, PIK3CA and HRAS, we identified mutations in FBXW7 and NOTCH1. Interestingly, nearly 40% of the 28 mutations identified in NOTCH1 were predicted to truncate the gene product, suggesting that NOTCH1 may function as a tumor suppressor gene rather than an oncogene in this tumor type.
While Wnt-Frizzled (Fzd) signaling is critical in the pathophysiology of carcinomas, its role in human breast cancer has been difficult to establish. We show here that the adaptor protein Na+/H+ Exchange Regulatory Factor1 (NHERF1), a protein abundantly expressed in normal mammary epithelium, regulates Wnt signaling, maintaining low levels of β-catenin activation. NHERF1’s effects are mediated by direct interactions between one of its PSD-95/Drosophila discs large/ZO-1 domains (PDZ domains) and the C-terminus of a subset of Fzd receptors. Loss of NHERF1 in breast cancer cell lines enhances canonical Wnt signaling and Wnt-dependent cell proliferation. Furthermore, the mammary glands of NHERF1 knockout mice exhibit increased mammary duct density accompanied by increased proliferation and β-catenin activity. Finally, we demonstrate a negative correlation between NHERF1 expression and nuclear β-catenin in human breast carcinomas. Taken together, these results provide novel insight into the regulation of Wnt signaling in normal and neoplastic breast tissues, and identify NHERF1 as an important regulator of the pathogenesis of breast tumors.
Exploring spatial-temporal patterns of disease incidence through cluster analysis identifies areas of significantly elevated or decreased risk, providing potential clues about disease risk factors. Little is known about the etiology of non-Hodgkin lymphoma (NHL), or the latency period that might be relevant for environmental exposures, and there are no published spatial-temporal cluster studies of NHL.
We conducted a population-based case-control study of NHL in four National Cancer Institute (NCI)-Surveillance, Epidemiology, and End Results (SEER) centers: Detroit, Iowa, Los Angeles, and Seattle during 1998-2000. Using 20-year residential histories, we used generalized additive models adjusted for known risk factors to model spatially the probability that an individual had NHL and to identify clusters of elevated or decreased NHL risk. We evaluated models at five different time periods to explore the presence of clusters in a time frame of etiologic relevance.
The best model fit was for residential locations 20 years prior to diagnosis in Detroit, Iowa, and Los Angeles. We found statistically significant areas of elevated risk of NHL in three of the four study areas (Detroit, Iowa, and Los Angeles) at a lag time of 20 years. The two areas of significantly elevated risk in the Los Angeles study area were detected only at a time lag of 20 years. Clusters in Detroit and Iowa were detected at several time points.
We found significant spatial clusters of NHL after allowing for disease latency and residential mobility. Our results show the importance of evaluating residential histories when studying spatial patterns of cancer.
Rhesus macaques are the most widely utilized nonhuman primate model in biomedical research. Previous efforts have validated fewer than 900 single nucleotide polymorphisms (SNPs) in this species, which limits opportunities for genetic studies related to health and disease. Extensive information about SNPs and other genetic variation in rhesus macaques would facilitate valuable genetic analyses, as well as provide markers for genome-wide linkage analysis and the genetic management of captive breeding colonies.
We used the available rhesus macaque draft genome sequence, new sequence data from unrelated individuals and existing published sequence data to create a genome-wide SNP resource for Indian-origin rhesus monkeys. The original reference animal and two additional Indian-origin individuals were resequenced to low coverage using SOLiD™ sequencing. We then used three strategies to validate SNPs: comparison of potential SNPs found in the same individual using two different sequencing chemistries, and comparison of potential SNPs in different individuals identified with either the same or different sequencing chemistries. Our approach validated approximately 3 million SNPs distributed across the genome. Preliminary analysis of SNP annotations suggests that a substantial number of these macaque SNPs may have functional effects. More than 700 non-synonymous SNPs were scored by Polyphen-2 as either possibly or probably damaging to protein function and these variants now constitute potential models for studying functional genetic variation relevant to human physiology and disease.
Resequencing of a small number of animals identified greater than 3 million SNPs. This provides a significant new information resource for rhesus macaques, an important research animal. The data also suggests that overall genetic variation is high in this species. We identified many potentially damaging non-synonymous coding SNPs, providing new opportunities to identify rhesus models for human disease.
single nucleotide polymorphism; common variants; SOLiD™; genetic variation; rhesus macaque
Lowering LDL cholesterol with statin regimens reduces the risk of myocardial infarction, ischaemic stroke, and the need for coronary revascularisation in people without kidney disease, but its effects in people with moderate-to-severe kidney disease are uncertain. The SHARP trial aimed to assess the efficacy and safety of the combination of simvastatin plus ezetimibe in such patients.
