The heart is sensitive to oxidative damage but a global view on how the cardiac proteome responds to oxidative stressors has yet to fully emerge. Using quantitative tandem mass spectrometry, we assessed the effects of acute exposure of the oxidative stress inducer paraquat on protein expression in mouse hearts. We observed widespread protein expression changes in the paraquat-exposed heart especially in organelle-containing subcellular fractions. During cardiac response to acute oxidative stress, proteome changes are consistent with a rapid reduction of mitochondrial metabolism, coupled with activation of multiple antioxidant proteins, reduction of protein synthesis and remediation of proteostasis. In addition to differential expression, we saw evidence of spatial reorganizations of the cardiac proteome including the translocation of hexokinase 2 to more soluble fractions. Treatment with the antioxidants Tempol and MitoTEMPO reversed many proteomic signatures of paraquat but this reversal was incomplete. We also identified a number of proteins with unknown function in the heart to be triggered by paraquat, suggesting they may have functions in oxidative stress response. Surprisingly, protein expression changes in the heart correlate poorly with those in the lung, consistent with differential sensitivity or stress response in these two organs. The results and data set here could provide insights into oxidative stress responses in the heart and avail the search for new therapeutic targets. Evidence of oxidative stress is commonly found in many animal models of heart diseases, but antioxidant therapeutics have only seen limited success and were in some cases harmful, suggesting further work is needed to understand stress response pathways and identify new therapeutic targets4. It is now known that acute oxidative stress triggers complex and multifactorial changes to the cardiac proteome including widespread chemical and enzymatic oxidative modifications6 as well as the activation of integrated stress response pathways7. Prior proteomics data on the global responses of cultured mammalian cells to hydrogen peroxide in vitro pointed to close connections between oxidative stress and the unfolded protein response, as well as a diverse number of regulatory pathways involved in metabolism and protein translation8. Whether similar proteome-wide responses are recapitulated in the heart of in vivo animal models remains unclear.Oxidative stress is widely implicated in cardiovascular disorders and other age-related diseasesN,N11. In humans, chronic environmental exposure to residues of paraquat used as pesticide has been linked to the development of Parkinson′s disease12, whereas in rare cases acute paraquat poisoning at higher dose has also been reported that leads to significant multi-organ toxicity and mortality14. In laboratory experiments, high doses of paraquat  were exposed to three doses of paraquat . The doses were chosen from a range previously reported for cardiac research models in the literature, with reports showing that exposure at 40–80 mg/kg for up to 48 hours induced acute oxidative stress responses as well as reduced contractile function and calcium handling in the mouse heart23. We first compared whether the applied paraquat doses elevated protein abundance of previously documented paraquat induced genes in the heart using mass spectrometry  and BCL2-like 1 (BCL2L1).Paraquat is a mitochondria-targeted redox-cycler that acts as a superoxide generator to produce ROS through interactions with Complex I within the inner mitochondrial matrix27. Under physiological conditions, NRF2 is continuously degraded and has a short half-life, but under stress conditions NRF2 accumulates and translocates into the nucleus to activate genes with antioxidant response element (ARE) sequences and thereby orchestrates the acute phase of cellular oxidative stress responses. At 24 h, we observed a robust induction of heme oxygenase 1 (HMOX1), a primary cardioprotective oxidative stress response protein in the heart  pathwayart Fig. a. In ourart Fig. b. Pre-trThe initial results above confirmed that exposure to moderate to high doses (50–75 mg/kg) of paraquat for 24–48 h robustly activated classic acute oxidative stress responses in the mouse heart, and at the same time induced other known paraquat-induced signatures. To further investigate how acute stress responses remodel the global cardiac proteome, we selected the medium dose paraquat exposure  and acquired deep quantitative proteome profiles using subcellular fractionation and two-dimensional peptide separation Fig. a–d. FromSurprisingly, we found that 50 mg/kg paraquat primarily induced differential expression of proteins localized in Fraction S2, with far fewer differentially regulated proteins in fractions S1 and S3 , suggesting PRR16 may be a downstream ER stress response factor. The function of PRR16 in the heart is unclear. (ii) Aldehyde dehydrogenase 16 family member A1 (ALDH16A1) is nominally up-regulated under paraquat exposure . Although a member of the aldehyde dehydrogenase family, ALDH16A1 contains aldehyde dehydrogenase domains that are thought to be inactive31, and its function in the heart remains unknown. It is ubiquitously expressed and has no clear co-expressed genes across human tissues, but previous ChIP-seq data has shown that its promoter region may bind to STAT332, which is also upregulated in the data . (iii) Signal transducing adaptor molecule (SH3 domain and ITAM motif) 1 (STAM) is conspicuously up-regulated following paraquat exposure  and is believed to be involved in cell growth33 and vesicular trafficking34, and is associated with brain neoplasm in the literature. Taken together, the data portray wide-ranging pathway-dependent and compartment-dependent consequences of paraquat exposure on the cardiac proteome, and nominate a number of novel acute response proteins for further characterization.Lastly, the proteomics data also uncovered the differential expression of a number of proteins with still unclear functions in the heart, implicating a potential function of these proteins in acute oxidative stress responses. For example: (i) Proline rich protein 16 (PRR16) is significantly activated after paraquat . Promoter binding analysis provides supporting evidence that the Myofibrillar disarray and alterations in cytoskeleton dynamics are commonly observed in oxidative damage models. To assess whether we could detect this process or other redistribution of proteins from the proteomics data, we examined whether some proteins can be observed to have differential subcellular fraction distribution in normal vs. paraquat treated hearts. We saw that overall the subcellular fraction distribution between the S2 and S1 fractions, and between the least soluble S3 and S1 fractions, are highly correlated in normal and paraquat samples  within 24 h is unlikely and the more parsimonious explanation of the data would be that it is consistent with the dissolution of myofibrils coupled with appearance in the more soluble fraction. Furthermore, we likewise observed a small but significant number of potential translocation events including prominently hexokinase 2 (HK2) . Considering an arbitrary set of proteins with apparent upward or downward trends following paraquat exposure  old Fig. a.Figure We also observed evidence that some protein changes were preferentially rescued by one antioxidant over the other. To investigate further, we used a linear discriminant analysis to identify proteins with high weights in discriminating between normal, stressed, and antioxidant treated samples Fig. b. We fouFinally, we performed a secondary experiment to evaluate how the observed proteome responses in the heart compare with the global response to paraquat in the animal. We collected the lungs from the cadavers of identical animals and subjected them also to isotope labeling tandem mass spectrometry analysis , CAT , and HMOX1  in the lung, indicating paraquat also induced an acute oxidative stress response in the lung of the examined animals. Comparison of organ-specific responses is made challenging by the different proteome composition and dynamic range of concentration between the organs, and moreover, each tissue may be differentially sensitive to paraquat or react in different time scales. Nevertheless, we were surprised to find that the fold-changes of proteins commonly quantified in both the heart and the lungs following paraquat treatment showed little to no correlation to each other  over the others in the heart. Differential detergent lysis is a common method to resolve partially overlapping protein fractions that correspond in part with specific cellular compartmentsMPO Fig. . Both Te42. A caveat of the current experimental design is that differential lysis provides a relatively crude method to separate cellular compartments based partly on membrane composition and compartment solubility, which could differ across cells and tissues. In fact, analysis of the proteomics results from different fractions in the heart shows that the cellular components in detergent fractions can overlap partially, and suggests direct mass spectrometry evidence should be sought when similar methods are applied to fractionate different cell type or samples. Future work may apply orthogonal methods such as ultracentrifugation in conjunction with detergent based methods to follow up on spatial proteomics findings and identify additional translocalization candidates.Surprisingly, we also found evidence that some proteins may redistribute in fractions upon paraquat challenge. These results therefore indicate that spatial dynamics may be a component of proteomic features during the acute stress response and echo the increasing number of reports that demonstrate proteome-wide spatial redistribution upon a variety of stimuli44. Future work might examine if the observed changes in metabolic and mitochondrial proteins were effectuated in the heart by autophagy of specific cellular components.Finally, the mechanisms behind the observed differential expression changes are not investigated in this study and could be an important future direction. The abundance of a protein pool may be controlled by both the protein’s synthesis rate and its degradation rate. In some instances where a protein requires fast abundance control in response to abrupt cellular and environmental changes, proteolysis provides a mechanism to quickly adjust protein abundance without requiring transcriptional regulation. This is evidenced by the activation mechanism of NRF2 in response to oxidative stress as well as other examples including PINK1 to mitochondrial membrane potential and HIF1a to oxygen level. Protein damage and unfolding may prompt targeted degradation, whereas autophagy is another prominent protein removal mechanism that is well described to respond to oxidative stressN,NAnimal experiments were approved and performed in accordance with the Institutional Animal Care and Use Committee (IACUC) guidelines at University of Colorado Denver/Anschutz Medical Campus. Twelve-week old male wild-type C57BL/6J mice were purchased from Jackson Laboratories  and housed in accordance with guidelines set by the National Institutes of Health (NIH) for the Care and Use of Laboratory Animals. The protocol for animal use was approved by the Animal Care and Use Committee of the University of Colorado School of Medicine. Animals were held in a temperature-controlled environment on a 12-h light/dark cycle and supplied with pellet chow and water ad libitum. In the first animal experiment, mice were randomized into six treatment groups (n = 4 per group each) and received either Dulbecco’s phosphate buffered saline (DPBS) . Mice in vehicle and paraquat exposure groups received either DPBS  using 3 pulses of 20 s each at setting 4. Subcellular fractions were extracted using differential lysis and solubilization with a commercial Millipore Compartmental Protein Extraction Kit (Millipore) according to manufacturer’s instructions. Briefly, compartments S1, S2, and S3 were attained through detergents supplied as buffer C , buffer N , and buffer M . Following extraction, total protein concentration from each fraction was determined by bicinchoninic acid assay (Thermo Pierce). We used a block multiplexing approach to design the randomization scheme and assign the comparison group samples to tandem mass tag channels. For each tissue, samples from all treatment groups and subcellular fractions were randomized to one of ten channels in one of six blocks using the RAND function in Excel with no control over randomization seed  system (Thermo Scientific), which was connected to a Q-Exactive HF hybrid quadrupole orbitrap mass spectrometer (Thermo Scientific) through an EasySpray interface (Thermo Scientific). Chromatographic separation was conducted on an EasySpray PepMap C18 column  32.Liquid chromatography and tandem mass spectrometry were used to analyze the fractionated labeled peptides as previously documented46 using ThermoRawFileParser v.1.2.047 with the options (-g -f 2). MS2 spectra were searched using SEQUEST algorithm implemented in Comet (v.2019.01 rev.4)48 against a .fasta database Uniprot/SwissProt mus musculus database accessed on 2020-03-19 49. Comet search parameters followed conventional standards including: peptide_mass_tolerance=10, peptide_mass_units=2, isotope_error=3, num_enzyme_termini= 1, allowed_missed_cleavage=2, fragment_bin_tol = 0.02. Variable methionine oxidation (M + 15.9949 Da), static cysteine carbamidomethylation (C + 57.021464 Da), and static tandem mass tag addition to lysine or the peptide N-terminus (nK + 229.16293 Da) were specified. Peptides were reranked and assigned with confidence of identification using the Crux/Percolator v.3.250 followed by protein inference, with peptides accepted for identification under 0.01 Percolator q-value.The Thermo raw spectrum files were converted to the open source .mzML format51, and returns an appended search result file with the channel intensities. It is compatible with MS2-level tandem mass tag experiments using TMT 0, 2, 6, 10, 11, and 16-plex tags and allows user-defined mass tolerance for integration (see Code Availability). Using py-tmt-quant we integrated the centroid peak intensity within 10 ppm of each channel m/z. For each peptide-charge combination, only the PSM with the best Percolator score per fraction is integrated. We corrected for isotope impurities using the contamination matrix from the manufacturer /Bioconductor (v.3.10)53. Spectrum-level variances were normalized using the median values of the internal reference standard channel intensities across each block, and finally the effective total intensities were normalized using trimmed means of m-values with singleton pairing as implemented in the edgeR package (v.3.29.1)54 in R/Bioconductor 63 was used to quantify the intensity of the bands of interest and normalized to Ponceau Stain64. Statistical tests on the densitometry values were performed using Welch’s t-test in R v.3.6.3.Additional validation experiments were performed using immunoblots. Protein lysates (50 g per lane), as determined by bicinchoninic acid assay (BCA) (Pierce) from two biological replicate animals each from compartments S1, S2, and S3 for vehicle and 50 mg/kg paraquat hearts were denatured by boiling and resolved on 12% sodium dodecyl sulfate-polyacrylamide gels. Proteins were then transferred to polyvinylidene fluoride membranes and stained with Ponceau S (Sigma). After de-staining, membranes were blocked in 5% bovine serum albumin at Supplementary Information.


