During the last several decades, the number of children who are overweight or obese has reached alarming levels worldwide. The purpose of the present study was to examine trends in measures of childhood obesity among Korean children aged 2–19 from 1998 to 2012.Height, weight, and waist circumference (WC) were measured, and body mass index (BMI) was calculated. Age-adjusted means of WC and BMI were compared between years. We used three international criteria  and a Korean national reference standard (Korea Centers for Disease Control and Prevention [KCDC]) to calculate age-standardized prevalence of childhood overweight and obesity.Despite differences in absolute prevalence of childhood overweight and obesity according to the four different criteria, the time trends of prevalence were generally similar across criteria. The prevalence of childhood overweight and obesity generally stabilized from 2001–2012 in both boys and girls. WC decreased from 2001–2012 in both boys and girls aged 2–19.Further studies exploring the factors causing plateaued trends of childhood obesity measures are needed to implement effective policies for reducing the prevalence of childhood overweight and obesity. However26 which used repeated cross-sectional nationally representative samples of 2- to 19-year-old Korean children and adolescents. In this study, we additionally present changes in WC and BMI Z-score over the years and update the recent prevalence of childhood overweight and obesity from 1998 to 2012, employing four different criteria: those of the IOTF, the U.S. CDC, WHO, and Korea Centers for Disease Control and Prevention (KCDC).This study is an extension of the above-mentioned study,27 The response rates for the K-NHANES were 86.5% in 1998, 77.3% in 2001, 70.2% in 2005, 74.5% in 2007–2009, and 76.5% in 2010–2012. Additional details of the study design and methods are described elsewhere.27,28Data were obtained from five waves of the Korean National Health and Nutrition Examination Survey (K-NHANES) conducted in 1998 (the first wave), 2001 (the second wave), 2005 (the third wave), 2007–09 (the fourth wave), and 2010–12 (the fifth wave). The participants in the K-NHANES were drawn as multi-stage clustered probability samples from Korean households representing the civilian, non-institutionalized population.A total of 18 174 participants (9493 boys and 8681 girls) aged 2–19  were available in the health examination survey, which included anthropometric data for each participant. In the 1998 K-NHANES, we were only able to use data from boys and girls aged 10–19, because the health examination survey including anthropometric measurement had not been conducted among children aged 2–9. Detailed numbers of study subjects by survey year, gender, and age groups are presented in 28 Body weight was measured to the nearest 0.1 kg on a calibrated balance-beam scale while the participants wore a lightweight gown or underwear. Height was measured in the upright position to the nearest 0.1 cm using a stadiometer. WC was measured at the midpoint between the lowest rib and the top of the iliac crest on the mid-axillary line to the nearest 0.1 cm with measurement tape placed horizontal to the floor without indenting the skin. BMI Z-scores were obtained using the method recommended by the U.S. Centers for Disease Control and Prevention.19Anthropometric measurements  were conducted with the same protocols and anthropometric measurement instruments for all waves of the K-NHANES.2) and obesity (30 kg/m2) from children living in six countries. We used IOTF cut-points at age 18, but for age 19, we used the same cut-points as for adults (overweight ≥25 kg/m2 and obesity ≥30 kg/m2), because no specific BMI cut-points have been established for age 19 in the IOTF criteria.20 The U.S. CDC growth charts were derived from five national surveys conducted between 1963 and 1994 in the U.S. The U.S. CDC defined overweight as BMI ≥85th percentile and obesity as BMI ≥95th percentile.19 The WHO criteria were generated from data collected from the WHO Multicentre Growth Reference Study (MGRS) in six countries.29 For ages less than 5, BMI values above +2 standard deviations are considered overweight, and values above +3 SD are considered obese.30 For ages 5–19, above +1 SD is equivalent to the overweight cut-points used for adults (≥25.0 kg/m2) and above +2 SD corresponds to the cut-points for obesity (≥30.0 kg/m2).21 The KCDC criteria also used age- and gender-specific BMI values and defined the 85th percentile as the cut-point for “overweight” and the 95th percentile as the cut-point for “obesity”.31 The KCDC criteria were based on the National Growth Survey for Korean children in 1997 and 2005, when increase in childhood overweight and obesity might have already begun. We used four different sets of criteria for childhood overweight and obesity based on BMI measurements: those of the IOTF, U.S. CDC, WHO, and KCDC. The IOTF criteria were adapted based on the extrapolation of adult BMI cut-off points for overweight  after taking into account the primary sampling units, stratification, and sample weights for K-NHANES. To analyze changes in the age-adjusted prevalence  of childhood overweight and obesity, we employed the direct standardization method using the 2010 Korean Census population as the standard population. Primary sampling units, stratification, and sample weights were also taken into account in this standardization. In this study, the prevalence of overweight includes the prevalence of obesity. We then used logistic regression analyses (PROC SURVEYLOGISTIC in SAS) to estimate P values for trends in the age-adjusted prevalence, after accounting for primary sampling units, stratification, and sample weights of the K-NHANES.Analyses were conducted according to sex (boys and girls) and age groups (2–9 and 10–19 years). All statistical analyses were performed using SAS version 9.1 . We analyzed changes in the age-adjusted least square mean (standard error) of BMI, BMI Z-score, and WC and presented P for trend = 0.005). Among boys aged 10–19, the increase in BMI was statistically significant from 1998–2012 (P for trend < 0.0001) but not from 2001–2012 (P for trend = 0.102). No significant increase in BMI was detected among girls aged 2–9 and 10–19. The increase in BMI Z-score among boys aged 2–9 from 2001–2012 was significant (P for trend = 0.011). Among boys aged 10–19, the increase in BMI Z-score from 1998–2012 was statistically significant (P for trend = 0.002), while the trend from 2001–2012 was not significant (P for trend = 0.090). Meanwhile, a significant decrease in BMI Z-score from 2001–2012 among girls aged 10–19 was observed (P for trend = 0.021) (Table Table Table P for trend = 0.032). Meanwhile, when the 1998 data were included in the time trend analysis, increasing trends of childhood overweight and obesity were statistically significant for all four sets of criteria among boys aged 10–19 (Table Table P for trend = 0.035) (Table Table 5) Table .26 but included more recent data from 2008–2012 and additionally examined trends in WC and BMI Z-score. BMI Z-scores and the changes in the BMI Z-score distribution are useful for assessing a population-wide shift in childhood adiposity over time.32This study examined trends in measures of childhood obesity from 1998–2012 using a representative sample of Korean children aged 2–19. This study is an extension of our prior study26In our calculations of age-standardized prevalences of childhood overweight and obesity, we also took into account primary sampling units and stratification, in addition to sample weights, and used more recent 2010 Korean Census data for standardizing the population, which resulted in slightly different prevalences than were presented in the prior study.33 Similarly, decreasing or plateaued trends in WC were observed in Brazilian and Spanish adolescents during the last decade.18,34 In contrast, WC and general obesity prevalence increased among Chinese children and adolescents aged 6–17 from 1993–2009.17The results of this study showed decreasing trends in WC from 2001–2012 in both boys and girls and for ages 2–9 and 10–19. Time trends in childhood WC have been explored in a few studies, but most of these studies compared only two time points. A study conducted in Switzerland showed a stabilized prevalence trend for the 85th and 95th percentiles of WC among boys and girls aged 6–12 from 1999–2012.Despite the decreasing trends in WC from 2001–2012 in both boys and girls aged 2–9 and 10–19, BMI Z-scores presented different trends by gender and age groups. BMI Z-scores plateaued among boys aged 10–19 and girls aged 2–9, decreased among girls aged 10–19, and increased among boys aged 2–9 from 2001–2012. Considering that body weight and height plateaued from 2001–2012 among boys aged 10–19 and girls aged 2–9 see , these rIn this study, we used four different sets of criteria for the prevalence of childhood overweight (including obesity) and obesity. The IOTF criteria resulted in the lowest prevalence of obesity for both boys and girls at ages 2–9 and 10–19, while the WHO MGRS criteria for boys and the KCDC criteria for girls produced the highest prevalence of obesity among the four sets of criteria. The absolute prevalence differences of childhood obesity were generally the smallest between the WHO MGRS and the CDC criteria compared to the prevalence differences using the other two criteria in both boys and girls and children aged 2–9 and 10–19. In contrast, the IOTF and WHO MGRS criteria produced the biggest differences in obesity prevalence among boys aged 2–9 and 10–19, whereas the IOTF and the KCDC criteria yielded the biggest difference in obesity prevalence among girls aged 2–9 and 10–19. These results from the four sets of criteria were expected, considering their use of different BMI cut-points for childhood obesity and overweight see .35,36 if different criteria were used. When we explored trends of overweight (including obesity) and obesity from 2001–2012, the results showed that the prevalence of overweight and obesity stabilized according to all four sets of criteria among boys and girls, at ages 2–9 and 10–19, except for the trend in obesity prevalence according to WHO MGRS criteria among boys aged 10–19. However, when we examined trends among boys and girls aged 10–19 from 1998–2012, overweight and obesity among boys according to all four sets of criteria and obesity among girls according to the IOTF criteria showed increasing trends. These results suggest that the prevalence of overweight and obesity among boys aged 10–19 might have increased from 1998–2001, and then stabilized from 2001–2012, while the prevalence of overweight and obesity among girls aged 10–19 generally stabilized across all the years surveyed.Despite the inconsistencies in the absolute prevalence of childhood overweight and obesity according to the four sets of criteria, time trends were similar for all four. When different childhood