Immunohistochemistry allowed us to identify proteins with distinct localisation to the glomeruli (n = 11), proximal tubules (n = 120), distal tubules (n = 9) or collecting ducts (n = 8). Among the identified kidney elevated transcripts, we found several proteins not previously characterised or identified as elevated in kidney. This description of the kidney specific transcriptome and proteome provides a resource for basic and clinical research to facilitate studies to understand kidney biology and disease.To understand renal functions and disease, it is important to define the molecular constituents of the various compartments of the kidney. Here, we used comparative transcriptomic analysis of all major organs and tissues in the human body, in combination with kidney tissue micro array based immunohistochemistry, to generate a comprehensive description of the kidney-specific transcriptome and proteome. A special emphasis was placed on the identification of genes and proteins that were elevated in specific kidney subcompartments. Our analysis identified close to 400 genes that had elevated expression in the kidney, as compared to the other analysed tissues, and these were further subdivided, depending on expression levels, into  Furthermore, it has an important role in elimination of organic compounds, metabolic waste products and many pharmaceutical drugs. A detailed knowledge of the proteins expressed by the cells comprising the different parts of the kidney forms the basis for understanding their functions, under both physiological and pathological conditions. Previously, molecular biology studies have lead to the identification of many proteins important for kidney function. These include nephrin (NPHS1), a primary component of slit-membrane in the glomerulus that functions as the glomerular ultrafiltration barrier www.proteinatlas.org) with more than 50,000 samples of kidney tissue analysed with immunohistochemistry and individually annotated by certified pathologists We recently performed a large RNAseq analysis on 27 human tissues, covering all other major organs The tissue samples used for transcript profiling of human kidney included histologically normal tissue from operated material from four individuals: Female, 58 years (Sample 1); female, 67 years (Sample 2); female, 55 years (Sample 3); male 46 years (Sample 4).The kidney tissue samples were collected from surgical specimens of resected kidneys from individuals operated for renal cell carcinoma. The tissue was sampled from the normal, healthy part of the kidney and was confirmed microscopically as having a normal morphology by a trained pathologist. The corresponding histology of each biopsy can be found in http://www.uppsalabiobank.uu.se/en/). All human tissue samples used in the present study were anonymised in accordance with approval and advisory report from the Uppsala Ethical Review Board (Dnr Ups 02-577 (protein) and Dnr 2011/473 (RNA)), and consequently the need for informed consent was waived by the ethics committee. The use and analyses based on human tissues has previously been described in Fagerberg L et al. The four individual kidney samples selected for RNA analysis comprise tissue from the cortex and medulla .. The ushttp://github.com/najoshi/sickle) was used to trim raw reads obtained from the sequencing system for low quality ends, using a phred quality threshold of 20. After discarding reads shorter than 54 bp, processed reads were mapped to the GRCh37 version of the human genome with Tophat v2.0.3 http://picard.sourceforge.net/). Quantification scores for all human genes, FPKM (fragments per kilobase of exon model per million mapped reads) values were calculated using Cufflinks v2.0.2 http://www.r-project.org/) with the addition of package ‘gplots’ (http://cran.r-project.org/web/packages/gplots/index.html). Network analysis was performed using Cytoscape 3.0 The software ‘Sickle’  “Not detected” - <1 FPKM in kidney; (2) “Highly kidney enriched” - 50-fold higher FPKM level in kidney compared to all other tissues; (3) “Moderately kidney enriched” - 5-fold higher FPKM level in kidney compared to all other tissues; (4) “Group enriched” – 5-fold higher average FPKM level in a group of 2–7 tissues including kidney compared to all other tissues; (5) “Expressed in all tissues” – detected in 27 tissues>1 FPKM; (6) “kidney enhanced” – 5-fold higher FPKM level in kidney compared to the average FPKM value of all 27 tissues; (7) “Mixed” – genes expressed in 1–26 tissues and in none of the above categories.Tissue microarrays (TMA) containing biological triplicate 1-mm cores of 46 different types of normal tissue from different individuals and duplicate 1-mm cores of 216 different cancer tissues representing the 20 most common forms of human cancer were generated as previously described A gene ontology analysis We queried the Human Protein Atlas database for proteins that