Plants respond to herbivore damage with the release of volatile organic compounds (VOCs). This indirect defense can cause ecological costs when herbivores themselves use VOCs as cues to localize suitable host plants. Can VOCs reliably indicate food plant quality to herbivores?Gynandrobrotica guerreroensis and Cerotoma ruficornis) when facing lima bean plants (Fabaceae: Phaseolus lunatus) with different cyanogenic potential, which is an important constitutive direct defense. Expression of inducible indirect defenses was experimentally manipulated by jasmonic acid treatment at different concentrations. The long-distance responses of male and female beetles to the resulting induced plant volatiles were investigated in olfactometer and free-flight experiments and compared to the short-distance decisions of the same beetles in feeding trials.We determined the choice behavior of herbivorous beetles (Chrysomelidae: Female beetles of both species were repelled by VOCs released from all induced plants independent of the level of induction. In contrast, male beetles were repelled by strongly induced plants, showed no significant differences in choice behavior towards moderately induced plants, but responded positively to VOCs released from little induced plants. Thus, beetle sex and plant VOCs had a significant effect on host searching behavior. By contrast, feeding behavior of both sexes was strongly determined by the cyanogenic potential of leaves, although females again responded more sensitively than males. Apparently, VOCs mainly provide information to these beetles that are not directly related to food quality. Being induced by herbivory and involved in indirect plant defense, such VOCs might indicate the presence of competitors and predators to herbivores. We conclude that plant quality as a food source and finding a potentially enemy-free space is more important for female than for male insect herbivores, whereas the presence of a slightly damaged plant can help males to localize putative mating partners. The factors that drive plant-herbivore interactions are complex and we are only beginning to understand their functional interplay in natural systems Sex-specific differences in the responses of herbivorous insects to plant VOCs have been reported for numerous insects belonging to various orders such as Lepidoptera Gynandrobrotica guerreroensis and Cerotoma ruficornis). In their natural habitat in southern Mexico, these oligophagous beetles feed on a small range of legumes, but with distinct preference for wild lima bean (Phaseolus lunatus). We used choice experiments to analyze the response of the insects over long and short distances to cues of lima bean plants. We focused on two plant traits, i) the release of induced VOCs and ii) the constitutive cyanogenic potential (HCNp) of leaves. The HCNp means the total amount of toxic cyanide (CN−), which can be released from preformed cyanogenic compounds  in response to cell damage. The induced VOCs have the potential to act over long distances. The HCNp, however, acts as a direct chemical defense that affects the feeding herbivore locally when free cyanide is released during the feeding process. In the present study, we experimentally induced the release of plant VOCs by application of jasmonic acid (JA) to lima bean foliage. Jasmonic acid is the central signaling molecule of the octadecanoid pathway To contribute in filling this gap in the understanding the sex-specific function of long- and short-distance plant cues, we used males and females of two chrysomelid herbivores (Phyllotetra spp.) Gynandrobrotica guerreroensis) Epitrix hirtipennis) In contrast to VOCs, the HCNp is not affected by JA application G. guerreroensis and C. ruficornis) to VOCs released from wildtype lima bean plants at different stages of development (mature and young plants) and different levels of induction ranging from little to strongly induced. We determined the long-distance responses of males and females to VOCs both in an olfactometer and in a large cage. The same individual beetles were consecutively used in feeding trials to assess their short distance choice behavior. The HCNp of the same leaves was determined as an important direct defense of lima bean In the present study, we analyzed the response of male and female chrysomelid beetles . Herbivorous insects naturally approaching the plants were manually removed twice a day to avoid any uncontrolled induction of herbivore-induced volatiles. At the time of the experiments, plants were 60–90 cm in height and had developed 10 to 15 leaves.Experiments were conducted from August to October 2006 in the coastal area of Oaxaca, Mexico. We conducted experiments with chrysomelid herbivores and wild lima bean plants. Plants and insects were collected directly before the experiments from a natural population 10 km W of Puerto Escondido . To consider potential effects on herbivores arising from ontogenetic variation of plant traits, experiments were carried out with shoot parts of mature plants as well as with young, intact potted plants that both were derived from the same site. Potted plants were grown from seedlings which were collected when unfolding their primary leaves. Seedlings were cultivated (3–4 weeks) in 250 ml plastic pots filled with soil from natural sites. Plants were exposed to natural conditions, watered twice a day and fertilized once a week with nitrogen-phosphate fertilizer  in a concentration of 0.25 mg LCerotoma ruficornis Olivier and Gynandrobrotica guerreroensis Jacoby  as herbivores. Beetles were randomly collected from host plants to represent natural ratios of sexes and ages. The insects were kept in transparent 250 ml plastic cups and were deprived of food for 1 d prior the experiment (water supplied on cotton). The sex of individual beetles was identified  after the experiments using a binocular microscope. In addition to locusts  and larvae of the bean leaf roller (Hesperidae: Urbanus proteus L.), these chrysomelid species were the most abundant herbivores on lima bean at the study site in the period of August to October. They were present throughout the day, exhibiting two peaks of feeding and moving activity in the first hours after dawn (8:00 a.m.–10:00 a.m.) and dusk (8:00 p.m.–11:00 p.m.) (pers. observ.). Chrysomelid species were identified by Astrid Eben .We used the phyllophagous beetles −1 JA) to analyze quantitative effects of experimental induction on VOC emission. Leaves were sprayed with JA solutions until completely moistened and allowed to dry (10 min) before being sprayed a second time. Plants were allowed to dry again and placed into PET bags  for volatile collection Herbivore-induced volatile organic compounds (VOCs) were induced by spraying foliage with aqueous solutions of jasmonic acid (JA) between 08:00 and 09:00 a.m. Plants used for VOC collection and choice experiments were treated identically and were randomly selected for the respective experiment. Control plants were treated the same way, but were sprayed with water instead of JA solution. We used different JA concentrations  and immediately transported to the field laboratory for further experiments. Plant material was transported under cooled conditions (<20°C) and water supply. Cutting off the shoots did not induce any detectable release of volatiles .Gynandrobrotica guerreroensis). Beetles were placed on potted plants and shoots between 08:00 and 09:00 a.m. Plant material was bagged with nets and beetles were allowed to feed on foliage for 24 h. The following day between 08:00 and 09:00 a.m., beetles and nets were removed and plants were bagged in PET foil for VOC collection. Experimental setup and number of treated and control plants (resp. shoot parts) were the same as described for experiments with JA.In addition to quantitative effects of JA-induced VOC emission on herbivore behavior, we analyzed the effects of VOCs released following insect leaf damage. For these experiments we used 10 adult beetles (−1) was provided by a ventilator at the end of the olfactometer. All experiments were conducted between 06:00 p.m. and 12:00 a.m. at a temperature of 28–30°C (air humidity >90%). Light conditions were standardized by placing the entire setup in a dark room and directional movement of the beetles was ensured by centering one lamp (40 W) 50 cm in front of the Y-olfactometer. In the olfactometer, individual beetle choice was recorded. Each beetle was tested eight times, in order to calculate a percent decision that allowed a direct and quantitative comparison with the data obtained in feedings trials. Only beetles entering one of the arms within 3 min were counted as having made a decision. We used different concentrations of jasmonic acid for induction of VOCs . The following choice situations were created for both mature shoot parts and potted plants: I (1.0):C, I (0.1):C, I (0.01):C and I (0.001):C. In addition to JA-induced lima beans, we used plants that were induced by herbivore-damage  and tested these plants against controls (HI∶C). We conducted controls, in which every beetle had the choice between an empty arm and a control shoot or plant (0∶C) as well as two empty arms (0∶0). In further controls, we tested induced shoots and induced intact young plants against each other [for each type of plant material: I (1.0)∶I (1.0)] as well as untreated shoots and plants , in order to evaluate potentially different effects of plant material  on choice behavior of insects. To prevent any bias due to possible unforeseen asymmetries in the experimental setup, after every ten trials, the odor source was changed and the odorant and control arms were reversed. Before changing the odor source, the glass tubes were washed with water and detergent, and then wiped with hexane and acetone. Olfactometer experiments were conducted over seven consecutive weeks between August and October 2006, each with different sets of plants and different sets of beetles. Altogether, n = 406 individuals of C. ruficornis  and n = 389 individuals of G. guerreroensis  were used in these experiments.Beetle behavior in response to VOCs released from JA-treated or herbivore-damaged plants was tested in a Y-olfactometer. The glass olfactometer consisted of a stem (length 16 cm) and two arms . Glass tubes had an inner diameter of 4 cm. Inflowing air was cleaned by charcoal filters  and then passed over the plants placed in PET foil bags for 1 h prior the experiment. Constant air-flow  and n = 299 individuals of G. guerreroensis . The number of beetles in olfactometer experiments does not match the number of beetles in feeding trials because additional beetles were used in the olfactometer for control experiments such as empty arms (0∶0) and empty arms vs. control plants (0∶C).Feeding experiments were conducted using the same beetle individuals and plant material as in the previously conducted olfactometer experiments. Single beetles were placed in 250 ml plastic cups, each containing one leaflet of each type of plant material from the olfactometer experiments creating a binary choice situation. We used completely intact leaflets of similar developmental stages for the feeding trials to reduce potential effects of ontogenetic variability in leaf structure. After 24 h, leaflets were digitally photographed  on a scale to calculate missing leaf area using the analySIS® software . After the feeding experiments, remaining leaf material was subjected to HCNp analysis (see below). In the feeding experiments we used n = 66 individuals of C. ruficornis  and n = 71 individuals of G. guerreroensis . When choice experiments were finished, young leaves of the experimental plants