CRP polymorphism, rs1205. Our aim was to explore the effects of additional CRP variants. The IBC (Cardiochip) genotyping microarray focuses on candidate genes and pathways related to the pathophysiology of cardiovascular disease.The etiology of pre-eclampsia (PE) is unknown; but it is accepted that normal pregnancy represents a distinctive challenge to the maternal immune system. C-reactive protein is a prominent component of the innate immune system; and we previously reported an association between PE and the CRP SNPs were analyzed. A replication sample of 178 cases and 427 controls of European ancestry was also genotyped.This study recruited 140 cases of PE and 270 matched controls, of which 95 cases met criteria as severe PE, from an American Indian community. IBC array genotypes from 10 suitable P<0.005) were seen for rs876538, rs2794521, and rs3091244. Univariate conditional logistic regression odds ratios (OR) were nominally significant for rs3093068 and rs876538 models only. Multivariate logistic models with adjustment for mother's age, nulliparity and BMI attenuated the effect  for rs876538 and  for rs3093068. An additive risk score of the above two risk genotypes shows a multivariate adjusted OR of 2.04 . The replication sample also demonstrated significant association between PE and the rs876538 allele . We also show putative functionality for the rs876538 and rs3093068 CRP variants.A nominally significant difference  was seen in the distribution of discordant matched pairs for rs3093068; and Bonferroni corrected differences (CRP variants, rs876538 and rs3093068, previously associated with other cardiovascular disease phenotypes, show suggestive association with PE in this American Indian population, further supporting a possible role for CRP in PE.The  Pre-eclampsia (PE) is a pathologic condition of pregnancy characterized by the onset of hypertension and proteinuria after 20 weeks of gestation.ARTS-1 in one of the few large well powered PE studies.Although there is significant phenotypic heterogeneity across the spectrum of PE, a number of studies have shown a 2–5 fold increase in risk amongst first-degree relatives of women with PE.CRP and PE.C-reactive protein (CRP) is a prominent component of the innate immune systemCRP and severe PE in an American Indian cohort;CRP captured on the IBC array are correlated with risk or severity of PE within this higher risk population.Previously we demonstrated an association between rs1205 in Approval was obtained from the Aberdeen Area IHS and University of North Dakota Institutional Review Boards (IRBs) and the tribal government. Written informed consent was obtained from each participant.The Institutional Review Board (IRB) of record for Massachusetts General Hospital and Brigham and Women's Hospital is Partners Human Research Committee; and for Beth Israel Deaconess Medical Center it is the Committee on Clinical Investigations. With IRB approval, the billing records of patients delivered at Massachusetts General Hospital and Brigham and Women's Hospital from 1995–2011 were queried for ICD9 codes suggestive of PE. Whenever a PE patient returned to a Partner's Hospital, blood samples at their point of discard  were collected for genetic analysis. Cases were validated for PE and its severity by electronic medical record/chart review in a 30 day time-limited link to protected health information. After links to protected health information were deleted, extracted DNAs of accessioned samples were released to the study staff in a de-identified manner for further investigation. Because the investigators do not interact with any individuals for the ascertainment of data or samples, informed consent was not obtained: the Partners Human Research Committee elected to waive the requirement to seek informed consent (as detailed by 45 CFR 46.116). The participants from Beth Israel Deaconess Medical Center provided written, informed consent.The Children's Hospital of Philadelphia (CHOP) IRB approved DNA collection from 73 mothers with PE and 353 control normotensive mothers from 2009–2012 at the Center for Applied Genomics (CAG) at CHOP through the Study of the Genetic Causes of Complex Pediatric Disorders (GCPD). Mothers of recruited children were asked between 2009–2012 if they had ever had PE during their pregnancy and gave written informed consent and HIPAA authorization to allow access to their medical records.Recruitment for the TMCC case and matched control study has been ongoing from 8/04 to 7/12. The federally funded Indian Health Service (IHS), through the hospital and clinic located in Belcourt, North Dakota, is the primary health care provider for eligible tribal members of the Turtle Mountain Band of Chippewa. Most potential cases (∼80%) were identified by automated query of an electronic medical record database  at this facility, using a relevant group of ICD9 codes, designed to be inclusive. Additional potential cases (∼20%) were “self-identified” among family members and acquaintances of cases and during the recruiting of controls.The medical records of all potential cases were abstracted for 78 clinically relevant factors, including the highest of up to 3 blood pressure (BP) measures between 20 weeks of gestation and 30 days postpartum and the highest of up to 2 measures of proteinuria in the same period. Cases were verified as meeting diagnostic criteria for PE if at least 2 of the following were identified:At least 3 BP values above either 140 mmHg systolic or 90 mmHg diastolic; and absence of a diagnosis of, or treatment for hypertension (during the year prior to conception and the first 20 weeks of gestation).Proteinuria as indicated by a 24 hour excretion of >300 mg, or at least two +1 dipstick measurements in the absence of prior proteinuria.A diagnosis of PE, eclampsia, or the hemolysis, elevated liver enzymes, low platelet (HELLP) syndrome by an attending physician after 20 weeks of gestation.These criteria were chosen to be compatible with the NHLBI Working Group on Research on Hypertension in Pregnancy (Working Group) definition Controls were ascertained by contact of the first individual to deliver before and after the index case. If a potential control declined participation, the woman delivering during the next prior or subsequent day was contacted; and this was continued until two controls were recruited, one before and one after the index case. This method of ascertaining controls was chosen as a convenient means of randomization and to control for possible seasonal influences on PE.Replication analysis was conducted on two groups assembled for a larger collaborative study, comprised of:The Boston area Pre-eclampsia Genetics Collection .Samples meeting ACOG 2002 criteria were included in the study (88 cases). Blood from 17 additional cases based on physician diagnosis and 74 normal term controls were collected from patients at the Beth Israel Deaconess Medical Center.Children's Hospital of Philadelphia Pre-eclampsia Genetics Collection.The definition of PE employed is defined as onset hypertension after 20 weeks of gestation with systolic BP≥140 or diastolic BP≥90 on two occasions at least 6 hours apart in the presence of proteinuria of 300 mg/dL on a 24 hour collection or at least +1 on a dipstick.For the American Indian population, prior to January of 2009, template DNA was provided by capillary blood samples collected on “FTA Classic Cards” (Whatman Inc) paper. Three 1.2 mm diameter “pellets” were punched from the cards and processed according to the manufacturer's recommendations. Recently template DNA has been collected and processed using salivary samples and the Oragene (DNA Genotek Inc) system; and the majority of those with capillary samples were re-consented and an additional salivary sample obtained. For genotyping of the replication cohort, DNA was extracted from blood or saliva using standard alkaline lysis methodologies and stored at −20°C.Genotyping was accomplished primarily (319 of 410 (78%) total samples) by microarray analysis on the ITMAT/Broad/CARe (IBCv1) at The Center for Applied Genomics at CHOP. This genotyping microarray selected >49,000 SNPs related to ∼2,000 genes and was guided by genome-wide association study data, pathway based approaches and comprehensive literature searches to variants with known relevance to cardiovascular, metabolic and inflammatory pathology.Results of the microarray genotyping will be available to qualified investigators with assurances that 1) no attempts will be made to identify individuals, 2) goals of the analysis are within the scope of the consent and 3) the results will not be used for commercial purposes.The remainder of the samples without salivary DNA were genotyped using pre-designed TaqMan genotyping assays and protocols were implemented for SNPs on a real-time, Mini-Opticon (Bio-Rad Laboratories Inc), four color thermocycler. Controls were identified for at least two of the three possible genotypes (and “blank” controls) for each SNP were included with each analysis. Control material of consistent genotype (replicated a minimum of ten times) for each genotype was run with each set of samples. In the case of rs876538, HapMap genotypes from eight Coriell Institute reference samples were confirmed in our laboratory.P<10−6. The rs1205 and rs876538 variants passed quality control but rs3093068 failed within this replication sample, therefore we used the proxy rs1206785 (r2 = 1 with rs3093068 in 1000 Genomes project [1KGP] CEU population).The replication genotyping for this study, European ancestry samples from Boston and Pennsylvania (n = 605), was also conducted on the IBC array with principal components analysis performed using standard Eigenstrat pipelines.CRP SNPs available on the IBCv1 array. Of these, eight were observed to be monomorphic in this population and another five were excluded for low prevalence. Another SNP exhibited a call rate of 60% and was considered unreliable for analysis, leaving ten SNPs remaining for analyses.The primary analysis of the American Indian samples chose all 24 P≤0.05, and study-wide statistical significance at P = 0.005 after Bonferroni correction. Principal component analysis was conducted using ancestry informative SNPs from the IBCv1 microarray.Statistical analysis was primarily carried out using SPSS version 10.1.0 software, with Egret version 2.0.31 used for the logistic regression analysis. Descriptive statistics report mean (+/− SD) for continuous variables and proportions with 95% CI for discrete variables. HWE analysis was assessed using standard chi-square methods. Due to the matched nature of case/control pairs, McNemar chi square analysis is required for genotype comparisons; and relies on evaluation of the two opposing sets of discordant genotypes. The null hypothesis holds that there should be equal numbers of discordant pairs where the case has genotype A and the matched control has genotype B; compared with the opposite situation, where the case has genotype B and the control, genotype A. The genotype risk score for McNemar analysis considered the presence of either identified risk genotype, rs3093068  or rs876538 , among pairs with both alleles available. The risk score for logistic regression analysis used a more powerful, ordinal variable, where both risk genotypes  = 2, either  = 1, and none  = 0. This ordinal risk score could not be used for the McNemar paired analysis because the available software will not support it and many statistical texts consider this analysis “beyond their scope”. Conditional logistic regression was used to explore the multivariate association of genotype and other variables with risk of PE. Statistical significance