were annotated as expressed either uniquely in kidney, or expressed in kidney and not more than 6 other tissues . These criteria were chosen to correspond to criteria applied to the transcriptome datasets to identify genes classified into the ‘Group Enriched’ and ‘Kidney Enriched’ gene categories.We have earlier described deep sequencing (RNAseq) analysis of 27 tissue types corresponding to all major tissues and organs in the human body, including four samples from kidney Histological analyses of all the kidney specimens used in the study were performed to verify that cell type compositions of samples included were representative of the respective tissue .. In theThe variation between individual kidney samples were analysed through pairwise correlations, plotting the expression level  of all protein coding genes. A strong correlation between samples was found  illustrated with an example of pair-wise comparison of two individuals .. The loAll putative protein coding genes  were classified into four categories; (1) not detected in kidney , (2) ‘kidney elevated’ (387 genes) - subdivided into ‘kidney enriched’ , group enriched  or ‘kidney enhanced’ , (3) a mixed group of genes that were found to be expressed in several, but not all tissue types, and furthermore did not full-fill the criteria for tissue-elevated genes , and (4) ‘housekeeping’ genes, defined as genes expressed in all 27 tissue types  . Analysi64 genes were found to be tissue-enriched  and 167 www.proteinatlas.org). The immunohistochemical staining was used to confirm kidney expression and to determinate protein localisation within different sub-compartments and cell types. Out of the 387 genes that we identified as elevated in kidney, the localisation of expression in kidney could be analysed using the HPA for 339 of the encoded proteins.In order to validate the translation of the kidney-specific genes identified, immunohistochemistry was used to stain kidney sections from various individuals for the corresponding proteins. This data was generated within our Human Protein Atlas project (HPA), which currently comprises over 13 million images from stained tissue microarray (TMA) cores from 46 different normal tissues from different individuals. The RNAseq data and immunohistochemistry images for the kidney genes are publically available in the Human Protein Atlas resource . Out of the 387 genes identified as ‘kidney elevated’, 148 could be localised to only one sub-segment of the nephron or collecting duct . These whttp://www.genecards.org/), and RP11-407N17.3 has been described as a tumor associated antigen, cTAGE-5, found in cutaneous T-cell lymphoma and several other cancers in vitro it has been shown to trap aggregation-prone denatured proteins (for review see Most of the ‘kidney elevated’ proteins (120) we identified were localised to the proximal tubule of which 59 had not been previously described in the context of proximal tubules . Our GenAmong the 9 distal tubulus-elevated proteins , the majOther proteins we identified as distal tubulus located include several membrane proteins for electrolyte transport, including potassium, sodium and calcium transporters, consistent with the function of the distal tubule as site of electrolyte reabsorption and exchange between blood and urine Proteins we identified as kidney elevated with expression localised to the collecting ducts were mainly involved in water and proton transport, consistent with the function of collecting ducts in water re-absorption and control of pH homeostasis We identified 11 glomerular elevated proteins  and the http://www.hkupp.org/index.htm) The low number of glomeruli specific proteins that we identified contrasts to the vast literature on the apparent ‘glomeruli specific proteome’ , to datasets of glomeruli proteins in the Human Kidney and Urinary Proteome Project, HKUPP, (http://www.hkupp.org/), and to datasets in the Urinary Exosome Protein Database (http://dir.nhlbi.nih.gov/papers/lkem/exosome/). The results are shown in Many kidney proteomic studies have focused on urine as sample material, however the specific origin of proteins and protein fragments in urine is difficult to determine. Tissue proteomics using methods for proteomic content enrichment can enable identification of the specific proteomes of different functional units, compartments and cells of the kidney. For example, isolating glomeruli followed by 2D SDS-PAGE and LC-MS/MS was successful to enable an in depth characterisation of the glomerular proteome The Human Protein Atlas database contains quantitative annotations of protein expression across the different tissues sections using IHC. These IHC annotations  are based on the evaluation of a pathologist and give a quantitative measure specified for different tissue parenchymal cells . The parenchyma specific annotations of protein expression in a tissue section could differ from the