International migrants are at heightened risk for tuberculosis (TB) disease. Intensified incarceration at international borders may compound population-wide TB risk. However, few studies have investigated the contributions of migration, local transmission, or prisons in driving incident TB at international borders. We conducted prospective population-based genomic surveillance in 3 cities along Brazil’s central western border from 2014–2017. Although most isolates  fell within genomic transmission clusters, genetically unique isolates disproportionately occurred among participants with recent international travel , suggesting that both local transmission and migration contribute to incident TB. Isolates from 40 participants with and 76 without an incarceration history clustered together throughout a maximum-likelihood phylogeny, indicating the close interrelatedness of prison and community epidemics. Our findings highlight the need for ongoing surveillance to control continued introductions of TB and reduce the disproportionate burden of TB in prisons at Brazil’s international borders. Mycobacterium tuberculosis acquired elsewhere to incident TB cases can inform public health responses. However, few studies have explored the drivers of incident TB along international borders.The global expansion and local spread of tuberculosis (TB) have been shaped by patterns of human migration ; and travel history. Recent immigrants were defined as participants with residency in Brazil for <2 years; recent travel was defined as international travel of any duration in the previous 5 years. We obtained additional sociodemographic and clinical data from the National Reporting System on Notifiable Diseases (SINAN). We stored and managed data in an electronic database . We used a 3-sample proportion test to determine whether study participants were representative of all notified TB cases with respect to incarceration status reported in SINAN; we compared the proportion incarcerated, not incarcerated, and with no information about incarceration status at the time of TB notification among study participants and all notified TB cases. We were not able to do a similar comparison for immigration history or recent travel, which were collected in questionnaires.We conducted a population-based prospective study of newly diagnosed and retreated pulmonary TB cases in 3 cities at Brazil’s borders with Paraguay and Bolivia during January 2014–April 2017. Patients with clinical suspicion of TB sought care at primary care providers or hospitals in Ponta Porã, Corumbá, and Ladário, Brazil. Diagnostic tests were done at Hospital Regional Dr. José de Simone Neto in Ponta Porã and the Laboratório Municipal de Corumbá, a public health diagnostic laboratory. Patients with nontuberculous mycobacteria or without positive cultures for All participants provided written consent. We obtained approval from the Research Ethics Committee of the Federal University of Grande Dourados (no. CAE 12676613.3.1001.5160) and Stanford University Institutional Review Board (IRB-40285).https://www.cepheid.com), processed with sodium hydroxide (NaOH), and inoculated in Ogawa-Kudoh culture medium. We incubated cultures at 37°C for <8 weeks and checked weekly for visible colonies at the participating laboratories. We determined microbial species using the MPT64 protein detection-based immunochromatographic rapid test . We performed phenotypic susceptibility testing of M. tuberculosis isolates using the BACTEC MGIT 960 system All sputum specimens collected in the participating laboratories were examined by microscopy or GeneXpert MTB/RIF . Sequence data are available on the Sequence Read Archive (accession no. PRJNA671770). We trimmed low-quality bases  and removed adapters with Trim Galore (stringency = 3)  method and sequenced the whole genome on an Illumina NextSeq (2 × 151-bp) .We applied variant calling methods closely following those described in Menardo et al. (We excluded single-nucleotide polymorphisms (SNPs) in previously defined repetitive regions   (We fit a Bayesian tree to the sequences from the multidrug resistance (MDR)-associated transmission cluster with BEAST 2.6.2  isolates were isoniazid resistant, and 3 isolates (2.2%) were multidrug resistant, resistant to both isoniazid and rifampin. Five of the 14 isoniazid monoresistant case-patients and 3 of the 5 isolates resistant to both isoniazid and streptomycin had been previously treated; the 3 MDR isolates were from newly notified cases.All of the 132 whole genome sequences were assigned to the European-American lineage and specifically to sublineages 4.1 (47), 4.3 (71), 4.4 (9), 4.8 (3), and 4.9 (2) with TBProfiler . Similarly, the observed number of clusters including only members currently or formerly incarcerated does not differ from what would be expected under proportionate mixing (p = 0.76). The observed patterns of clustering indicate that transmission networks inside and outside prisons are closely related.To further investigate genetic structure among sampled isolates, we identified the most closely related isolate to each study isolate, including multiple isolates when there were multiple nearest genetic neighbors. Many isolates from participants reporting no incarceration history were most closely related to isolates from participants who were currently or previously incarcerated  or participants reporting contact with those incarcerated  . SimilarPatients with a recent history of travel, defined as travel within the previous 5 years, were significantly more likely to be infected with an unclustered or unique isolate (17/36) than patients with no history of travel (25/92) , potential evidence that they were infected outside of Brazil. These participants reported travel to Bolivia (9), Spain (1), Paraguay (5), Paraguay and Argentina (1) and Paraguay and Bolivia (1). All 4 isolates from recent immigrants to Brazil were unique and fell outside of predicted transmission clusters; again, possibly indicating they were infected outside of Brazil. However, limited sampling constrains our ability to predict whether genetically unique isolates represent imported lineages or locally circulating but unsampled lineages.. Similarly, streptomycin-resistant isolates occurred in 2 predicted transmission clusters, each including 1 person incarcerated at the time of TB diagnosis and a single unique isolate, which was evidence of transmitted resistance. In contrast, the 2 pyrazinamide-resistant isolates were unique.We then examined the distribution of drug resistance across predicted transmission clusters. The 22 isoniazid-resistant isolates occurred within 4 distinct transmission clusters; 1 isolate fell outside of the clusters, suggesting that isoniazid resistance has emerged or been introduced several times within the sampled isolates. The genetic clustering of resistance indicates that the majority of isoniazid resistance in this study was transmitted rather than acquired de novoinhA S94A mutation. In addition, the 3 MDR isolates all shared the rpoB S450L, rifampin-conferring mutation. Two of the MDR isolates occurred among persons who were previously incarcerated, and one was from a participant with an incarcerated family member. The mean pairwise distance between MDR isolates was 4 SNPs (range 3–5 SNPs), suggesting that MDR was transmitted (primary MDR) rather than acquired de novo.The 3 MDR isolates fell within a single predicted transmission cluster of 14 isolates from a single city, Corumbá (>6 years (the time between the MRCA of the MDR clade and the most recent date of sampling).We more closely examined the MDR-containing cluster by fitting a Bayesian timed tree . The mosIn recent years, while TB incidence declined nationally in Brazil, TB notifications have increased in its Central West region border cities. We found evidence that both local transmission and travel-associated introductions contribute to incident cases. In addition, we found that many genomic clusters involved persons with and without an incarceration history, evidence that prison and community epidemics of TB are closely interrelated in cities at Brazil’s Central West border. During 2005–2017, the state’s incarcerated population more than doubled  (M. tuberculosis clone associated with prisons that circulated locally for >6 years. Our findings highlight the critical need for the early detection of drug-resistant TB to prevent ongoing transmission.The prevalence of primary isoniazid resistance and MDR TB have increased across Brazil over the past 20 years (M. tuberculosis diversity observed within this study with a larger sample of genomes sampled from across Mato Grosso do Sul state, Paraguay, and Bolivia, we could better characterize the contributions of local transmission and importation of lineages into Brazil’s border cities. Finally, because of incomplete epidemic sampling and within-host diversity, phylogenetic trees constructed from consensus genomes do not represent actual transmission histories, but instead, the evolutionary histories of sampled M. tuberculosis. Phylogenetic trees enable us to characterize genetic structure in our study sample yet cannot be used to directly assess the probability of individual transmission events nor to quantify the role of high-transmission environments in driving the local epidemic.Our investigation of TB transmission at Brazil’s Central Western international borders has several limitations. In a setting characterized by frequent population movement, it is possible that many persons are not linked to healthcare, and TB may be undiagnosed, unnotified, or notified elsewhere. Further, more complete sampling among notified cases would enable a more complete portrait of transmission in border cities. For example, additional sampling could reveal that isolates we identified as genetically distinct do indeed fall within local transmission clusters; our estimates of the contribution of ongoing local transmission are likely conservative. In addition, selection bias could have been introduced if enrolled culture-positive participants were demographically different from the total population with TB. Although we did not find a difference in the proportion of incarcerated patients among study participants and all notified TB patients during the study period, we were unable to compare other characteristics such as recent travel history or migration history. It is possible that recent immigrants may have limited access to healthcare and therefore were undersampled; if so, the result would be underestimation of the role of travel-associated importation in incident TB. More complete information about study participants’ residential and travel histories could inform inferences of where transmission occurred. By contextualizing the Our findings indicate that both local transmission and long-distance importation of TB drive local TB incidence in Brazil’s Central Western border cities. Prison and community TB epidemics are interrelated, and prisons are associated with ongoing transmission of drug-resistant strains. The conditions for transmission and spread of TB in these border communities and congregate settings may undermine the broader national progress in TB control. Our study highlights the need for heightened surveillance and transmission-blocking interventions to prevent continued transmission of drug-sensitive and drug-resistant TB strains.