overweight and obesity criteria are used, different prevalences of childhood overweight and obesity will inevitably result. However, it was uncertain how the time trends of childhood overweight and obesity would compare when different cut-points were used. The present findings suggest that analyses using different criteria are not crucial to determine time trends of childhood overweight and obesity. In addition, similar time trends in childhood overweight and obesity would have been produced in other studies37 Similarly, stabilized trends have been observed in other countries, such as Australia,38 France,39 Greece,40 and Germany.41 Recent plateaued trends of childhood obesity may be explained by public awareness of childhood obesity and interventions at both the individual and policy levels. Stamatakis and colleagues have suggested that massive media attention on obesity and widespread health concerns, as well as policies aimed at reducing obesity, may be related to the stabilization of obesity in England.37 Olds and colleagues have indicated that interventions on physical activity and nutrition at the home, school, community, state, and national level resulted in a flattening prevalence of obesity in Australia.38 In Korea, Park and colleagues have argued that regular weekly exercise increased from 2001–2005 among Korean adolescents aged 10–19.42 Changes in early life nutritional factors in recent decades may also explain plateaued trends in childhood obesity.43 Wabitsch et al have noted that childhood obesity rates have stabilized in developed countries and that the possible causes of these trends are the cumulative effects of interventions, such as reducing television viewing duration and consumption of energy-dense foods, as well as increasing daily physical activity.44 In addition to the aforementioned explanations, selection bias may play a role in the observed stabilization of obesity rates. As Marild et al have suggested,45 if people with a higher prevalence of overweight and obesity have tended to avoid participating in more recent surveys, plateaued or even decreasing prevalences of childhood overweight and obesity may result. Further studies examining these possibilities should be conducted to explain the plateaued or even decreasing prevalence of childhood obesity found in this study.A plateaued trend in childhood obesity has been observed in recent years in many developed countries. For example, in England, the prevalence of overweight and obesity among children aged 5–10 increased from 1997–2002 and then stabilized from 2002–2007.The present study has several strengths. First, we used five sets of nationally representative data, which contained consistent anthropometric measurements of height, weight, and WC examined by the same protocols and anthropometric measurement instruments from samples of 2- to 19-year-old Korean children. Second, to the best of our knowledge, this is one of the first studies to compare the trends of childhood overweight and obesity using four different sets of criteria. The time trend of childhood obesity according to WHO MGRS criteria showed a significantly decreasing trend among boys aged 10–19, while the other criteria did not present such significant trends. This study also presented dissimilar prevalences of childhood overweight and obesity according to these criteria.However, our study also has several limitations. First, since K-NHANES began in 1998, we could not examine childhood overweight and obesity trends before 1998. Second, we could not use anthropometric data for children aged 2–9 for the 1998 K-NHANES, because this age group was not included in the first wave of K-NHANES. Third, the possibility of selection bias associated with declining survey participation rates among overweight and obese individuals should be considered in interpretation of the results.46 This study showed that the prevalence of overweight and obesity among Korean children stabilized over the last decade and that a decreasing trend in WC was observed. However, there is scarce evidence explaining these recent trends toward stabilization among Korean children. We surmise that increased awareness of childhood obesity, which leads to improved quality of dietary intake and physical activity, could be one possible explanation. Considering the still-high absolute prevalence of childhood overweight, further studies exploring factors causing trends to level off are needed to implement effective policies for lowering rates of childhood overweight and obesity.In summary, we examined trends in childhood overweight and obesity in nationally representative samples of South Korean children from 1998–2012 using four different sets of criteria for childhood overweight and obesity. The results indicate that the absolute differences in childhood overweight and obesity prevalence by different criteria were relatively large, although the time trends of these prevalences were generally similar among the criteria. Thus, clarification of the criteria and use of two or more sets of criteria in determining the prevalence of childhood overweight and obesity should be considered when reporting such prevalence measures to the public and monitoring the prevalences and time trends of childhood overweight and obesity to avoid any potential confusion. Moreover, investigations into health outcomes associated with these different cut-off points are of critical importance.eTable 1. eTable 2. eTable 3. eFigure 1.


Poecilia parae, a fish species with five distinct male color morphs: a drab , a striped, and three colorful  morphs. Then, using physiological models, we assessed how male color patterns can be perceived in their natural visual habitats by conspecific females and a common cichlid predator, Aequidens tetramerus. Our estimates of chromatic and luminance contrasts suggest that the three most colorful morphs were consistently the most conspicuous across all habitats. However, variation in the visual background resulted in variation in which morph was the most conspicuous to females at each locality. Likewise, the most colorful morphs were the most conspicuous morphs to cichlid predators. If females are able to discriminate between conspicuous prospective mates and those preferred males are also more vulnerable to predation, variable visual habitats could influence the direction and strength of natural and sexual selection, thereby allowing for the persistence of color polymorphisms in natural environments.The conspicuousness of animal signals is influenced by their contrast against the background. As such, signal conspicuousness will tend to vary in nature because habitats are composed of a mosaic of backgrounds. Variation in attractiveness could result in variation in conspecific mate choice and risk of predation, which, in turn, may create opportunities for balancing selection to maintain distinct polymorphisms. We quantified male coloration, the absorbance spectrum of visual pigments and the photic environment of  The expression of exaggerated male traits, such as colorful ornaments or elaborate songs, often evolves under conflicting selective pressures The perception of elaborate signals depends on the physical properties of the habitat  and the sensory parameters of the receivers assessing the signals Although there is an established association between the photic environment and sensory physiology e.g., , only fePoecilia parae exhibit five discrete Y-linked color morphs  parae, the largest males that exhibit a striped tail and black vertical body bars that intensify during social interactions, and the (iii) blue, (iv) red, and (v) yellow males with colorful body flanks and intermediate body size Males of the poeciliid fish r morphs  [34]. ThP. parae is stable across time −1 m−2 mimicking natural, clear day light spectrum) revealed repeatable and variable female mating preferences for the colorful red, yellow and blue morphs. That is, most test females strongly preferred either red or yellow males but a few showed consistent preferences for blue males Aequidens tetramerus, a common visual cichlid predator of P. parae, exhibited a visual bias for red and yellow males, suggesting that selective predation may offset the mating advantages of red and yellow males thus keeping them relatively rare in natural populations Field and laboratory studies suggest that the male color polymorphism in Poecilia parae, one of the most common poeciliids inhabiting freshwater coastal areas and did not involve any endangered or protected species.The research was conducted under the Syracuse University Institutional Animal Care and Use Committee (IACUC) protocol number 06-014. Permit for field studies and collection of live individuals was issued by the Environmental Protection Agency, Republic of Guyana (Ref. 111207 BR 086). The present study only included the use of P. parae is found. Although both populations are contiguous, the east and west populations are separated by the Demerara River (ca. 1851 m wide), the third largest river in the Republic of Guyana that likely reduces gene flow between individuals of P. parae inhabiting the east and west populations. In January 2006, we established 15 permanent sampling sites  at each population. Sampling sites were ca. 100–150 m apart from each other. From January to August 2006, we recorded the relative abundance of the five P. parae male color morphs, and that of their common predator, Aequidens tetramerus at 15 sites in the east and 15 sites in the west study populations. In January 2010, with the exception of individuals used for reflectance measurements (see below), males and females of P. parae were collected from the sampling sites and were transported to our laboratory at Syracuse University. Fish were maintained in 20 gal aquaria with treated water at 27±1°C, on a 12∶12 h light: dark cycle, and fed daily with live brine shrimp and Tetra-Min  flakes two times per day until used for microspectrophotometry analysis.Study populations were located on the east  and west  sides of the Demerara River, Republic of Guyana . The anaesthetized fish was then placed on its right flank on ice separated by a wet cloth, and its color patches on the flanks were measured using a portable spectrophotometer. We focused on color patches that are conspicuous, used during courtships and preferred by females  and 10 adult females were used for microspectrophotometry (MSP) analyses at Cornell University, Ithaca, New York. Methods for MSP readings are described in detail in ©, USA). All samples were prepared under infrared illumination  using image converters. The eyes were hemisected and the retinae removed while in buffer solution (cold phosphate buffer solution pH 7.4 supplemented with 6% sucrose). Retinae were then separated from the retinal pigment epithelium and macerated using razor blade fragments and tungsten needles 2 rectangular measuring aperture was produced by demagnification using a Leitz  180X quartz mirror objective. A