were used for feeding experiments with individual beetles and HCNp as well as leaf area consumption were determined as described above.Free-flight choice experiments were conducted in a cage (length 1.25× width 0.70× heights 1.00 m) between 3:00 and 6:00 p.m. in the open. We used young potted plants, which were placed at each end of the cage with a distance of 1 m. Beetles were placed in the middle between both plants through a small opening in the front. Each beetle was tested individually eight times, and only beetles landing on one of the plants within 10 min were considered as having made a decision. Control versus JA-induced plants were tested at two different concentrations [I (1.0)∶C and I (0.001)∶C] as well as control vs. herbivore-damaged plants (HI∶C). In a control setup two undamaged plants were tested against each other (C∶C). In free-flight experiments we used −1) had been added as an internal standard (IS) −1 to 200°C, then 30°C min−1 to 280°C with He (constant flow 1.5 mL min−1) as carrier gas.Plant volatiles (VOCs) that were released from mature shoots and young potted plants in response to the different treatments were collected in a closed-loop stripping system (24 h) on charcoal filters . Volatiles were dissolved from the charcoal filters with dichloromethane to which n-bromodecane (200 ng µLHevea brasiliensisn = 21), the position of leaflets analyzed after the different time periods was set at random  of leaf material was quantified by enzymatic degradation of cyanide containing precursors  by adding specific β-glucosidase from rubber tree, t random .post hoc analysis  after univariate ANOVA. To test for significant differences of VOC emissions of mature shoot parts and young intact plants within one treatment group, Wilcoxon signed-rank tests were applied. Friedman-tests were applied to test for time-depended changes in the cyanide concentrations in leaflets of individual trifoliate leaves after incubating the leaflets for 0, 12, and 24 h. In the Y-tube olfactometer experiments, a percentage decision of individual beetles for the different odor sources was calculated . In the olfactometer and free-flight cage experiments, the differences in the percentage decisions of individual beetles between induced  plant material (mature shoots and potted young plants) and the controls were analyzed with Wilcoxon signed rank tests after arcsine transformation of data Cerotoma ruficornis or Gynandrobrotica guerreroensis), ‘Sex of beetle’, and ‘Beetle species×Sex of beetle’ we used a general linear model (GLM). Correlations of insect choice behavior in olfactometer and free-flight experiments with actual feeding decisions in subsequent feeding trials were analyzed with Pearson's correlations. For statistical analyses we used SPSS (IBM SPSS Statistics 19).The differences among different jasmonic acid (JA) treatments in the amounts of released VOCs were analyzed with a −1 resulted in the highest release of VOCs for both mature shoots and potted plants, while induction with JA solutions at lower concentrations  resulted in lower amounts of VOCs released  after one-way ANOVA  treatment . The amoreleased . Plant mreleased . For eactest; −1 .n = 696) µmol HCN g−1 fresh wt. for shoots and 0.20 to 64.69  µmol HCN g−1 fresh wt. for potted plants. Comparative analyses of leaflets of individual trifoliate leaves after 0, 12, and 24 h  revealed no significant differences of HCNp among leaflets depending on experimental time period  concentrations . In contrast to females, males of both species showed no significant preference for both odor sources when they had the choice between plant material treated with 0.1 and 0.01 mmol L−1 JA and the respective controls. Males of C. ruficornis, however, represent an exception as they significantly preferred young intact plants induced with 0.01 mmol L−1 JA over the controls  to attraction by plants induced with low concentrations of JA (0.001 mmol L−1 JA) and herbivore-damaged plants, whereas females consistently preferred the controls over induced plant material. However, among all experiments in which beetles of both species had the choice to select between JA treated and herbivore damaged plant material we observed significant sex-dependent differences even when both sexes showed the same qualitative response (according to Mann Whitney U-test).In olfactometer experiments, eroensis  of both e shoots  and intae shoots  treated When given the choice to select between untreated plant material (from mature shoots or potted young plants) and an empty arm, beetles of both species and sexes significantly preferred plant material . However, in contrast to olfactometer experiments, we found no significant differences in choice behavior between males and females when offering highly induced (1.0 mmol L−1) and control plants  and between herbivore-damaged and control plants (HI∶C), male beetles preferred induced plants over the controls, whereas females preferred the controls  to localize host plants and whether long-distance responses towards these VOCs can serve as a reliable predictor of feeding choice. Supporting our initial predictions, we found that the release of JA-induced plant volatiles affected beetle behavior in a concentration- dependent manner. Female beetles were repelled by VOCs that had been induced both by high and low JA concentrations. Males, however, were repelled by the VOC spectra of plants treated with high JA concentrations, but were attracted to the VOCs released from plants treated with low JA- concentrations. Furthermore, we demonstrated that factors directly determining food quality are strong predictors of insect feeding decisions. The feeding behavior of both sexes was strongly determined by the cyanogenic potential of leaves. Again, females responded more sensitively than males.Chrysomela populi on leaves of Populus×euroamericanaHeliothis virescens on Nicotiana tabacumFor herbivorous insects, induced plant VOCs convey a broad range of information. In addition to indicating the mere presence of the plant, the specific chemical composition of VOCs can signal plant quality, as well as other vitally important factors such as, the presence of mating partners, competitors, or enemies In the present study, we used jasmonic acid (JA) at different concentrations to induce the release of VOCs in order to create significant variability in this trait without having VOCs in the system that might be directly derived from feeding herbivores. Cyanogenesis was determined as the most important direct defense that does not respond to JA treatment −1  of leaflets was the crucial parameter that determined feeding choice, confirming earlier results Gynandrobrotica guerreroensis paralleled the amount of VOCs released following plant treatments with JA solutions in concentrations of 0.001 and 0.01 mmol L−1 (G. guerreroensis and Cerotoma ruficornis) were repelled by VOCs at these concentrations, whereas male beetles were attracted to the induced plants  resulted in amounts of volatiles which—despite substantial variation—frequently reached levels as seen following plant treatments with JA −1 can also occur in the plant-herbivore system.The amount of VOCs released in response to leaf damage by mmol L−1 . Female d plants . HoweverAt natural sites, both chrysomelid species have been observed to feed on a small range of wild and cultivated fabaceous plants (pers. observation by authors). However, lima beans are the preferred food plants and the insects can therefore be considered well adapted to feeding on this cyanogenic plant species Conversely, damaged plants indicate the potential presence of competitors and the induction of VOCs and extrafloral nectar attracts ants and other carnivores to lima bean in nature Cerotoma ruficornis and Gynandrobrotica guerreroensis) to herbivore-induced plant VOCs varied substantially depending on the amount of volatiles released and sex of the herbivores. Females were repelled by both low and high concentrations of VOCs, whereas males were repelled by high concentrations but attracted to plants releasing low amounts of VOCs. However, the information carried by VOCs must not necessarily reflect food plant quality, since feeding choice of insects was determined by the cyanogenic potential of leaves Studies on the function of induced plant volatiles (VOCs) for host plant location of chrysomelid herbivores have yielded inconsistent results. Induced VOCs have been demonstrated to attract chrysomelid herbivores to their host plants Table S1Treatment effects on the quantitative emission of volatile organic compounds (VOCs). We statistically compared the VOC emission of tendrils with potted plants using Mann Whitney U-tests for each treatment. Plants were induced with different concentrations of jasmonic acid (JA), by feeding damage through herbivorous beetles (Feeding damage) or treated with water and served as control (Control).(DOC)Click here for additional data file.Table S2Effect of induced plant volatiles on the choice behavior of Cerotoma ruficornis and Gynandrobrotica guerreroensis. Decisions made by male and female beetles in olfactometer (mature lima bean shoots and young intact plants) and free flight experiments (young intact plants) were tested for significant differences by Wilcoxon signed rank tests. The plant material used in the choice experiments was induced for release of volatiles by different treatments: I (1.0) = 1.0 mmol L−1 jasmonic acid (JA); I (0.1) = 0.1 mmol L−1 JA; I (0.01) = 0.01 mmol L−1 JA; I (0.001) = 0.001 mmol L−1 JA; HI = herbivore-induced (G. guerreroensis) plant material. Plant material sprayed with water instead of JA (C) and empty olfactometer arms (0) served as controls.(DOC)Click here for additional data file.Table S3Ontogenetic effects of plant volatiles on the choice behavior of Cerotoma ruficornis and Gynandrobrotica guerreroensis. In olfactometer choice experiments with induced (sprayed with 1 mmol L−1 jasmonic acid) and untreated lima bean plants (mature shoots and young intact plants), decisions made by male and female beetles for the one or the other source were tested for significant differences with Wilcoxon signed rank tests. In these control experiments plant material of the same state of induction but various ontogenetic developmental stage was tested against each other.(DOC)Click here for additional data file.Table S4Sex-specific choice behavior of Cerotoma ruficornis and Gynandrobrotica guerreroensis. Choice behavior of beetles observed in olfactometer and free flight experiments using mature lima bean shoots and intact young plants as odor source was tested for sex-specific differences by Mann-Whitney U tests. Plant material used in the choice experiments was induced by various treatments: I (1.0) = spraying with 1.0 mmol L−1 jasmonic acid (JA); I (0.1) = 0.1 mmol L−1 JA; I (0.01) = 0.01 mmol L−1 JA; I (0.001) = 0.001 mmol L−1 JA; HI = herbivore-induced (G. gynandrobrotica) plant material. Untreated plant material (C) and empty olfactometer arms (0) served as controls.(DOC)Click here for additional data file.Table S5Effects of ontogenetic plant develeopmental stage on sex-specific choice behavior of Cerotoma ruficornis and Gynandrobrotica guerreroensis. Choice behavior of beetles observed in olfactometer experiments with induced (sprayed with 1 mmol L−1 jasmonic acid) and untreated mature lima bean shoots and intact young lima bean plants was tested for sex-specific differences with Mann-Whitney U tests. In these control experiments plant material of the same state of induction but various ontogenetic developmental stages was tested against each other.(DOC)Click here for additional data file.