quantitative average measure of mRNA transcription that is captured by RNAseq analysis, as the proportion of parenchymal cells relative to other cell types  differ between tissues and sections. For comparison, we queried the HPA database of IHC-based annotations of protein expression with search criteria that would correspond to either the ‘Group enriched’ or ‘Kidney enriched’ categories in the transcriptome dataset. This query identified 12 proteins annotated as uniquely found in kidney tissue, of which 6 corresponded to genes found among the 20 ‘highly kidney enriched genes’ in the transcriptome analysis (data not shown). The total overlap between the query-based analysis of the quantitative IHC-annotations in the HPA database and the transcriptome analysis was 35 proteins/genes. These 35 genes were classified as ‘Kidney enriched’ (11 genes), ‘Group enriched’ (14 genes) and ‘Kidney enhanced’ (10 genes) in the transcriptomics analysis. Thus, while IHC analysis provide more specific information of protein expression in parenchyma cells in individual tissues, the combination of RNA seq followed by IHC-based confirmation of protein expression and localisation enabled a more comprehensive identification of kidney enriched genes on genome wide level.Here, we have comprehensively described the kidney elevated transcriptome and proteome. By comparing the kidney transcriptome against the transcriptomes from 26 other organs, we were able to produce a kidney specific profile of gene expression. To our knowledge, this is the first study utilising RNA-seq in an integrated ‘omics’ approach with supplementary immunohistochemistry data to provide a comprehensive characterisation of protein expression in normal human tissues. We show how this combined approach adds value to the IHC-based information of protein expression in the Human Protein Atlas when aiming to identify kidney enriched genes on a genome wide scale.Our RNAseq analysis is based on kidney tissue from only four individuals, but the high correlation in mRNA profile between the four samples . suggestWe validated this profile using immunohistochemical staining, which allowed us to identify specific sub-compartments or cell types where these proteins were expressed . ImportaFurthermore, our results can also provide a resource for more clinically applied research since expression specificity of a protein is useful in the quest for specific markers of injury or disease of a particular tissue or cell type, e.g. elevated plasma levels. From this perspective, our list of identified kidney-enriched proteins with specific compartment location  provide To conclude, we here provide a comprehensive knowledge resource identifying the kidney transcriptome and, importantly, the specific localisation of expression of the corresponding proteins. We envisage that this will be a useful tool for researchers working in both basic and clinical nephrology research.S1 FigHistology of the samples used for RNA-Seq. Hematoxylin-eosin stained frozen sections of kidney tissue, showing the histology of the four individual kidney samples used for RNA-Seq preparation. All four samples display histologically normal kidney tissue. Numbers indicate the percentage of glomeruli, tubule, endothelial, fibroblasts, smooth muscle and inflammatory cells in each biopsy section chosen for RNA seq.(TIF)Click here for additional data file.S2 FigCorrelation of genes expressed in kidney in relation to gene expression in other tissues. (A) The correlation between two kidney samples from different individuals shown as a scatterplot of FPKM values for all detected genes. The Spearman pairwise correlations across all genes ranged from 0.96 to 0.98 between different kidney samples. (B) Scatter plot of average FPKM values for all detected genes in four kidney samples (x-axis) and seven testis samples (y-axis) from different individuals (pairwise Spearman correlation coefficient of 0.69).(TIF)Click here for additional data file.S1 TableTop 30 expressed genes in kidney (based on FPKM).(PDF)Click here for additional data file.S2 TableAll kidney enriched genes.(PDF)Click here for additional data file.S3 TableThe 39 shared proteins between kidney and liver.(PDF)Click here for additional data file.S4 TableThe nephron segment and collecting duct specific proteins identified in this study.(PDF)Click here for additional data file.S5 TableGO analyses of the different nephron segment and collecting duct specific proteins.(PDF)Click here for additional data file.S6 TableGO analyses of the group enriched proteins.(PDF)Click here for additional data file.S1 FileKidney enhanced genes.(XLS)Click here for additional data file.S2 FileGroup enriched genes.(XLS)Click here for additional data file.S3 FileThe highly kidney enriched genes of  with measures of variance and individual FPKM values of four individual kidney samples.(XLS)Click here for additional data file.