We investigated whether glycemic control affects the relation between endothelial dysfunction and coronary artery disease in patients with type 2 diabetes mellitus (T2DM).In 102 type 2 diabetic patients with stable angina, endothelial function was evaluated using brachial artery flow-mediated dilation (FMD) with high-resolution ultrasound, and significant stenosis of major epicardial coronary arteries (≥ 50% diameter narrowing) and degree of coronary atherosclerosis (Gensini score and SYNTAX score) were determined. The status of glycemic control was assessed by blood concentration of glycated hemoglobin (HbA1c).The prevalence of significant coronary artery stenosis  and degree of coronary atherosclerosis  were higher and FMD was lower  in diabetic patients with poor glycemic control  compared to those with good glycemic control . Multivariate regression analysis revealed that tertile of FMD was an independent determinant of presence of significant coronary artery stenosis , Gensini score  and SYNTAX score  in diabetic patients with poor glycemic control but not for those with good glycemic control (P > 0.05).Poor glycemic control negatively influences the association of endothelial dysfunction and coronary artery disease in T2DM patients. Endothelial dysfunction occurs early in the chain of atherosclerotic process and is mainly caused by systemic effects of common risk factors for coronary artery disease , 2. In cType 2 diabetes mellitus (T2DM) is independently associated with an increased risk for cardiovascular diseases that is primarily due to the early development of advanced atherosclerotic vascular changes . RecentlIt is well-recognized that chronic hyperglycemia could further accelerate the development of endothelial dysfunction and the pathological process of atherosclerosis, resulting in more diffuse coronary artery lesions and worse clinical outcomes , 20. NguThis study complied with the Declaration of Helsinki. The study protocol was approved by the local hospital ethics committee, and written informed consent was obtained from all participants.A total of 311 consecutive patients with T2DM and chest pain on exertion referred for diagnostic coronary angiography from January 2018 to May 2019 were enrolled. The diagnosis of T2DM was made according to the criteria of American Diabetes Association . HyperteCoronary angiography was performed through radial or femoral approach. Quantitative coronary angiography was performed using the Cardiovascular Measurement System version 3.0 software  by two interventional cardiologists who were blinded to the study protocol. Significant coronary artery disease was diagnosed if luminal diameter narrowing was estimated as ≥ 50% in a major epicardial coronary artery. Left main coronary artery stenosis ≥ 50% was considered as 2-vessel disease. The SYNTAX score and Gensini score were calculated and used as indexes of the anatomic extension and severity of coronary atherosclerosis , 26.Brachial artery FMD was assessed in the right arm using a high-resolution ultrasound machine with a 10‐MHz linear array probe and the GE Vivid 7 Imaging System following the recommended protocol . BrieflyContinuous variables are presented as mean ± standard deviation (SD) and median (25th–75th percentile) for normal and non-normal distribution, respectively, and categorical data are summarized as frequencies (percentages). We evaluated the differences in categorical variables between groups with a chi-square test. We evaluated the normality of distribution with the Kolmogorov–Smirnov test and applied logarithmic or square-root transformations to continuous variables showing a non-normal distribution. The differences between groups for continuous variables were analyzed by Student t test. We constructed multivariable logistic regression models to assess the independent determinants of coronary artery disease without (Model 1) and with (Model 2) FMD. All analyses used 2-sided tests with an overall significance level  of 0.05, and all tests were performed with SPSS 25.0 for Windows .Patients with poor glycemic control were older (P = 0.026) and exhibited higher serum levels of fasting blood glucose (P = 0.009), HbA1c (P < 0.001) and high-sensitivity C- reactive protein (hsCRP) (P = 0.002) compared to those with good glycemic control. However, there were no significant differences between the two groups in gender distribution, body mass index, proportion of cigarette smoking and hypertension, brachial blood pressure, kidney function and lipid profiles   and the severity of coronary atherosclerosis was more prominent  in patients with poor glycemic control than those with good glycemic control. Consistently, FMD was also significantly lower in patients with poor than those with good glycemic control . We arbitrarily categorized FMD values of all patients into three groups according to tertile of FMD . In patients with poor glycemic control (HbA1c ≥ 7.0%), a significant decrease in the prevalence of significant coronary artery disease, Gensini score and SYNTAX score was observed across the three tertiles of FMD , while no such trends were found in those with good glycemic control  . In multiple linear regression analysis, tertile of FMD was independently correlated with Gensini score  and SYNTAX score , respectively, in patients with poor glycemic control , tertile of FMD  remained to be independent determinants for coronary artery disease in patients with poor glycemic control (Model 2B)  in the assessment of risk of cardiovascular disease for patients with T2DM –52. LikeThis study indicates that poor glycemic control exerts an adverse effect on endothelial function, and aggravates coronary atherosclerosis in T2DM. These observations, at least partly, provide substantial insight into the management of type 2 diabetic patients with coronary artery disease.


We proved that Fyn tyrosine kinase elicits APP phosphorylation on Tyr682 residue, and we reported increased levels of APP Tyr682 and Fyn overactivation in AD neurons. Here, we want to contemplate the possibility of using fibroblasts as tools to assess APP Tyr682 phosphorylation in AD patients, thus making the changes in APP Tyr682 phosphorylation levels a potential diagnostic strategy to detect early pathological alterations present in the peripheral cells of AD patients’ AD brains.Alzheimer’s disease (AD) is a devastating neurodegenerative disorder with no cure and no effective diagnostic criteria. The greatest challenge in effectively treating AD is identifying biomarkers specific for each patient when neurodegenerative processes have not yet begun, an outcome that would allow the design of a personalised therapeutic approach for each patient and the monitoring of the therapeutic response during the treatment. We found that the excessive phosphorylation of the amyloid precursor protein (APP) Tyr Alzheimer’s disease (AD) is a prominent neurodegenerative disease which takes a major toll on the elderly and places an enormous burden on the healthcare system.The amyloid precursor protein (APP) is one of most extensively studied molecules in AD, and its cleavage, mediated by β-site APP-cleaving enzyme 1 (BACE1 or β-secretase) and by γ-secretase, leads to the dysregulated production of Aβ peptides, which mostly accumulate at central synapses [682 residue in the processes responsible for the generation of Aβ peptides in human neurons [682 residue is located in the highly conserved 682YENPTY687 motif, which binds specific adaptor proteins depending on its phosphorylation/dephosphorylation state [682 phosphorylation prevents the APP from binding to the Clathrin and AP2 endocytic proteins [682 residue in neurons differentiated from neuronal stem cells of AD patients [682 phosphorylation might be an early marker of neuronal degeneration [682 phosphorylation, that in turn triggers biochemical events downstream, finally resulting in Aβ production and neuronal death [682 phosphorylation and Aβ release in AD neurons [682 residue as a biomarker to identify patients with high levels of APP Tyr682 phosphorylation that may benefit from personalised treatments with Fyn TKI.Our group have previously underlined the crucial role of APP Tyr neurons . APP Tyron state . The APPon state ,6,7. In proteins , and affproteins . Consequproteins . Notablypatients ,10 preceneration ,6. Furth neurons . These fWe found elevated APP Tyr phosphorylation levels and Fyn overactivation both in skin fibroblasts and cortical tissues of minipigs carrying PSEN1 M146I mutation . The inchttps://catalog.coriell.org/). All fibroblast cultures derived from an ante mortem skin biopsy from the forearm, and had approximately the same biological age in culture (number of passages of cell lines and cell divisions). Fibroblast cell cultures grew very slow, but they all showed the same morphology and shape in culture. No differences were found in the extent of neuronal survival or proliferation. In addition, Aβ40 and Aβ42 were undetectable in media of fibroblast cultures, because the levels appeared below the sensitivity of a commercially available ELISA kit (data not shown).Human fibroblasts were purchased from the Coriell Institute  and cultured following the guidelines reported on the manufacturer’s web site , using the source number (#) reported in Some more information about patient clinical features and specific characteristics of the fibroblast cultures are available on the Coriell online catalogue , mouse anti-p-Tyr  and monoclonal anti-β-actin-peroxidase  antibodies.Fibroblasts were kept in culture until cell confluence was reached. Lysates were firstly processed for IP (pTyr) using mouse anti-p-Tyr magnetic beads, and subsequently analysed by WB. For pTyr protein enrichment, we used Phospho-Tyrosine Mouse mAb Magnetic Bead Conjugate (P-Tyr-100) . The samples were loaded on a 4–20% precast gel. Western blot analysis was performed using anti APP, clone Y188 , rabbit anti-pan Fyn , rabbit anti-Src pTyrData are expressed as mean ± standard error of the means. All the experiments were performed at least three times. The appropriate statistical test was selected using GraphPad Prism software (version 9.0c) ; details are reported in the legend for each figure.In the study, we included fibroblasts from ante mortem skin biopsies of 14 AD patients, six unaffected spouses, and four healthy donors with a familiar history of AD. In addition, four patients with a diagnosis of dementia, three associated to Parkinson’s disease (PD) and one to epilepsy were included. Post-mortem histopathology brain examinations were performed in all patients with mild cognitive impairment or dementia diagnosis to confirm AD.682 residue (APP has indeed other two Tyr(s) whose changes in the phosphorylation can also easily be detected by the same procedure), previous findings from our group proved that only APP Tyr682 residue phosphorylation is crucial in initiating APP amyloidogenic processes in AD neurons, whereas Tyr phosphorylation at different APP sites