Genomic instability and copy number alterations in cancer are generally associated with poor prognosis; however, recent studies have suggested that extreme levels of genomic aberrations may be beneficial for the survival outcome for patients with specific tumour types. We investigated the extent of genomic instability in predominantly high-grade serous ovarian cancers (SOC) using two independent datasets, generated in Norway (n = 74) and Australia (n = 70), respectively. Genomic instability was quantified by the Total Aberration Index (TAI), a measure of the abundance and genomic size of copy number changes in a tumour. In the Norwegian cohort, patients with TAI above the median revealed significantly prolonged overall survival (p<0.001) and progression-free survival (p<0.05). In the Australian cohort, patients with above median TAI showed prolonged overall survival (p<0.05) and moderately, but not significantly, prolonged progression-free survival. Results were confirmed by univariate and multivariate Cox regression analyses with TAI as a continuous variable. Our results provide further evidence supporting an association between high level of genomic instability and prolonged survival of high-grade SOC patients, possibly as disturbed genome integrity may lead to increased sensitivity to chemotherapeutic agents. Clinical and pathological classification methods, including tumour grade and the extent of surgical debulking, still fail to fully predict disease progression and patient outcome.Serous ovarian cancers (SOC) are highly aggressive but often chemosensitive tumours, characterised by substantial morphological heterogeneity, frequent genomic aberrations, and genomic instability , to assess the level of genomic aberrations in SOC. Based on high-throughput DNA copy number data, we investigated the relationship between survival and the degree of genomic instability within two independent datasets of predominantly high-grade SOC patients.The study including patients of the Norwegian cohort was approved by the Regional Committees for Medical and Health Research Ethics (REC) board (Reference No: S-01127). Exception from written informed consent was given from the REC authorities based on patients being deceased and all materials used were remaining material after diagnosis. The study including patients of the Australian cohort was approved by the Human Research Ethics Committees at the Peter MacCallum Cancer Centre, Queensland Institute of Medical Research, University of Melbourne and all participating hospitals. Written informed consent was obtained from all participants in this study.The Norwegian cohort, diagnosed and treated at the Department of Gynecological Oncology at the Oslo University Hospital The Norwegian Radiumhospital during the period May 1992 to February 2003, consisted of 74 patients diagnosed with SOC on routine pathology reports. All patients underwent primary surgery, followed by adjuvant platinum-based chemotherapy. A summary of the clinicopathological characteristics is shown in The second cohort, originally analysed in Australia www.microarray.org/sfgf/jsp/home.jsp; for details see Haematoxylin and Eosin stained sections from frozen tissue were used to evaluate the percentage of tumour cells in tissue samples. The percentage of tumour cells in the samples in the Norwegian cohort ranged from 20% to 90% with a median of 70%. In the Norwegian cohort, genomic DNA was extracted from 10–15 serial frozen tissue sections (each 50 µm thick) using proteinase K digestion and phenol/chloroform in an ABI DNA extractor  following standard protocols. Copy number profiles of all samples were obtained with the Stanford 42k cDNA aCGH platform (XbaI single nucleotide polymorphism (SNP)  mapping arrays were applied to obtain copy number profiles  according to the manufacturer's protocol. Affymetrix 50 k To segment the copy number data the Piecewise Constant Fitting (PCF) algorithm The degree of genomic instability in a tumour was quantified by the total aberration level, using a similar method as described previously Thus, TAI is basically a weighted sum of the segment averages and represents the absolute deviation from the normal copy number state, averaged over all genomic locations .Comprehensive germ-line testing for the Australian cohort was completed in a certified diagnostic pathology laboratory using sequencing and multiplex ligation-dependent probe amplification The analysis of copy number data in serous ovarian cancers revealed that the aberrations in the Norwegian and Australian cohorts were broadly concordant , with thThe clinicopathological characteristics age, stage, grade, chemotherapy response , and progression were analyzed in relation to differences in genomic instability . PatientBRCA1 or BRCA2 in SOC patients is associated with favourable clinical outcome BRCA1 and BRCA2 mutation status the germline carrier status was not significantly associated with the extent of genomic instability (using Fisher's exact test). Average TAI was 0.28 (SD = 0.06) for patients with germline mutation in BRCA1 (n = 6), 0.27 (0.05) for patients with mutation in BRCA2 (n = 2), 0.22 (0.03) for patients with unclassified variants of BRCA genes (n = 3), and 0.25 (0.05) for patients with wild type BRCA genes (n = 24).Given that a germline mutation in either We investigated the association between genomic instability and survival of predominantly high-grade SOC in two independent study cohorts from Norway and Australia and found that patients with high level of genomic instability, as measured by TAI, had a more favourable outcome. The results were confirmed by univariate and multivariate Cox analysis, were TAI was included as a continuous variable. The aberration patterns in the two cohorts, determined by two different gene-centred platforms, were highly concordant and consistent with those reported by others Only few publications have investigated the relationship between complex rearrangements and survival in ovarian cancer. A previous study, primarily focused on breast cancer but also considering ovarian tumours, provided some evidence that high levels of rearrangement in tumours may lead to better clinical outcome The presented study is based on the analysis of high-resolution DNA copy number data and the application of a robust and easily interpretable measure of genomic instability (TAI). TAI assesses the deviation of the estimated copy number curve from the zero-line , and thuGenomic instability causes disturbed mitoses, segregation, and spindle assembly Click here for additional data file.Figure S1Model of the Total Aberration Index algorithm.(PDF)Click here for additional data file.Table S1Additional clinical data for the Norwegian cohort.(XLSX)Click here for additional data file.Table S2Additional clinical data for the Australian cohort.(XLSX)Click here for additional data file.Abstract S1Abstract in German.(PDF)Click here for additional data file.Abstract S2Abstract in Norwegian.(PDF)Click here for additional data file.