Malus × domestica) rootstock F1 breeding population, including resistance to powdery mildew (Podosphaera leucotricha) disease and woolly apple aphid (Eriosoma lanigerum).Apple tree breeding is slow and difficult due to long generation times, self-incompatibility, and complex genetics. The identification of molecular markers linked to traits of interest is a way to expedite the breeding process. In the present study, we aimed to identify genes whose steady-state transcript abundance was associated with inheritance of specific traits segregating in an apple (1 apple trees from a cross of two highly heterozygous parents, using RNA isolated from healthy, actively-growing shoot tips and a custom apple DNA oligonucleotide microarray representing 26,000 unique transcripts. Genome-wide expression profiles were not clear indicators of powdery mildew or woolly apple aphid resistance phenotype. However, standard differential gene expression analysis between phenotypic groups of trees revealed relatively small sets of genes with trait-associated expression levels. For example, thirty genes were identified that were differentially expressed between trees resistant and susceptible to powdery mildew. Interestingly, the genes encoding twenty-four of these transcripts were physically clustered on chromosome 12. Similarly, seven genes were identified that were differentially expressed between trees resistant and susceptible to woolly apple aphid, and the genes encoding five of these transcripts were also clustered, this time on chromosome 17. In each case, the gene clusters were in the vicinity of previously identified major quantitative trait loci for the corresponding trait. Similar results were obtained for a series of molecular traits. Several of the differentially expressed genes were used to develop DNA polymorphism markers linked to powdery mildew disease and woolly apple aphid resistance.Transcription profiling was performed for 48 individual F1 population readily identified genes physically linked to powdery mildew disease resistance and woolly apple aphid resistance loci. This result was especially useful in apple, where extreme levels of heterozygosity make the development of reliable DNA markers quite difficult. The results suggest that this approach could prove effective in crops with complicated genetics, or for which few genomic information resources are available.Gene expression profiling and trait-associated transcript analysis using an apple F The advent of large-scale transcription profiling technologies, such as DNA microarrays  and RNAsApple tree breeding is a slow and difficult process for reasons that include long juvenility periods, large size of mature plants, inbreeding depression, reproductive self-incompatibility, and complex phenotypes related to grafted trees . The use1 generation due to the self-incompatibility of apple [1 population from a cross of two highly heterozygous rootstock parents, ‘Ottawa 3’ (O3) and ‘Robusta 5’ (R5). This is a phenotypically diverse and well-characterized population that is segregating for numerous traits of interest to apple growers. The segregating traits include resistance to biotic stresses such as fire blight  and powotricha; ) diseasenigerum; ,10) pest1 trees from the O3 × R5 cross and identified transcripts with expression levels associated with PM disease and WAA pest resistance phenotypes. When the genes encoding these transcripts were mapped to the apple genome, they were found to be physically clustered. This is similar to the phenomenon described for single nucleotide polymorphisms and gene expression markers in Brassica napus[We applied DNA microarray transcription profiling to 48 individual Fica napus. The uti1 trees from the O3 × R5 cross population growing in a rootstock production stool bed in Geneva, NY. The RNA was used to probe 24 microarrays in multi-plex format, using two different color probes per array, so that all 48 RNA samples could be assayed on the 24 microarrays. The microarrays were clustered based on their expression profiles using the hierarchical clustering function in R  analysis using the O3 and R5 parents and a group of FMalus × domestica contig MDC015568.236 , which contained the best matching DNA sequence according to BLAST analysis. Several polymorphisms were identified within this region when comparing the O3 haplo-contigs with the R5 haplo-contigs. Of particular interest for easy marker development were two microsatellite regions between bases 2,500 - 3,500 of contig MDC015568.236, for which PCR primer pairs were designed  and tested with parent DNA with annealing temperature gradient PCR  and GD153  [As a proof of concept, DNA markers were developed for WAA resistance-associated gene APPLE0FR00068101. The sequence of APPLE0FR00068101 was used to identify 1 individuals yielded relatively small numbers of genes that were differentially expressed between the phenotypic groups, and these differentially expressed genes displayed a remarkable degree of physical clustering on the apple genome. The clustered genes were typically in the physical vicinity of the major locus controlling the trait, in the case of PM and WAA resistance, or in the vicinity of the gene expression marker used as the molecular trait. Clusters at locations unlinked to their corresponding trait of interest  and gene expression markers [1 population, while the other study used a collection of accessions.The clustering pattern of differentially expressed genes is consistent with the relative expression levels of these genes being inherited from a parent. This is different from genome neighborhood effects, where groups of linked genes are typically up- or down-regulated together . Just as markers . However1 population. The development of PCR based molecular markers associated with several of the differentially expressed sequences was in many cases successful because sequence mutations such as large INDELs and microsatellite variation was discovered within or nearby the target genes. While several methods are available to detect polymorphisms in marker assisted breeding, markers based on the polymerase chain reaction are still the most accessible and least expensive for small scale breeding programs. The combination of expression analysis for target identification and sequence based marker development proved a good strategy as the PCR markers developed in this study have been routinely proven useful