does not affect the extent of A𝛽 production or accumulation [420 phosphorylation was also assessed as an indirect measure of Fyn TK activity [The levels of phosphorylation of APP Tyr were assessed using the same procedure previously applied to neurons ,3 and comulation . The incactivity , and theWe found a significant increase in the level of APP Tyr phosphorylation in the majority of fibroblasts from AD patients when compared to healthy donors, as well as patients with other dementias . In part420 phosphorylation levels  and interacting directly with tau [18 [682 residue, which would then induce the amyloidogenic cleavage [682 phosphorylation levels are elevated, thus preventing off-target effects, and improving the outcome of the disease.It is well established that Fyn can play a dual role in both the Aβ and tau pathologies observed in AD , exacerbwith tau  by phosp tau [18 . We prevcleavage . This cacleavage . This ob682 phosphorylation in AD neurons opens the scenario to either test already commercially available Fyn TKIs or develop more specific compounds that can potentially control Fyn hyperactivity in AD patients and prevent Aβ42 production in patients in whom APP Tyr682 phosphorylation is increased.Indeed, the identification of Fyn as an actor in APP Tyr682 phosphorylation.Overall, the observations reported here prospect the possibility that events related to APP Tyr phosphorylation in fibroblasts may reflect Aβ related abnormalities in the brain, and that, if correlated with changes in cognitive performance and with other biomarkers including neuroimaging approaches or to the levels of Aβ and phosphorylated tau in biofluids, may allow the identification of patients who could benefit from a personalised pharmacologic approach, by using Fyn TKI. Indeed, these results underline the need to extend this evidence to a larger number of samples/fibroblasts, as well as to develop a procedure selective and quantitative for the detection of the levels of APP Tyr682 phosphorylation levels in peripheric cells, such as fibroblasts, as a potential diagnostic approach to design personalised therapy in patients in which APP Tyr682 phosphorylation is increased. Indeed, further studies will be necessary to extend this analysis to a larger number of patients.In conclusion, this study proposes to use changes in APP Tyr


There remains up to a 50% recurrence rate in advanced p16- head and neck squamous cell carcinoma with current standard of care treatment. In an attempt to improve survival, multiple trials administering induction or neoadjuvant chemotherapy have been conducted but none demonstrated improved overall survival. The established efficacy of immune checkpoint inhibitors in the recurrent and metastatic setting has produced widespread interest in their neoadjuvant use.To survey the landscape of active neoadjuvant immunotherapy trials in head and neck squamous cell carcinoma and summarize and synthesize currently available outcomes from these trials.Neoadjuvant immunotherapy has proven safe and well tolerated in head and neck squamous cell carcinoma with encouraging efficacy results, including relatively high rates of pathologic response. Ongoing studies offer an opportunity to study immune responses in vivo. PD-L1 positivity, high tumor mutational burden and infiltration of NK cells, CD8, CD26 and Tim3 positive lymphocytes at time of surgery have been correlated with pathologic responses. We await updated reports of disease free survival and overall survival data and results of ongoing phase III studies utilizing neoadjuvant immunotherapy to determine if this treatment paradigm will have a place in the standard of care treatment in head and neck squamous cell carcinoma. Current standard of care treatment for patients with locoregionally advanced head and neck squamous cell carcinoma (HNSCC) is either definitive chemoradiotherapy (CRT) or surgical resection followed by adjuvant radiation with the addition of radiosensitizing chemotherapy when high risk features are noted on final pathology. Even with this aggressive approach, there remains up to a 50% recurrence rate in advanced Human papillomavirus (HPV) unrelated, p16- HNSCC. P16+ HNSCC has a far more favorable response to therapy with significantly lower rates of recurrence reflected in the development of updated staging reflected in the AJCC 8th edition .p = 0.002). The most common grade 3 and 4 toxicities during induction were febrile neutropenia (11%) and mucositis (9%). Impact of induction chemotherapy on absolute neutrophil count persisted through CRT with grade 3 neutropenia in 14% of the induction arm and 3.8% in the control arm (p = 0.039). These studies were both unfortunately underpowered due to failure to meet target enrollment.With a goal toward improving survival outcomes in HNSCC there has historically been interest in the application of induction chemotherapy. Results of 2 major well designed trials (PARADIGM and DeCIDE) failed to demonstrate improved overall survival (OS) with the addition of induction chemotherapy to CRT , 4. ConcHitt et al. enrolled 439 patients with locally advanced, unresectable, HNSCC and randomized to induction TPF  followed by CRT, PF (cisplatin and 5-FU) followed by CRT, or CRT alone and found no difference in progression free survival (PFS) or time to treatment failure , 7.n = 26, 31% in CRT arm, n = 19, 21% in TPF and cetuximab RT arm), but results were consistent with the overall population. A significant increase in grade 3 and 4 fever, neutropenia, and febrile neutropenia was also observed. Despite the recommended prophylactic use of lenograstim and the systemic use of ciprofloxacin, 12 patients died during or in the 30 days after TPF (6.6%). All deaths, except one, were considered to be related to TPF. The decrease in DM noted in the GORTEC 2007–02 study and the trend to lower DM noted in the DeCIDE trial suggests there may yet be a subset of patients who could benefit from induction chemotherapy, but this group remains inadequately defined.More recently, GORTEC 2007–02 showed no improvement in PFS or OS with induction chemotherapy followed by cetuximab radiotherapy compared to chemoradiotherapy, though there was a reduction in the rate of distant metastases (DM) in the induction chemotherapy arm . p16 staMost induction chemotherapy studies were designed prior to the widespread incorporation of p16 testing and stratification into prospective studies of HNSCC. Given the improved prognosis related to HPV initiated HNSCC, interest in induction therapy has primarily been related to the idea of chemoselection – with a goal toward treatment de-escalation . ECOG 13Induction chemotherapy may still be considered in select clinical situations after multi-disciplinary discussion, but its routine use is not supported by the available data, except as a treatment option in larynx preservation . When inp = 0.0004). A phase III trial demonstrated an excellent response rate of 80.6%, but did not show a survival benefit with neoadjuvant TPF in stage III or IVA OSCC in patients undergoing radical surgery and postoperative radiation therapy [n = 52, 20.3%) with cN2 HNSCC in the study and the small number of events related to the assessed clinical outcomes.As with induction chemotherapy, there is a lack of evidence to support the routine administration of chemotherapy prior to surgery (neoadjuvant chemotherapy) in patients with resectable HNSCC. A study of patients with stage T2-T4, N0-N2 previously untreated SCC of the oral cavity (OSCC) failed to show a significant difference in locoregional relapse or survival with the addition of neoadjuvant PF . Of inte therapy . Clinica therapy . SubgrouThe induction/neoadjuvant treatment paradigm in HNSCC remains alluring, both as a part of treatment intensification in HPV- HNSCC and a potential driver of deintensification in HPV+ HNSCC. The vast majority of ongoing trials of neoadjuvant therapy in HNSCC now are designed with immunotherapy as the foundation. This foundation is built on the success of immunotherapy in the recurrent and metastatic setting.Immunotherapy has a well-established role in recurrent and metastatic HNSCC as a second line agent following platinum-based chemotherapy. Checkmate 141 demonstrated superiority of anti-programmed death 1 (PD-1) monoclonal antibody nivolumab versus standard single agent therapy of the investigator’s choice in recurrent or metastatic HNSCC refractory to platinum chemotherapy . Median More recently, data from KEYNOTE-048 support first line use of pembrolizumab with or without chemotherapy as first line therapy in recurrent/metastatic head and neck cancer , 22. In The established efficacy of immune checkpoint inhibitors in the recurrent and metastatic setting has produced widespread interest in their neoadjuvant use. Neoadjuvant immunotherapy in HNSCC is appealing for a host of reasons. Incorporation of immunotherapy in the curative setting might reduce the risk of local recurrence and distant metastasis thereby improving overall survival. Opportunities to potentially de-escalate adjuvant therapy or the potential to reduce the morbidity and necessary extent of surgical resection are intriguing as well. Investigational use of neoadjuvant immunotherapy also offers an ideal platform to perform correlative studies to assess whether improved predictors of response to immunotherapy might be found. Finally, neoadjuvant immunotherapy is expected to be more easily tolerated than TPF and as a result would be unlikely to limit dose intensity of adjuvant cisplatin based chemoradiation when indicated.The purpose of this review is to survey the field of active neoadjuvant immunotherapy trials in HNSCC and summarize currently available outcomes from these trials. Most of these trials are investigating efficacy and safety of immune checkpoint inhibitors, but cytokine therapy and vaccine studies are also ongoing Table .Table 1Prior to the immune checkpoint inhibitor era, Stefani et al. reported improved OS and disease free survival utilizing neoadjuvant perilymphatic interleukin 2 (IL-2) in patients with resectable squamous cell carcinoma of the oral cavity and oropharynx . PatientFindings from NCT02296684, a trial of neoadjuvant pembrolizumab in surgically resectable stage III and IV HPV- head and neck cancer were presented at the American Society of Clinical Oncology (ASCO) 2017 annual meeting . To our Results reported thus far are favorable. There were no surgical delays or unexpected adverse events (specific observed immunotherapy-related adverse events have not yet been reported). There were no reported hyperprogressions or pseudoprogressions. The majority of patients in the study were locally advanced with stage IV disease. Despite this, there were no locoregional recurrences or distant metastases at the time of 1 year follow up from surgery in the first 14 patients for whom 1 year follow up data was available. 