Comprehensive High-throughput Arrays for Relative Methylation (CHARM) platform, covering 3.5 million CpGs. CHARM identified 224 candidate regions with DNAm differences >10%. These regions are highly enriched for neuronal growth and development genes. Ten of 17 regions for which validation was attempted showed true DNAm differences; the greatest were in PRIMA1, with 12–15% increased DNAm in MDD (p = 0.0002–0.0003), and a concomitant decrease in gene expression. These results must be considered pilot data, however, as we could only test replication in a small number of additional brain samples (n = 16), which showed no significant difference in PRIMA1. Because PRIMA1 anchors acetylcholinesterase in neuronal membranes, decreased expression could result in decreased enzyme function and increased cholinergic transmission, consistent with a role in MDD. We observed decreased immunoreactivity for acetylcholinesterase in MDD brain with increased PRIMA1 DNAm, non-significant at p = 0.08.While genome-wide association studies are ongoing to identify sequence variation influencing susceptibility to major depressive disorder (MDD), epigenetic marks, such as DNA methylation, which can be influenced by environment, might also play a role. Here we present the first genome-wide DNA methylation (DNAm) scan in MDD. We compared 39 postmortem frontal cortex MDD samples to 26 controls. DNA was hybridized to our PRIMA1 DNAm in MDD, the involvement of neuronal development genes across the set showing differential methylation suggests a role for epigenetics in the illness. Further studies using limbic system brain regions might shed additional light on this role.While we cannot draw firm conclusions about  Family studies show that siblings of probands with major depressive disorder (MDD) have about a three-fold elevated risk of illness, while the estimated heritability of MDD from twin studies is about 37% There are at least two other major kinds of explanations for this variation in susceptibility. One is that environmental factors such as stressful life events play a significant role in triggering MDD Epigenetics, which has been frequently implicated in cancers Bdnf in rats There are several examples of epigenetic variation in candidate MDD genes and in DNA treated with medications used for MDD. For example, early life adversity increased DNAm in HpaII and McrBC to prepare DNA, which they hybridized to a 12,192 CpG-island microarray.Despite the availability of an essentially complete genome sequence for several years, understanding of the methylome has progressed more slowly, largely due to limitations in technology affecting sensitivity, specificity, throughput, quantitation, and cost among the previously used detection methods. Microarray-based methods can interrogate much larger numbers of CpGs than other approaches. One study to date has reported on a genome-wide DNAm study in psychiatric disorders, demonstrating differences in the 4–9% range between DNA from bipolar disorder or schizophrenia brain samples vs. controls McrBC, though we have implemented it on a microarray platform (CHARM), which is not biased towards CpG islands, but rather has features chosen agnostically based on high CpG density. We have shown that CHARM robustly distinguishes tissue types based on differential DNAm profiles, and can also discriminate between colon cancer and normal colon tissues We have similarly used a methylation-sensitive restriction enzyme-based method focused on We have now used CHARM analysis to study genome-wide DNAm variation in 39 MDD and 26 control brains. Here we report results of this experiment and of follow-up pyrosequencing experiments to attempt to validate the initial findings and to correlate DNAm differences with gene expression. While these data should be followed up on a much larger replication set, the absence of large DNAm differences in the brains of MDD patients is itself important in considering the epigenetic hypothesis. These results suggest that if DNAm plays a role in MDD, the most critical target may not be the frontal cortex, but other regions, such as hippocampus and amygdala, key components of the limbic system, in which epigenetic changes have been shown to influence cognitive and behavioral phenotypes The Johns Hopkins University IRB approved all research involving human participants. Subjects gave written informed consent under the IRB-approved protocol.Postmortem frontal cortex brain tissue, Brodmann area 10, from 39 individuals with MDD and 27 matched controls were donated by the Stanley Medical Research Institute, in two batches. The first sample set consisted of 12 psychotic depression cases, 12 non-psychotic depression cases and 12 age and sex matched controls. A second set consisted of 15 non-psychotic depression cases and 15 age and sex matched controls. To increase power, the two samples were analyzed together. A structured interview-based DSM-IV diagnosis was assigned to each sample independently by two senior psychiatrists, based on available medical records and a series of interviews conducted with the family Cases of MDD (N = 30) were selected from the Genetics of Recurrent Early Onset Depression (GenRED) study. Clinical methods have been described elsewhere McrBC (NEB). Following digestion cut and uncut fractions from the same sample were electrophoresed in adjacent wells of a 1% agarose gel. Areas corresponding to the 1.6 kb–3 kb regions were excised and purified using Qiagen Spin Gel Purification columns. The gel-purified DNA was quantified on a spectrophotometer and 30 ng of DNA from each fraction was amplified using a GenomePlex Whole Genome Amplification Kit (SIGMA). The amplified DNA was then isolated with a Qiagen PCR Purification column, then quantified using a spectrophotometer. The untreated, total DNA fraction was labeled with Cy3 and the methyl-depleted DNA fraction was labeled with Cy5 and hybridized onto the custom NimbleGen 2.1 M feature CHARM microarray (design previously described The CHARM assay was performed as described previously 1 µg of genomic DNA was bisulfite treated using the Epitect kit (Qiagen). CpG unbiased primers were designed to PCR amplify 92 CpG sites in 17 genes. Nested PCR was performed. Amplicons were analyzed on a PSQ HS 96 pyrosequencer (Biotage), and CpG sites were quantified, from 0% to 100% methylation, using Pyro Q-CpG software RNA was extracted from frontal cortex using the RNAeasy Kit (Qiagen). MonsterScript 1st – Strand cDNA Synthesis Kit (Epicentre) was used to generate cDNA for subsequent quantitative real-time PCR. Negative RT samples were used to ensure the absence of contamination. All reactions were carried out in triplicate using 1× TaqMan master mix (Applied Biosystems), 1× TaqMan probe for each gene, and 10 ng of template in a volume of 20 µL. Real-time reactions were performed on an Applied Biosystems 7900HT Real-Time PCR System. Each set of triplicates was checked to ensure that the threshold cycle (Ct) values were all within 1 Ct of each other. The delta-delta-Ct method was used to determine sample quantity.Hybridization quality was assessed by comparing the untreated fraction signal intensity for each genomic probe to that of background (anti-genomic) probes, with the expectation that the genomic probes should register significantly higher signals. Poor hybridization was indicated by genomic probe signal levels not being significantly higher than background probe levels. Using this metric eight arrays were identified as having failed hybridization and discarded.Normalized methylation log-ratios were smoothed using a weighted sliding window as previously described We sought to determine whether our nominally significant differentially methylated regions were in or near genes that clustered together functionally. We determined the nearest gene for each differential DNAm region and thus created a list of genes with differential DNAm. We then asked whether this gene list was enriched for GO Biological Process categories PRIMA1 each of these was added as a covariate into a regression equation with case status as the primary independent variable and DNAm as the dependent variable.For each of the 17 most differentially methylated regions, we assessed pyrosequencing data based on primers designed across the most CpG dense part of the region implicated by CHARM. A linear regression model was used to assess the statistical significance of the effect of case-control status on DNAm. These were then corrected at two levels of stringency: 1) taking the best p-value for each gene and correcting for 17 tests (the number of regions tested); and 2) taking all p-values and correcting for 92 tests (the number of CpGs tested). We then tested DNAm levels at all CpGs against a number of additional sample variables including: pH, postmorterm interval, age, sex, side of brain assayed, smoking at time of death, and lifetime alcohol use, using a univariate regression model. Resulting p-values were corrected for the number of tests performed (92). For Ten micron-thick paraffin sections from four subjects with MDD and five controls were processed for AChE immunostaining. Sections were incubated with a rabbit polyclonal antibody targeting a signature epitope of an AChE precursor recombinant protein, particularly suitable for tissue immunohistochemistry , at a 1∶25 dilution and subsequently processed by a modification of the avidin-biotin-peroxidase method as we have previously described Characteristics of postmortem brain samples are provided in We assessed the DNAm differences between MDD and controls using the Biological Processes categories of the Gene Ontology database LASS2, CPSF3, ZNF263, and PRIMA1 .We attempted validation for 17 DMRs chosen because they were among those showing the greatest DNAm differences between MDD and controls, and were in or near genes. Within these regions bisulfite pyrosequencing was conducted across 92 CpG dinucleotides. We observed nominally significant DNAm differences in 10 of the regions. The four regions with the strongest results, those in or near the genes d PRIMA1 , remainePRIMA1, two variables predicted DNAm for CpG-2 at a nominal level of significance: increased age was associated with decreased DNAm (p = 0.04), as was lower pH (p = 0.03) (PRIMA1 in MDD samples by focusing on the subset of seven samples that were medication free. DNAm for these were 3–6% greater than for the remaining 32 MDD samples (p = 0.15), suggesting that medication was not responsible for the difference between MDD and controls.For all of the 17 regions tested, we tested the impact of additional demographic, clinical, and biologic variables on DNAm . After c = 0.03) . When thPRIMA1 DNAm in MDD, we tested mRNA levels of the gene in the same brain samples that were used for the DNAm experiments. Levels were altered in the MDD brain samples in the expected direction, being decreased 53% (p = 0.047).To assess the potential functional impact of increased PRIMA1 DNAm differences could be detected between subjects from our GenRED study as compared to normal controls collected for genetic studies. We used DNA from these subjects' lymphoblastoid cell lines and saw results similar to those in brain. DNAm was increased in MDD subjects as compared to controls  for three of the four PRIMA1 CpGs . We also observed that subjects with MDD had a larger number of superficial pyramidal neuron perikaryal staining, despite overall reduction in neuropil immunoreactivity, suggesting redistribution of AChE towards the cell bodies Click here for additional data file.Table S1The results of the primary experiment using CHARM to compare postmortem brain samples between MDD cases and controls.(DOC)Click here for additional data file.Table S2The result of taking all genes in or near nominally significant differentially methylated regions and examining their representation in Gene Ontology Categories.(DOC)Click here for additional data file.Table S3Bisulfite pyrosequencing was used to experimentally validate some of the regions that showed differential methylation between MDD and controls by CHARM analysis. This table shows those that were nominally validated. P-values for regression of pyrosequencing methylation at individual CpGs (rows) on 6 covariates (columns). The last column shows the F-statistic p-value for the multiple regression of methylation on all 6 covariates.(DOC)Click here for additional data file.Supporting Information S1Supplementary Tables S4, S5, S6 show results of replication attempts in postmortem brain and in lymphoblastoid cell lines.(DOCX)Click here for additional data file.