Zeiss 100X Ultrafluar (0.85 NA) collected the transmitted light and focused it onto the photomultiplier photocathode. Retinal cells were selected individually. Determination of double cones was based on intact pairs of examined cells only. MSP scans were collected at 1 nm intervals scanning from 750 to 350 nm, and back from 350 to 750 nm To characterize the spectral sensitivity of max  smooth the data, (ii) determine the peak absorbance (Xmax), (iii) normalize the absorbance curve, (iv) fit the templates, (v) calculate the standard deviation of λmax, and (vi) compare with the actual data and choose the best fit Xmax) used in the normalization represented the calculated maximum of the best fit Gaussian to the data points 20 nm either side of the estimated-by-eye absorbance maximum of the alpha band. Using Xmax, the data were then normalized using standard methods 1 templates We used template fitting to determine the λP. parae was common and all male color morphs present. Measurements were taken early in the morning (600–800 h) when male and female P. parae are most socially active P. parae inhabits shallow waters and most social and foraging activities occur within this depth We collected irradiance  and radiance  scans at all sampling sites (east: n = 15 sites and west: n = 15 sites), where P. parae was observed or likely to interact with females. The aim of these radiance scans was to measure the average visual background against which an individual would be perceived by a conspecific or predator. To allow for direct comparison among scans, the spectrophotometer fitted with the irradiance and radiance probes was calibrated with a standard light source  so that only light from a small solid angle can reach the detector surface. Radiance scans were taken every 15° angle until completing a semicircle, starting at an approximate location where an individual of  = 15) and west populations (n = 15).To characterize the spectral distribution of the ambient light and background for each sampling site in the east and west populations we calculated the spectral index λp50, the wavelength that halves the total number of photons between 300–700 nm S) and luminance (L) contrasts of male color morphs as viewed through the eyes of P. parae conspecifics, we used the (i) reflectance spectra of a color patch  raP. parae has seven types of cone photoreceptors located within single and double cones (see results section). Because it remains unclear if all seven of these cones are used in color or luminance discrimination, and if the double cones interact, we first modeled the visual system of P. parae as pentachromatic, assuming that double cones are neurally linked and able to operate as a single receptor channel  the relative frequencies of the cone classes and (ii) estimation of the Weber fraction for LWS cones in S) and luminance (L) contrasts of male color phenotypes as estimated by penta- and hexachromatic eye models using one-way ANOVAs. The pentachromatic model should represent a visual system with a limited subset of MSW/LWS photoreceptors compared to a hexachromatic model. Thus, we ask whether the exclusion of a MSW/LWS visual photoreceptor  will result in differences in color and luminance discrimination. We did not find any significant differences in color  and luminance  discrimination between pentachromatic and hexachromatic eye models. For simplicity, we therefore use the ΔS and L estimations based on hexachromatic eye for exploring the role of variable visual habitats in mediating variation in perceived conspicuousness of male signals (see below).We evaluated the results of chromatic (ΔS) and luminance (L) contrast of the five morphs and females using nested ANOVAs. The independent variables included: population , sites  and ‘morphs’ . The dependent variables were the color (ΔS) and luminance (L) contrasts estimated for each male color morph. ‘Population’ and ‘morph’ were considered as fixed factors given that we sampled two populations in Guyana, and that all male color morphs were present at all sampling sites. ‘Sampling sites’ were nested within ‘population’ and considered as a random factor. We used Fisher’s LSD pairwise tests to test for posthoc differences in the perceived conspicuousness or luminance of P. parae color morphs by conspecifics.To test for perceived differences in conspicuousness of male color morphs within- and between populations by conspecifics, we used the 355–407–456–526–533–543 hexachromatic eye. We compared the color  whether predators may play a role in mediating selection. Using the Vorobyev-Osorio trichromatic visual model S) and luminance (L) contrasts of male color morphs against natural backgrounds as viewed through the eyes of a cichlild predator.Cichlid fishes are common visual predators of colorful poeciliids and so oppose positive sexual selection e.g., . CurrentA. tetramerus cones, we used published data from A. pulcher, a closely related, voracious predator of adult guppies in Trinidadian streams A. pulcher cone spectral sensitivity: SWS = 453 nm; MWS = 530 nm; LWS = 570 nm  contrast values of the particular male color morph at the sampling sites’ visual environments perceived by conspecifics or predators, as well as the relative abundance of predators. Morph frequency and predator abundance data were arcsine square root and log transformed, respectively. For each of the models, we inspected the variance inflation factors (VIF), which indicated little evidence of collinearity. All variables included in the model did not deviate significantly from normality .As an indirect assessment of the effects of variable mate choice and predator susceptibility on the frequency of male color morphs, we constructed a backward, stepwise multiple regression model for each of the five morphs using morph frequencies as the dependent variable and the estimates of male conspicuous to conspecifics and predators as possible predictors. Morph frequency was estimated by dividing the total number of males counted for each morph by the total number of all males counted at each sampling site. As predictor variables, we used the color (ΔS) and luminance (Poecilia parae  fit well with vitamin A1 pigment templates. Our MSP study revealed the presence of seven spectrally distinct types of cone photoreceptor arranged either as single or double cones. Most notably, the retina of Poecilia parae contains single cones with UV absorption at 355±1.4 nm  and a violet/blue at 407±1.6 nm . Values for λmax of double cones ranged from 456 to 553 nm: 456±1 nm , 526±0.8 nm , 533±2 nm , 543±1 nm , and 553±1.9 nm . The different types of double and twin cones were commonly found as 456/526 nm, 456/533 nm, 456/543 nm, 526/533 nm, 533/533, 543/543, and 553/553. Rod cells were also observed and had an estimated λmax 503±1.2 nm . Example MSP absorbance curves for these photoreceptor types are provided in The absorbance spectra of all the cone classes and individual rod cells from P. parae visual habitats are shown in F1,28 = 2.78, p = 0.11). However, the λp50 of the visual background (radiance) significantly differed between the east (545±2.68 nm) and west (562±4.45 nm) populations , indicating more yellow shifted light in the west compared to the east population. The intensity of ambient light  and visual background  was consistently lower in the east population  populations indicate significant variation in the conspicuousness of each male morph at each site, suggesting a strong effect of the ambient light and/or visual background. For instance, in the east population, blue males were more conspicuous than red males in localities 4, 7, 11, 12 and 13, while red males were more conspicuous than blue males in localities 2, 5, 6, 8, 10 and 14  and adult females , which is consistent with the hypothesis that immaculata males are perceived as females by conspecifics When viewed through 0 and 14 . No signP. parae females are also more conspicuous to cichlid predator, as estimated by perceived color  and between  the east and west populations. Moreover, in ca. 45% of sampling sites in the east  found also in guppies and other poeciliids max of 512 nm, whereas the S180 LWS may have a λmax of 560 nm. From these two opsins only the latter appears to differ among populations P. parae.At the molecular level, there is evidence indicating that Xiphophorus spp) appear to work as a ‘private channel’ of communication between conspecifics, as predators do not see well in the UV spectrum Chrenicicla frenata) lacks SWS1 opsin; therefore, this species may be relatively insensitive to the UV visual spectrum Poecilia parae remains an interesting open question to be resolved.Courtship traits are expected to evolve under conflicting selective forces. Sexual selection should favor the evolution of conspicuous traits, whereas natural selection via predation should agonistically work to decrease trait conspicuousness P. parae, laboratory experiments of predator preferences show consistent bias for the carotenoid-based red and yellow males, which are also attractive to females  are strongly preferred by females as potential mates under standardized laboratory conditions. Red and yellow males, however, are also preferentially targeted by visual predators under the same visual conditions P. parae color morphs in nature.Combining our current results with our previous work Poecilia reticulate the visual background varies with the lighting environment in natural streams, affecting the spatial and temporal operation of sexual selection. This, in turn, generates opportunities for the maintenance of quantitative variation in male coloration Telmatherina sarasinorum, yellow and blue males are highly abundant in habitats that enhance their contrast from the visual background, which presumably augments their reproductive fitness P. parae, conspecifics perceive red males as the most conspicuous color morph, followed by blue then yellow males. However, variation in the visual habitat results in the blue morph being more conspicuous than red and yellow males, which in turn, predicts their relative abundances across sampling sites. Sexual selection favoring the most conspicuous males can therefore favor different color morphs under variable lighting conditions. That is, variation in the visual environment could lead to changes in the strength and direction of sexual selection. Our study therefore suggests an important role for environmental heterogeneity in favoring the maintenance of striking color polymorphisms.Recent studies provide support for the role of the visual background in generating and/or maintaining color polymorphisms by altering the direction of natural and sexual selection. In the guppy Figure S1Example absorbance spectra for visual pigments of the rod photoreceptor cells of Poecilia parae. The raw absorbance spectra, derived by MSP, are overlain with smooth curves calculated from the best-fitting A2-type chromophore visual pigment curve.(DOCX)Click here for additional data file.