43% of patients had a pathologic treatment response (> 10% tumor necrosis and/or giant cell/histiocytic reaction to keratinous debris) at the time of surgery following a single dose of pembrolizumab. Pathologic treatment effect (TE) in ≥70% of the resected tumor or lymph node tissue area occurred in 6/21 pts. (29%). Pre-treatment tumor cell PD-L1 staining correlated with the percent treatment effect. Additionally, 48% of patients had clinical to pathological treatment downstaging.One patient whose contralateral neck dissection was performed weeks after his initial surgery demonstrated an additional treatment response in the contralateral lymph nodes. This could represent a delayed treatment effect from his initial pembrolizumab dose. It is also possible that surgery itself promoted a further immune-mediated anti-tumor response.Initial results from a multisite phase II trial investigating neoadjuvant and adjuvant pembrolizumab combined with adjuvant radiation plus or minus cisplatin NCT02641093) were presented at the ASCO 2018 annual meeting [41093 werIn the completed safety lead in including 16 patients no toxicities occurred resulting in delayed surgery, radiation, or chemotherapy. Enrollment was ongoing at the time of data presentation. In the first 34 patients, grade 3 pembrolizumab-associated toxicities included 1 event each of autoimmune colitis, duodenal hemorrhage, mucositis, nausea, vomiting, and syncope. There were no grade 4 pembrolizumab-associated toxicities. 14/27 (52%) patient’s tumors demonstrated a pathological response, defined as ≥10% tumor necrosis and/or histiocytic reaction to keratinaceous debris or ≥ 10% decrease in viable tumor. One patient had a complete pathologic response after one dose of pembrolizumab. Many tumors  were down-staged based on clinical to pathologic stage comparison. There were no reported hyperprogressions related to immunotherapy. Clinical response was assessed at 3 months post radiation or at the time of relapse with imaging and exam. 5 patients developed progressive disease or recurrence following surgery and RT or CRT with pembrolizumab. None of the patients who developed progressive or recurrent disease post-operatively by clinical response assessment had a pathologic response at the time of initial surgery. Pathological response was associated with robust immune cell infiltration, particularly CD8 positive T cells at time of surgery and PD-L1 and PD-L2 positivity at both pre-treatment biopsy and time of surgery.A randomized phase III trial utilizing a slightly modified treatment protocol in the experimental arm is now underway (NCT03765918).Results for the first stage of a planned two-stage, single arm, phase II clinical trial utilizing neoadjuvant nivolumab in oral cavity squamous cell carcinoma (OCSCC) (NCT03021993) were recently presented . Nine paThe primary endpoint was response to treatment. Response to treatment was defined as a pathologic complete response or pathologic partial response . Secondary endpoints included safety, feasibility, and memory phenotype and surface marker expression of ex vivo expanded tumor infiltrating lymphocytes (TILs).There was a 44% (4/9) objective overall response rate. Each of the 4 patients with response had a partial response. There were no pathologic complete responses (CRs). 1 additional patient had stable disease. The remaining 4 patients progressed through treatment. One patient’s tumor was felt to experience hyperprogression, having nearly doubled in size, but was still able to undergo definitive surgical resection. Anecdotally, those patients with responses had clinical findings of reduced cancer associated pain, trismus, or reduced extent of the primary tumor at 2 weeks after the first treatment. At a median follow up of 10 months, 3 patients had recurred with 1 death due to recurrence. The remaining 6 were recurrence free. There were no delays in surgical treatment. Negative margins were achieved in all patients. There were no grade 3 or 4 adverse events (specific adverse events are not yet reported). There were no treatment interruptions.Clinical response correlated with pathologic response. All patients who had a pathologic response had also demonstrated response on their pre-surgery interval CT scan. This is in line with observations in the recurrent and metastatic setting. While pseudo-progression is a concern in other cancer types with immune checkpoint inhibitors, it appears to be quite uncommon in head and neck cancers .An increased lymphocytic infiltrate was observed in pathology from responders compared to non-responders. The authors were able to demonstrate an immune profile of TILs in surgical specimens . There wPreliminary results from the head and neck cohort of Checkmate 358 were presented at the 2017 ESMO Conference. CheckMate 358 (NCT02488759) is exploring the safety and feasibility of neoadjuvant nivolumab in patients with resectable HPV+ or HPV- HNSCC . Patientn = 6, 21%) or stage IVA . Nine patients (31%) had locally recurrent disease were included in the study. The majority of patients were p16 positive . 96% were male and 76% were non-smokers.Findings of the Checkpoint Inhibitors Assessment in Oropharynx Carcinoma Study (CIAO) (NCT03144778) were presented at ASCO 2019 . Twenty-Durvalumab plus tremelimumab did not increase CD8+ TIL or response compared to durvalumab alone. The majority of patients  received the 2 planned immunotherapy cycles. Two patients progressed during treatment. These two patients, along with a third who had stable disease and was considered borderline resectable, were switched to treatment with neoadjuvant chemotherapy and, therefore, were excluded from the pathologic response and primary response assessments. All patients underwent the planned surgery, though some surgeries  did take place outside the planned window. Most patients did not receive adjuvant radiation therapy (57%). There was no difference in the toxicity profile between the durvalumab group and durvalumab plus tremelimumab group. Grade 2 and 3 toxicities included increased AST/ALT, increased lipase, and hypothyroidism. 4 (13.7%) of patients experienced grade 3 toxicities . Mean average quality-of-life scores using the MDASI-HN 28 item multi-symptom inventory were low at baseline and throughout treatment in both arms.Two patients had a pathologic CR at the primary tumor . 32% had a major pathological response, defined as less than or equal to 10% viable tumor cells in the primary or nodal disease site. The overall response rate by RECIST was 43% in both groups, including a 50% response in patients with recurrent disease. Within the primary tumor there was excellent correlation between response defined by imaging and pathologic response. Imaging was not as strongly predictive of nodal response. No patients had recurred at the overall median follow up of 10.5 months.Results from the phase 1b portion of immunomodulation by the combination of ipilimumab and nivolumab neoadjuvant to  surgery in advanced or recurrent head and neck carcinoma, IMCISION (NCT03003637) were also presented at ASCO 2019 . Twelve There were no surgical delays. One patient with recurrent disease after definitive radiotherapy had a greater than 50% increase from baseline local tumor size. In total, grade 1–4 immune-related toxicity occurred in 4/6 (67%) patients in arm A and 3/6 (50%) in arm B. One patient developed grade 3–4 colitis in the nivolumab arm. In the ipi/nivo arm there were 2 grade 3–4 toxicities, 1 incident of hepatitis and 1 of colitis. No unexpected wound healing problems were observed.1/6 (17%) patients in the nivolumab arm had a pathologic response . 3/6 (50%) in the combined ipi/nivo arm had a pathologic response (> 50% pathologic response) with 2 near pCRs. No patients with a near pCR had a recurrence at latest follow up (median 15 months). It is important to note the increased proportion of patients with recurrent disease undergoing salvage surgery in the nivolumab arm.n = 3) had 1–50% PD-L1 (n = 2) and/or high tumor mutational burden (TMB) (n = 2). TMB was a significant predictor for pathological response (p < 0.05). Increased B7-H3 gene expression was detected in non-responders before treatment. Increased NK cell gene expression was detected in responders post treatment. Intra-tumoral NK cell influx was confirmed by immunohistochemistry.Patients with a near pCR (In addition to these studies highlighted, numerous other studies Table  will repNotably, response rates, though generally impressive appear lower than those seen with TPF chemotherapy, but significantly greater than those seen with single agent immunotherapy in the recurrent and metastatic setting . ResponsPredictors of response to neoadjuvant therapy in HNSCC are lacking, as are those of response to immunotherapy writ large. Many of these studies have built in robust pre-specified correlative analyses to potentially identify signatures that predict response or lack thereof to immunotherapy. While much data remains preliminary, there are early signals toward association of response to therapy with PD-L1 expression and high pre-treatment TMB . Of inteThe optimal treatment for those patients who do recur after neoadjuvant immunotherapy will be an increasingly important question to study. It is unclear if recurrent tumors would retain responsiveness to PD-1/PD-L1 antibodies or if those that did not respond in the neoadjuvant setting might prove more responsive in R/M setting. In the metastatic setting, there are multiple drugs undergoing investigation seeking to recapture therapeutic benefit with immunotherapy in patients who have previously responded .Neoadjuvant immunotherapy has proven safe and well tolerated in HNSCC with encouraging efficacy results, to date. We await further reports of DFS and OS data and results of ongoing phase III studies utilizing neoadjuvant immunotherapy to help determine if this treatment paradigm will have a place in the standard of care treatment in HNSCC. Should a role for neoadjuvant immunotherapy be established there will still be many questions to answer including: duration of neoadjuvant treatment, whether there is a role for the addition of dual checkpoint inhibition or chemo-immunotherapy, and whether the addition of adjuvant immunotherapy provides any additional benefit. Unfortunately, many ongoing neoadjuvant studies feature adjuvant immunotherapy, which will make it more difficult to determine the optimal sequencing of immunotherapy in HNSCC. In an increasingly cost-conscious era of medicine, the optimal sequencing and duration of immunotherapy in the curative setting will be an exceedingly important question to answer. Finally, the promise of defining predictors of response to immunotherapy is increasingly real and may help select patients who would benefit from neoadjuvant immunotherapy and those who should instead proceed to current standard of care treatments. Immunotherapy has and is continuing to change the treatment landscape of recurrent and metastatic disease and now promises to change the face of curative intent therapy as well.