We present GStream, a method that combines genome-wide SNP and CNV genotyping in the Illumina microarray platform with unprecedented accuracy. This new method outperforms previous well-established SNP genotyping software. More importantly, the CNV calling algorithm of GStream dramatically improves the results obtained by previous state-of-the-art methods and yields an accuracy that is close to that obtained by purely CNV-oriented technologies like Comparative Genomic Hybridization (CGH). We demonstrate the superior performance of GStream using microarray data generated from HapMap samples. Using the reference CNV calls generated by the 1000 Genomes Project (1KGP) and well-known studies on whole genome CNV characterization based either on CGH or genotyping microarray technologies, we show that GStream can increase the number of reliably detected variants up to 25% compared to previously developed methods. Furthermore, the increased genome coverage provided by GStream allows the discovery of CNVs in close linkage disequilibrium with SNPs, previously associated with disease risk in published Genome-Wide Association Studies (GWAS). These results could provide important insights into the biological mechanism underlying the detected disease risk association. With GStream, large-scale GWAS will not only benefit from the combined genotyping of SNPs and CNVs at an unprecedented accuracy, but will also take advantage of the computational efficiency of the method. Over the last years, Genome-Wide Association Studies (GWAS) using microarray-based technology have played an important role in the identification of common genetic variations and their relationship with disease susceptibility In addition to SNPs, Copy Number Variants (CNVs) have shown to play an important role in disease susceptibility The development of the two mentioned technologies (CGH arrays and genotyping microarrays) for high throughput CNV screening has prompted the inclusion of CNVs in GWAS studies In this study we present GStream, a method for SNP and CNV/CNP genotyping that is tailored to GWAS objectives. GStream integrates a substantially improved version of our previous CNV calling software CNstream In addition to presenting this new method and demonstrating its superior performance over reference datasets, we have also performed different relational analyses concerning previously known risk loci. Using GStream we have been able to identify, for the first time, several CNVs in strong linkage disequilibrium (LD) with risk-associated SNPs We first introduce the Illumina BeadChip microarrays and describe the algorithms for SNP and CNV genotyping. Next, we provide information about the datasets used in this study and the implemented metrics for evaluating SNP and CNV genotyping accuracy. Finally, we describe the methods used for the CNV association studies that we have run using the GWAS catalog Illumina BeadChips have been largely used in large-scale genome-wide association studies and are based on the Infinium assay technology Before identifying the clusters corresponding to the AA, AB and BB genotypes at each probe, raw intensity data of each probe must be normalized in order to equalize the overall sample intensity distribution at each channel . This stOnce the intensities from both A and B channels have been normalized, GStream proceeds to identify the clusters corresponding to each SNP genotype . Developing an accurate SNP genotyping method is crucial not only for SNP analysis itself, but also because GStream CNV genotyping method uses this information to improve the accuracy of the CNV call. GStream applies the following procedure to assign a SNP genotype to each sample at each marker:Channel A and channel B intensities from the analyzed marker are transformed to B allele frequencies (BAF) and absolute intensities Absolute intensities are used to detect samples without any allele (homozygous deletions) which are characterized by very low intensities at both channels. In order to compute the absolute intensity threshold between homozygous deletion samples and the other samples, the absolute intensities are sorted and then differences between each pair of consecutive intensities are computed. If high intensity differences are found within the expected threshold range , the zero-threshold is fixed to the corresponding intensities .The BAF probability density function (PDF) is estimated by computing the scaled histogram of all the sample BAF values. Peaks over this PDF corresponding to genotype clusters will be identified sequentially starting by the peak generated by the major allele frequency homozygote cluster. The algorithm establishes a minimum separation between peaks in order to assign them to different genotype clusters and it stops when three peaks have been found or when no more peaks are found. Once genotype peaks have been found, genotype limits are computed by finding the PDF minimum between each consecutive pair of centres . These lIf the number of genotype peaks identified is less than three, each genotype cluster is re-analyzed with a better resolution (i.e. increasing the number of histogram bins to estimate the BAF PDF) in order to identify sub-clusters corresponding to different genotypes. This procedure avoids common errors seen in others algorithms where, for example, genotypes of SNPs with highly discordant sensitivities at each channel are incorrectly assigned.Finally, a global genotyping quality score and an individual score for each sample genotype are computed . The gloBoth genotypes and quality control measurements for each sample are extracted by GStream to the output files. This information is also required by the CNV genotyping method, which is based both on the normalized channel intensities and the SNP genotype information. Further algorithm details are given in CNV identification and genotyping is one of the principal contributions of GStream to the current state-of-the-art microarray genotyping methodology, clearly outperforming previous approaches. Although this method has been based on our previous CNstream method GStream uses normalized intensities and SNP genotypes computed in the SNP genotyping stage to identify the presence of deletions and amplifications characterized by variable clustering patterns on the intensity data (i.e. high frequency CNVs) or by slight deviations from the diploid distribution (i.e. low frequency CNVs).One of the improvements incorporated in the algorithm is that each SNP genotype cluster is independently analyzed taking only into account the intensity channel that carries valuable information. This way, the CNV algorithm is divided in four parallel steps :Analysis of channel A intensities from the samples previously genotyped as AA homozygotes.Analysis of channel B intensities from the samples previously genotyped as BB homozygotes.Analysis of channel A intensities from the samples previously genotyped as heterozygotes (i.e. AB).Analysis of channel B intensities from the samples previously genotyped as heterozygotes (i.e. AB).As well as dividing the analysis in four independent steps, the algorithm is based on the following assumptions:Homozygous deletions  are previously detected during the SNP genotyping stage.Due to the technical limitations of genotyping microarrays, the intensity measurements show a saturation effect when amplifications are found. For this reason, intensity clustering patterns corresponding to amplifications are very rare and hard to detect unless they span multiple probes Samples categorized as homozygote samples (i.e. AA and BB) can correspond to hemizygous deletions (i.e. A and B) or amplifications (i.e. AA+ and BB+). Due to the saturation effect the algorithm does not stratify amplifications by the number of allele copies.Samples characterized as heterozygotes (i.e. AB) can have two or more copies . The total number of copies can be inferred by independently computing the number of copies of each allele and then adding the results for each sample.Below we describe the procedure for determining the CNV genotypes from the set of channel intensities of each one of the four analysis steps.For each SNP genotype, the algorithm starts identifying clusters over the channel intensities that carry the corresponding allele information . Due to the mentioned saturation effects, it is very uncommon to observe more than two intensity clusters in microarray data and, for this reason, only two models will be fitted to the intensity data: a one- and a two-component Gaussian mixture model (GMM). The first one will be fitted using the mean and the variance of all the intensities while the second one will be fitted using the Expectation-Maximization algorithm If the two-component model has been selected, a copy number category will be assigned to each one of the two components. As no prior knowledge is available to assign the two components either to a deletion pattern (i.e. CN = 1 and CN = 2) or to an amplification pattern (i.e. CN = 2 and CN = 3), a disambiguation method is necessary. GStream bases the component labelling both on the relative weight of each component  and on the presence of homozygous deletions . When thi a score S between 0 and 3 depending on its copy number posterior probabilities (Outlier identification is intended to capture low frequency CNVs that are not captured by a two-component GMM and is based on identifying samples showing high or low deviations from the intensity distributions defined by the selected model. CNV scoring assigns to each sample bilities . At the bilities . Additiohttp://www.ncbi.nlm.nih.gov/geo/) In order to evaluate and compare the performance of GStream SNP and CNV genotyping methods we have used raw Illumina microarray data from HapMap samples available at the Gene Expression Omnibus . For this study, we downloaded the genotypes corresponding to the samples having available microarray data and used them as the golden standard. SNPs used for performance evaluation were chosen in order to fulfil three criteria: (i) to have available Build37 mapping information, (ii) to be present both in the analyzed microarray platform and in the golden standard HapMap dataset, and (iii) to have concordant reference alleles both in the microarray and in the golden standard annotations  GenoSNP Copy number evaluation was performed at two levels: Evaluation of the GStream ability to detect structural variation obtained from the 1000 Genomes Project ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/release). From all the variants included in the 1KGP release we chose those that corresponded to structural variations (i.e. CNVs) and we filtered out variants with a MAF lower than 2% within all the populations. The resulting