in apple rootstock breeding program in Geneva, NY. As RNAseq methods become more refined, it may be possible to find differentially expressed genes associated with traits of interest and at the same time leverage polymorphisms contained in the expressed sequences for haplotype specific breeding marker development.Validation of the expression patterns using qPCR indicated that most of the genes indeed had patterns of expression consistent with the array data. The congruence of DNA microarray and qPCR data for selected differentially expressed genes and gene expression markers provided strong validation for the DNA microarray data. qPCR validation was successful using a different set of individuals from the same cross in a different environment and year from those used for the microarray, indicating that the differentially expressed genes had relative expression levels consistent across different growing conditions and years and between different groups of individuals from the O3 × R5 FWe have shown in a segregating population from the cross of a highly heterozygous plant that gene expression analysis can result in identification of differentially expressed genes that are physically linked to one another. Gene expression heritability as a method to detect genes physically linked to trait loci of interest could be useful in crops for which few genetic and genomic information resources are available. Even in the absence of a genetic map, molecular markers linked to traits of interest could be developed using transcriptome profiles of segregating populations, since a substantial proportion of the differentially expressed genes would be expected to cluster in the vicinity of the trait of interest. While there may be no causal link between the differentially expressed genes and their associated traits, they do provide an excellent starting point for development of DNA markers linked to segregating traits of interest.1 population from an O3 × R5 cross and were grown in an orchard in Geneva, NY [The trees used for the DNA microarray analysis were from a segregating Fneva, NY . SamplesFor the qPCR analyses, shoot tip samples were collected in late spring of 2013 from a separate group of 46 individuals belonging to the same O3 × R5 population from clonally propagated material in a replicate orchard in Geneva, NY. Shoot tips from the population parents (O3 and R5) were collected from trees at the apple collection of the USDA ARS Plant Genetic Resources Unit in Geneva, NY.1 tree were analyzed using a single array only; however, analyses were conducted using pooled data from trees with similar phenotypes, which represented pseudo-replicates in this context [Total RNA was isolated from whole apple shoot tips as previously described . The mic context .While the parents of the breeding population were different from the varieties used to design the DNA microarray, this did not interfere with probe performance or account for patterns. Any nucleotide polymorphisms between the probes and the samples, when present, did not affect hybridization, as the other probes for the same transcripts, which had no polymorphisms, gave similar intensity values  value for each transcript was then calculated separately in each phenotypic group, and then the M-value (log2 fold difference in expression) for each transcript was computed as the difference in the mean log2 (expression) value for each transcript between the two groups of trees. Empirical Bayes ANOVA analysis [The gene expression data from the DNA microarray hybridization experiments were previously normalized using R software . To idenanalysis  was perfanalysis  as part analysis . P-valueanalysis  as impleanalysis . Differe2 (expression) value for each transcript in the entire F1 population. Next, the divergence in expression for each individual tree compared to the average was determined for each transcript. GEMs were identified as transcripts that could divide the trees into groups of roughly equal size based on having at least a 1.5-fold difference in expression levels between the groups  following the manufacturer’s instructions, with the addition of DNAse I  to the RNA wash buffer in the kit as recommended in the manufacturer’s protocol. cDNA synthesis was performed using the QuantiTect Reverse Transcription kit  according to the manufacturer’s instructions. PCR primers for selected differentially expressed genes and gene expression markers were optimized using parental genomic DNA with Annealing Temperature Gradient PCR . Contig MDC015568.236 contained sequences most similar to the R5 next-gen sequences, and was therefore chosen for further analysis. Unique single nucleotide polymorphisms (SNPs), simple sequence repeats (SSRs), and haplotypes were identified for the R5 (WAA-resistant) parent. Several 18–21 bp PCR primers were designed to match unique SNP haplotypes at the 3′ end, and primer pairs were tested with parental DNAs using annealing temperature gradient PCR (45°C to 65°C) to verify genotype specificity, stability and reproducibility of amplicons. Amplicons were resolved on 2% EtBr-stained agarose gels and visualized with an AlphaImager HP gel documentation system (ProteinSimple). In addition, some primers were designed flanking microsatellite SSRs. Genotype-specific amplicons were then tested on segregating individuals in the O3 × R5 population and a diversity panel of apple rootstocks to verify genetic inheritance, linkage to other markers, and haplotype uniqueness.The sequence of gene APPLE0FR00068101, whose expression was associated with WAA resistance, was compared to the ‘Golden Delicious’ apple genome hosted by the Genome Database for Rosaceae  using thMicroarray data are available through the GEO website using accession number GSE43268.ANOVA: Analysis of variance; ATG-PCR: Annealing temperature gradient PCR; BLAST: Basic local alignment search tool; bp: Base pairs of DNA; cDNA: Complementary DNA; DNA: Deoxyribonucleic acid; EtBr: Ethidium bromide; kb: Kilobase pairs; O3: ‘Ottawa 3’; PCR: Polymerase chain reaction; PM: Powdery mildew; qPCR: Quantitative PCR; QTL: Quantitative trait locus; R5: ‘Robusta 5’; RNA: Ribonucleic acid; RNAseq: RNA sequencing; SNP: Single nucleotide polymorphism; SSR: Simple sequence repeats; USDA: United States Department of Agriculture; WAA: Woolly apple aphid.The authors declare that they have no competing interests.PJJ performed the microarray experiments and analyzed