was set at The replication analysis was performed using logistic regression with SNPs coded in an additive genetic model, and with adjustment for study site and 10 principal components computed as described above to correct for population structure.2 LD>0.5. LD was defined in the three HapMap population groups using the HaploReg tool which includes LD data derived from Phase I 1000 genomes project. Population groups were CEU (Utah Residents (CEPH) with Northern and Western European ancestry), ASN  and AMR . The full genomic context of the CRP locus defined by this process is shown in The extended locus around each associated SNP was defined by identification of all SNPs showing rAmong the 140 cases of the discovery sample, 52 (37%) utilized the clinical diagnosis as one of only two qualifying criteria. Of these 52, all but 11 (8%) would have met either the minimal standards for BP or proteinuria of the Working Group or ASH; and all of those 11 met at least the more stringent BP/proteinuria standard of the current study, in addition to a clinical diagnosis of PE. Five of these 52 cases also had objective signs of end organ involvement as defined below and three had seizures.3, creatinine greater than 1.3 mg/dl, liver transaminases over two times the upper limit of normal for the hospital laboratory, or experienced a seizure during parturition, also meeting criteria for severe PE.At least one of the American College of Obstetricians and Gynecologists' (ACOG) defining criteria CRP, all of the SNPs (except rs3093070 – which had design/synthesis issues) have been captured.P<0.05), whereas rs3091244 and rs3093068 showed marginally significant differences between cases and controls. When comparing allelic frequency of severe PE cases and controls, none of the SNPs retained significance. Significant differences between all cases and controls were noted for nulliparity, weeks of gestation at delivery, body mass index (BMI), gestational diabetes, weight at first prenatal visit, and both systolic and diastolic blood pressure. Differences in near term delivery, birth weight of infants and BPs were not included in further models and felt likely to be consequences of PE or the applied diagnostic criteria, rather than etiologic. Principal component analysis of the ten vectors showed no significant difference between cases and controls.Pertinent characteristics of all paired cases and controls are summarized in P = 4×10−5). For analyses considering only severe cases, the only comparisons that retained significance were rs876538 (C recessive), nominal; and the dichotomous risk score (P<0.0001), significant after Bonferroni adjustment. The two SNPs of primary interest are modestly correlated with each other  and with rs1205 .P = 0.117) when included in a multivariate model with age at delivery, nulliparity and BMI. The SNP rs876538 showed nominal significance in analysis of all cases; and the additive risk score was nearly significant with a Bonferroni adjusted p value of 0.009, whereas rs2794521, rs3091244, and rs3093068, demonstrated only marginal significance  in univariate analysis. Models utilizing multivariate conditional logistic regression (P = 0.066). The additive risk score showed nominally significant association with PE among all cases and controls (P = 0.016).Univariate conditional logistic regression results are shown in gression  continueP = 0.04, 95%CI 1.03–5.42); but not with any of the other single SNPs genotypes. The additive risk score demonstrated nominal significance . Multivariate conditional logistic regression results showed marginal, independent association for rs3093068, p = 0.050, but lack of significance for the other individual SNPs. The additive risk score attained nominal significance, with an OR = 2.03, p = 0.013, and 95% CI 1.16–3.56.Univariate analysis of the 95 cases meeting the definition of severe PE and their 186 matched controls did not change any of the previously mentioned univariate relationships with clinical factors; and now showed nominally significant association with rs3093068 , in HapMap CEU, ASN and AMR populations respectively. Review of Haploreg and Regulomedb results showed multiple variants are predicted to be located in regulatory active regions  that were PE-associated at nominal significance using both McNemar chi-square and logistic analysis; and three  attained Bonferroni adjusted significance in the chi square, paired analysis. An additive risk score using these two alleles showed Bonferroni adjusted significance in both chi square  and significant P values of 0.009 and 0.01 in univariate and multivariate logistic analysis respectively. While these variants in the CRP gene have been investigated for their association with other inflammatory conditions, to our knowledge, this is the first report of association between these two SNPs (in addition to rs1205) and PE. The replication of these findings in a population of European ancestry increases our confidence in these results.Among many proposed theories related to the etiology of PE, dysfunctional immune responses have played prominent roles.2 = 0.792) and the Asian population referenced above (r2 = 0.764 in 1000 G HapMap Asians (CHB/JPT)).The extent and specifics of genetic influences on serum CRP levels remains unclear; but a number of studies have identified important associations with various SNPs, 2 = 0.63, d′ = 1) affects CRP expression.The other SNP with significant findings of association in the present study, rs876538, has a prevalence very similar to the 22.1% reported for Europeans.In evaluating the findings of the present analysis and comparison with other association studies of CRP levels and PE, consideration must be given to genetic background effects such as: 1) differences in linkage disequilibrium between populations, causing discordant results due to the presence or absence of linkage to an unknown causative SNP, 2) differences in power to detect association due to different population prevalence for the SNP of interest and 3) differential effects of the SNP of interest based on interaction between the SNP of interest and an unknown genetic variant with population-specific effects. Furthermore, environmental factors can vary substantially between populations and overwhelm the genetic signal, resulting in a loss of power, or differential SNP-environment effects may influence the comparison between populations. The adjustment for age, BMI and gender in the present primary study may increase the likelihood that the identified SNPs are influencing the immune response, provided the modeled adjustment is accurate .CRP SNPs in a large cohort study of older Americans. Although rs876538 was included in the analysis, it did not retain significance after multivariate adjustment.Although the heritability of CRP in another American Indian population was found to be approximately 40% The association between PE and inflammation has suggested CRP levels as an important marker of this disease. None of these purported associations, either cross-sectional or prospective, prove that CRP is in the direct causal chain for PE, it may be simply a marker of inflammation initiated by current or still latent primary factors causing PE. The results of genetic association studies such as this do contribute to our understanding, in so far as the genetic variants are obviously present prior to the establishment of any disease state. None the less, CRP variants could still be either simple biomarkers, indicating a genetic heightened sensitivity to the primary cause(s) of PE; or they could be involved in pathogenesis as a modifier of response to an actual cause of the condition. Two publications make a similar observation when they suggest that CRP may be an intermediary between BMI and hypertension.CRP gene warrants further investigation. The fact that 24% of our controls were recruited in a slightly different fashion and the difference between case and control delivery dates was slightly over 9 days greater than the other case/control pairs could have possibly introduced a subtle, unrecognized bias, although concern for seasonality effects of this degree seems unwarranted. The inability to adjust the replication sample for clinical covariates was also a limitation but we again emphasize that this is a unique study population. Strengths of this study include a well-defined phenotype of PE and the study design which employed an unbiased ascertainment of cases and controls. The fact that the present study focused on CRP gene variants rather than circulating CRP levels, should reduce the extraneous effects of multiple environmental factors on CRP expression.Limitations of this study include limited power due to modest sample size which is common to many investigations of PE, although we emphasize that this is the largest PE study to date in American Indians. Our multivariate logistic regression results nearly reached study-wide significance after conservative, Bonferroni adjustment for multiple testing only for the additive risk score among severe cases. However, our previous finding of association with rs1205 in this cohort, replication of results for rs876538 in another population, and nominally significant findings in two of ten tested SNPs, certainly suggests that the CRP gene variants provide suggestive findings that may warrant further expression studies and replication in other cohorts. Although there is a significant correlation between these SNPs, the combined strength of association for both rs3093068 and rs876538 with severe PE risk were enhanced, compared with either assessed separately suggesting that these gene variants do not merely share a haplotype. Until our publication reporting the significant association of the CRP SNP rs1205Our findings related to these 2 previously unreported Figure S1Genomic context of CRP, Chromosome 1 extended locus. Data is visualized using the UCSC human genome browser and custom track data, presented in the following order: i) AMR LD proxy SNPs (r2 LD>0.5), ii) ASN LD proxy SNPs (r2 LD>0.5), iii) CEU LD proxy SNPs (r2 LD>0.5), iv) Directly associated SNPs, v) Liver EST sequence BG616599, vi) Published studies of CRP including downstream regions of functional characterization, vi) ENCODE: RNA-seq assayed in 9 cell lines, vii) ENCODE: H3K4Me3 histone marks representing probable promoter activity, viii) ENCODE: H3KMe1 histone marks presenting probable regulatory enhancer activity, ix) ENCODE: H3K27Ac histone marks presenting probable regulatory enhancer activity, x)ENCODE: DNase I hypersensitive regions, indicating DNA binding activity, xi) Liver tissue RNA-seq from the Burge lab, xii) Mammalian conservation.(TIF)Click here for additional data file.Figure S2Chromosome 1 directly associated SNPs. Data is visualized using the UCSC human genome browser and custom track data, presented in the following order: i) AMR LD proxy SNPs (r2 LD>0.5), ii) ASN LD proxy SNPs (r2 LD>0.5), iii) CEU LD proxy SNPs (r2 LD>0.5), iv) Directly associated SNPs, v) Liver EST sequence BG616599, vi) Published studies of CRP including downstream regions of functional characterization, vi) ENCODE: RNA-seq assayed in 9 cell lines, vii) ENCODE: H3K4Me3 histone marks representing probable promoter activity, viii) ENCODE: H3KMe1 histone marks presenting probable regulatory enhancer activity, ix) ENCODE: H3K27Ac histone marks presenting probable regulatory enhancer activity, x)ENCODE: DNase I hypersensitive regions, indicating DNA binding activity, xi) Liver tissue RNA-seq from the Burge lab, xii) Mammalian conservation.(TIF)Click here for additional data file.Table S1Further detail concerning SNPs shown in (XLS)Click here for additional data file.