Peripheral blood is a highly accessible biofluid providing a rich source of information about human physiology and health status. However, for studies of the blood transcriptome with RNA sequencing (RNA-Seq) techniques, high levels of hemoglobin mRNAs (hgbRNA) present in blood can occupy valuable sequencing space, impacting detection and quantification of non-hgbRNAs. In this study, we evaluated two methods for preparing ribosomal RNA (rRNA)-depleted sequencing libraries for RNA-Seq of whole blood, one of which is also designed to deplete hgbRNAs. Two experiments were performed: one evaluating library performance across 6 human blood samples and the other examining library reproducibility and performance in a two-subject subset. We find that addition of hgbRNA depletion to the rRNA-depletion protocol for library preparation from blood RNA effectively reduces highly abundant hgbRNA reads; however, it does not result in a statistically significant increase in differentially expressed genes in our patient-control study. Bioinformatic removal of globin gene counts in non-hgbRNA depleted libraries provides improvement in overall performance of these libraries. We conclude that use of a standard ribosomal RNA depletion method for library preparation coupled with bioinformatic removal of globin gene counts is sufficient for reproducible and sensitive measurement of both coding and noncoding RNAs in the blood transcriptome. These high abundance transcripts can interfere with sensitive measurement of the rest of the blood transcriptome4.Blood is a relatively easy to access biofluid that contains information on the physiology and health status of the individual. As a primary conduit of nutrients, cells, small vesicles and molecular signals throughout the body, blood can serve as a reporter for both systemic disease and localized pathological activity associated with many different tissues. Analysis of the blood transcriptome has potential to identify gene expression signatures and other biomarkers for diagnosis, prognosis, and monitoring of treatment response, and to improve understanding of disease mechanisms. Because peripheral whole blood collection is a standardized and routine procedure, the use of whole blood samples in multicenter gene expression studies is more likely to produce reproducible results than the use of time consuming and potentially variable procedures to isolate specific cell populations following blood collection. In addition, whole blood collection and storage products such as PAXgene or Tempus Blood Tubes limit RNA degradation and minimize the risk of cell activation that can occur during a blood cell fractionation step post-collection. However, RNA extracted from whole blood samples contains high levels of hemoglobin RNAs (hgbRNA)7. More recently, it has been reported that globin RNA depletion from blood RNA prior to preparation of polyA+ RNA sequencing libraries results in the detection of thousands of additional transcripts with RNA-seq8. While use of polyA+ RNA libraries allows comprehensive coverage of polyA+ protein-coding RNAs, these libraries do not provide information on polyA− mRNAs or many noncoding RNAs. For our studies of the blood transcriptome we wished to identify an efficient and reproducible library preparation method that would support measurement of a broad range of blood RNAs, including noncoding RNAs and low abundance transcripts.Studies utilizing microarrays for whole blood expression profiling have demonstrated that sample preparation methods that reduce or block hgbRNAs increase sensitivity of gene detection9. Routine implementations of these methods, however, do not remove highly abundant protein-coding RNAs such as the hemoglobin gene transcripts present in whole blood. A library preparation method that simultaneously removes both hgbRNA transcripts and rRNAs, Ribo-Zero Globin (Illumina) is available. Ribo-Zero Globin  is a modification of the Ribo-Zero Gold rRNA removal protocol which incorporates a panel of human hgbRNA oligos along with nuclear and mitochondrial rRNA oligos to allow simultaneous removal of both hgbRNA transcripts and rRNAs. These oligos are hybridized with total RNA followed by magnetic bead-based removal of the resulting complexes prior to RNA fragmentation and cDNA synthesis for sequencing library preparation. In the present study we wished to determine if the Ribo-Zero Globin (Globin-Zero) library method provided an effective approach to RNA-Seq of whole blood that would allow measurement of protein-coding and non-coding RNAs in a reproducible and sensitive manner. We hypothesized that removal of hgbRNAs along with rRNAs for RNA-Seq of blood would result in detection of more genes and less variation among samples due to large differences in hgbRNA levels. Therefore, we compared Globin-Zero to the standard Ribo-Zero Gold method to determine if Globin-Zero effectively removed hgbRNA transcripts and produced reproducible libraries that allow detection of additional genes.Sequencing library methods have evolved to effectively remove highly abundant ribosomal RNA (rRNA) species through pre-selection of poly A+RNAs or capture and removal of rRNA during library preparation10. We examined which hgbRNAs were removed by the Globin-Zero method, effects of reducing RNA input to 250 ng from 900 ng, and the impact of library method on gene detection and differentially expressed genes (DEG). In a separate experiment, we further examined RNA-Seq reproducibility with the different methods by preparing technical replicate libraries from blood RNA of one male and one female subject. We also evaluated differences in detection sensitivity with and without bioinformatic removal of hemoglobin gene counts from the RNA-Seq data. The results of our study confirm that the Globin-Zero method is very effective at reducing hgbRNA transcripts in human blood samples. However, we were unable to demonstrate an improvement in DE gene measurement with the Globin-Zero library method compared to Ribo-Zero Gold in our multi-sample gene expression study.We designed a small exemplar study utilizing blood samples collected from an age and diagnostically mixed donor group to evaluate library method effectiveness in revealing gene expression differences among biological replicates. We included samples from healthy controls and patients with sarcoidosis, an inflammatory disease in which we have previously demonstrated characteristic gene expression profiles in blood with potential for molecular diagnosisBlood was collected in PAXGene Blood RNA Tubes from three sarcoid patients (P) and three healthy volunteers (C). Total RNA isolated from two PAXGene tubes per donor was pooled followed by a second DNase treatment Fig. . RNA ext11) for sequencing  and Globin-Zero (Ribo-Zero Globin) and use the term ‘library method’ to refer to all steps in library generation from RNA depletion to PCR-amplified DNA library. The methods evaluation study was divided into two experiments. Experiment 1 examined the impact of library method and RNA input on RNA-Seq performance across six independent blood samples  using a balanced block design  in triplicate and sequenced in a block design Fig. .Sequence data from both experiments were aligned, quality-checked and analyzed as outlined in Fig. et al.12, utilizing both FastQC13 and RNA-SeQC14. Mapping rates of total reads to both rRNA and hgbRNA were estimated by using the rRNA estimation functionality in RNA-SeQC which allowed the inclusion of both uniquely mapped and multi-mapped reads for these two gene families  expression levels of hemoglobin genes in Experiment 2 libraries were determined and average log2 (CPM + 1) are shown in Fig. Following read alignment and gene quantification with STAR18 biotype categories. Biotype categorization of raw gene counts  for the Experiment 2 C1 and C2 libraries showed that functional distributions were similar for the two library methods  and generated MA plots with and without bioinformatic removal of hemoglobin gene counts. MA plots between methods were similar across the 6 samples so the averaged paired logFC (RZG/GZ) by average logCPM is shown Fig. . The loe2 CPM values from three technical replicates with each method for samples C1 and C2  in raw gene counts per gene. A small, but significant, increase in variation with Globin-Zero library preparation (p < 0.001 by the Wilcoxon signed-rank test) was observed. The median CV across technical replicates for RZG libraries was 0.24 for C1 and 0.22 for C2, and across Globin-Zero libraries, 0.26 and 0.33, respectively.et al.19 recommends filtering genes that do not have at least 10–15 counts or equivalent CPM values. We selected a filter of >1 CPM (corresponding to approximately 15 to 25 raw counts in our dataset) as our threshold for detection. Genes are considered uniquely detected if present at >1 CPM in all libraries prepared with one of the input amounts and measured at ≤1 CPM in at least one of the alternative input libraries. In a combined analysis of all Experiment 1 control libraries (Globin-Zero and RZG), 0.3% more genes (46) were detected with 900 ng RNA input compared to 250 ng out of approximately 14,000 genes detected. While this result suggests a slight increase in gene detection with 900 ng RNA, the actual increase in gene number is very small . Therefore, we elected to prepare libraries with 250 ng RNA input in Experiment 2. We reasoned that the further evaluation of libraries prepared with 250 ng RNA would provide useful information for whole blood transcriptome studies in which RNA is limited.The impact of RNA input amount on transcript detection sensitivity in Experiment 1 was examined by counting the number of genes above a fixed CPM threshold in each library. Genes with consistently low counts across all libraries are usually removed for downstream analysis since they are unlikely to be biologically important or to be assessed as significantly DE. Chen Selected library method pairs (one Globin-Zero and one RZG library) for each of the 6 samples in Experiment 1 were used to evaluate gene detection using detection criteria of >1 CPM in at least one of the libraries. Figure Using Experiment 2 data we determined reproducible gene detection with each library method. In total, 36,395 genes were counted at least once (CPM > 0) in the Experiment 2 data set. Applying a relatively stringent filter of CPM > 1 in all 3 method replicates for each sample, 14,087 genes were reproducibly detected in C1 with both library preparations and 14,019 genes in C2 Fig. . Globin-We further examined how library method affected gene measurements by performing a paired analysis (RZG × Globin-Zero) of the 6 sample method pairs from Experiment 1. We found 2,138 genes measured at significantly different levels between methods  to explore the impact of RNA input and library method on measurable differences between the groups. Genes were considered unexpressed if measured at ≤1 CPM in all 31 libraries and removed prior to DE analysis. Significant DE was defined as the false discovery rate (FDR) adjusted p-value < 0.05 and fold change (FC) > 1.5.There were 2,694 DEG  in the patient group Fig. , howeverIn a further use of our technical replicate study to explore library-associated differences in gene expression measurements between the C1 and C2 samples, we performed DE analyses of the replicates with each library method. In independent difference assessments, 2,005 and 2,320 DEG were found between C1 and C2 with RZG and Globin-Zero, respectively  produced high quality libraries. However, up to one-fifth of all sequence reads corresponded to hgbRNAs. Furthermore, up to 33% of uniquely mapped gene counts