the results; GF performed phenotypic characterizations of apple trees and performed the qPCR validation of selected transcripts and developed the DNA markers based on the differentially expressed genes; NA provided statistical advice and assisted with data analysis; CP performed microarray hybridizations and provided advice for experiment design and data interpretation; TWM initiated, developed, and supervised the project; all authors contributed to the writing of the manuscript and have approved the manuscript.1 population from an ‘Ottawa3’ × ‘Robusta 5’ cross. Table S2. Correlation between Microarray Gene Expression Values for WAA resistance differentially expressed genes APPLE0FR00068101, and associated features APPLE00R00024612, APPLE0F000011491, APPLE0F000050102. Table S3. Correlation between Relative Gene Expression values (Target Gene/Actin Reference Gene) in qPCR Basic Relative Quantification outputs for the following targets: APPLE24612, APPLE50102, APPLE11491 and WAA resistance differentially expressed gene APPLE68101. Table S4. Individual probe data and SNP counts for transcripts in the region 29.0 to 30.2 Mb on Chromosome 12. Table S5. qPCR conditions and primers for gene expression validation.Transcripts identified as having expression patterns associated with powdery mildew resistance, woolly apple aphid resistance, and the gene expression marker traits analyzed in a segregating FClick here for fileFigure S2. Relative quantification results for qPCR of differentially expressed gene APPLE0FR00048809 (associated with PM resistance) relative to actin. Figure S3. Visualization of qPCR amplicons of gene APPLE0F000001977, showing clear segregation (presence/absence) of amplified target cDNA in selected progeny. Figure S4. Annealing temperature gradient amplification (65°C - 45°C) of differentially expressed gene APPLE0FR00068101 derived markers on parental DNAs. Figure S5. Alignment of microarray feature APPLE0FR00068101 to Chromosome 17 of the apple genome.Example gene expression marker (GEM) trait. Click here for file


Vdr ablated C57Bl6 mice to assess fetal growth, morphological parameters and global gene expression in Vdr null placentae. Twelve Vdr+/- dams were mated at 10–12 weeks of age with Vdr+/- males. At day 18.5 of the 19.5 day gestation in our colony, females were euthanised and placental and fetal samples were collected, weighed and subsequently genotyped as either Vdr+/+, Vdr+/-or Vdr-/-. Morphological assessment of placentae using immunohistochemistry was performed and RNA was extracted and subject to microarray analysis. This revealed 25 genes that were significantly differentially expressed between Vdr+/+ and Vdr-/- placentae. The greatest difference was a 6.47-fold change in expression of Cyp24a1 which was significantly lower in the Vdr-/- placentae (P<0.01). Other differentially expressed genes in Vdr-/- placentae included those involved in RNA modification (Snord123), autophagy (Atg4b), cytoskeletal modification (Shroom4), cell signalling  and mammalian target of rapamycin (mTOR) signalling (Deptor and Prr5). Interrogation of the upstream sequence of differentially expressed genes identified that many contain putative vitamin D receptor elements (VDREs). Despite the gene expression differences, this did not contribute to any differences in overall placental morphology, nor was function affected as there was no difference in fetal growth as determined by fetal weight near term. Given our dams still expressed a functional VDR gene, our results suggest that cross-talk between the maternal decidua and the placenta, as well as maternal vitamin D status, may be more important in determining pregnancy outcome than conceptus expression of VDR.Vitamin D deficiency has been implicated in the pathogenesis of several pregnancy complications attributed to impaired or abnormal placental function, but there are few clues indicating the mechanistic role of vitamin D in their pathogenesis. To further understand the role of vitamin D receptor (VDR)-mediated activity in placental function, we used heterozygous  Normal fetal development is undoubtedly underpinned by normal placental function. The placental vascular network provides an interface between the fetus and mother for the exchange of gases, nutrients and wastes . Additio3 2D3) increase by 2 to 5-fold  was greater in Vdr-/- placentae when compared to Vdr+/+. Pex5 plays a central role in the function of peroxisomes which are present in cells to clear reactive oxygen species (ROS) like hydrogen peroxide [Vdr-/- placentae may be indicative of increased ROS, therefore increased oxidative stress, which has been hypothesized to be an underlying factor in the development of pregnancy complications like preeclampsia [Vdr-/- placentae.Expression of both peroxide . Tin2 isperoxide  potentiaclampsia . HoweverDeptor expression in Vdr-/- placentae and higher expression of Prr5 when compared to Vdr+/+. Both genes are components of the mTOR signaling pathway. During pregnancy, placental mTOR signaling plays an important role in the regulation of fetal growth, particularly as a maternal nutrient and growth factor sensor [DEPTOR and PRR5 have been shown to be highly expressed within the placenta [Prr5 is a component of the mTORC2 complex that promotes cell growth through its interaction with Rictor [2D, through VDR signaling, can suppress downstream mTOR signaling [Vdr-/- placentae decreased Deptor and increased Prr5 indicates activation of the mTOR pathway. Thus, mTOR activation may explain why there was no difference in fetal weight and placental structure as there would be a drive for growth which may normalize any differences between the genotypes D serum level <20 ng/mL)[Our results indicate that VDR signaling in the placenta is not essential for pregnancy success. This is supported by recent studies assessing placental VDR expression and polymorphisms in complicated pregnancies. VDR polymorphisms do not appear to predispose women to preeclampsia and gestational hypertension  and VDR 20 ng/mL). Materna20 ng/mL) suggestiVdr+/- dams to assess the effect of vitamin D on placental and fetal development without the confounding factor of poor maternal health seen in Vdr-/- mice [In this study, we used -/- mice . DespiteS1 FileVdr genotyping PCR primers and conditions [nditions  (Table Anditions  (Table B(DOCX)Click here for additional data file.S2 File(HTML)Click here for additional data file.