Associated anomalies have been reported in around 20% of Hirschsprung patients but many Authors suggested a measure of underestimation. We therefore implemented a prospective observational study on 106 consecutive HSCR patients aimed at defining the percentage of associated anomalies and implementing a personalized and up-to-date diagnostic algorithm.After Institutional Ethical Committee approval, 106 consecutive Hirschsprung patients admitted to our Institution between January 2010 and December 2012 were included. All families were asked to sign a specific Informed Consent form and in case of acceptance each patient underwent an advanced diagnostic algorithm, including renal ultrasound scan (US), cardiologic assessment with cardiac US, cerebral US, audiometry, ENT and ophthalmologic assessments plus further specialist evaluations based on specific clinical features.Male to female ratio of our series of patients was 3,4:1. Aganglionosis was confined to the rectosigmoid colon (classic forms) in 74,5% of cases. We detected 112 associated anomalies in 61  patients. The percentage did not significantly differ according to gender or length of aganglionosis. Overall, 43,4% of patients complained ophthalmologic issues , 9,4% visual impairment, 20,7% congenital anomalies of the kidney and urinary tract, 4,7% congenital heart disease, 4,7% hearing impairment or deafness, 2,3% central nervous system anomalies, 8,5% chromosomal abnormalities or syndromes and 12,3% other associated anomalies.Our study confirmed the underestimation of certain associated anomalies in Hirschsprung patients, such as hearing impairment and congenital anomalies of the kidney and urinary tract. Subsequently, based on our results we strongly suggest performing renal US and audiometry in all patients. Conversely, ophthalmologic assessment and cerebral and heart US can be performed according to guidelines applied to the general population or in case of patients with suspected clinical features or chromosomal abnormalities. This updated diagnostic algorithm aims at improving overall outcome thanks to better prognostic expectations, prevention strategies and early rehabilitation modalities. The investigation of genetic background of patients with associated anomalies might be the next step to explore this intriguing multifactorial congenital disease. Hirschsprung’s disease (HSCR) is a congenital multifactorial developmental disorder of the enteric nervous system characterized by the absence of ganglion cells in the hindgut with variable distal bowel involvement. This is a rare disease, which occurs with an incidence of 1 into 5000 live births as a consequence of premature arrest of cranio-caudal migration of neural crest derived neuroblasts (NCN) in the hindgut, and is therefore regarded as a neurocristopathy . The neuAssociated anomalies have been detected in between 20% and 30% of HSCR patients according to literature ,9,10. InAll consecutive HSCR patients who were admitted to Giannina Gaslini Research Institute between January 2010 and December 2012 were eligible for this prospective observational cross-sectional study. Inclusion criterion was the presence of a reliable diagnosis of HSCR achieved with adequate pathological assessment . Exclusith of October 2009. After enrolment, patients and parents were interviewed to collect demographic data, personal and family history and any information regarding known associated anomalies. All families were asked to sign a specific Informed Consent form and in case of acceptance each patient underwent an advanced non-invasive diagnostic algorithm, including ultrasound scan (US) of the kidney and urinary tract, cardiologic assessment with cardiac US, audiometry and ENT assessment, ophthalmologic assessment, cerebral US (< 1 year-old), and further specialist investigations based on clinical features. All these non-invasive investigations were performed according to widely accepted guidelines. The following data were collected according to the Personal Data Protection Act:This study received Institution Review Board approval on the 15• Demographics• Personal and family history.• Length of aganglionosis [8]. ○ S-HSCR (aganglionosis extending up to the left descending colon). ○ L-HSCR . ○ TCSA (aganglionosis involving the whole colon). ○ TIA (less than 20 cm of normoganglionic bowel).• Associated anomalies detected during the phenotype screening.• Other associated anomalies or syndromes not included in the phenotype screening.All patients underwent renal US + urinalysis and colture. In case of positive findings we applied widely accepted diagnostic guidelines to properly investigate the patients . To perfThe cardiovascular screening included personal medical history, physical examination, non invasive blood pressure measurement, a twelve lead electrocardiogram and echocardiogram. Detailed US was performed with a pulsed, continuous, and color-Doppler provided US system (Philips iE33), using a 5 or 8 MHz transducer. Echo-scan was done by a transthoracic approach with the patient in the supine and left decubitus position. Morphological variables were measured offline by two observers (G.T. and M.D.) who used the so called Z score for normalization of the cardiac structures dimensions to the body size -14.Cardiac anatomy was routinely assessed by a segmental approach that included the abdominal view, the subcostal short and long axis view, the apical four-chambers view, the parasternal short axis view at the level both of the papillary muscles and of the mitral valve, the parasternal long axis view and the suprasternal view. Two-dimensional echocardiographic measurements included the end-systolic diameter of the aortic annulus of the Valsalva sinuses and of the ascending aorta. These dimensions were measured from the parasternal long-axis view with the “inner edge convention”, which uses the innermost bright edge reflection as a contour. End-diastolic posterior wall thickness, left ventricular internal dimension and interventricular septal thickness were measured through the M-mode technique, which was performed also for calculating the shortening fraction. Diastolic ventricular function as well as valves function was assessed by both pulsed and continuous Doppler technique -18.We assessed the possible presence of audiologic risk factors as those indicated by the Joint Committee of Infant Hearing, including in-utero infections, craniofacial anomalies, physical findings suggesting syndromes known to include a sensorineural or permanent conductive hearing loss, neonatal intensive care (NICU) admission longer than 5 consecutive days, exposure to ototoxic medications, family history and prematurity . The tes• From birth to 6 months of age - We used the Automated Auditory Brain System (AABR). This methodology measures cochlear response in the 1 to 4-kHz range with a broadband click stimulus in each ear. The automated screener provides a pass-fail report; no test interpretation by an audiologist is required. A “fail” report on an AABR implies a new non-automatic ABR test associated to ENT clinical assessment.• From 6 months to 3 years of age - We used the Visual Reinforcement Audiometry (VRA), a behavioural test measuring responses of the child to speech and frequency-specific stimuli presented through speakers or insert earphones.• From 3 years to 6 years of age - We used the Conditioned Play Audiometry (CPA) through earphones; in this test the child is conditioned to respond when stimulus tone is heard, such as to put a peg in a pegboard or drop a block in a box.• From 6 years of age onwards - We used the Conventional Audiometry (CA) in which the patient is instructed to raise a hand in case of stimulus is heard. Deafness or hearing impairment were defined and graded according to widely accepted international standards.We recorded detailed information regarding family history and any risk factors for visual impairment, including length of stay in NICU and gestational age and weight at birth. Methodology differed according to the age. Visual impairment (VI) was defined as uncorrected visual acuity (VA) < 20/50 and was assessed only in patients older than 36 months of age. In younger patients we addressed strabismus or other VA indirect measures.• Before 3 years of age, we performed only an objective evaluation due to the lack of child compliance. After a good visual inspection to exclude eye and eyelid malformations, anatomic abnormality, evident strabismus, anomalies of eye motility and head posture, we assessed: ○ Anterior segment and pupillary evaluations. The anterior segment was evaluated using a handheld or stand-mounted slit lamp. Pupillary responses were tested with a hand light. ○ Cycloplegic autorefraction and keratometry, with Plus Optix autorefractor  or Retinomax autorefractor, . Refractory defects were defined and graded according to widely accepted international standards [22]. ○ Fundus with cycloplegia.• After 3 years of age, throughout adolescence and adulthood we also performed visual acuity assessment, orthoptist screening with distance acuity test, cover test, extrinsic ocular movements, prism test, and Lang-stereotest. Moreover, we also looked for the presence of amblyopia. In this age group we resorted to subjective components thanks to reasonably good patients’ compliance.Patients were divided into three different age groups: younger than 3 (infants), between 3 and 6 (pre-school children), and older than 6 years of age (school children) to compare our results to literature data -24.Studies were performed with convex and linear-array 7,5 MHz transducers via the anterior fontanel in coronal and sagittal planes. Further screening images were obtained via other supplemental fontanels (posterior and mastoid). The investigation was performed in all patients younger than 12 months and/or with adequate echoic windows. We assessed subarachnoid spaces, lobes, ventricles and midline structures to rule out major congenital malformations .Further investigations were performed basing on clinical features detected during the first detailed physical examination aimed at ruling out major gross abnormalities, including skull and facial malformations, cleft lip or palate, gross skeletal malformations, skin pigmentation disorders, external genitalia abnormalities, neurodevelopmental delay, etc. All information regarding other associated anomalies or syndromes reported by parents or patients were recorded. In case of need, patients were investigated in details according to widely accepted standards of care. In particular, chromosome analysis, array-CGH or direct sequencing of targeted genes was performed basing on genetist’s suggestion after appropriate dysmorphology assessment.Chi-square test. Comparison of continuous data was performed using the 2-tailed unpaired t test. In case of scant data or non-normal distribution, non parametric tests (Man-Whitney) were used. A p value lower than 0.05 was considered as statistically significant. Analyses were performed using Stata for Windows statistical package .Descriptive statistics were reported as percentages with 95% confidence interval (CI), when indicated, in case of categorical variables. Median and range was used for age, given the wide variability in our series. Differences in the frequencies of each categorical variable were evaluated by the p = 0,8209 and 1,0000, respectively). Twenty-eight patients  were already aware of their association when they were enrolled in the study  patients met the inclusion criteria, were enrolled and underwent the entire phenotype screening during the study period. Median age was 2,4 year (range 3 months to 25 years). Male to female ratio was 3,4:1. Nineteen patients  had ultralong HSCR (either TCSA or TIA), 8  had L- HSCR, and 79  had S- HSCR. A total of 112 associated anomalies were detected in 57,5%  of the patients (61 out of 106) with a proportion that did not significantly differ either according to length of