in RZG libraries mapped to the hemoglobin genes, and hgbRNA levels varied up to 3-fold among subjects. We found that Globin-Zero was highly effective at removing hgbRNA transcripts from RNA-Seq libraries. This resulted in an increase in unique gene detection based on a commonly used gene count threshold (>1CPM), but fewer additional genes than anticipated. In addition, when all hemoglobin gene counts were bioinformatically removed from the Globin-Zero and RZG data, the detection rate advantage seen with the Globin-Zero method was lost. In another methods comparison study working with whole blood samples, sequencing libraries were prepared using polyA+ RNA with and without the prior removal of hgbRNA with GLOBINclear. The authors report that several thousand additional genes were detected with GLOBINclear removal of hgbRNA compared to non-depleted RNA in a pair of libraries prepared from a single sampleet al.8 in their study using GLOBINclear to remove hgbRNA transcripts prior to polyA+ selection and library preparation. We suspect that these reductions in other transcripts are due to cross-hybridization with the α- and β-globin probes present in the Globin-Zero and GLOBINclear kits.We find a large number of genes, predominately protein-coding, are present at reduced levels in Globin-Zero libraries relative to RZG . This suggests that the addition of an hgbRNA removal step with Globin-Zero results in the reduction of reads for some non-hgbRNAs as well. A broad reduction in counts for genes other than the hemoglobin genes was also reported by Shin DE analyses of libraries prepared in Experiment 1 showed that the removal of hgbRNAs with Globin-Zero did not result in an increase in identification of DEG between patients and controls compared to the standard RZG protocol used in this study. While our subject sample number is small, if one library method considerably improved DEG sensitivity in Experiment 1, our design should have detected it. Independent comparisons of blood RNA samples from two individuals in Experiment 2 using method replicates showed only a small increase in differentially-measured genes with the Globin-Zero method (approximately 8%). Our assessment of sequencing library performance characteristics suggests that one of the reasons the Globin-Zero method did not result in a large increase in gene detection and DEG may be a concomitant reduction in efficiency of rRNA removal, resulting in relatively fewer exonic reads with the Globin-Zero protocol compared to RZG. DEG analysis with the Globin-Zero method may be further impacted by variability in the amount of rRNA retained in the GZ libraries. Reduction in the levels of many non-hgbRNAs with Globin-Zero may also contribute to its inability to detect more DE genes.Our results confirm that Globin-Zero effectively removes high abundance human hgbRNA transcripts from RNA-Seq libraries. However, increases in rRNA content in Globin-Zero libraries relative to the RZG method and a small increase in technical variation with Globin-Zero protocol are also seen. Nonetheless, Globin-Zero library preparation results in the detection of additional genes compared to RZG libraries when routine data filtering protocols are applied, if hemoglobin gene counts are not removed prior to data normalization. A small increase in DE sensitivity with Globin-Zero is seen in a two-sample technical replication experiment, but this increase in sensitivity with Globin-Zero did not translate to our patient-control study. In addition, we observed an impact of the hgbRNA removal protocol on the measured abundance levels of hundreds of genes in addition to the hemoglobin genes.Overall, our results indicate that the benefit of the combined hgbRNA and rRNA removal with Globin-Zero on gene expression measurements is small. Alternative approaches that remove hgbRNAs and rRNA in separate steps prior to cDNA synthesis may offer useful options for those seeking to profile the expression of both polyA+ and polyA− genes in whole blood samples, but the two-step depletion/enrichment process adds time and cost, and increases risk of RNA degradation and loss. In our study both the Globin-Zero and RZG methods produced informative and largely overlapping RNA-Seq data for whole blood samples. We find that RZG when combined with bioinformatic removal of hemoglobin gene counts is an effective library preparation method for the analysis of blood RNA.Blood collection from patients and healthy controls was performed in accordance with a protocol approved by the Institutional Review Board of Oregon Health & Science University. Blood was collected in PAXgene Blood RNA Tubes  and processed according to manufacturer recommendations prior to storage at −80 °C. Blood was collected from three sarcoid patients  and three healthy volunteers . All subjects were female except C2. Two blood tubes were collected per donor.PAXgene tubes were thawed and incubated at room temperature overnight. RNA was extracted according to manufacturer instructions using PAXgene Blood RNA Kits (Qiagen) and a QIACube sample-processing robot (Qiagen). Isolated RNA from the same donor was pooled, and a second DNase treatment was performed using DNase 1 (Zymo Research) according to manufacturer instructions followed by RNA repurification and concentration with RNA Clean & Concentrator kits (Zymo Research). RNA concentrations were initially determined by UV measurement. RNA integrity and concentration for library preparation were determined by analysis on an RNA 6000 NanoChip using a 2100 Bioanalyzer (Agilent Technologies). RIN values were 8.6, 8.3, 8.8 for control (C) samples and 7.7, 8, 8.5 for patient (P) samples.Libraries were prepared with total RNA according to manufacturer instructions with either the TruSeq Stranded Total RNA Library Prep Kit with Ribo-Zero Gold  or the TruSeq Stranded Total RNA Library Prep Kit with Ribo-Zero Globin Set A . Experiment 1 libraries were prepared with either 900 ng or 250 ng of total RNA. All Experiment 2 libraries were prepared with 250 ng of total RNA.Library quality was confirmed by size analysis on 2200 Tapestation with D1000 ScreenTape. All libraries were within an average size range of 360 to 380 bp. Library concentrations were determined using the KAPA Library Quantification Kit for Illumina platforms (KapaBiosystems) on a StepOne or a StepOnePlus Real Time PCR Workstation (Thermo Fisher) and pooled at 4 samples per lane. Single-end, 100 cycle sequencing was performed on a HiSeq 2500 platform (Illumina). Libraries were pooled according to block designs shown in Supplementary Fig. 13 combined with MultiQC23 (http://multiqc.info/). Sequence files were imported into and processed through our LabKey24 and DISCVR-Seq-based system (https://github.com/BimberLab/discvr-seq). Trimmomatic25 was used to remove any remaining Illumina adapters. Reads were aligned to the Homo_sapiens.GRCh38.p10 genome assembly in Ensembl along with its corresponding annotation, release 91. The program STAR (v020201)17 was used to align the reads to the genome using default settings and two-pass mode. Since STAR utilizes the gene annotation gtf file, it calculated the number of reads aligned to each gene. Stranded counts were selected.Sequence data quality was evaluated using FastQC14 was utilized to calculate post-alignment QC metrics. To estimate the proportion of reads belonging to rRNA, the rRNA sequences provided with RNA-SeQC were utilized. RNA-SeQC selects a random subset of the reads and aligns them to the rRNA sequences to estimate the proportion of reads that belong to rRNAs. To similarly calculate a proportion of reads belonging to hgbRNAs, sequences from 12 hgbRNA genes (Supplementary Table RNA-SeQC (v1.1.8.1)Bioinformatic removal of hgbRNA reads was performed after alignment and gene count calculation by removing 12 hgbRNA genes  and scatter plots were used to visualize measurement similarities among libraries using log2(CPM)15. The coefficients of variation (CV) were computed for each library using raw counts. The Wilcoxon signed rank-test was used to compare the medians of CV values.Exploratory data analysis was primarily performed on loghttps://uswest.ensembl.org/info/genome/genebuild/biotypes.html). The biotypes were binned into a reduced number of more general categories for Fig. https://github.com/dpryan79/Answers/blob/master/SEQanswers_42420/GTF2LengthGC.R) and were used to calculate Transcripts Per Million (TPM) values.For analyses utilizing biotype, biotype classifications were retrieved from Ensembl (r91)  or low (250 ng) RNA inputs, we filtered for detected genes using a threshold of >1 CPM in all Control donor libraries with high or low input in Experiment 1 and ≤ 1 CPM in at least one of the alternative input group.Uniquely-mapped gene counts from STAR were imported into the R statistical computing environment and normalized by the weighted trimmed mean of M-values (TMM)RNA-Seq data from 6 Globin-Zero libraries and 6 RZG libraries from Experiment 1 libraries were selected for a pairwise comparison of library effect on detection with each donor sample. In the paired analysis of Experiment 1, genes were filtered using a threshold of >1 CPM with at least one of library methods. In Experiment 2, we filtered for detected genes using a threshold of >1 CPM in all three technical replicates of at least one sample.27. For analysis of Experiment 1 we kept a gene if its uniquely mapped count was larger than 1 CPM in at least one library; this threshold corresponds to about 15 to 25 raw counts. The negative binomial regression model with quasi-likelihood test implemented in the edgeR19 R package was employed to find significantly DE genes for the multi-factor analyses. A gene was declared as significantly DE when the associated false discovery (FDR) adjusted p-value was <0.05 and FC > 1.5. In the model for Experiment 1, we included the interaction effect of library preparation methods by diagnosis as well as lane assignment as a blocking factor in addition to all main effects . The interaction effect was included to determine which additional genes would be identified by the Globin-Zero library method in the patient group. The blocking factor (lane effects) was included to account for potential lane and other batch biases.For DE analysis, we used uniquely-mapped gene read counts from STAR imported into R and normalized by the weighted trimmed mean of M-values (TMM)DE analysis of Experiment 2 libraries was performed independently to assess overlap of DEG with the two library methods. Library replicates for C1 and C2 were filtered and analyzed separately for each library method group. A gene was kept and analyzed if it was measured at CPM > 1 in at least one out of the six libraries. EdgeR negative binomial regression models were fitted separately to 6 libraries in a same method group. That is, DE was tested comparing C1 vs C2 using technical replicates within each library method.All protocols were approved by the Institutional Review Board of Oregon Health & Science University. Informed consent was received from all participants in the study.Supplementary Figures.Supplementary Table S1Supplementary Table S2Supplementary Table S3Supplementary Table S4Supplementary Table S5Supplementary Table S6