SIRPA transgene have previously been shown to support improved human cell hematopoiesis. Here, we have generated for the first time, human SIRPA BAC transgenic rats, for which the gene is faithfully expressed, functionally active, and germline transmissible. To do this, human SIRPA BAC was modified with elements to work in coordination with genome engineering technologies-piggyBac, CRISPR/Cas9 or TALEN. Our findings show that piggyBac transposition is a more efficient approach than the classical BAC transgenesis, resulting in complete BAC integration with predictable end sequences, thereby permitting precise assessment of the integration site. Neither CRISPR/Cas9 nor TALEN increased BAC transgenesis. Therefore, an efficient generation of human SIRPA transgenic rats using piggyBac opens opportunities for expansion of humanized transgenic rat models in the future to advance biomedical research and therapeutic applications.BAC transgenic mammalian systems offer an important platform for recapitulating human gene expression and disease modeling. While the larger body mass, and greater genetic and physiologic similarity to humans render rats well suited for reproducing human immune diseases and evaluating therapeutic strategies, difficulties of generating BAC transgenic rats have hindered progress. Thus, an efficient method for BAC transgenesis in rats would be valuable. Immunodeficient mice carrying a human  E. coli based large insert DNA clones capable of carrying genomic fragments ranging in size between 150–300 kb124Mammalian model systems provide an essential platform in biomedical research for deciphering the complexities underlying the pathogenesis of human disease, and for developing the applicative and translational potential of new therapies. Bacterial artificial chromosomes (BACs) have played an important role in these endeavors by providing the DNA source material with which transgenic animals are derived. BACs are in vivo, this quality also makes efficient integration of BACs into host genomes challenging. Furthermore, the BAC integration site is difficult to determine. In spite of these limitations, BACs continue to be widely used, mainly for the production of transgenic mice. In contrast, BAC transgenesis in rats has been less successful, due to inherent physiological differences between the two rodent speciesWhile the large genomic insert size of BACs is beneficial for creating animals with transgenes that are integration site independent and accurately expressed contingent on copy number Sirpa) locus supported human hematopoiesis through enhanced binding of the SIRPα protein to the human CD47 ligand. Once bound, SIRPα acts as an inhibitory receptor for mouse macrophages, allowing human hematopoietic cells to escape host cell phagocytosis161718et al.SIRPA BAC transgenic mice on mixed 129/BALB/c background, and demonstrated that the expression of human SIRPA enhanced human cell engraftment and improved functionality of human adaptive immune system in vivo. For these reasons, an efficient method for generating human SIRPA BAC transgenic rats would allow for the building of a repository of humanized SIRPA rats on various immune-deficient rat strainsAn area of research where BAC transgenic rat model system would be particularly useful is the humanization of the immune system. While immune-deficient mouse and rat strains devoid of key lymphoid components have played an important role in biomedical sciences by making feasible studies involving the engraftment of human cells, tissues and organs91011piggyBac transposon, CRISPR/Cas9 and TALEN mediated approaches, as they have emerged as powerful tools for manipulating the genome. The piggyBac transposon system is a genetic element capable of mobilizing a segment of DNA encased between terminal inverse repeat (TIR) elements in the presence of transposase proteins22232526piggyBac system’s “cut and paste” mechanism, researchers have utilized the TIR elements to design strategies for carrying out high throughput insertional mutagenesis for cancer research283032Here, we aim to generate BAC transgenic rats faithfully expressing human SIRPα. To do this, we seek to develop strategies utilizing genome engineering technologies reported to be highly efficient for generating transgenic animal models. Specifically, we examined piggyBac mediated approach, CRISPR/Cas9 and TALEN are a family of endonucleases capable of inducing double stranded break (DSB) at precise locations in the genome, initiating the stimulation of two different pathways of repair