MTHFR) C677T genotype, with the birth weight of offspring.We used Mendelian randomization analysis to investigate the causal relationship between maternal homocysteine level, as represented by maternal methylenetetrahydrofolate reductase . Birth weight decreased from 120 to 130 grams as maternal homocysteine level increased, while controlling for confounding factors; however, the association was of marginal significance (P = 0.06).Our results suggest an adverse relationship between maternal homocysteine level and birth weight. A reduction in homocysteine levels might positively affect birth outcomes. In addition to these risk factors, homocysteine concentration was reported to be positively associated with the risk of pregnancy complications2 and low birth weight.3 In general, homocysteine concentration remains low during pregnancy.2 However, abnormal homocysteine levels in the intrauterine environment could induce adverse birth outcomes such as pregnancy loss,4,5 placental abruption,5 and intrauterine growth retardation.3The extent of intrauterine growth can determine lifelong health and the health of future generations. During recent decades, a number of studies have confirmed Barker’s hypothesis regarding the fetal origin of adult disease.MTHFR) genotype.2MTHFR is a critical enzyme required for the synthesis of 5-methyltetrahydrofolate, an active form of folic acid in blood. It is involved in converting homocysteine to methionine,6 and a known polymorphism of MTHFR is a transition of C to T at nucleotide 677 of the MTHFR gene, which is a cause of reduced MTHFR activity7 and hyperhomocysteinemia.8 Moreover, the Hordaland Homocysteine Study reported a trend toward restricted intrauterine growth as the number of T alleles increased.9 In addition, previous studies reported that women homozygous for C677T in MTHFR had an increased risk of preeclampsia and placental vasculopathy.2,3 Although previous studies showed that MTHFR C677T mutation could lead to hyperhomocysteinemia and retardation of intrauterine growth, there is no direct evidence that variation in maternal homocysteine due to MTHFR genotype affects offspring growth in utero. One approach to investigate causal inference, Mendelian randomization, is an application of instrumental variable (IV) analysis that uses genetic instruments, due to the principal of independent assortment of an allele during meiosis. The use of Mendelian randomization in an observational study was introduced by Katan.10 In the present study, we use maternal genotype as an unconfounded and unbiased instrument for homocysteine level in maternal blood. The application of IV analysis using maternal genetic variants allows us to estimate intergenerational effects.Homocysteine metabolism is affected by many factors, including age, gender, current diet, and methylenetetrahydrofolate reductase .n = 720). For all participants, venous blood was collected from the antecubital vein during the morning after an overnight fast, and all participants responded to structured questionnaires. We excluded women who had twin births (n = 30), gestational diabetes (n = 23), hypertension, or chronic renal disease (n = 13), those with no data on homocysteine or folate level due to inadequate blood sampling (n = 92), and those with incomplete questionnaires (n = 89). Therefore, a total of 473 newborns were analyzed for this study . There was no difference between participants and excluded subjects in the frequencies of the CC, CT, and TT genotypes . This study was approved by the Institutional Review Board of Ewha Womans University Hospital.We recruited women between 24 and 28 weeks’ gestation who visited Ewha Womans University Hospital in Seoul, Republic of Korea for prenatal care during the period from August 2001 to December 2003. All participants gave their informed consent for study participation. Our analysis of deliveries during the period from October 2001 to February 2004  showed that 2481 women gave birth in Ewha Womans University Hospital. About 30% of them were included in this study (MTHFR C677T genotypes were identified by polymerase chain reaction (PCR) amplification and digestion with Hinf1. The PCR product resulted in synthesis of 198 bp fragments, which contain a C to T substitution at nucleotide 677 of the MTHFR gene. The fragments were cut into 175 and 23 bp fragments by the derived T allele and were visualized on 2.5% polyacrylamide gel with ethidium bromide staining after electrophoresis.Extraction of DNA from fasting venous blood was done using a QIAmp DNA blood kit  according to the protocol recommend by the manufacturer, and samples were stored at −20°C until analysis. 18 column. Folate was measured by a radioimmunoassay kit .High-performance liquid chromatography was used to measure homocysteine. Briefly, we mixed 100 µL of plasma with 10 µL of 10% tri-n-butylphosphine and stored the sample for 30 min at 4°C. Deproteinization was performed by adding 100 µL of 10% trichloroacetic acid, followed by centrifugation for 5 minutes at 3000 g. The supernatant was mixed with 250 µL of 0.125 M borate buffer and 100 µL of ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate acid. After incubation for 1 hour at 60°C, the mixture was filtered with a 0.45-µm syringe filter . Fluorescence detection was performed  using an Xterra RPBirth weight and length were measured in the delivery room by trained nurses using digital scales. Gestational age was calculated based on the maternal report of the date of last menstrual period and ultrasound measurement performed by an obstetrician.T alleles (in the codominant model) and as a continuous variable (in the additive model). We used a general linear model and the χ2 test to assess associations of confounding variables with the different MTHFR genotypes.Data analysis was conducted using the statistical package STATA (version 11.0). In descriptive analysis, continuous variables were presented as mean with SD and categorical variables as frequencies. Genotype was analyzed both as a categorical variable based on number of MTHFR C677T genotype was used as an IV to estimate the association between intrauterine homocysteine environment and the birth weight of offspring. Gestational age, the sex of the offspring, maternal age, and folate concentration were analyzed as confounding variables. We used the Durbin form of the Durbin–Wu–Hausman statistic to compare estimates of the effects of homocysteine on birth weight obtained from ordinary least-squares linear regression and IV analysis.11Mendelian randomization analysis was done using the “ivreg” command in STATA. Two-stage least-squares estimates were used to fit the IV analysis. Maternal CC, CT, and TT in the included  and excluded  subjects were similar and were in Hardy–Weinberg equilibrium (P > 0.05). The mean homocysteine concentration of the subjects was 5.76 µmol/L and did not significantly differ from that of excluded subjects  , and mean birth weight was 3299 grams. The genotype frequencies of .2%, and MTHFR C677T genotype and showed a dose–response association for each additional T allele: namely, CC (5.47 µmol/L), CT (5.75 µmol/L), and TT (6.48 µmol/L)  after controlling for gestational age and sex. In particular, adjusted mean birth weight for the TT allele was lower than that for the CC allele . However, homocysteine level was not significantly associated with birth weight, even after adjustment for confounding variables (data not shown).Birth weight significantly differed in relation to P = 0.06). Ordinary least-squares regression analysis showed a clear distinction in the direction of the associations, and the results of IV analysis and the Hausman test were statistically significant (P < 0.05). This suggests that, as compared with the ordinary least-squares method, IV analysis is more suitable for analyzing the relationship of maternal homocysteine and birth weight (data not shown).Table MTHFR C677T genotype and homocysteine level. There was a dose–response association between homocysteine and homozygosity for MTHFR C677T (TT), ie, homocysteine level significantly increased with the number of T alleles (P for trend <0.01). Furthermore, the findings of our advanced statistical approach suggest that maternal homocysteine concentration during pregnancy has an adverse effect on the birth weight of offspring, although the result was of marginal significance (P = 0.06), perhaps due to limited statistical power or the small sample size. To our knowledge, no other study has used an advanced statistical approach to investigate the association of intrauterine homocysteine exposure, as determined by maternal MTHFR genotype, with offspring birth weight.We found a strong association between MTHFR genotype to estimate the causal effect of variation in maternal homocysteine. The characteristics of Mendelian randomization permit genetic variants to be investigated in an observational study. Because such studies have properties similar to those of intention-to-treat analyses in randomized controlled trials,12 the problems of observational studies, eg, reverse causality and bias due to confounding factors, are avoided.13 In addition, genetic variants have larger effects on intermediate features than on the disease itself.14 The MTHFR C677T polymorphism is already established as an instrument: a study using Mendelian randomization reported that homocysteine level, as determined by MTHFR C677T gene variant, was associated with an increased risk for stroke.15As mentioned above, the use of maternal gene polymorphism as an instrumental variable in our analysis allowed us to use 3 A causal role for homocysteine in the pathogenesis of metabolic disorders has been suggested in hypertension, congenital heart disease, and cardiovascular disease.3,8,15 In addition, many previous studies have reported that homocysteine was associated with adverse effects on pregnancy and birth outcomes2,5,16; however, opinions differ in relation to causality. Regarding the genetic effect on homocysteine concentration, a transition of C to T at nucleotide 677 of the MTHFR gene was shown to cause hyperhomocysteinemia, which could inhibit MTHFR enzyme activity. Moreover, this inhibition of MTHFR enzyme activity might be amplified by folate deficiency.2 As in previous studies, we found that homocysteine negatively correlated with folate concentration in blood  and that homocysteine level was inversely associated with offspring birth weight, even after controlling for folate concentration in blood and other potential confounders. Studies reported that babies with MTHFR mutation were small and had low birth weights, due to growth restriction caused by MTHFR dysfunction.17,18 Moreover, several studies reported that variation in MTHFR C677T genotype resulted in different pregnancy outcomes. Newborns homozygous for C677T have approximately double the risk of adverse birth outcomes .19 Therefore, future studies should examine whether co-occurrence of MTHFR C677T in mother and fetus increases the risk of undesirable pregnancy outcomes.Homocysteine is normally found in blood, and it has been suggested that increased levels impair endothelial vasomotor function and facilitate platelet production, thereby damaging arteries and blood flow.20 For that reason, a number of studies have used birth weight as a main outcome. In our previous study, we found that birth weights of offspring from mothers with hyperhomocysteinemia (≥15 µmol/L) were significant lower than those of offspring from mothers with normal homocysteine concentrations.21 However, that study did not adjust for relevant confounding factors and did not use an adequate statistical approach, such as multilevel modeling for path construction. Folic acid supplementation has been proposed as a treatment of hyperhomocysteinemia. A randomized control trial observed that oral folic supplementation significantly reduced plasma homocysteine level.22 Other studies reported that consumption of folic acid attenuated the risk of preterm birth in women with pre-eclampsia.16,23 However, lowering homocysteine level does not decrease disease risk. Hence, future studies should examine the true effect of folic acid supplementation on adverse pregnancy outcomes.The importance of birth weight to lifelong health has been a consistent theme during the last few decades. Birth weight is a good surrogate marker of intrauterine growth retardation caused by diverse factors. Low birth weight increases the risk of chronic diseases and adversely affects the health of subsequent generations.This study had several limitations. First, in a genetic epidemiology study the results of Mendelian randomization may be confounded by genetic variants in linkage disequilibrium with the variant of interest. In addition, unmeasured and residual confounding factors, such as nutritional intake during pregnancy, may have affected the results. A number of factors in the model, including homocysteine and maternal age, are associated with miscarriage and adverse birth outcomes. Because only women who successfully completed pregnancy were included in the present study, there is a possibility of selection bias. Last, due to lack of statistical power, generalizability of the results is problematic—similar previous studies enrolled more than 1000 people. Despite the relative small size of the present study, we obtained valuable results from IV analysis. Future studies should investigate the effect of homocysteine on birth outcomes by considering the genotypes of many people.24 The present advanced statistical method also allows for inference of causality. In addition, by considering a genetic polymorphism as an instrumental variable, it can assess unmeasurable effects. In conclusion, our results provide more evidence of an adverse relationship between maternal homocysteine level and birth outcomes and suggest that reduction of homocysteine levels during pregnancy could positively affect birth outcomes.A strength of the present study is that it used an innovative study methodology that differed from the designs of previous studies, which were mostly case-control studies.