Germline copy number variants (CNVs) increase risk for many diseases, yet detection of CNVs and quantifying their contribution to disease risk in large-scale studies is challenging due to biological and technical sources of heterogeneity that vary across the genome within and between samples.We developed an approach called CNPBayes to identify latent batch effects in genome-wide association studies involving copy number, to provide probabilistic estimates of integer copy number across the estimated batches, and to fully integrate the copy number uncertainty in the association model for disease.MYC and of Tumor Suppressor Candidate 3 (TUSC3).Applying a hidden Markov model (HMM) to identify CNVs in a large multi-site Pancreatic Cancer Case Control study (PanC4) of 7598 participants, we found CNV inference was highly sensitive to technical noise that varied appreciably among participants. Applying CNPBayes to this dataset, we found that the major sources of technical variation were linked to sample processing by the centralized laboratory and not the individual study sites. Modeling the latent batch effects at each CNV region hierarchically, we developed probabilistic estimates of copy number that were directly incorporated in a Bayesian regression model for pancreatic cancer risk. Candidate associations aided by this approach include deletions of 8q24 near regulatory elements of the tumor oncogene Laboratory effects may not account for the major sources of technical variation in genome-wide association studies. This study provides a robust Bayesian inferential framework for identifying latent batch effects, estimating copy number, and evaluating the role of copy number in heritable diseases. Germline copy number variants (CNVs) can be identified from hybridization-based arrays and capture-based sequencing with measures of abundance derived from intensities and normalized read depth, respectively. Biological and technical sources of heterogeneity of these measurements are intricately related. For example, the GC composition of genomic DNA effects PCR efficiency and leads to autocorrelated measures of DNA abundance across the genome –4. TheseFor studies with hundreds to thousands of samples, estimation of copy number at regions known to harbor CNVs has the potential to improve sensitivity and specificity as technical sources of variation across the genome are largely controlled when limited to a focal genomic region (less than 1 MB) and variation between samples can be explicitly modeled –12. SuchHere, we performed genome-wide copy number analysis for 3,974 cases and 3,624 controls in PanC4 using Illumina’s OmniExpress Exome array. We developed methods for identifying latent batch effects at CNV regions from commonly available experimental data on the samples. The effects of copy number and batch on measures of DNA abundance were modeled hierarchically through implementation of Bayesian finite mixture models. For the association model, we used Markov Chain Monte Carlo (MCMC) to incorporate the uncertainty of the integer copy numbers in a logistic regression model of pancreatic cancer risk.The Pancreatic Cancer Case and Control ConsortiumR ratios and probe GC content, and predicted the log2R ratios for the full probeset from the LOESS model. For spatial correction, we applied LOESS to the GC-corrected log2R ratios at single nucleotide polymorphisms (SNPs) with balanced allele frequencies  ordered by genomic position within each chromosome arm and predicted the GC-corrected log2R ratios for the full probeset, including SNPs with imbalanced allele frequencies. The residuals from the spatial LOESS were used in all downstream analyses with CNPBayes.Clinical and demographic characteristics of the cases and controls in PanC4 and recruitment methods have been previously described . All samCNV regions:CNV regions identified for further analysis by CNPBayes were obtained from the collection of CNVs identified from a hidden Markov model as well as known CNV regions from the 1000 Genomes Project. For the former, we fit a 5-state hidden Markov model implemented in the R package VanillaICE (version 1.40.0) using default parameter settings . To obtaCNV regions were defined by the set of non-overlapping disjoint intervals across the pooled set of all CNVs from cases and controls. We computed the number of subjects with a CNV overlapping each disjoint interval, retaining intervals where CNVs were identified in at least 150 participants. Among the disjoint intervals, we defined the CNV region as the minimum start and maximum end for which at least 50 percent of the copy number altered samples had a CNV. For CNV regions obtained from the 1000 Genomes Project, we excluded regions that did not span at least 4 markers on the OmniExpress array.Batch effects:R ratio between two batches (excluding samples with log2R ratio <−1) by the Kolmogorov-Smirnov (K-S) test statistic. For two batches without a statistically significant difference in the K-S statistic at a type 1 error of 0.01, the batches were combined into a single new batch. This procedure was applied recursively at each CNV region until no further grouping of batch surrogates could be obtained.We evaluated both chemistry plate and DNA extraction method as surrogates for batch effects. With provisionally defined batches by plate or extraction method, we compared the empirical cummulative distribution function (eCDF) of the mean log2Hierarchical Bayesian mixture model:t-distributions were used to cluster median log2R ratios within a CNV region. Let rib and zib denote the observed one-dimensional summary of log2 ratios measured from the array and the true (but latent) mixture component, respectively, for the ith individual in batch b. Given zib is some integer h , our sampling model for the observed data is a shifted and scaled t-distribution with d degrees of freedom, mean θhb, and standard deviation σhb that depends on batch:Hierarchical Bayesian mixtures of d controls robustness to outliers with larger values approximating a mixture of normal distributions , was defined as 1 for samples in this set and 0 otherwise. As the integer copy number Ci was not observed, we treated Ci as a parameter measured with error given by the aggregated posterior probabilities of the mixture component indices after genotyping, All continuous independent variables were mean centered, including PC1, PC2, and PC3 denoting the first three principal components of the SNP genotype matrix in PanC4 . An indi by MCMC .Scalability and software:https://github.com/scristia/CNPBayes).Hierarchical mixture models were fit to a random sample of 1000 observations from the 7,598 available participants at each CNV region, and to all samples with apparent homozygous deletions. We parallelized our analysis so that all regions were evaluated simultaneously. Bayesian logistic regression models fit independently to each CNV region were also evaluated in parallel. CNPBayes is available from github . Following the spatial correction, nearly all samples (≈ 98%) had low autocorrelation . Additionally, we identified 46 regions for which deletions or duplications were identified in at least 2% of the PanC4 participants by the HMM applied to the spatially corrected log2R ratio eCDF  and stratified the PC1 summaries by study site . Here, DNA samples were collected from multiple study sites and processed on 94 chemistry plates at a central lab. To identify batch surrogates for the central lab, we developed an approach for grouping chemistry plates with a similar median log2ite Fig. a or PCR ite Fig. b. While ite Fig. b. As PCRite Fig. .Fig. 2IR ratio is a mixture of t distributions with component-specific means and variances modeled hierarchically across batches  and diploid copy numbers (n=5647)  that capture a range of data quality and copy number states Figures –S9. For s Figure . CNVCALLs Figure  and deteSimulationn = 990) processed on 16 chemistry plates and hybridized to Affymetrix 6.0 chips. A 109 kb region on chr 4 containing 1 SNP and 53 nonpolymorphic markers spans a deletion polymorphism with an allele frequency near 22%. We increased the level of difficulty for genotyping these samples by increasing the variance and/or shifting the location of the probe-level data in a subset of the chemistry plates. For each simulated dataset, we fit both CNVCALL and CNPBayes. While we did not provide the true batch labels to either method, CNPBayes estimated the batches from the plate surrogates. With no simulated batch effects, CNPBayes and CNVCALL had nearly identical performance with near perfect sensitivity and specificity (area under the receiver operator characteristic curve (AUC) > 0.99). However, for simulated datasets with batch effects in the mean or variance, accuracy of CNVCALL decreased by an average of 25% while performance characteristics of CNPBayes remained qualitatively similar  for odds ratio: 1.01 - 1.39). While the direction of this effect is inconsistent with its putative role as a tumor suppressor, up-regulation of TUSC3 and possible oncogenic roles for this gene have been reported in cancers including non-small cell lung cancer, colorectal, thyroid, and head and neck cancers [MYC located at 128,748,315-128,753,680 bp [TUSC3 and near MYC were positive and highly correlated for both the low and high quality sample collections, an unstratified analysis using all 7598 participants doubled the posterior probability of association for these genes , we identified and adjusted for batch effects in a region-dependent manner in contrast to alternative methods. Using this approach, nearly 70% of CNV regions analyzed had multiple batches that were related to chemistry plates and not the individual laboratories that contributed samples.TUSC3 and near MYC were more prevalent among participants without pancreatic cancer. Germline deletions of these genes have not been previously implicated in pancreatic cancer, though upregulation of expression of these genes have been implicated in some cancers in an apparent tissue-dependent manner. Although this study did not evaluate whether deletions at these loci were well tagged by neighboring SNPs, phasing the nearby SNPs would allow direct inference for whether variation in copy number is associated with pancreatic cancer risk among participants with the same SNP haplotype [Using the methods outlined in this study, we found that germline deletions of aplotype , 40. Whiaplotype –44.Finally, we assumed an additive model for integer copy number and the log odds of cancer risk. Dominant and recessive mechanisms of genotype-phenotype associations are possible and the evidence for these models using Bayes factors could be averaged with weights reflecting our a priori beliefs.Statistical inference predicated on measures of abundance such as DNA copy number are highly susceptible to batch effects, and the sources of these effects are not generally known. As studies become increasingly large-scale with inevitable batch effects and heterogeneity in sample quality, the scalable approach provided by CNPBayes will be helpful for modeling unwanted technical variation and avoiding the potential confounding between batch effects and copy number when evaluating disease risk.Additional file 1 Figure S1: Median absolute deviation and autocorrelation of autosomal log2R ratios. Figure S2: Preprocessing and quality control analyses. Figure S3: Frequency of CNV regions with 1 to 7 batches identified by grouping the eCDFs of the log2R summaries. Figure S4: Number of additional CNVs identified from the Bayesian mixture model. Figure S5: Technical variation within and between samples obscures identification of hemizygous deletions. Figure S6: A deletion polymorphism at CNP_121. Figure S7: A deletion polymorphism at CNP_128. Figure S8: A duplication polymorphism at CNP_100. Figure S9: A CNV region with both deletions and duplications evident in the high quality samples. Figure S10: Performance of CNV detection methods on HapMap data.Additional file 2 Supplemental Tables for Bayesian copy number detection and association in large-scale studies.