aganglionosis or gender  or blindness was recorded. Refractive error or ophthalmologic issues were detected in 43,4%  of the patients  of the patients. Male to female ratio was 4,5:1. One patient had a clear familial history of CAKUT. We observed a total of 26 abnormalities with a slight right-sided preponderance   of the patients  of the patients with slight female preponderance. Two further patients  had congenital preauricular fistulas (Ear pit)  of the patients . No other major brain abnormalities were detected. We identified ventricle asymmetry or subarachnoid spaces dilatation in 7 patients (16%) all with left side involvement. Moreover, we detected a mild hydromielic spinal cavity in a patient who underwent MRI for other reasons and congenital thalamic calcifications of unclear aetiology in another.A further 18 associated anomalies have been detected in 12,3%  of the patients from our series . Amongst the various associated anomalies detected in our study outside the phenotype screening, endocrinologic and/or metabolic issues accounted for 4,7% of the patients 5/106), gastrointestinal abnormalities for 2,8% (3/106) and genital abnormalities and tumors (FMTC) for 1,9% each (2/106). One patient with severe developmental delay and one with corpus callosum agenesis underwent molecular genetic analysis of ZFHX1B/SIP1 to rule out Mowat-Wilson syndrome. See Table , gastroiChromosomal abnormalities or known syndromes  have been detected in 9,4%  of the patients  had ≥ 2 associated anomalies or chromosomal abnormalities. Twelve  had ≥ 3 and 3  had ≥ 4. The latter patients all had known syndromes .This is the first prospective observational study aimed at defining the spectrum of possible phenotypes in a large cohort of HSCR patients. A reason why none previously addressed this issue probably relies on the rarity of the disease and on the difficulty to organize and perform such complex multidisciplinary prospective clinical assessments. In fact, literature reports regarding associated anomalies in HSCR are merely retrospective assessments of surgical series or systematic literature reviews ,9,10.The results of our study are remarkable and confirmed a measure of underestimation already suggested by some -10,26,27Delayed diagnosis or mild symptoms can explain the reason for such a discrepancy. In fact, refractive anomalies or visual impairment (VI), mild CAKUT or unilateral hearing impairment (HI) can be diagnosed with significant delay in the absence of specific screening programmes ,19,28.With regard to phenotype considerations, our study confirmed the high percentage of CAKUT in HSCR patients, as previously published . In factWe could not detect any major ocular anomaly in our series of HSCR patients but nearly 10% of VI and more than 40% of refraction abnormalities. The latter are frequent in HSCR patients as well as in the general population. In 2009, the Baltimore Pediatric Eye Disease Study indicated in 0,7% the prevalence of myopia and nearly 9% that of hyperopia, in 1030 white Americans younger than 6 years of age . SimilarThe prevalence of major or moderate CHD has been reported to be around 7,2 per 1000 live birth in the general population , whereasIn the general population, the incidence of HI in subjects without audiological risk factor is around 1,5‰, whereas that of subjects with audiological risk factors is 4,5% ,33. WithAlthough Moore reported a relatively high proportion of brain abnormalities in HSCR patients, we could only confirm a case of corpus callosum agenesis in a patient of ours who was already diagnosed with prenatal US ,26. The Amongst other associated anomalies Table  tumors wThe percentage of anomalies that could have been tracked down retrospectively by notes reviews or questionnaire administration resembles that of literature reports ,9,10. InBesides providing a novel view on clinical associations and variable symptoms of HSCR disease in children, the present data prompt us to deepen into follow-up data from adults who were treated for HSCR, which are lacking at the moment. Indeed, we cannot exclude that additional organs or systems can be involved and become impaired later in adult life. Indeed, typical adulthood associated diseases have not been described yet and are unknown at the moment. For instance, association with specific cancers, nervous degeneration, intestinal chronic inflammation and/or other late-onset disorders cannot be ruled out and further investigation in an adult set of HSCR patients would be advisable.Our Institution is known to be a referral centre for HSCR with an intrinsic subsequent risk of inclusion BIAS. This concern was confirmed by the higher than expected percentage of ultralong forms of the disease in our series 17,9%). Nonetheless, given the fact that the percentage and type of associated anomalies did not correlate to gender or length of aganglionosis, the results of our study maintained their strength. Furthermore, the percentage of Down Syndrome as well as the strong male preponderance observed in our series of patients are coherent with literature data and confirm that the results of our study are representative of the whole HSCR population ,9,10.%. NonethThe relatively small population of this observational study represents another potential limitation. As a consequence, the estimates we could provide have the intrinsic limitation of a wide variability and should be taken with care  . Figure Early diagnosis of associated anomalies provides more reliable prognostic expectations, prompt establishment of prevention strategies and implementation of adequate rehabilitation treatments. In accordance to our previous publication and to present results, we hypothesize that intestinal aganglionosis in HSCR patients represents the intestinal phenotype of a more complex syndrome driven by the interaction of neural crest maldevelopment and predisposing genetic background -10,29,31HSCR: Hirschsprung; NCN: neural crest derived neuroblasts; CAKUT: Congenital anomalies of the Kidney and Urinary Tract; US: Ultrasound scan; ENT: Ear nose and throat; NICU: Neonatal intensive care unit; HI: Hearing impairment; AABR: Automated auditory brainstem response; VRA: Visual reinforcement audiometry; CPA: Conditioned play audiometry; CA: Conventional audiometry; VI: Visual impairment; VA: Visual acuity; CCHS: Congenital central hypoventilation syndrome; CHD: Congenital heart disease.The authors declare that they have no competing interests.APP conceived the study, supervised clinical screening and drafted the manuscript. IC, DM, PG, VJ, PB, GM participated in the acquisition of most of clinical data and in drafting the manuscript. VR, MM, CH participated in the acquisition of clinical data. GT, MD, MM, GM, CG, GG, EP, LS, AP participated in the design of the study and in the acquisition of clinical data. FL performed the statistical analysis and participated in drafting the manuscript. All authors read and approved the final manuscript.


Felis catus v8.0), which incorporated ∼20 × coverage of Illumina fragment reads. The new genome assembly shows substantial improvements in contiguity, with a nearly fourfold increase in N50 scaffold size to 18 Mb. We use this map to report probable structural errors in previous maps and assemblies, and to describe features of the recombination landscape, including a massive (∼50 Mb) recombination desert  on the X chromosome that parallels a similar desert on the porcine X chromosome in both size and physical location.High-resolution genetic and physical maps are invaluable tools for building accurate genome assemblies, and interpreting results of genome-wide association studies (GWAS). Previous genetic and physical maps anchored good quality draft assemblies of the domestic cat genome, enabling the discovery of numerous genes underlying hereditary disease and phenotypes of interest to the biomedical science and breeding communities. However, these maps lacked sufficient marker density to order thousands of shorter scaffolds in earlier assemblies, which instead relied heavily on comparative mapping with related species. A high-resolution map would aid in validating and ordering chromosome scaffolds from existing and new genome assemblies. Here, we describe a high-resolution genetic linkage map of the domestic cat genome based on genotyping 453 domestic cats from several multi-generational pedigrees on the Illumina 63K SNP array. The final maps include 58,055 SNP markers placed relative to 6637 markers with unique positions, distributed across all autosomes and the X chromosome. Our final sex-averaged maps span a total autosomal length of 4464 cM, the longest described linkage map for any mammal, confirming length estimates from a previous microsatellite-based map. The linkage map was used to order and orient the scaffolds from a substantially more contiguous domestic cat genome assembly ( We used the flips option  to evaluate and refine local marker ordering at each step of the process. We also iteratively examined maps for putative “expander” markers (those that inflated maps > 3 cM), identified by larger-than-average flanking marker intervals. Local maps were rebuilt following removal of each expander marker, and map distances recalculated and compared with the previous map. In the final stage, we placed all remaining candidate markers relative to their most likely marker interval. The final maps and source data may be found in Supplemental Material, File S1 and Table S1.Given the large number of markers assayed on the SNP array for each chromosome, we constructed an initial framework map that was then used to successively position more markers at less stringent thresholds of marker inclusion and number of informative meiosis (IM). The Felis silvestris lybica). The data from prior maps included ∼2 × whole genome coverage of Sanger-based sequencing  (de novo assembly (v7.0) using the MaSuRCA assembler version 1.9.2  to align flanking probe sequences for the Illumina SNP markers (used in the linkage map construction) to the v8.0 genome assembly scaffolds. In addition, the assembled cat genome was broken into 1 kb segments and aligned against the v6.2 domestic cat assembly, the dog genome (canFam3), and the human genome (hg19) using BLAT  with ideBLASTZ and BLAT alignments of the v8.0 assembly to the dog and human genomes were then used to refine the order and orientation information, as well as to insert additional scaffolds that may not have been tagged by markers on the linkage map. These new scaffolds were placed into the conditional scaffold framework provided by the initial v6.2/v8.0 cat alignments and marker assignments. Alignments between the cat, dog, and human genomes were used to more precisely identify the breakpoints within scaffolds when marker and alignment information confirmed a false join within the genome assembly. In addition, 98 finished BAC clone sequences  were also incorporated into the genome assembly. Satellite sequences were identified in the v8.0 assembly, and centromeres were placed along each chromosome using the localization data from the radiation hybrid (RH) map of The authors state that all data necessary for confirming the conclusions presented in the article are represented fully within the article.We removed 35 markers with Mendelian inheritance violations, and 2478 markers with fewer than 100 IM during the marker ordering step. The final maps included 58,055 markers, including 8402 unique map positions at LOD ≥ 0.5 that were ordered relative to an initial framework map of 2204 LOD > 3.0 markers distributed across the 18 autosomes and X chromosome. 