Intake of monounsaturated fatty acids has been reported to reduce oxidative stress, insulin resistance and related inflammatory processes and may thus protect from skin photoaging. The objective of this study was to investigate the association between the risk of photoaging, monounsaturated fatty acids intake and the sources of monounsaturated fatty acids.A cross sectional study was conducted within the framework of the SUVIMAX cohort. The survey included 1264 women and 1655 men aged between 45 and 60 years old. Dietary monounsaturated fatty acids intakes were estimated by dietary source through at least ten 24-h diet records completed during the first 2.5 years of the follow-up period. Severity of facial skin photoaging was graded by trained investigators at baseline during a clinical examination using a 6-grade scale illustrated by photographs. A lower risk of severe photoaging was associated with higher intakes of monounsaturated fatty acids from olive oil in both sexes. Strikingly, no association was found with intake of monounsaturated fatty acids from animal sources whether from dairy products, meat or processed meat.These findings support the beneficial effect of dietary olive oil or healthy diet habits associated with olive oil consumption on the severity of facial photoaging. In the past century, life-expectancy has increased in most developed countries Skin aging is driven by both intrinsic and extrinsic factors. Intrinsic aging, also referred to as chronological skin aging, is an ineluctable process Numerous studies have focused on the possible role of diet in the capacity of the skin to resist damage induced by UV radiation In this context, we have performed an analysis within the framework of the SU.VI.MAX cohort designed to explore possible associations between the severity of facial skin photoaging, MUFA intake and the sources of MUFA. Photoaging was measured using a 6-grade scale specially developed and validated for assessing the overall severity of photodamage, including pigmentation abnormalities, wrinkling and tissue slackening Supplémentation en Vitamines et Minéraux Antioxydants) study, a double blind, placebo-controlled primary prevention trial evaluating the effect of antioxidant supplementation  on the incidence of ischemic heart diseases and of cancers in a population of adult men and women. A total of 13,017 volunteers, 7876 women and 5141 men were included in 1994–1995 with a planned follow-up of eight years. Men were aged 45–60 years and women 35–60 years at enrolment. The design, objectives and methodology of the study have been described in extenso elsewhere ad hoc ethical committees, the “Comité Consultatif de Protection des Personnes dans la Recherche Biomédicale” , and the “Commission Nationale de l’Informatique et des Libertés” (CNIL no. 334641). The study was registered at clinicaltrials.gov as NCT00272428 Subjects were participants in the SU.VI.MAX . Dietary data were collected using the Minitel Telematic Network, a small terminal that was widely used as an adjunct to the telephone in France at the beginning of the study in the 1990s. This 24h record included about 900 items relating to food and drink for each of three meals  and for four other possible occasions for food intake (snacks). For each item, the subjects were requested to indicate the portion size consumed. To improve the quality of data collected, subjects received an instruction manual at the start of the study, including photographs of three portion sizes of about 250 foods and drinks. The use of the manual has been validated elsewhere Food composition was determined using the SU.VI.MAX food composition table The severity of facial skin photoaging was assessed at baseline by trained investigators using a six-grade ordinal scale, each grade being depicted by three photographs to illustrate the diversity and the range of manifestations within each grade 2, 25–30 kg/m2, ≥30 kg/m2. For geographical location, France was arbitrarily divided into North and South areas using the northern frontier of Aquitaine, Limousin, Auvergne, and Rhône-Alpes regions. In addition, skin phototype was determined at baseline according to the classification proposed by Césarini: I, II, IIIa, IIIb, IV, V, VI Data on age, geographical location (postcode), smoking habits , physical activity , educational level , and hormonal status  were collected through a self-administrated questionnaire at inclusion. Height and weight were measured using standardised procedures in subjects wearing undergarments. Body mass index (BMI) was calculated as weight (in kilograms) divided by height , and categorized into three groups: <25 kg/mFor the present analysis, we included 1264 women and 1655 men aged between 45 and 60 years old, from both placebo and intervention groups, with data for dietary intake and skin photodamage .Separate analyses were conducted for each sex. First, nutrient density was calculated by expressing MUFA as a percentage of total energy intake (TEI) and this nutrient density was then categorized into quartiles. Individual MUFA densities from the main food sources and the intake of the three most frequently consumed oils containing large amounts of MUFA were similarly categorized into quartiles.Due to the ordinal nature of the outcome variable All tests were two-sided and type I error was set at P<0.05. Statistical analyses were carried out using SAS® software release 9.1.3 .The distribution of the women and men enrolled according to photoaging severity and by demographic, medical and behavioral variables is presented in vs lowest quartile of MUFA: AOR = 0.76, 95%CI (0.57–1.00), p = 0.03). For both sexes, a higher consumption of MUFA provided by vegetable oils was found to be associated with a lower risk of severe photoaging (for women: 0.63 (0.44–0.90), p = 0.01; for men: 0.55 (0.40–0.76), p = 0.0004). No association was found with MUFA intake from dairy products, meats and processed meats. Finally, of the three most frequently consumed oils , a significant association was found for olive oil. A higher intake of olive oil was significantly associated with a lower risk of severe photoaging (for women: 0.69 (0.50–0.95), p = 0.03; for men: 0.58 (0.43–0.77), p = 0.0002). In our population, olive oil was the main source of vegetable oil MUFA , whereas sunflower and peanut oils provided only 15% and 13% of vegetable oil MUFA.After adjustment for possible cofounders, a significant association was found in men between severity of photoaging and dietary intake of MUFA . Higher In this study we report a significant association between total intake of MUFA and skin photoaging in men but not in women. When the individual contribution of each source of MUFA was considered, higher intakes of MUFA from vegetable oil were however found to be negatively associated with severe skin photoaging independently of environmental factors known to cause premature and accelerated skin aging in both sexes, whereas intake of MUFA from animal products  was not significantly associated with skin photoaging. In particular, a higher consumption of olive oil was inversely correlated with the severity of skin photoaging. Olive oil was the only one of the three vegetable oil sources of MUFA usually consumed in our study population  to present such a protective effect. These findings are consistent with previous studies which have addressed individual aspects of this relationship The observed negative association between olive oil intake and severe photoaging may be due to its specific fatty acid profile with a high amount of MUFA and a low ratio of n-6 PUFA/n-3 PUFA Our study has both strengths and limitations. The strengths encompass the assessment of dietary intake based on a mean of ten computerized 24-hour diet records in order to take into account weekly and seasonal intra-individual variability in the intake of the monounsaturated fatty acids, which may be considerable In conclusion, our findings provide support for a beneficial role of olive oil in preventing severe facial photoaging. This result should be supported by further mechanistic studies taking into account the relationship between a diet rich in MUFA, subcutaneous fat and the overall aging process, of which skin photodamage may be a readily observable surrogate marker. Finally, our findings provide a useful insight into the beneficial effect of olive oil, as the main source of dietary fat, as promoted in a diversified diet, although the only way to demonstrate that olive oil can prevent photodamage is to perform an interventional study.