This randomised double-blind trial included 9270 patients with chronic kidney disease (3023 on dialysis and 6247 not) with no known history of myocardial infarction or coronary revascularisation. Patients were randomly assigned to simvastatin 20 mg plus ezetimibe 10 mg daily versus matching placebo. The key prespecified outcome was first major atherosclerotic event (non-fatal myocardial infarction or coronary death, non-haemorrhagic stroke, or any arterial revascularisation procedure). All analyses were by intention to treat. This trial is registered at ClinicalTrials.gov, NCT00125593, and ISRCTN54137607.
4650 patients were assigned to receive simvastatin plus ezetimibe and 4620 to placebo. Allocation to simvastatin plus ezetimibe yielded an average LDL cholesterol difference of 0·85 mmol/L (SE 0·02; with about two-thirds compliance) during a median follow-up of 4·9 years and produced a 17% proportional reduction in major atherosclerotic events (526 [11·3%] simvastatin plus ezetimibe vs 619 [13·4%] placebo; rate ratio [RR] 0·83, 95% CI 0·74–0·94; log-rank p=0·0021). Non-significantly fewer patients allocated to simvastatin plus ezetimibe had a non-fatal myocardial infarction or died from coronary heart disease (213 [4·6%] vs 230 [5·0%]; RR 0·92, 95% CI 0·76–1·11; p=0·37) and there were significant reductions in non-haemorrhagic stroke (131 [2·8%] vs 174 [3·8%]; RR 0·75, 95% CI 0·60–0·94; p=0·01) and arterial revascularisation procedures (284 [6·1%] vs 352 [7·6%]; RR 0·79, 95% CI 0·68–0·93; p=0·0036). After weighting for subgroup-specific reductions in LDL cholesterol, there was no good evidence that the proportional effects on major atherosclerotic events differed from the summary rate ratio in any subgroup examined, and, in particular, they were similar in patients on dialysis and those who were not. The excess risk of myopathy was only two per 10 000 patients per year of treatment with this combination (9 [0·2%] vs 5 [0·1%]). There was no evidence of excess risks of hepatitis (21 [0·5%] vs 18 [0·4%]), gallstones (106 [2·3%] vs 106 [2·3%]), or cancer (438 [9·4%] vs 439 [9·5%], p=0·89) and there was no significant excess of death from any non-vascular cause (668 [14·4%] vs 612 [13·2%], p=0·13).
Reduction of LDL cholesterol with simvastatin 20 mg plus ezetimibe 10 mg daily safely reduced the incidence of major atherosclerotic events in a wide range of patients with advanced chronic kidney disease.
Merck/Schering-Plough Pharmaceuticals; Australian National Health and Medical Research Council; British Heart Foundation; UK Medical Research Council.
Hybridization of heterologous (non-specific) nucleic acids onto arrays designed for model-organisms has been proposed as a viable genomic resource for estimating sequence variation and gene expression in non-model organisms. However, conventional methods of normalization that assume equivalent distributions (such as quantile normalization) are inappropriate when applied to non-specific (heterologous) hybridization. We propose an algorithm for normalizing and centering intensity data from heterologous hybridization that makes no prior assumptions of distribution, reduces the false appearance of homology, and provides a way for researchers to confirm whether heterologous hybridization is suitable.
Data are normalized by adjusting for Gibbs free energy binding, and centered by adjusting for the median of a common set of control probes assumed to be equivalently dissimilar for all species. This procedure was compared to existing approaches and found to be as successful as Loess normalization at detecting sequence variations (deletions) and even more successful than quantile normalization at reducing the accumulation of false positive probe matches between two related nematode species, Caenorhabditis elegans and C. briggsae. Despite the improvements, we still found that probe fluorescence intensity was too poorly correlated with sequence similarity to result in reliable detection of matching probe sequence.
Cross-species hybridizations can be a way to adapt genome-enabled tools for closely related non-model organisms, but data must be appropriately normalized and centered in a way that accommodates hybridization of nucleic acids with diverged sequence. For short, 25-mer probes, hybridization intensity alone may be insufficiently correlated with sequence similarity to allow reliable inference of homology at the probe level.