6659 of the 8402 markers were positioned at LOD > 1.0 and 4978 at LOD > 2.0. The final map provided an ordered and positioned density of ∼24 markers/Mb .The sex-averaged length of the autosomal map was 4464 cM, just 2% longer than the 4370 cM map length based on microsatellite genotyping . The ovee.g., The sex-averaged recombination rate across the genome was 1.9 cM/Mb, with rates varying from a high of 2.3 cM/Mb on chromosome D4, to a low of 0.85 cM/Mb on the X chromosome outside of the pseudoautosomal region (PAR) . RecombiThe cat X chromosome possessed a large recombination coldspot that spans ∼50 Mb, including the centromere. Notably, this same coldspot was “painted” in FISH mapping experiments that hybridized multiple, individual BAC clones selected from within this general region of the X chromosome to feline metaphase spreads . We thervs. 40 Mb) because more sequence was incorporated into the assembly. The v8.0 assembly contig and scaffold N50 lengths were 45 kb and 18 Mb, respectively, an increase of nearly 2.1 × and 3.8 × over the v6.2 assembly (The new v8.0 genome assembly incorporates ∼2.56 Gb of sequence (excluding Ns in gaps) that were ordered and oriented on the 18 autosomes and X chromosome . An addiassembly .We examined the integrity of the v8/linkage map chromosome marker orders by aligning the SNP-based linkage map with published RH and microsatellite-based linkage maps, which were previously used to guide the assembly of the domestic genome sequence (v6.2) . The newProduction of “draft” quality mammalian genome assemblies has grown tremendously over the last few years with reduced costs of next-generation sequence data production. Despite the increase in throughput, these initial “blueprints” are often insufficient for detailed genetic investigations of the molecular signatures of selection or disease-causing alleles of particularly complex traits (F. lybica)  produced a map of 3300 cM, also relatively longer than linkage maps of most other eutherian species  within regions of high differentiation between wild and domestic cats. The authors also identified a putative case of adaptive linkage  and a candidate domestication gene (PLEKHH1). Considering these facts, together with the evidence presented here that the domestic cat recombination rate is higher than any other studied eutherian mammal, our data lend support to the hypothesis that the process of domestication may have driven an increase in the domestic cat’s rate of recombination. Future analyses of chiasma frequencies in wild cat predecessors of the domestic cat would test this hypothesis.Previous studies have hypothesized that selection during domestication events could potentially result in elevated recombination rates , althouge sensu,  between The domestic cat genome assembly is unique among many other mammalian genome assemblies, being based on a large variety of sequencing read lengths, including a combination of legacy and NGS technology, and is anchored by a variety of physical mapping resources, including 2 × Sanger sequence data, multiple genetic linkage and RH maps, and fosmid and BAC-end sequences. This combination of mapping technologies has resulted in an extremely well-validated chromosome level assembly. These improvements to the assembly, coupled with whole genome SNP arrays, have led to a surge in trait and disease mapping efforts in cats, similar to advances in canine trait and disease mapping following the release of a high coverage draft assembly .de novo genome assemblies , which are known to inhibit the construction of highly contiguous i.e., duplications) are important contributors to health-related phenotypes, possibly due to overlapping, interchangeable, or even novel tissue-specific functions of slightly differentiated, physically separated sequences that regulate gene expression  and other emerging scaffolding technologies  will lead to additional improvements in the quality and completeness of the domestic cat genome (A growing body of evidence suggests that redundant genomic elements (pression . Many ph


During the FMD outbreak in Ireland and the UK in 2001, there was significant uncertainty amongstveterinary practitioners and government veterinary inspectors surrounding the clinical diagnosis of FMD insheep. This situation was complicated by reports of idiopathic oral ulcers that closely resembled FMD ongross appearance which at that time were referred to as ovine mouth and gum obscure disease.A field and abattoir study was carried out to determine the frequency, appearance and significance of oraland digital lesions in sheep in Ireland. A total of 3, 263 sheep were examined in 22 flocks, including 1, 969lambs and 1, 294 adults. A further 2,403 animals were examined by abattoir inspections. Animals bearing lesions of interest were identified, samples of the lesions were taken and subsequently examined by bacteriology, electron microscopy, serology, immunohistochemistry and histopathology.Forty four oral and 20 digital lesions were identified and characterised. Oral lesions were recorded mostfrequently in lambs, where the most common cause was orf virus infection. The majority of the oral lesions recorded in the adults was idiopathic and consistent with a diagnosis of idiopathic oral ulceration. A variety of digital lesions was observed, consistent with scald, foot-rot and contagious ovine digital dermatitis (CODD). All of the animals with lesions were seronegative to FMD virus (FMDV).There was no difficulty in differentiating these lesions from those caused by FMDV on the basis of flockhistory and careful clinical examination.The online version of this article (doi:10.1186/s13620-015-0055-0) contains supplementary material, which is available to authorized users. Foot-and-mouth disease (FMD) is a highly contagious viral disease of ungulates that has a very significant economic impact : the cosDuring the FMD outbreak in Ireland and the UK in 2001, there was significant uncertainty amongst veterinary practitioners and government veterinary inspectors surrounding the clinical diagnosis of FMD in sheep. This situation was complicated by reports of idiopathic oral ulcers that closely resembled FMD on gross appearance, then referred to as ovine mouth and gum obscure disease .This diagnostic uncertainty highlighted the lack of published information surrounding the gross appearance and prevalence of lesions seen in the FMD-free sheep population. The objectives of this study were to address this deficiency by systematically identifying, describing and investigating the cause of the range of background oral and digital lesions in FMD-free sheep in Ireland. It was anticipated that the information gleaned from this work would greatly assist veterinary practitioners and inspectors in reaching a more informed diagnosis when confronted with such lesions ‘in the field’.An abattoir study was undertaken to mimic the conditions under which private veterinary practitioners, in their role as temporary veterinary inspectors acting on behalf of the Department of Agriculture, Food and the Marine, were experiencing pressure in diagnosing particlularly oral lesions of unknown aetiology.ante mortem were identified and all four feet as well as the heads were collected post mortem for sampling and study.Three abattoirs were chosen based on their high throughput of sheep and their proximity to University College Dublin. The abattoirs slaughtered sheep from all over the Republic of Irelend but the majority of their intake was from the province of Leinster. A total of 2,403 sheep of all ages were examined for oral and digital lesions in the lairages of these facilities between November 2002 and November 2003. Sheep were examined at random and those displaying lesions Twenty-two commercial flocks were sourced ranging in size from 122–600 ewes. Of the 22, three of the flocks were hill flocks, 18 were lowland and one was mixed. The majority of the flocks were in Leinster, in counties Meath, Wicklow and Carlow. A small number of flocks in Cavan and Monaghan were also included. The most common breeds of ewe kept on the 22 farms were Suffolk, Cheviot and Texel, while the most common ram breeds were Suffolk, Charollais and Texel. Each flock was visited on three or four occasions during the investigation. Flocks were selected based mainly on the reality of sheep farmers who were willing to cooperate with the study and the significant inconvenience that the visits would cause. Flocks were recruited with the good will of farmers with the support of Teagasc, the Irish Agricultural advisory service. Visits were designed to inspect sheep at different stages of the production cycle: adults while housed prior to lambing, lambs at six to ten weeks of age, lambs on pasture ready for slaughter, adults at pasture. During each visit a sample of 10-20 % of the flock was chosen at random by gathering the sheep and after counting the number of sheep, 10-20 % was calculated. Sheep were selected by walking through the group and every 5–10 sheep marking one with spray. A count was kept of marked sheep. Once the required total was marked, the marked sheep were separated and examined. Each animal within that sample group was examined for evidence of peri-oral, oral and digital lesions. The number of sheep examined was dictated by the size of the flock, the handling facilities and the willingness of the flock owners. In larger flocks (>250 ewes) 10-15 % of the flock was examined, while in smaller flocks (<250 ewes), 20 % of the flock was examined; a total of 3,263 sheep were examined for the presence of lesions. Examination consisted of individually casting sheep and examining the peri-oral region of each animal visually and by palpation. Subsequently, the mouth was opened manually and with the aid of a mouth gag as appropriate, the mucosae, gingivae and tongue were visually inspected. All four feet were examined, with emphasis on the interdigital space, horn and coronary band. All sheep exhibiting lesions were subjected to a full clinical examination including the recording of rectal temperature and sampling procedure as described below.Samples taken from each lesion consisted of a swab for bacteriological examination, a tissue sample for histopathological examination and a tissue sample for electron microscopy (EM). The swab was placed in Amies transport medium  for transport to the laboratory. Following local regional analgesia using 2 % lignocaine , sedation using 2 % xylazine  or general anaesthesia using ketamine 10 %  as required, a biopsy was taken from each lesion using an 8 mm circular biopsy punch . Half of the biopsy was placed in a universal container and stored at −70 °C for examination by electron microscopy and the other half was placed in a universal container containing 10 % formalin for histopathological examination. In addition, a clotted blood sample was taken from each sheep with a lesion by jugular venipuncture.Samples were incubated at 37 °C under three different atmospheric conditions: aerobic, microaerophilic and anaerobic. Samples for aerobic incubation were cultured on McConkey agar and blood agar containing 5 % defibrinated sheep blood with colistin and nalidixic acid  . Microaeet al. [Samples were prepared and examined for the presence of viral particles as described by Harkness et al. .Formalin-fixed samples were embedded in paraffin wax using standard procedures, cut into 3-5 μm thick sections, mounted on slides and stained with haematoxylin and eosin (HE) .Tissue sections were selected for confirmatory immunohistochemical labeling on the basis of histopathological and electron microscopy results. Unstained tissue sections were placed on ‘sticky’ slides and labelled with a murine monoclonal antibody 2E5  specificCollected sera were stored at −70 °C and the sera were batch-tested by liquid phase blocking (LPB) ELISA to detect antibodies to serotype O of FMDV . The LPBSixty four individual animal lesions were recorded during the entire study, 52 (81 %) in the flock study and 12 (19 %) in the abattoir study, respectively. For simplicity of classification, lesions located within the oral cavity and around the oral cavity  were classified as oral lesions.A total of 2,403 sheep were examined during the abattoir study and 12 (0.5 %) oral lesions were identified, these