To detail the highly variable ocular phenotypes of a French family affected with an autosomal dominantly inherited vitreoretinopathy and to identify the disease gene.VCAN) transcripts was performed after mutation detection in the VCAN gene.Sixteen family members with ten affected individuals underwent detailed ophthalmic evaluation. Genetic linkage analysis and gene screening were undertaken for genes known to be involved in degenerative and exudative vitreoretinopathies. Qualitative reverse transcriptase-PCR analysis of the versiscan , which produced aberrantly spliced VCAN transcripts.The first index patient of this French family was referred to us because of a chronic uveitis since infancy; this uveitis was associated with exudative retinal detachment in the context of a severe uncharacterized familial vitreoretinopathy. Genetic linkage was obtained to the Extensive molecular investigation allowed us to classify this familial vitreoretinopathy as Wagner syndrome. This study illustrates the need to confirm clinical diagnosis by molecular genetic testing and adds new ocular phenotypes to the Wagner syndrome, such as vascular and inflammatory features. Structurally, the core protein of versican is organized as a tridomain with two globular domains G1 and G3 at the N and C-terminals, respectively, and two CS attachment domains CSα and CSβ in its central area. The N- and C-terminal regions of versican contain multiple motifs that can interact with diverse extracellular matrix and cell-surface structural molecules [VCAN gene, coding for the glycoaminoglycan (GAG)-α and GAG-β domains respectively, generates four isoforms of versican  with a different number of chondroitin sulfate chains and whose expressions are temporally and spatially regulated in a specific manner [The disease gene for Wagner syndrome, recently referred to as olecules ,9. Alterc manner -12. The c manner , whereasc manner . The trac manner ,16.There is considerable evidence that differential expression of the lectican family of chondrotin sulfate proteoglycans may be important factors for modulating cell adhesion as well as axonal growth and guidance during neural development . In the VCAN gene. A total of six different causative mutations, all affecting either the conserved acceptor splice site of intron 7 or the donor splice site of intron 8 of the VCAN gene, have been identified [To date, only nine Wagner families have been reported with the identification of the molecular defect in the entified -22. It hentified ,21. TherVCAN gene (c.4004–2A>T) that produces aberrantly spliced VCAN transcripts identical to those found in the Japanese Wagner family with the (c.4004–2A>G) VCAN splice site mutation [We report a four-generation French family presenting a severe vitreoretinal disorder inherited as an autosomal dominant trait without evident systemic clinical features. The first index patient was referred to us because of a chronic uveitis associated with exudative retinal detachment in the context of a severe uncharacterized familial vitreoretinopathy. The clinical diagnosis of the disease presented by this family was controversial, but genetic linkage was obtained to the gene for Wagner syndrome. Subsequently, we identified a novel acceptor splice site mutation in intron 7 of the mutation . Here, wThe study followed the tenets of the Declaration of Helsinki and was approved by the ethics review committee of the Institutional Review Board of Cochin Hospital . Informed consents were obtained from all participating patients before donation of blood samples. Complete ophthalmologic examinations were performed in affected and unaffected family members. Kinetic perimetry was tested with the Goldmann perimeter , and fundus photography was performed when pupil dilatation was sufficient . Optical coherence tomography imaging was obtained with the Stratus OCT  and full-field electroretinography was performed on selected affected patients with the MonElec2 Monitor Model . Anterior chamber flare was quantified with the Kowa FM 500 flare meter .Marshfield human genetic linkage map. These markers are closed to the genes for Collagen, type II, α-1 , Frizzled, Drosophila, homolog of, 4 , low density lipoprotein receptor-related protein 5 , and versican . Linkage analysis was performed under the assumption of a dominant model with 100% penetrance and 0.0001 frequency of the disease allele. Two-point linkage analyses were performed using the MLINK program from the Linkage package, version 5.1.Genotyping was performed using fluorescently tagged microsatellite markers, which were previously reported or were selected from the FZD4 , LRPR5 , norrin , tetraspanin 12  genes and exons 6, 7, 8, and 9 of the VCAN gene were amplified by PCR. PCR primers for VCAN, FZD4, and NDP exons were developed using the Primer 3 software, and all these sequences are listed in TSPAN12 and LRP5 exons were designed as described previously [DNAs were extracted from peripheral blood samples, using the QIAamp DNA mini kit . Exons and intron–exon junctions for the eviously . SequencTotal RNA was extracted from cultured lymphoblastoid cells from two affected family member’s (V.5 and IV.8) and one control unaffected subject (V.4), with a commercial kit . cDNA was synthesized from 1 µg total RNA using random hexamers and M-MLV reverse transcriptase . RT–PCR was performed using different sets of primers previously designed for specific amplification of each of the four versican isoforms .Patient V.5, our index case, was a 29-year-old female patient who had been referred to our ophthalmologic center for repetitive anterior uveitis with iris–lens synechiae . She wasPatient V.2, a 31-year-old female first cousin of the index case, had a BCVA of 0.4 in her right pseudophakic eye and 0.25 in her left eye. Because of her family medical history, her first ophthalmologic examination was performed at the age of 1 year and showed vitreoretinal exudative lesions; the disease was classified as familial exudative vitreoretinopathy. Bilateral peripheral cryotherapy was performed at the age of 9 years and was followed by an RD in her left eye, which was treated by a scleral buckling procedure. An RD of the left eye was treated by vitrectomy and cryotherapy at the age of 13 years. Cataract surgery by phacoemulsification with placement of a posterior chamber IOL was performed at the age of 29 years in her right eye. At the time of our examination, her IOP was 12 mmHg OD and 16 mmHg OS. Laser flare photometry was minimally increased to 14 photons/ms OD and more significantly to 39 photons/ms OS. Fundus examination showed a bilateral nasal stretching of retinal vessels with ectopic foveae and nasal pigment clumping . The vitPatient V.6, a 36-year-old female first cousin of the index case, had a BCVA of 0.6 OD and no light perception in her left eye. She had a history of RD in her left eye, which was diagnosed at age 6 and which was unsuccessfully operated on. Although she had no light perception in that eye, a white cataract was removed at the age of 33 years for esthetic purposes, without placement of an IOL. Cryotherapy in her right eye was performed at the age of 8 and 12 years, and this was later followed by laser photocoagulation of the retinal periphery. At the time of our examination, laser flare photometry was minimally increased to 12.5 photons/ms OD and more significantly to 32.3 photons/ms in her left aphakic eye. Examination of the fundus showed a clear vitreous without strands or veils, and vessels did not appear stretched. Optic disc pallor was observed in the left eye only. In the right fundus, retinal atrophy was seen in the peripapillary area and along the temporal vascular arcades, and this was associated with pigment clumping . In the The medical history and ocular phenotypes of affected family members of the fourth generation are summarized in As a first step toward identifying the causative gene in this four-generation French family, we performed linkage analyses. Sixteen family members, including ten affected individuals and six unaffected individuals, were genotyped, and significant evidence of linkage was shown with markers D5S641  and D5S2103 , indicating a critical disease region for the WGN1 locus on 5q13-q14 .VCAN gene from the index patient (V.5) DNA sample revealed a novel heterozygous A>T transversion at the second base of the 3′-acceptor splice site of intron 7  and one control subject (V.4). Results of RT–PCR indicated that the c.4004–2A>T mutation activates a cryptic acceptor splice site within exon 8, located 39 bp downstream from the authentic 3′ splice acceptor site. Indeed, electrophoresis in 3% agarose Nusieve gel of RT–PCR products obtained with primer pairs 7F/8R revealed a band of 372 bp and a slightly shorter fragment of 333 bp in RNA sample from the index patient, but only one fragment of 372 bp in the control RNA sample (The effects of this novel A sample . Direct A sample . AdditioVCAN gene (c.4004–2A>T). This new single base pair substitution affects the same nucleotide of the intron 7 splice acceptor site that had been substituted into a guanine (c.4004–2A>G) in a previously reported Japanese Wagner family [VCAN transcripts identical to those found in the Japanese Wagner family [We report the first French family with ocular features of Wagner syndrome segregating with a adenine to thymine transversion at the highly conserved splice acceptor site in intron 7 of the r family . In the r family .VCAN gene was achieved. Exudative vascular features are unusual phenotypic findings in Wagner syndrome, and the exudative component was significant enough that the initial diagnosis of autosomal dominant exudative vitreoretinopathy (FEVR) in our index case was highly suspected. Indeed, this patient, who was born at term with normal birthweight and who did not received oxygen therapy, showed peripheral retinal vascular abnormalities with severe retinal exudates leading to exudative retinal detachment, telangiectatic neovascularization, peripheral chorioretinal pigmentation, straightening of the retinal blood vessels, and situs inversus similar to a FEVR-like phenotype. To exclude genetic heterogeneity in the kindred studied here or a synergistic effect of an additional mutation in a gene implicated in FEVR, we performed mutation screening of FZD4, LRP5, NDP, and TSPAN12 genes, which are known to be responsible for FEVR. We finally demonstrated that these heterogeneous clinical presentations exhibited by this single French kindred are linked to the same molecular defect in the VCAN gene. The visual impairment of the disease was highly significant in our pedigree since, among ten affected family members, three are totally blind and five other patients have completely lost vision in one eye. RDs were particularly frequent and occurred at an early onset in the family studied here, and the RDs also had a poor surgical prognosis. Therefore, RD in Wagner syndrome appears to be a more common feature and one that occurs earlier than previously suggested [This large single kindred demonstrated a range of highly variable phenotypes, from exudative vascular abnormalities and diffuse retinal atrophy with pigment clumping to nasally deviated retinal vessels with ectopic foveae, leading to different initial diagnoses of the disease until characterization of the molecular defect in the uggested . Our firuggested . Therefouggested ,25,26. Auggested . Moreoveuggested .In conclusion, molecular investigation of this French family allowed us to classify this severe uncharacterized familial vitreoretinopathy as Wagner syndrome. This study illustrates the necessity to confirm clinical diagnosis by molecular genetic testing and adds new ocular phenotypes to the Wagner syndrome, such as vascular and inflammatory features. Therefore, phenotype–genotype studies of additional Wagner patients are necessary to establish whether such vascular and inflammatory features are consistent observations in Wagner disease.