set included 2,531 structural variants (SV) with their respective calls over N = 353 unrelated HapMap samples. From these 2,531 loci, 1,956 are covered by HumanOmni1-Quad markers and GStream calls were available for 149 out of the 353 1KGP samples, which jointly formed the final evaluation set.In order to test the ability of GStream to detect copy number variation, we have used the HumanOmni1-Quad GStream calls and a golden standard dataset from a public release of the 1KGP. HumanOmni1-Quad platform was chosen due to its highest coverage and resolution which allowed an evaluation over a major number of loci. The golden standard dataset consisted of the last variant call files that have been released by the 1KGP  and without (i.e. Human1M) a specific design to cover CNV.The genome-wide association study over CNV markers was performed at the population level aiming to identify CNVs significantly associated to specific populations and comparing the association statistics with those obtained from golden standard datasets.The CNV algorithms used are described below:PennCNV QuantiSNP is also one of the well-known methods for CNV analysis over Illumina microarrays. It is based on an Objective Bayes Hidden-Markov Model that is used to set certain hyperparameters in the HMM priors  in any population test  were performed using the calls from the three golden standard datasets. Loci that either were not covered by the microarray platform or did not obtain significant associations  In order to identify putative causal CNVs we have analyzed the LD patterns between all the trait-associated SNPs reported by the catalog of published genome-wide association studies .Correlation coefficient rDistance between the CNV marker and the nearest gene not greater than 100 kb or CNV marker spanning binding transcription factor regions as defined by the Transcription Factor ChIP-seq track on the UCSC browser From the 7,571 trait-associated SNPs, 382 were paired with one or more CNV markers fulfilling these conditions. A final set of 333 SNP-CNV pairs was obtained after filtering out repeated associations of SNPs with the same trait by different GWAS studies.http://www.omim.org) In this second approach, we examined the CNV variants called by GStream spanning genes known to be involved in disease based on the OMIM database  only those located less than 15 kb away from an OMIM gene and with at least two more CNV markers covering this gene were selected .2>0.7) spanning the same OMIM gene. After applying this filter we obtained a final set of 212 CNV loci spanning OMIM genes.We defined CNV loci as sets of three or more nearer CNV markers (i.e. distance between them not greater than 5 kb) in high LD  were compared with their respective GStream calls in order to measure the ability to detect this type of variation using GStream on microarray data. For each SV , we computed the CNV genotyping accuracy by finding the maximum LD measurement between its golden standard calls and the GStream calls over the HumanOmni1-Quad markers covering the region. The results showed a high correlation between GStream and 1KGP calls: 75.7% of the SVs were captured by GStream with an r2>0.8, 18.3% with an r2<0.8 and only 6.0% were not detected by GStream , obtaining CNV calls with an rIn order to evaluate the power to detect CNV associations we have performed a pairwise association study between three HapMap populations using golden standard data from three reference studies 10P-value ratio between the association values obtained by the calls of each method and those obtained from the golden standard calls. Under this metric, ratios near 1 represent a good performance for the method since the obtained association P-value is very similar to the golden standard. A performance summary statistic was computed as the percentage of −log10P-value ratios giving values between 0.9 and 1.1 over all the associated CNV loci.The main metric used to test CNV methods was the −logFirstly, the obtained results showed a high performance decrease in the detection of CNV associations when comparing HumanOmni1-Quad results with Human1M-Duo . This loP-value obtained by the golden standard calls  as the P-value ranges decreased (i.e. greater evidence of association). Interquartile ranges obtained by the other methods were at least twice the obtained by GStream and increased as the P-value ranges decreased, losing performance when comparing higher associated loci. Poorer ratio medians were also obtained when using the other methods.HumanOmni1-Quad results show that GStream is able to precisely capture an average of 23.6% more associated loci than the other methods  and thatrd calls . GStream10P-value ratio distributions by their respective golden standard association P-values, ratio medians were only maintained near one when using GStream  has been associated to Rheumatoid Arthritis (RA) in a previous GWAS  using a Japanese cohort (1247 RA cases and 1486 controls) PADI4 intron (length  = 800 bp) in high LD with rs2240335 both on CEU (r2 = 0.82) and CHB+JPT (r2 = 0.95) HapMap populations.A synonymous exonic SNP rs2240335 (TMEM18 gene) has been associated to body mass index in previous GWAS  2 = 1 both on CEU and CHB+JPT populations) with this SNP.An intergenic SNP rs2867125  Levels of glycated hemoglobin have been associated to the MAEA gene has been associated  with type 2 diabetes Intronic SNP rs6815464 on HCG9 gene) has been previously associated with Vitiligo  HCG9 gene found in high LD with this SNP.5′UTR SNP rs6904029 (HLA-DPA1 gene) has been recently associated to chronic Hepatitis B  on a Japanese cohort HLA-DPA1 exons has been identified to be highly correlated with this SNP both on the CHB+JPT and the YRI populations.Intronic SNP rs3077 (MLIP gene) has been identified spanning 5 kb MLIP intron. This SNP has been associated with liver enzyme levels  A deletion highly correlated with intronic SNP rs9296736 (ZAN gene) has been associated to red blood cell count  on a GWAS exploring erythrocyte phenotypes 2 = 0.87) and the CHB+JPT (r2 = 0.91) populations.Intronic SNP rs2075671 (ZNF490 gene), modestly associated with bipolar disorder  ZNF490 intron.Finally, the 3′UTR SNP rs7247513  deletion has been identified by GStream within eight microarray probes spanning two gene exons and two gene introns . Since mutations within this gene have been previously associated to uric acid concentration SLC2A9 gene) is predominantly expressed in the kidney and expression association signals reported by Doring et al. DAZL (OMIM 601486) deletion was identified over 4 microarray probes spanning 5.8 kb . This deletion could affect multiple gene exons resulting in a drastic functional modification. Previous studies MBL2 gene. MBL2 mutations and the consequent Mannose Binding Lectin deficit have been previously associated with cystic fibrosis MBL2 gene expression and subsequently involve a Mannose Binding Lectin deficit whose association has also been demonstrated.Furthermore, as CNV are known to span from hundreds of bases to multiple kilobases, it is interesting to analyze not only those that correlate previously associated SNPs, but also those that overlap coding sequences of genes that have been previously associated to human disease (i.e. OMIM genes). The results from this analysis include several known CNV associated loci, as those spanning In a time of rapidly evolving technologies and where Next-Generation Sequencing is becoming available for the study of common diseases, microarray-based technologies are still a commonly used strategy to identify the genetic basis of human traits. First, they allow the analysis of large sample collections at an affordable cost and, second, they have anincreasing global genome coverage, expanding their analysis scope to rarer variants. Therefore, accurate genotyping methods are basic to discover new associated loci that can be then further studied in more detail using Next-Generation Sequencing. The tool that we present in this study, GStream, provides an unprecedented accuracy when analyzing GWAS data from previous and recent Illumina microarray platforms. Furthermore, our software tool implementation allows large-scale GWAS projects to be analyzed in a very short time, providing both SNP and CNV in a single analysis. With these results, we encourage researchers conducting GWAS on these genotyping platforms to use GStream in order to leverage the power of their SNP and CNV loci association analyses.Figure S1Example of raw intensity normalization. The intensity distribution of candidate homozygote samples (i.e. AA) across its specific allele channel (i.e. channel A) is plotted together with its centroid scaling value as computed by Peiffer et al.(PDF)Click here for additional data file.Figure S2Example of how zero-threshold is computed. (A) BAF and absolute intensities of an example marker where some homozygous deletion samples with low intensity values can be observed. (B) Absolute intensities are sorted and differences between consecutive sorted intensities normalized to one. The observed peak over these differences points to the intensity value that will be set as threshold.(PDF)Click here for additional data file.Figure S3CNV labelling and scoring. (A). Category disambiguation when the two-component model is selected. The leftmost graph shows a case where the higher intensity component (blue) is more frequent and it is assigned to the diploid state while the lower intensity component (red) is assigned to the deletion state. This assignment is due to the fact that high frequency amplifications are very uncommon and undetectable with this technology. The centre graph shows a case where the higher intensity component is less frequent and homozygous deletion samples have been found (magenta). In this case, the higher component (blue) is assigned to the diploid state and the lower component (red) to the deletion state fulfilling the expected Hardy-Weinberg equilibrium frequencies. Finally, the rightmost graph shows the last case where the higher intensity component is less frequent and no homozygous deletion samples have been found. In this case the higher component is assigned to the amplification state and the lower component to the diploid state. (B) Posterior probability of each copy number depending on the score assigned by GStream: From 0 to 0.5 samples can be categorized as homozygous deletion, from 0.5 to 1.5 as hemizygous deletion, from 1.5 to 2.5 as diploid and from 2.5 to 3 as amplification.(PDF)Click here for additional data file.Figure S4Genotyping performance. (A) Genotyping performance depending on the drop rate, where calls dropped from the accuracy analysis were selected according to the genotype call quality score. (B) Genotyping performance depending on the SNP minor allele frequency. (C) Genotyping accuracy of GStream at different sample sizes  compared to the accuracies obtained by GenoSNP and M3 with the highest sample size (N = 225).