were investigated in detail. Lesions found during this study were most commonly located on the dental pad .In the course of the flock study, 3,263 sheep were examined comprising 1,264 adults and 1,969 lambs. Of the 3,263 sheep, 32 (1 %) sheep displayed oral lesions, none exhibited pyrexia or any other significant clinical abnormalities. Of the 3,263 sheep, 20 (0.6 %) sheep bore digital lesions, six were overtly lame on one limb, three of which were non-weight bearing.Twelve lesions (0.9 %) were detected amongst the surveyed adult population, seven (0.5 %) oral lesions and 5 (0.4 %) digital lesions. During the flock study, numerous lame sheep were detected but due to the limited resources available, the most representative digital lesions were sampled.Among the lambs, 40 (2 %) animals with lesions were detected comprising 25 (1.3 %) oral lesions and 15 (0.7 %) digital lesions. Of the 25 oral lesions, 12 (48 %) were confirmed as orf by EM, immunohistochemistry and histopathology.In total, 64 lesions were described, there were 44 oral lesions (25 erosive/ulcerative and 19 exophytic) and 20 digital (13 erosive/ulcerative and 7 exophytic).Oral and digital lesions from both studies were divided on the basis of morphological characteristics into two categories, erosive/ulcerative and exophytic lesions. Descriptions of the size, shape, anatomical location and number of lesions are shown in Tables Twenty-five oral lesions from both studies were included in the erosive/ulcerative category Figs. , 3, 4, 5Staphylococcus species, Streptococcus species and Mannheimia haemolytica [Orf virus was not detected from any of these lesions, whilst bacterial species isolated were considered to be commensals at this location and included: molytica . In one molytica .Fig. 6PhExophytic lesions were defined as lesions which were growing or extending outwards from the skin/mucosal surface . There wFusobacterium necrophorum , Escherichia coli , Peptostreptococcus indolicus , Trueperella pyogenes  and Bacillus licheniformis . No viruses were detected in any samples.The 20 digital lesions identified in the two studies were also classified as either erosive/ulcerative or exophytic. There were 13 (65 %) and 7 (35 %) lesions in the erosive/ulcerative and exophytic categories, respectively. Diagnoses were as follows: foot-rot , white line disease (shelly hoof) , interdigital dermatitis  , interdigital fibroma  and toe fibroma . Of the 20 sheep affected, three (15 %) were lame, whilst eight (40 %) displayed evidence of localised lymph node enlargement  and the prescapular lymph node ). The most commonly isolated bacteria represented in the two categories of lesions were During the FMD outbreak in the UK and Ireland in 2001, there was considerable uncertainty surrounding the diagnosis of FMD on clinical grounds in sheep , 25, 26.Examination of 5,666 sheep in this survey revealed the presence of oral lesions in 0.8 % of animals. A higher lesion prevalence was found in lambs (1.3 %) compared to adult animals (0.7 %). The lesions could be divided morphologically into erosive/ulcerative and exophytic types. Evidence of orf virus was detected in the majority of the latter type of lesions while the aetiology of the erosive/ulcerative oral lesions was not determined. All of the animals with lesions were seronegative for antibodies to FMDV. There was no difficulty in differentiating these lesions from those caused by FMDV Fig.  on the bCrataegus monogyna) or rushes (Juncus effusus) [A number of hypotheses as to the pathogenesis of the idiopathic oral ulcerative lesions have been proposed. Direct trauma to the oral mucosa may occur due to grazing short grass on rough ground or possibly as a result of dosing gun injuries . It was effusus) .Idiopathic oral ulceration has been shown to have a low prevalence ranging from 0.95 % to 1.15 % in the UK , whilst Idiopathic lesions of oral ulceration were described in previous studies as solitary or multiple, red, raw ulcerated areas surrounded by a hyperaemic border on the lower gum ventral to the incisor teeth with lesions less frequently seen on the upper gum, dental pad, hard palate and tongue , 31. In The histopathological appearance of lesions in the oral erosive/ulcerative (idiopathic) category in the present study suggested bacterial infection. The bacteria isolated were consistent with opportunistic commensal organisms . Many ofIn the UK, where these idiopathic oral ulcerative lesions were identified, foot lesions were not recorded in the same animals . HoweverContagious pustular dermatitis (orf) was the most commonly diagnosed cause of oral lesions in the present study, confirmed in 12 out of 44 cases (27 %) i.e. 12 out of 19 oral exophytic lesions (63 %). Whilst the prevalence of orf amongst lambs in Irish flocks is unknown, studies establishing prevalence of orf in New Zealand (4.1 %)  and in TThe important differential diagnoses, in the oral region, in Ireland, for FMD include contagious pustular dermatitis, idiopathic oral erosion or ulceration, junctional epidermolysis bullosa and the exotic diseases bluetongue and vesicular stomatitis. The differential diagnoses for FMD in the foot region include contagious pustular dermatitis, foot-rot, contagious ovine digital dermatitis, junctional epidermolysis bullosa and the exotic disease bluetongue.The presence of pyrexia with sudden onset severe lameness characterised by vesicle formation tends to eliminate other common causes of foot lameness such as virulent foot-rot . HoweverThe relevant differential diagnoses of FMD in sheep include the exotic disease of bluetongue infection (BT). Signs of BT include initial pyrexia , 37 leadIn conclusion, whilst the lesions profiled in the present study bore little resemblance to the classical oral lesions described in cases of FMD in sheep, the presence of idiopathic oral ulcers should be considered in the context of the differential diagnosis of FMD in the field.


The heterogeneous biology of breast cancer leads to high diversity in prognosis and response to treatment, even for patients with similar clinical diagnosis, histology, and stage of disease. Identifying mechanisms contributing to this heterogeneity may reveal new cancer targets or clinically relevant subgroups for treatment stratification. In this study, we have merged metabolite, protein, and gene expression data from breast cancer patients to examine the heterogeneity at a molecular level.The study included primary tumor samples from 228 non-treated breast cancer patients. High-resolution magic-angle spinning magnetic resonance spectroscopy (HR MAS MRS) was performed to extract the tumors metabolic profiles further used for hierarchical cluster analysis resulting in three significantly different metabolic clusters . The clusters were further combined with gene and protein expression data.Our result revealed distinct differences in the metabolic profile of the three metabolic clusters. Among the most interesting differences, Mc1 had the highest levels of glycerophosphocholine (GPC) and phosphocholine (PCho), Mc2 had the highest levels of glucose, and Mc3 had the highest levels of lactate and alanine. Integrated pathway analysis of metabolite and gene expression data uncovered differences in glycolysis/gluconeogenesis and glycerophospholipid metabolism between the clusters. All three clusters had significant differences in the distribution of protein subtypes classified by the expression of breast cancer-related proteins. Genes related to collagens and extracellular matrix were downregulated in Mc1 and consequently upregulated in Mc2 and Mc3, underpinning the differences in protein subtypes within the metabolic clusters. Genetic subtypes were evenly distributed among the three metabolic clusters and could therefore contribute to additional explanation of breast cancer heterogeneity.Three naturally occurring metabolic clusters of breast cancer were detected among primary tumors from non-treated breast cancer patients. The clusters expressed differences in breast cancer-related protein as well as genes related to extracellular matrix and metabolic pathways known to be aberrant in cancer. Analyses of metabolic activity combined with gene and protein expression provide new information about the heterogeneity of breast tumors and, importantly, the metabolic differences infer that the clusters may be susceptible to different metabolically targeted drugs.The online version of this article (doi:10.1186/s40170-016-0152-x) contains supplementary material, which is available to authorized users. Breast cancer accounts for 25 % of newly diagnosed cancers and 15 % of cancer deaths among women worldwide –5.Searching for genetic features causing the variation in breast cancers, Perou et al. used gene expression analyses followed by hierarchical clustering and defined naturally occurring molecular subtypes , 6. ThesProteins are the ultimate cellular effectors of pathways and networks within cells, tissues, and organisms. Although protein levels are dependent on mRNA expression, not all mRNA will be translated into protein and further protein levels are also influenced by protein stability. In a study by Myhre et al. only 22 of 52 quantified breast cancer-related proteins were found to correlate with mRNA expression levels  and simiThe chemical processes controlled by proteins involve metabolites as intermediates or end-products. In metabolomics, metabolite levels are measured to gather the final downstream information of ongoing cellular processes. Which processes are active at a specific time point is strongly influenced by environmental factors like diet and drugs as well as disease state. Well-established metabolic differences have been observed when comparing cancer cells to normal cells. Cancer cell energy production frequently depends on increased glycolysis and production of lactate from glucose regardless of access to oxygen, in contrast to normal cells which produce pyruvate and lactate in aerobic conditions . Also, tMerging transcriptomics and metabolomics led to the discovery of three luminal A subgroups with distinct metabolic profiles and significant differences within gene set expression in a study by Borgan et al. . The aimPrimary breast carcinoma samples from 228 patients at the Oslo University Hospital  were collected in the time period 2006–2009 as part of the Oslo2 study. The samples were fresh frozen after surgery and stored at −80 °C. The tumors were divided into smaller pieces depending on their size, and one of them was selected for this study. The samples were cut into three sections where the edges of the two outer pieces were used for histological evaluation (including estrogen receptor (ER) status and tumor cell percentage), and an adequate part of the mid pieces were used for HR MAS MRS experiments to obtain metabolic profiles. The remnants of all three pieces were pooled and cut into smaller pieces with scalpel, depending on the size of the tumor and divided into fractions used for extraction of DNA, RNA, and protein. Due to high lipid content, HR MAS MRS was performed on a second piece from the same tumor for 13 of the samples. A total of 228 samples were analyzed by MR spectroscopy, of which 201 and 217 were analyzed for gene expression by arrays and protein expression using RPPA, respectively, leaving a total of 191 samples analyzed by all three methods. Patient and tumor characteristics are shown in Table HR MAS MRS spectra were acquired from tissue samples (mean sample weight 7.3 mg ± 2.6 mg) on a Bruker Avance III 600 MHz/54 mm US  equipped with a 1H/13C MAS probe with gradient aligned with the magic angle . Spin-echo spectra were recorded using a Carr-Purcell-Meiboom-Gill (cpmg) pulse sequence . For experimental details and information about data processing, see Additional file Forty-three samples were excluded from the original sample cohort of 271 samples due to large lipid content. The spectral region between 1.40 