EGFR and KRAS mutations in non-small cell lung cancer (NSCLC) have predominantly been determined in East Asian and North American populations, showing large differences between these populations. The aim of the present study was to determine the frequency of EGFR and KRAS mutations in NSCLC in the West European Dutch population in primary carcinomas and different metastatic locations.Frequencies of EGFR  and KRAS (exons 2 and 3) mutation test results of NSCLC samples of patients in 13 hospitals were collected. The tests were performed on paraffin-embedded tissue or cytological material of primary and metastatic lung carcinomas.EGFR mutations were detected in 71/778 (9.1 %) tested patients; in 66/620 (10.6 %) adenocarcinomas. EGFR mutations were significantly more often detected in female than in male patients . KRAS mutations were found in 277 out of 832 (33.3 %) tested patients; in 244/662 (36.9 %) adenocarcinomas. A significantly increased frequency of EGFR mutations was observed in patients with malignant pleural/pericardial effusions  2.80, 95 % confidence interval (CI) 1.22–6.41), whereas the frequency of KRAS mutations was significantly decreased .EGFR mutations in lung adenocarcinomas in this West European population is within the frequency range of North American and South European populations, whereas KRAS mutation frequency is higher than in any population described to date.In the investigated Dutch cohort, patients with malignant pleural/pericardial effusion of lung adenocarcinoma have an increased frequency of EGFR mutations. The overall frequency of  EGFR gene, whereas tumors harboring activating mutations in the KRAS gene do not respond to this treatment [Non-small-cell lung cancer (NSCLC) often presents at an advanced stage with no options for curative treatment. Response to chemotherapy is rather poor, resulting in a median overall survival of approximately 1 year. New therapies, targeting specific signaling pathways, have been developed over the last years. These include the small-molecule tyrosine kinase inhibitors (TKIs) erlotinib and gefitinib, blocking signaling through the epidermal growth factor receptor (EGFR). Treatment with these TKIs seems to be especially effective in tumors with activating mutations in the EGFR mutations has been studied most extensively in East Asian populations, where it varies from 36.4 to 66.3 % in adenocarcinoma (ADC) [KRAS the situation is reversed, with a low mutation frequency of 2.3 to 9.4 % in East Asian [EGFR and KRAS mutations has not been studied in large numbers of patients.The frequency of ma (ADC) , 3. Studma (ADC) . For KRAst Asian , 8. HoweEGFR mutation rate. EGFR mutations in these populations are associated with female patients, patients who have never smoked and patients with an adenocarcinoma [EGFR mutations are usually studied in tissue from the primary lung tumor. Although the association between patient characteristics and mutation status has been investigated extensively, the association between different metastatic locations and EGFR status has remained relatively underexplored.In East Asian and South European populations it has been demonstrated that certain patient characteristics are associated with an increased arcinoma , 9, 10. EGFR and KRAS mutations in the Dutch population in both primary NSCLCs and different metastatic locations.The aim of the present study was to determine the frequency of EGFR and KRAS mutation analysis from 13 hospitals in the Netherlands from January 2008 to April 2011. The participating hospitals were St. Antonius Hospital, Nieuwegein; University Medical Center Utrecht; Rijnstate Hospital, Arnhem; Medical Center Alkmaar; Jeroen Bosch Hospital, ‘s Hertogenbosch; Radboud University Nijmegen Medical Center; Zuwe Hofpoort Hospital, Woerden; Slingeland Hospital, Doetinchem; Gemini Hospital, Den Helder; Hospital Gelderse Vallei, Ede; Hospital Bernhoven, Oss and Veghel; Maas Hospital Pantein, Boxmeer; and Elkerliek Hospital, Helmond. Mutation analysis was centrally performed in four Pathology Departments .We collected data of all NSCLC samples used for routine diagnostic The results were obtained using formalin-fixed paraffin-embedded tissue or cytological slides of primary lung tumors and metastases. The patients’ smoking status was included for a subgroup of patients whose samples were analyzed at St. Antonius Hospital, Nieuwegein and University Medical Center, Utrecht. Patients were categorized as never smokers (<100 lifetime cigarettes), former smokers (≥1 year since cessation), or current smokers (still smoking or <1 year since cessation). When available, the number of packyears was included in the smoking history.Specific approval of the ethics committee was not necessary for this study, since all mutation analyses were part of the routine diagnostic procedure and all patient and tumor characteristics were collected anonymously.EGFR and KRAS mutation analysis. In most cases mutation analysis was performed by polymerase chain reaction followed by sequencing of the EGFR  and KRAS (exons 2 and 3) genes, using GenBank Accession Numbers NM_005228.3 and NM_004985.3 as a reference. The samples of the patients from the hospitals in Utrecht, Alkmaar, and ‘s Hertogenbosch were tested using high resolution melting analysis, followed by sequencing only if the obtained melting curve was abnormal, as described previously [Two methods were used for eviously .EGFR and KRAS status and patient and tumor characteristics were analyzed using the Fisher’s exact test. Normal distribution of age and number of packyears was tested using the Kolmogorov-Smirnov test and means were compared using the Mann–Whitney test or the T test when appropriate. Binary logistic regression analysis was used to compare the mutation frequency of different metastatic sites to the group of primary tumors. Odds ratio (OR) and 95 % confidence interval (CI) were calculated. In all tests, two-sided p-values of less than 0.05 were considered statistically significant.Statistical calculations were performed using SPSS software (version 15.0). Associations between EGFR mutation status was determined in 816 samples, 791 of which had an interpretable result. The samples consisted of 655 cases of ADC, 42 cases of squamous cell carcinoma (SCC), and 119 others . Of these samples, 462 were derived from primary tumors and 342 from different metastatic sites. For 12 pleural biopsies/excisions, distinction between a primary tumor and a metastasis was not possible. The samples were derived from 803 individual patients, of whom there was an interpretable result in 778 patients. For 13 patients the mutation status of both their primary tumors and a metastasis was determined. These patients were considered EGFR mutation positive if either the primary tumor or the metastasis was tested positive. EGFR mutations were detected in 71 (9.1 %) individual patients, 66 of whom had a diagnosis of ADC (frequency in ADC 10.6 %). The other 5 mutations were found in 3 large cell carcinomas, one adenosquamous carcinoma, and one sarcomatoid carcinoma (subtype spindle cell carcinoma). EGFR mutations were significantly more often observed in ADC than in SCC or other tumor types  and more frequently in female than in male patients  . Within the group of former and current smokers, the average number of packyears smoking history was significantly lower in EGFR mutation positive compared to EGFR mutation negative patients .EGFR mutations, deletions in exon 19 were the most common , followed by the L858R point mutation in exon 21  . The single nucletoid polymorphism A840T in exon 21 was detected twice.Among the KRAS mutation status was determined in 873 samples , 845 of which could be interpreted. Of these samples, 497 were derived from primary tumors and 363 from different metastatic sites. For 13 pleural biopsies/excisions, distinction between a primary tumor and a metastasis was not possible. The samples were derived from 860 individual patients, of whom there was an interpretable result in 832 patients. For 13 patients the mutation status of both their primary tumor and a metastasis was determined. These patients were considered KRAS mutation positive if either the primary tumor or the metastasis was tested positive. KRAS mutations were detected in 277 (33.3 %) patients, 244 of whom had a diagnosis of ADC (frequency in ADC 36.9 %). Like EGFR mutations, KRAS mutations were significantly more common in ADC than in SCC or other tumor types  and there was a trend towards more mutations in female compared to male patients , but this did not reach statistical significance . There was no difference in average number of packyears between KRAS mutation positive and KRAS mutation negative patients . The KRAS mutations consisted of point mutations in codon 12 , codon 13  and codon 61   of combined EGFR and KRAS mutations were detected: EGFR exon 20 S768N combined with KRAS exon 2 G12A and EGFR exon 20 G779S combined with KRAS exon 2 G12C. These were both cases of ADC in males, detected in bronchial biopsies.For 752 patients both the EGFR and KRAS mutations were found in ADC, we performed these calculations for ADC only. Pleural biopsies and excisions were excluded from these calculations, since discrimination between outgrowth of a primary tumor or a metastasis was not always feasible. The EGFR status was determined in 360 primary tumors and 261 metastatic sites, the KRAS status in 394 primary tumors and 268 metastatic sites. For EGFR mutations, no statistically significant difference in frequency in primary tumors compared to metastases  was observed  . Binary logistic regression analysis was performed to test which category or categories was responsible for this difference. Both for EGFR and KRAS the category pleural and pericardial effusions was associated with a difference in mutation frequency compared to primary tumors (EGFR OR = 2.80 (95 % CI 1.22-6.41), p = 0.015; KRAS OR = 0.35 (95 % CI 0.14–0.86), p = 0.022). The frequency of EGFR mutations in this category was 26.5 %; the frequency of KRAS mutations was 18.8 %. Also brain metastases showed a trend towards an increase in EGFR mutation frequency and a decrease in KRAS mutation frequency  compared to the group of primary tumors, but this did not reach statistical significance.Next, we divided the metastasis locations in different tissue types, using main categories with at least 10 samples each. The categories were lymph node, pleural and pericardial effusion, liver, brain, bone, soft tissue and others. The results are summarized in Tables EGFR and/or KRAS mutation status in 4 out of these 13 (30.8 %) pairs, leading to a clinically discordant mutation status in 3/13 (23.1 %) pairs: one KRAS mutation was present in the metastasis but not in the primary tumor; one EGFR mutation was present in the metastasis but not in the primary tumor, while this primary tumor showed a KRAS mutation that was not present in the metastasis; and one primary tumor showed both a deletion in exon 19 and the T790M point mutation in exon 20 of EGFR, whereas the metastasis only showed the deletion in exon 19. In one pair the primary tumor and the metastasis each contained a different KRAS mutation .The present study population contained 13 pairs of matched primary tumors and metastases. There was discordance of the EGFR mutation rate and metastatic location in patients with lung adenocaricnoma in the European population. We found an increased EGFR mutation frequency (27 %) and a decreased KRAS mutation frequency (19 %) in patients with malignant pleural or pericardial effusions. A similar trend was present in patients with brain metastases, although this did not reach statistical significance. These results correspond with two studies from East Asia, showing significantly higher EGFR mutation rates in tumors with malignant pleural effusions compared to those without [EGFR mutation rate in brain metastases [EGFR and KRAS mutation status could very well influence the pattern of metastasis formation, a topic that merits further investigation.To our best knowledge this is the first study showing an association between  without , and a rtastases . These oEGFR and KRAS mutations between primary tumors and matched metastases [Several studies have shown discordance in the presence of tastases , 15. In EGFR and KRAS mutations has mainly been studied in East Asian and North American and to a lesser extent in South European populations, showing large differences between these populations. This study is the first to address this topic in a large West European cohort. In adenocarcinomas, we found EGFR mutations in 11 % of cases. This is far below the 36–66 % reported for East Asia [EGFR gene was not included in the analysis, because this exon was not routinely investigated in all participating laboratories. This may have led to an underestimation of the EGFR mutation frequency, although exon 18 mutations have been reported to account for only a small minority of total EGFR mutations [The overall frequency of ast Asia , 3, but ast Asia , 5, 6–16ast Asia , 16, andast Asia . This siutations , 18.KRAS mutations were detected in 37 % of ADC, far above the 2–9 % reported for East Asia [KRAS mutation frequency in our West European cohort is even higher than the 12–31 % and 11–29 % reported for North America [KRAS mutations [ast Asia . The KRA America , 8 and S America . A possiutations . In addiEGFR mutations were twice as often observed in female compared to male patients . This observation confirms