(PDF)Click here for additional data file.Figure S5Microarray coverage density. Coverage density of each microarray platform over the CNV regions defined by each reference study. There are major differences between the first Infinum HD platforms (Human610-Quad and Human1M-Duo) and their succesors including specific CNV coverage (Human660W-Quad and HumanOmni1-Quad). Both Human610-Quad and Human1M-Duo have a mean number of ∼10 markers covering CNV regions, while Human660W was designed with a highest coverage (∼20 markers/region) for almost 50% of the regions. Finally, HumanOmni1-Quad increased the global coverage to ∼20 markers/region.(PDF)Click here for additional data file.Figure S6Missed associations. Percentage of associations  that were not captured by the different methods tested .(PDF)Click here for additional data file.Figure S7HumanOmni1-Quad P-value distributions. Distributions of the P-value association ratios depending on the golden standard dataset used for evaluation (i.e. represented by different colours) and on the P-value range obtained over the golden standard calls .(PDF)Click here for additional data file.Figure S81M-Duo P-value distributions. Distributions of the P-value association ratios depending on the golden standard dataset used for evaluation (i.e. represented by different colours) and on the P-value range obtained over the golden standard calls .(PDF)Click here for additional data file.Figure S9Previously reported CNV associations detected by LD analysis between GStream CNV genotypes and trait-associated SNPs. (A) LCE gene cluster deletion associated with Psoriasis risk. (B) NEGR1 deletion associated with body mass index. (C) IRGM deletion associated with Chrohn's disease. (D) ARMS2 deletion associated with age-related macular degeneration. (E) GPRC5B upstream deletion associated with body mass index.(PDF)Click here for additional data file.Figure S10Interesting CNV associations detected by LD analysis between GStream CNV genotypes and trait-associated SNPs. (A) PADI4 gene deletion associated with Rheumatoid Arthritis. (B) TMEM18 downstream deletion associated with body mass index. (C) 3′-deletion of gene ABCB11 associated with glycated hemoglobin levels. (D) MAEA gene intron deletion associated with type 2 diabetes. (E) HCG9 deletion associated with Vitiligo. (F) HLA-DPA1 deletion associated with Hepatitis B. (G) MLIP intron deletion associated with liver enzyme levels. (H) ZAN gene deletion associated with red blood cell count. (I) ZNF490 intron deletion associated with bipolar disorder.(PDF)Click here for additional data file.Figure S11GStream detected CNP loci spanning disease-related genes (OMIM) where CNVs have been previously associated with disease. (A) CNP spanning CFHR1 and CFHR3 previously associated to age-related macular degeneration. (B) Deletion of GHR exon 3 that has been previously associated with increased responsiveness to growth hormone and Laron dwarfism. (C) Detected SMN gene deletion previously associated with spinal muscular atrophy. (D) PKHD1 deletion associated with polycystic kidney. (E) CCL3L1/CCL3L3 deletion previously associated with susceptibility to HIV/AIDS.(PDF)Click here for additional data file.Figure S12GStream calls across consecutive markers spanning the same CNV loci. These 6 microarray probes cover the same CNV loci but show very different CNV intensity patterns. GStream is completely adapted to these types of variations and its calling procedure is able to obtain very concordant calls when analyzing probes spanning the same CNV.(PDF)Click here for additional data file.Table S1CNV markers in high LD with trait-associated SNPs reported in the GWAS catalog.(XLSX)Click here for additional data file.Table S2Set of 149 CNV consistent loci spanning OMIM genes.(XLSX)Click here for additional data file.Text S1GStream algorithm details.(PDF)Click here for additional data file.


Background. Breast conservation therapy (BCT) is the standard treatment for breast cancer; however, 32–63% of procedures have a positive margin leading to secondary procedures. The standard of care to evaluate surgical margins is based on permanent section. Imprint cytology (IC) has been used to evaluate surgical samples but is limited by excessive cauterization thus requiring experienced cytopathologist for interpretation. An automated image screening process has been developed to detect cancerous cells from IC on cauterized margins. Methods. IC was prospectively performed on margins during lumpectomy operations for breast cancer in addition to permanent section on 127 patients. An 8-slide training subset and 8-slide testing subset were culled. H&E IC automated analysis, based on linear discriminant analysis, was compared to manual pathologist interpretation. Results. The most important descriptors, from highest to lowest performance, are nucleus color (23%), cytoplasm color (15%), shape (12%), grey intensity (9%), and local area (5%). There was 100% agreement between automated and manual interpretation of IC slides. Conclusion. Although limited by IC sampling variability, an automated system for accurate IC cancer cell identification system is demonstrated, with high correlation to manual analysis, even in the face of cauterization effects which supplement permanent section analysis. Approximately 32–63% of breast conservation therapy surgeries result in positive margins . To contIC is an alternative intraoperative technique to detect cancer cells at the margin surface. Following tumor excision, the sample surface is imprinted on glass slides while keeping track of the margin location , 22. CelFurthermore, the imprinting process as well as the cauterization during tumor excision tends to introduce staining and drying artifacts depending on environmental and sample conditions thus rending interpretation difficult , 27. ForIntraoperative margin evaluation reduces reexcision rates , 12, 28,2 (25 mm × 50 mm × 6) of slide area per tissue excision which is immensely time consuming and impractical for real time accurate interpretation in intraoperative settings. Furthermore, because of the sparse nature of the imprint without any of the original architectural information, interpretation based purely on cytological features is required at modest to high magnification level  by a highly trained cytopathologist to identify cancerous cells from other types of debris and artifacts. For a standard field of view of 1.4 mm by 1.0 mm (10x magnification), it is expected that 5357 fields of view must be thoroughly inspected to accurately determine margin status.The challenge of the imprinting process is that it creates a specimen that may be covered by epithelial cells (including cancer cells if present), erythrocytes, leukocytes, cellular debris (organelles and cellular substructures), scrape debris, cauterized debris, and staining and drying artifacts such as bubbles, stained noncellular tissue, and so forth. A standard microscope slide (25 mm × 50 mm) is sufficient to imprint a margin surface. A reasonably sized excision tissue with 6 separate margin surfaces  would therefore require the manual search of 7500 mmTo overcome these limitations, an automated analysis has been developed to detect and identify positive cancer cells at the margin via IC. Current slide scanning technology has progressed to being able to quickly scan through slides. Additionally, the sparse nature of the IC surface implies that parallel image processing would be exploitable to analyze the entire slide. This enables the use of modern multiprocessor computing architectures to process the slides with reduced processing time in intraoperative margin assessment. Furthermore, the same technology could be employed with minor modification for core biopsy analysis or even permanent section analysis where the issues of cellular debris are much reduced.This paper reports upon the development and evaluation of an automated imprint cytology analysis tool using retrospectively collected samples to provide directly comparable performance characteristics of automated assist versus manual techniques. Experimental methods were designed to provide results which are easily transferable and applicable to an intraoperative setting without disrupting accepted surgical and pathologic protocols. This included (1) working with primary human breast tissue, (2) excision of tumors using standard electrosurgical techniques, and (3) measurement of system performance using true cauterized margins. It is noted that the most successful study of manual margin interpretation by IC employed uncauterized margins . HoweverImprint cytology specimen collection was approved by the Institutional Review Board of the University of California, San Diego and was performed in accordance with all accepted standards for human clinical research. All patients gave written informed consent. A retrospective analysis of 127 patients for whom IC slide was prepared on their excised tumors was performed to identify specimens with positive or close permanent section diagnosis. 16 positive/close margin cases were selected and an 8 slide training subset was culled which consisted of 4 slides with positive margin determination and 4 slides with close margin determination . An addiMultiple 1-inch × 3-inch glass microscope slides coated with poly-L-lysine (PLL)  were used for imprint cytology. Six (6) margin faces  oriented by suture placement during initial excision were used for imprint cytology. A no.10 scalpel blade  was placed perpendicular to the tumor surface and dragged across the tissue in a scraping fashion to remove surface cautery artifact. Immediately following scraping, a PLL-coated slide was pressed onto the scraped margin face for 10 seconds and immediately fixed in pure ethanol for 24 hours, after which the slide was stained with Hæmatoxylin and Eosin (H&E).