and 4.70 ppm was chosen for further analysis excluding lipid peaks at 4.36–4.27, 2.88–2.70, 2.30–2.20, 2.09–1.93, and 1.67–1.50 ppm. After removal of the lipid residuals, the spectra were mean normalized by dividing each spectral variable to the average spectral intensity. This is done to account for differences in tumor cell percentage and sample weight, as it can be assumed that most of the lipid signals from breast samples do not originate from cancer cells.Protein levels were determined using reverse phase protein array (RPPA), a platform where single protein levels can be measured across a series of samples simultaneously . One hunThe samples underwent consensus clustering with an option for four or five groups. The best fit on consensus clustering identified five groups, luminal, HER2, basal, and reactive I and II subsets as defined in The Cancer Genome Atlas Network data set .Total RNA was isolated with TRIzol . Expression of mRNA was measured using SurePrint G3 Human GE 8x60K (Aglient Technologies) according to the manufactory’s protocol  and 100 ng RNA was used as input for labeling. Arrays were log2-transformed, quantile normalized, and hospital adjusted . Values The PAM50 subtype algorithm  was usedHierarchical cluster analysis (HCA) was performed with Euclidean distance as the distance parameter and Ward’s method (furthest inner square distance) as the clustering distance  on the preprocessed metabolic spectra. Similar spectra based on the distance measures cluster together. The dendrogram was cut to give three clusters. To evaluate the robustness of the three HCA clusters, partial least square discriminant analysis (PLS-DA) model, using the cluster group for classification was carried out and classification accuracy was evaluated. For details, see Additional file p values were corrected for multiple testing by The Benjamini Hochberg false discovery rate (FDR) in Matlab, and the differences were considered statistically significant for adjusted p ≤ 0.05.Metabolite assignments were performed based on literature values . Relativp values were corrected for multiple testing by The Benjamini Hochberg FDR, and the differences were considered statistically significant for adjusted p < 0.05.Differences in the distributions of RPPA and PAM50 subtype as well as that of other clinical characteristics of the tumors between the different metabolic clusters were tested for significance using Fisher’s exact test for count data (R 2.15.2). Calculated T statistics/Wilcoxon statistics were calculated using multiclass comparisons and two-class unpaired tests while comparing two clusters. The differences were considered statistically significant for adjusted p < 0.01.Significance analysis of microarrays (SAM) was used to identify differentially expressed gene between the metabolic clusters . SAM anaDAVID, an online network analysis tool , was useμ and σ are the mean and standard deviation, respectively.Gene set enrichment analysis (GSEA) was used to identifying sets of genes that were enriched in the metabolic clusters , 28. DurHigh absolute signal-to-noise ratio will represent genes that are more likely to be “class markers” in the comparison because of high difference in expression.The gene set C5 (gene ontology (GO) gene sets) available from the Molecular Signatures Database (MSigDB)  from Thep < 0.05 from SAM analysis and metabolites differently expressed between the clusters were used as input. Pathways with p values ≤0.05 were interpreted as significant.To combine transcriptomics and metabolic data the “Integrated pathway analysis” tool in MetaboAnalyst 3.0 software was used . Genes wFrom the spectral data of 228 breast tumors, hierarchical clustering gave a dendrogram divided in three metabolic clusters (Mc) Fig.  Mc1, Mc2p < 0.001). The regression vectors for each of the clusters  between at least two of the clusters , where 11 of 21 lobular tumors and all ductal carcinoma in situ (DCIS) (n = 4) were classified as Mc2  were analyzed for differences in distribution among the metabolic clusters. Only histology was found to be significantly different between the clusters (adjusted p = 0.138), RPPA distribution was significantly different  with only 9 % of the RPPA reactive I and II samples being classified as Mc1, and 44 % of Mc2 samples subtyped as reactive I. The complete distribution of PAM50 and RPPA subtypes is listed in Table The metabolic clusters were investigated for differences in distribution of PAM50 and RPPA subtypes. While PAM50 subtypes did not show increased frequency of occurrence in any of the metabolic clusters,  between Mc1 and Mc2  between Mc1 and Mc3  is converted into PtdCho. Lower expression of this gene may explain why the GPC precursor is directed to the production of GPC instead of PtdCho. The same hits were obtained when Mc1 was compared to Mc3. In addition, PLA2G5, one of the enzymes degrading PtdCho to acyl-GPC, is downregulated in Mc1 compared to Mc3, further supporting that Mc1 has an altered PtdCho metabolism.Of the three metabolic clusters, Mc1 was on a separate branch in the dendrogram indicating that the metabolic profile of this cluster was the most different. This cluster is defined by significantly higher levels of GPC and PCho, two choline-containing metabolites involved in the synthesis and degradation of phosphatidylcholine (PtdCho), a major component of cell membranes . AlteredCHKA) could be detected in the SAM analysis. However, univariate analysis confirmed that CHKA expression was significantly higher in Mc1. This is in agreement with previous findings revealing a positive correlation between levels of PCho and GPC and expression of CHKA [.The levels of PCho and GPC were higher in Mc1 compared to the two other clusters, but no significant difference in the expression of choline kinase alpha  was significantly different between the metabolic clusters, whereas no significant differences in the distribution of PAM50 subtypes were found. Thus, the metabolic difference between Mc1, Mc2, and Mc3 is not a result of intrinsic subtypes and might therefore contain additional information for understanding breast cancer heterogeneity. Among the tumors clustered in Mc1, 12 % were classified as RPPA-reactive (either I or II) while 49 % were classified as RPPA-luminal. The reactive RPPA subtypes have a characteristic protein expression pattern probably produced by the microenvironment , these gp = 0.056), supporting a higher glycolytic rate in Mc1 and that the pyruvate produced is not directed to metabolism in the TCA cycle. The significantly lower acetate levels in Mc1 compared to Mc2 could be linked to ALDH1A3 and ALDH2 downregulation, since the enzymatic product of these genes catalyzes the reversible reaction where acetaldehyde is converted to acetate.Mc2 has a metabolic profile with significant higher glucose level and at the same time lower levels of most of the other metabolites compared to one or both of the remaining clusters. High glucose level could reflect lower glucose consumption, inferring a lower demand for energy within these tumors. Glycolysis/gluconeogenesis came out as a significant pathway when Mc1 was compared to Mc2 during integrated pathway analysis with two metabolite hits and five gene hits. For the most significant metabolite, glucose, the levels are higher in Mc2 compared to Mc1. Glucose is the main source of energy for mammalian cells, either through aerobic glycolysis (production of lactate even in the presence of oxygen) or tricarboxylic acid (TCA) cycle and oxidative phosphorylation. For normal proliferating cells and cancer cells, which both have an increased energy demand, a glycolytic switch is often observed (higher glycolytic rate) . The incBoth DAVID and GSEA showed that many of the genes found to be downregulated in Mc1 and consequently upregulated in Mc2 were related to ECM activity. Mc2 had the highest percentage of RPPA-reactive I with 44 % of Mc2 tumors classified as this protein subtype, also related to stromal changes. Together with the metabolic finding, this implies that Mc2 tumors have cancer cells with low proliferating rate and at the same time ongoing changes within the ECM of the stroma. Mc2 tumors also had a higher frequency of lobular and ductal carcinoma in situ, indicating metabolic differences between histological subtypes of breast cancer which should be further investigated.AGPAT4, PPAP2B, PPAP2A, LCAT, and PLD1. Of these, LCAT and PLD1 are directly involved in choline metabolism. LCAT catalyze the conversion of PtdCho to acyl-GPC while PLD1 catalyzes the conversion of PtdCho to choline. Higher GPC levels, but no difference in choline levels in Mc3 compared to Mc1 indicates that a higher amount of GPC is converted to choline in Mc3, and further contributing to higher glycine levels through choline degradation.Mc3 has the highest lactate levels of all three clusters and higher glycine level than Mc2. These metabolites have been related to poor prognosis in ER positive patients , and higMc3 shares similarities with a previously reported metabolic subgroup of luminal A tumors with significantly lower levels of glucose, higher levels of alanine, and nearly significantly higher lactate levels . In Mc3,The majority of Mc3 tumors were classified as RPPA-luminal, similar to Mc1. In contrast to Mc1, Mc3 had a higher percentage of RPPA–reactive II tumors, probably linked to changes in stromal content. Also, gene expression wise, this was observed by significantly different gene expressions linked to ECM activity and the gene expression profile of Mc3 was found similar to the previously reported ECM3 or ECM4 subtypes .CHKA, the enzyme responsible for PCho production from choline, causes tumor growth arrest and apoptosis in preclinical models [In this study, information flow between the transcriptomics, proteomics, and metabolomics levels is illustrated; at the transcriptomics level, only one of the metabolic clusters shows difference in gene expression compared to the two others, while at the proteomics level, there is difference between all three clusters. Combining these findings, Mc1 is expected to have the worst prognosis due to the distinct gene expression profile and the alterations in both glycerophospholipid metabolism and evidence of increased glycolytic rate. However, this has to be validated when 5-year follow-up of this cohort is available. The main metabolic characteristics, especially of Mc1 and Mc3, have been proposed as treatment targets that could improve the therapeutic effect . Cancer l models , while tl models . MetabolWe have here identified three metabolic clusters of breast cancer, also characterized with differences at the proteomic and transcriptomic level. The metabolic clusters are not reflecting the intrinsic genetic subtypes and may give important additional information for understanding breast cancer heterogeneity. Gene enrichment analysis revealed diverse ECM characteristics among these clusters in accordance with RPPA-subtyping. The approach of combining information from several -omics levels in the same tumor shows promise in improving the understanding of breast cancer heterogeneity potentially leading to more patient specific treatment.Ala, alanine; Cr, creatine; DCIS, ductal carcinoma in situ; ECM, extracellular matrix; ER, estrogen receptor; FDR, false discovery rate; Gly, glycine; GPC, glycerophosphocholine; GSEA, gene set enrichment analysis; HCA, hierarchical cluster analysis; HR MAS MRS, high resolution magic angle spinning magnetic resonance spectroscopy; Lac, lactate; Mc, metabolic cluster; MRS, magnetic resonance spectroscopy; MSigDB, molecular signatures database; PAM50, prediction analysis of microarrays 50; PCho, phosphocholine; PET, positron emission tomography; PLS-DA, partial least square discriminant analysis; PtdCho, phosphatidylcholine; RPPA, reverse phase protein array; SAM, significance analysis of microarrays; Tau, taurine; TCA, tricarboxylic acid; β-Glc, β-glucose