that the difference in EGFR mutation frequency between male and female patients that previously has been described in Asian and South European populations, is also present in the West European population [KRAS mutations in female compared to male patients (36 % vs. 31 %), but this did not reach statistical significance. Previous studies also did not show a statistically significant correlation between KRAS mutation status and sex, though some studies showed a trend towards more mutations in males [KRAS mutations was very low as these were performed in East Asian populations.pulation , 16. We in males . HoweverEGFR mutation frequency was over five times higher in never smokers compared to former and current smokers. This strong negative association with smoking history has also been observed in Asian, North American, and South European populations [KRAS mutations and smoking has also been reported before [The ulations , 9, 16. d before .EGFR and KRAS mutations were present more commonly in ADC compared to SCC and other tumor types. This observation confirms previous results [Both  results .EGFR mutations consisted mainly of deletions in exon 19 and the L858R point mutation in exon 21. These two types of mutations combined formed 81 % of all EGFR mutations in our cohort, which approximates the 85 to 90 % mentioned in previous studies [The  studies . Possibl studies , we foun studies , 23–27,  studies , 29. ForEGFR mutation (one deletion in exon 19 and two L858R point mutations in exon 21). The T790M point mutation is considered to cause secondary resistance to treatment with TKIs and thus is expected to be present after treatment only [Three T790M point mutations in exon 20, known to cause resistance to treatment with TKIs, were detected, all in combination with an activating ent only , althougent only . The insent only , 6. Thisent only , 32.EGFR and KRAS mutations are generally considered to be mutually exclusive [EGFR and KRAS mutations [EGFR and KRAS mutations. Interestingly, in both cases the EGFR mutation was a rare point mutation (S768N and G779S) with unknown clinical significance. Possibly these mutations do not cause constitutional activation of EGFR and thus have no clinical significance. This could also explain why they have often been found in combination with known activating EGFR mutations, as described previously [xclusive –4, 9, thutations . We founeviously , 26.A limitation of the present study is the fact that it does not provide a true cross-section of non-small cell lung carcinomas, since most analyses were requested by the treating physician. This introduces a selection bias towards more advanced disease stages, because the treating physician will generally not request the analysis for patients who have been cured surgically. However, this corresponds to daily clinical practice, and the population studied here, is the population that would potentially benefit from treatment with TKIs, which still makes the data relevant.EGFR and KRAS mutation frequencies are not evenly distributed among different metastatic sites, with a relative increase in EGFR mutation frequency and a decrease in KRAS mutation frequency in pleural and pericardial effusions compared to primary tumors, which could have implications for patient management. In the West European Dutch population the overall EGFR mutation frequency lies within the range of frequencies described for North American and South European populations. For KRAS the frequencies are considerably higher than those described in any other population studied to date. Both EGFR and KRAS mutations were found more often in ADC than in other tumor types and EGFR mutations were more common in females compared to males.In conclusion,


Soil microorganisms are key players in biogeochemical cycles. Yet, there is no consistent view on the significance of microbial biodiversity for soil ecosystem functioning. According to the insurance hypothesis, declines in ecosystem functioning due to reduced biodiversity are more likely to occur under fluctuating, extreme or rapidly changing environmental conditions. Here, we compare the functional operating range, a new concept defined as the complete range of environmental conditions under which soil microbial communities are able to maintain their functions, between four naturally assembled soil communities from a long-term fertilization experiment. A functional trait approach was adopted with denitrifiers involved in nitrogen cycling as our model soil community. Using short-term temperature and salt gradients, we show that the functional operating range was broader and process rates were higher when the soil community was phylogenetically more diverse. However, key bacterial genotypes played an important role for maintaining denitrification as an ecosystem functioning under certain conditions. Rapid decrease in biodiversity due to human activities has lead to a large body of research focusing on the relationship between biodiversity and ecosystem functioning The positive effects of biodiversity on ecosystem functioning are attributed to complementarities among species that would enhance resource use or simply to the increased probability of finding a few high-performing key species within the community 2O. In the present study, denitrification was used as a proxy for ecosystem functioning and denitrification rates were monitored over broad temperature and salt gradients. Denitrifier biodiversity in terms of genotypic dissimilarities was assessed using traditional biodiversity indices and phylogenetic diversity metrics in order to more fully evaluate the link between the denitrification operating range and the biodiversity of the denitrifying community.Here, we used the insurance hypothesis to address the role of microbial biodiversity for ecosystem functioning using naturally assembled soil microbial communities as our model system. Maherali and Klironomas To sample naturally assembled denitrifier communities, we used soil from the Ultuna long-term soil organic matter experiment in Uppsala, Sweden established in 1956 from the following treatments: unfertilized bare fallow (A), planted with maize and unfertilized (B), fertilized with calcium nitrate (C) and fertilized with cattle manure (J). The set up is a block design with three independent replicate blocks for each soil treatment partitioned into 2 by 2 m plots. Composite samples of ten soil cores  were taken from each treatment plot (n = 12) in between rows in October 2007 after harvest, sieved (4-mm mesh) and stored at −20°C. Access to the site and data on soil properties were provided by The Department of Soil and Environment, Swedish University of Agricultural Sciences, Uppsala  were added to 8 g soil in flasks that were sealed and then purged five times, by evacuating the ambient air and filling with N2. Acetylene was added to reach 0.1 atm partial pressure to block N2O conversion to N2. The soils were then incubated under agitation at 2, 10, 20, 30, 37, 45, 53, and 60°C. Another set of soil microcosms were set-up similarly to assess denitrification activity in a salt gradient created by addition of NaCl to a final concentration of 0, 0.5, 0.75, 1, 1.5, 2, 3, and 4% w/v and then incubated under agitation at 30°C. For both the temperature and salt gradient experiments, gas samples were collected every half hour during 3 hours of incubation. Nitrous oxide in the gas samples was analyzed on a gas chromatograph  equipped with a 63Ni electron capture detector. The amount of N2O produced was corrected for the amount dissolved in the liquid at each test temperature The functional operating range for the microbial process denitrification, used as a proxy for ecosystem functioning, was assessed by measuring potential denitrification activity across temperature and salt gradients for each of the three field replicates from each soil community according to Pell et al 2O produced during incubation using the statistical software R . To characterize the functional operating range of each soil community, the data on effects of temperature and salt concentration on the denitrification activity were modeled using the statistical software SigmaPlot 12 . Briefly, the potential denitrification rates under the temperature gradient were fitted with the Gaussian function:v is the rate obtained at the temperature T, maxv the maximum rate obtained at the temperature mT, and w multiplied by 2.355 gives the full width at maxv/2. Modeled rates according to this equation gave good fits 2, and to start decreasing from the uninhibited rate 0v at the threshold concentration c = [k/(0v+ a)]2. Fits of the modeled rates  was carried out followed by a Tukey’s HSD test using R at nosZ gene encoding the nitrous oxide reductase catalyzing the last step of the denitrification pathway as a molecular marker of the genotypic dissimilarity and phylogenetic diversity of the denitrifier community. The nosZ gene is the molecular marker among denitrifiers that is most congruent with taxa affiliation We used the nucleotide variation in the nosZ gene was amplified using primers nosZ-F and nosZ-R DNA was extracted in duplicate from 250 mg of soil from each of the soil replicates using the ISO 11063 soil DNA extraction method nosZ PCR products from each soil sample (n = 12) were gel purified using the MinElute purification kit  and cloned using the pGEM-T Easy Vector System . Approximately 100 clones from each of the 12 libraries were randomly selected for amplification using the T7 and SP6 vector primers. The PCR products were then digested with the restriction endonuclease AluI as previously described AluI was selected after being tested in silico on a diverse set of nosZ fragments downloaded from public databases. The restriction fragments were resolved by electrophoresis in a 3% high-resolution agarose gel  and clones were grouped according to their restriction profile. Randomly selected clones from each RFLP group were sequenced by Beckman  using the vector primer T7. In addition to phylogenetic analysis, the nosZ sequences were used to verify the RFLP restriction profiles and ensure representative sampling of the soil treatments. The sequences were analyzed for restriction sites using Geneious v 5.0.4 . To display how the RFLP groups correspond to the nosZ phylogeny, a maximum likelihood (ML) phylogenetic analyses (see below) of representative sequences from each RFLP group and similar sequences from pure cultures and other soil clones retrieved from Genbank was performed. The 400 nosZ sequences were deposited in Genbank under accession numbers JF310276 to JF310675.We used cloning followed by restriction fragment length polymorphisms (RFLP) analysis and sequencing to assess denitrifier diversity. The H’) indices for each soil community were calculated based on RFLP patterns To explore differences in genotypic dissimilarities among soil communities, shared RFLP groups were visualized in a Venn diagram using the software mothur nosZ nucleotide sequences were translated into protein residues, aligned using CLUSTAL X in Geneious v 5.5  and then back-translated into aligned nucleotides using MacClade v 4.05 . Maximum likelihood (ML) phylogenetic analyses for this dataset of 400 sequences of 708 bp were completed using the RAxML-HPC Blackbox (v 7.2.6) program www.phylo.org). The ML tree was displayed and annotated by treatment using the Interactive Tree OF Life (iTOL) nosZ genotypes calculated as the sum of branch lengths separating genotypes in the phylogeny. Thus, the total amount of phylogenetic distance among genotypes in a specific community is estimated, which is influenced both by how related genotypes are to each other on average and how many that are present. If we assume that closely related genotypes are more similar and have similar niches, PD integrates several aspects of biodiversity. In addition to PD, net relatedness index (NRI) testing basal relationships and nearest taxon index (NTI) testing relatedness at the tips of the tree were calculated to gauge the degree of phylogenetic clustering or overdispersion for sequences by replicate and soil community across the phylogeny The phylogenetic diversity metric and phylogeny based community composition were calculated using the 90, 99, 77 and 134 sequences representing the RFLP groups from soil communities A, B, C and J, respectively. Phylogeny-based community composition among the 12 soil communities were tested using unweighted UniFrac analysis, which calculates the unique fraction of shared branches on the ML tree pairwise for each replicate based on the presence or absence of shared lineages in the tree w) of the denitrification rate curves in the temperature gradient was broader in community J (P<0.1) than in the other communities, for which no significant differences were observed. Similarly, the optimum temperature for denitrification (mT) was not different for communities A, B, and C, but was significantly higher for community J.Significant differences in denitrification activity were observed between the soil communities. Community J had the highest activity in both the temperature and salt gradient experiments with lower rates in decreasing order for C, B, and A coincident with significant decreases in soil carbon and nitrogen content . To testmunity J ; P<0.1 tP<0.1). However, comparison of the extrapolated salt concentrations when the denitrification rates were completely inhibited (SA0) indicated significant differences between community J and the other