μm/pixel) and saved as SVS files with JPEG 2000 compression. An IC image processing system was developed based on ImageJ [Whole imprint cytology slides were digitized on a ScanScope XT slide scanner  at 40x  an outlining phase in which raw pixel data was converted to objects representing nuclei, debris, and artifacts; (2) an object analysis phase in which individual objects were identified and quantified across the entire slide. The software was written and designed to learn and identify cancer cells and cancer cell clusters. Training was performed on a representative set of labeled slides selected by the authors (M. E. Ruidíaz and J. Wang-Rodriguez) which contained known tumor cell clusters, debris, adipose tissue, and white blood cells. After optimizing for cancer cells, the software algorithm was tested on unlabeled samples as a measure of system performance. μm2 and ≥360 μm2 to remove noncell like objects. (6) Object outlining: store object location and outline for measurement phase.An outlining algorithm was developed and optimized for the detection of nucleated objects from cauterized and scraped imprint cytology specimens. Briefly: Each scanned whole-slide IC slide image was processed in 2000 × 2000 pixel subtitle increments with 100 pixel overlap. Each subtitle was processed in 6 serial steps: (1) Red channel filtering: mainly to remove red blood cells from the image. (2) Laplacian of Gaussian thresholding: identify object boundaries on the image. (3) Binary shape corrections: reduce noise, smooth out and improve object boundaries. (4) Watershed filtering: separate falsely concatenated nuclei. (5) Object selection: discard objects ≤10 Individual objects were measured for 179 characteristics in the following categories. (1) Grey descriptors: grey scale intensity and texture features of the imaged object. (2) Shape descriptors: size and dimensional characteristics of outline shape. (3) Intensity and texture features within (nuclear) each object in two color models (RGB and YUV). (4) Intensity and texture features in the area immediately outside (cytoplasm) each object in RGB and YUV.MASS package), was used to create the training set for IC object classification. Recursive feature elimination was performed with the caret package. For error estimation, the errorest function was used in the ipred package. Cross validation was used to provide estimates of variable performance for classification and for the determination of which variables provided the greatest prognostic significance.Statistical analysis was performed using the R statistical computing environment version 2.13.0. Raw values were examined to elicit sources of maximal variance for data classification studies. Multivariate linear discriminant analysis  to train an LDA classifier based on the previously described classes. Additionally, worse case (randomized data) classification and full (combined descriptors) classification performance was evaluated. Accuracy was estimated by a 10-fold cross validation and relative accuracy performance improvements are displayed in To further elucidate which individual measurements provided the greatest discrimination, an analysis of the respective contribution to the overall accuracy of the classifier was performed by evaluation of each class as a binary classification by recursive feature elimination. The variable importance in decreasing order for each class is as follows: cancer , noncancer cell , debris/artifact .Validation of the analysis system was performed using an 8-case testing subset, listed in As a proof of concept, an automated image analysis tool has been developed which enables whole slide scanning microscopes with automated computational analysis to provide a method for cancer cell detection in a background of debris and noncancerous cells using a novel analysis software to evaluate multiparameter cytologic features. Analysis was performed on prelabeled training data with subsequent validation on an independent testing set, to give a sense of true system performance. The training set was based on an 8 IC slides obtained from resection margins and correlated with confirmed positive or “close” margin on permanent sections. M. E. Ruidíaz and J. Wang-Rodriguez manually identified cells belonging to the class of cancer cells, normal cells, or debris/artifact objects . In evalColorimetric information (RGB and YUV color spaces) from both the nuclear area and the cytoplasm area provides the highest degree classification performance improvement. This is reflected visually with cell-like objects, such as cancer cells and normal cells generally having a dark nuclear area with a lighter cytoplasmic surrounding, while debris/artifacts are more variable and often are within much darker stained surroundings. Furthermore, in normal cells, a well-organized clusters and extremely dark nuclear appearance corresponding to condensed chromatin are observed, while a much larger, diffuse and lighter stained nucleus is present for cancer cells.Shape information is important primarily in the separation of the debris/artifact class from the biologically derived classes due to cell-like objects having a round nuclear configuration, while generally debris/artifact objects having non-circular configurations. Therefore, a high degree of separation is observed when looking at shape alone. Additionally, it is noted that shape discrimination elucidates a degree of separation between the cancer class and the noncancer cell class. This can be explained due to cancer cells having larger and/or irregularly shaped nuclei than normal cell types.Identification of specific measurements which best separate each of the classes of objects on an IC specimen was determined by binary classification based recursive feature elimination. Analysis of the top 5 important features for class discrimination revealed the following: (1) cancer cell discrimination is primarily due to the integrated density along with the area. (2) Noncancer cell discrimination is mainly based on the degree of redness of the cell. (3) Debris/artifact discrimination also lies in the red color of the object with less emphasis on the peripheral staining color which is likely due to the lack of cytoplasmic staining of a debris/artifact object. The similar importance of the red color metrics on both (2) noncancer cells and (3) debris/artifact implies a separation is present with noncancer cells having a less red staining while debris/artifact imparts a brighter red profile.The detection of cancer cells on IC specimens which were judged to be negative (close or clear designation) based on permanent section  illustrates the utility of this analysis as an intraoperative positive tumor margin detection system and as a permanent section verification tool. However, the failure of both manual and automated analysis to identify cancer cells on margins judged positive by permanent section shows the sampling variation which limits the utility of IC specimens intraoperatively. Improved system performance is expected, with minor software modifications, when applied to various cytology specimens such as fine needle aspiration or core tissue biopsy. It is additionally expected that improvements in IC technology, such as cell adherent coatings, would further increase the number of detected positive specimens during cauterized margin analysis.The evolution of high speed slide scanning technology along with the increased ubiquity of computing resources enables a new class of automated analysis tools to discriminate between cancer cells and nonepithelial cells and debris in cytologic evaluation. The system presented in this report to has a 100% agreement between manual and automated analysis, as validated by a small sample set. The study indicates that an automated analysis approach performs well in identification of cancer cells on IC specimens with performance primarily being limited by IC sampling variability due to the heterogeneous nature of specimens with cauterized margins. This project demonstrated the proof of concept that an automated system can be successfully utilized to match human interpretation of IC especially when challenged with the difficult situation of identifying rare tumor cells picked up by IC on cauterized surgical margins. The technique is also able to identify some positive margins missed by permanent section allowing for postoperative verification by permanent section analysis. It is expected that other cell based cytologies, such as fine needle aspiration, core biopsy analysis, and even permanent section analysis could be automated with a similar algorithm.


We recently demonstrated immunophenotyping of SIVrtTA vaccinees prior to wt challenge drives global polarisation of a T cell memory phenotype, particularly in the CD4 population, toward an effector memory response.Vaccination with live attenuated SIV confers potent protection against wild-type SIV challenge in the SIV/macaque model of HIV. Although safety concerns preclude direct application of this vaccine approach in humans, a clearer understanding of the mechanistic processes at play may lead to better HIV vaccines. A novel live attenuated SIVmac239Δnef vaccine (SIVrtTA), dependent on doxycycline (dox) administration for replication, has been evaluated for its ability to protect Initially, two groups of six Indian rhesus macaques (RM) were vaccinated with SIVrtTA with an oral doxycycline dosing regime for 6 months. In Group A, doxycycline was stopped 8 weeks before homologous wild-type SIVmac239 challenge but continued in Group B. Separately, Mauritian cynomolgus macaques (CM) were vaccinated with SIVrtTA in three groups  and challenged with the heterologous wild-type SIVsmE660. Kinetics of vaccine virus replication and superinfection resistance outcomes were determined by sensitive and specific molecular and virological assays and detailed immunological studies performed.EM was associated with protection but was not necessarily essential. In Mauritian CM there was a clear correlation between persistence of the vaccine virus and protection with 6/6 protected against detectable SIVsmE660 infection after 20 weeks vaccination (dox ‘on’), with 1/6 protected after 3 weeks vaccination (dox ‘on’) and 3/6 dox ‘on’ for 3 weeks followed by 17 weeks dox ‘off’.Following wild-type challenge with either SIVmac239 or SIVsmE660, all SIVrtTA vaccinees exhibited significantly lower levels of breakthrough virus which in 2/8 SIVrtTA vaccinees challenged with SIVmac239 and 10/18 challenged with SIVsmE660 were protected from detectable wt virus. While the kinetics of vaccine virus replication differed between species, in Indian RM enhanced control of wild-type virus was correlated with a transitory shoulder of sustained plasma viral RNA load in the period up to 100 days post-vaccine delivery. On the day of challenge, a high frequency of circulating CD8+ TTogether, these data suggest a maturation of protection with longer periods of vaccination is beneficial, even when the vaccine-virus is apparently ‘switched-off’. Since the genetics and antigenic relatedness of vaccine and challenge virus does not appear to predict the outcome of these vaccine studies, the kinetics and relative persistence of vaccine virus replication in contributing to adaptive and innate immune responses are being evaluated. The outcome of these studies will inform rational HIV/AIDS vaccine design