communities, with J being active up to 5.9% salt, whereas communities A, B, and C ceased denitrification activity at 2.9, 2.4, and 4.2% respectively.Extrapolated values for salt concentrations when inhibition of the denitrification activity (SAI) began showed that communities A and B were significantly different from C and J, which had higher salt tolerance ; P<0.1. nosZ clones from the 12 different libraries could be separated into 64 RFLP groups reflecting the genotypic dissimilarities. They were distributed among community A, B, C and J each with 30, 34, 23 and 44 groups, respectively  and accounted for 17% of all the variation present in the dataset.Denitrifier community diversity in terms of richness, evenness, and phylogenetic diversity differed between the soil communities, as well as the community composition based on genotypic dissimilarties  and phylectively  and S2. ectively . All comectively . Chao ane lowest . The AMOnosZ gene sequences . The only diversity metric that correlated with parameters from the modeled denitrification rates was PD and optimum temperature .Phylogenetic diversity based on ML phylogenetic analyses of equences  revealedequences . The PD equences . Both NRequences . In pairr = 0.62, P = 0.001). Vectors in the NMS indicate that diversity metrics and modeled rate parameters were significantly correlated with the ordination  and harbored the highest number of total and unique community members. This supports that the insurance hypothesis, stating that biodiversity is important for ecosystem functioning under changing environmental conditions The genotypic dissimilarities among the four soil communities are the result of 50 years of different fertilization regimes. These results are consistent with a large body of literature emphasizing the impact of agricultural practices on microbial communities, including nitrogen cycling communities and denitrifiers, through alteration of soil properties, e.g. If we assume that distantly related genotypes co-exist due to less competition, increased phylogenetic dissimilarity should reflect increased functional dissimilarity. This has been shown true for plant, fungal and bacterial communities Our results do not establish a straightforward relationship between the functional operating range and a level of biodiversity, but rather show complex patterns that vary with the community composition and environmental conditions. Recent work has demonstrated that phylogenetic dissimilarity rather than species richness is positively linked with community functioning in microbial communities Figure S1Denitrification rates at different temperatures and salt concentrations. The rates for each soil community replicate were modeled using a Gaussian function and a power equation for temperature (a) and salt concentration (b) gradients, respectively. The fitted curves and data points are colored by community and soil treatment: red, community A, fallow; green, community B, unfertilized; blue, community C, nitrate fertilized; and black, community J, cattle manure fertilized. Each field replicate within a treatment/community is represented by different symbols.(DOCX)Click here for additional data file.Figure S2nosZ clones in the 64 RFLP groups detected among the soil communities. Community A had 30, community B had 34, community C had 23 and community J had 44 groups. Data are normalized by percent of total number of clones for each soil community.Distribution of 1152 (DOCX)Click here for additional data file.Figure S3nosZ gene sequences (708 bp) from soil communities. Community and soil treatment are colored: red, community A, fallow; green, community B, unfertilized; blue, community C, nitrate fertilized; and purple, community J, cattle manure fertilized. The tree was displayed and colored by treatment using the iTOL web-based tool.Maximum likelihood phylogenetic analysis of 400 (DOCX)Click here for additional data file.Figure S4nosZ gene sequences (713 bp) from soil clones selected to represent each of the 64 RFLP groups described in Maximum likelihood phylogenetic analysis of 100 (DOCX)Click here for additional data file.Table S1p<0.05).Soil properties for the Ultuna long-term soil organic matter experiment . Values followed by the same letter indicate treatments without significant differences ((PDF)Click here for additional data file.Table S2w = measure of the width of the curve. All model parameters were statistically significant (p<0.0007).Goodness of fit and model parameters for denitrification rates in soil communities at different temperatures modeled for each field replicate using the following Gaussian equation: Denitrification rate =  (PDF)Click here for additional data file.Table S3k/√(c) - a, where k = slope, c = % salt concentration and a = intercept. The salt concentration when denitrification begins to be inhibited (SAI) and when the rate reaches zero (SA0) was extrapolated using the equation.Goodness of fit and model parameters for denitrification rates in soil communities at different NaCl concentrations and modeled for each field replicate using the following power equation: Denitrification rate = (PDF)Click here for additional data file.Table S4H’) indices or by comparative sequence analysis for phylogenetic diversity (PD), net relatedness index (NRI), and nearest taxon index (NTI) based on nosZ gene clones from each soil community replicate.Diversity indices of denitrifying bacterial communities determined using RFLP group membership for Chao and Shannons ((PDF)Click here for additional data file.


Corvus brachyrhynchos), a North American species that breeds in both urban and agricultural landscapes. We monitored 195 nestlings in 106 nests across an urban and agricultural gradient in the Sacramento Valley, California, USA. We found that 85.2% of crow nests contained anthropogenic material, and 11 of 195 nestlings (5.6%) were entangled in their nests. The length of the material was greater in nests in agricultural territories than in urban territories, and the odds of entanglement increased 7.55 times for each meter of anthropogenic material in the nest. Fledging success was significantly lower for entangled than for unentangled nestlings. In all environments, particularly urban, agricultural, and marine, careful disposal of potential hazards  could reduce the occurrence of entanglement of nestling birds.Much attention has been paid to the impacts of plastics and other debris on marine organisms, but the effects of plastic on terrestrial organisms have been largely ignored. Detrimental effects of terrestrial plastic could be most pronounced in intensively human-modified landscapes , which are a source of much anthropogenic debris. Here, we examine the occurrence, types, landscape associations, and consequences of anthropogenic nest material in the American crow ( The consequences of plastic accumulation for marine organisms are well-documented and have received wide attention In contrast, impacts of debris on terrestrial organisms are poorly documented and have been largely ignored, perhaps because terrestrial debris is less conspicuous Pandion haliaetus(Pycnonotus sinensis), for example, showed that the proportion of anthropogenic material in nests increased with degree of urbanization Potential effects of debris on terrestrial organisms could be most substantial in intensively human-modified landscapes . One of the few previous studies of nest entanglement in terrestrial birds, for example, reported that 4.6% of Osprey , a North American species that breeds in both urban and agricultural landscapes. This study is the first to explicitly document the links between terrestrial land cover, anthropogenic nest material, and nest entanglement in a terrestrial songbird.Herein, we examine the occurrence, types, landscape associations, and consequences of anthropogenic nest material in the American crow (n = 106 nests) and accessed by boom lift. Nestlings were checked for entanglement in the nest either once  or twice . Entanglements were removed from live nestlings. Surviving nestlings were marked 25 days after hatching with a unique combination of color bands and a USGS band. After banding, nests were checked daily to monitor success or failure. The encounter rate of entangled nestlings represents a minimum entanglement rate, because dead, entangled nestlings could have been removed by parents or predators prior to nest checks.In 2012 and 2013, we monitored success of 106 crow nests across an urban to agricultural gradient in Davis, California. The study site spanned the urban campus of the University of California, Davis into the surrounding campus-owned agricultural areas  of each piece of anthropogenic material longer than 5 cm from the lining of each nest. Corresponding nestling entanglement data were available from 32 of these nests. We had no corresponding entanglement data from 22 of these nests because the broods failed  prior to the first nestling check.n = 64 nestlings from 32 nests).To assess how the probability of entanglement varied with amount of anthropogenic nest material, we analyzed entanglement of individual nestlings (0/1) as the response in a generalized linear mixed model , with total length of anthropogenic nest material in the nest as the predictor. We specified nest as a random effect to account for non-independence among nestlings within a nest. We included in this analysis the subset of nestlings for which we had corresponding nest material data . All work was conducted on the private property of the University of California, Davis. No protected species were sampled.n = 36 nests; mean total length urban = 163.60±43.64 cm; n = 18 nests; β (urban) = −6.03±2.81; t (−2.15); p = 0.04), and the longest piece of anthropogenic material was significantly longer, on average, in agricultural nests than in urban nests  = −2.18±1.02; t (−2.15); p = 0.04).Anthropogenic material greater than 10 cm was detected in 85.2% (46/54) of dissected nests. Only three of 36 nests in primarily agricultural territories (8.3%) and five of 18 nests in urban territories (27.8%) contained no anthropogenic material. The mean total length of anthropogenic material in these 54 nests was 292.0±49.3 cm (range: 0–1858.3 cm). Material included synthetic string, twine, or rope; plastic tape; strips of plastic or cloth , fishing line, balloon string, unraveled nets, and wire mesh . Amount t (2.53); p = 0.017): the odds of entanglement increased 7.55 times for each meter of anthropogenic material in the nest. Nine nestlings were discovered entangled while still in the nest, and two were discovered dead below their nest tree. Entanglements were removed from all live nestlings, but in most cases, birds showed evidence of entanglement-related injury  = 0.009; 2-sample test for equality of proportions with continuity correction).Eleven of 195 nestlings (5.6%) were entangled . The likiotarsus ; malformIn an early review of entanglement and ingestion of plastic debris by marine organisms, Laist (1987) listed three reasons why the significance of marine debris had been disregarded, all of which could apply to terrestrial organisms today: (1) the mechanics of entanglement were so straight-forward that they lacked “hidden mysteries;” (2) encounters between debris and wildlife could have been rare; and (3) the paucity of published reports appeared to confirm this overall rarity. Since that review, however, awareness and concern about the prevalence and problems associated with plastics in the marine environments have grown rapidly, and entire journal issues have been devoted to the topic Charadrius vociferousZenaida macrouraCardinalis cardinalisSturnus vulgarisPasser domesticusand/or urban exploiters and adaptersAeronautes saxatalisPoecile rufescensPsaltriparus minimusMimus polyglottos Calypte annaHaemorhous mexicanusand Melozone crissalisWe have shown that 85.2% of crow nests along an urban to agricultural gradient contained anthropogenic material, that the amount of material was higher in nests in agricultural areas, and that the likelihood of entanglement increased with length of anthropogenic material. All entangled nestlings (5.6% of the nestlings marked in this study) failed to fledge. Potential for entanglement and associated mortality could be widespread in birds in highly human-dominated  landscapes. Anecdotal descriptions of anthropogenic nest material, including string, balloon ribbon, fishing line, plastic bags, paper, and dental floss, have been reported for many of the North American avian species defined as farmland species Birds in urban and agricultural settings may use hazardous anthropogenic materials because they resemble their preferred, natural nest material , analogous to marine turtles ingesting plastic bags because they resemble their jellyfish prey In some situations, anthropogenic nest material could be a beneficial resource, enabling nest construction in places where natural materials are limited We found that the amount of anthropogenic nest material was greater in the agricultural landscape than the urban landscape, likely due to the ready availability of twine and shade cloth wire in the agricultural settings. The majority of crow nests across this urban and agricultural gradient contained some anthropogenic material, however, and nestling entanglement occurred across this land use gradient. In all environments, therefore, particularly urban, agricultural, and marine, careful disposal potential hazards  could reduce the occurrence of nestling entanglement.