mechanism–non-homologous end-joining (NHEJ) and homology-directed repair (HDR)3536373839Rosa26 locus through HDR.In contrast to the SIRPA carrying BAC to work in coordination with piggyBac transposition, CRISPR/Cas9 or TALEN mediated approaches for the generation of humanized BAC transgenic rats. Hence, the objective of this study is twofold: 1) to develop an efficient method for generating BAC transgenic rats by evaluating the piggyBac, CRISPR/Cas9 and TALEN mediated approaches in the zygote; and 2) to generate humanized SIRPA rats that are functionally viable by expressing the protein in leukocytes and actively interacting with human CD47 ligand.Encouraged by these findings, we seek to modify the human piggyBac mediated BAC transposition system involves converting the large circular DNA into a transposon capable of being recognized and mobilized by the transposase proteins  elements flanking the spectinomycin resistance gene, by replacing the chloramphenicol resistance gene in the vector backbone (hSIRPA-BAC will be referred to as hSIRPA-BAC-TIRs DNA). The positioning of the TIR elements in the BAC vector backbone was designed to mimic the inherent ‘cut and paste’ mechanism of piggyBac-based gene transfer, for which DNA segment encapsulated between the two TIR elements is seamlessly excised out of the genome in the presence of the transposase proteins, and transpositioned into a different location in genomic sites containing the TTAA sequence  mRNA into the pronucleus of rat zygotes  while increasing the amount of hyPBase co-injected with 100 ng may give rise to higher number of pups relative to the number of embryos injected, the genotyping result indicates a higher percentage of PCR positive pups relative to the total number of pups when the amount of hyPBase injected is decreased to 25 ng.The implementation of the  zygotes . Co-injeRPA gene . In bothRPA gene . As a conscripts . In the nscripts . These fpiggyBac TIR retrofitted hSIRPA BAC with hyPBase mRNA transcripts increases the number BAC insertions relative to the negative control, we sought to determine whether these insertions are transposition events mediated by piggyBac transposase. To do this, splinkerette PCR was utilized to: 1) identify the precise location of the insertion sites; and 2) determine whether the BAC transposon ends are flanked by TTAA sequence, since the hallmark feature of piggyBac mediated transposition is the mobilization of the transposon from one TTAA region into another, by duplicating the tetranucleotide sequence, and inserting itself in between the duplicated sequences , 6 were found to have duplicated TTAA sequences, each tetranucleotide residing immediately adjacent to the 5′ and 3′ TIR elements   . Detaileectively .hSIRPA-BAC-TIRs DNA are fully intact, a series of primer pairs were designed at approximately 20 kb intervals, along the BAC insert  pup 4.2 was PCR positive only at primer pair 5, suggesting random integration of a fragmented BAC consistent with the absence of TIRs; whereas 2) pup 5.5 showed random insertion of a full BAC DNA without transposition , as well as the pup with random hSIRPA-BAC-TIRs DNA insertion (5.5), using qPCR on genomic DNA. When normalized to the Ct values derived from the endogenous rat Gapdh gene, for which there are two copies, we found that all of the founders carry a single copy of BAC integration  were mated with wild-type animals to obtain F1 offspring, and analyzed for the rate of BAC transmission  (data not shown). Furthermore, in mice transgenic for human SIRPα expression, human SIRPα detection was restricted to mouse monocytesIn order to evaluate the functionality of ransgene  and then+ cells, while the genotyped negative offspring (1.6F1.4) showed expression of only the rat SIRPα protein  genes as recipients for immune humanization experiments. These findings indicate that piggyBac mediated hSIRPA-BAC-TIRs transpositioned rats give rise to progeny that faithfully express functionally viable human SIRPα protein in rat monocytes.We also evaluated the expression of hSIRPα in rat monocytes by staining with anti-human and rat specific SIRPα monoclonal antibodies. The results confirmed that genotyped positive offspring (1.6F1.3) faithfully expressed both human and rat SIRPα protein on CD11b protein . Additio protein , suggestSIRPA transgenic rats can be generated when mediated by piggyBac transposition. While the BAC insertion sites can be readily determined using splinkerette PCR, one of the major limitations of the piggyBac mediated approach is that the BAC can be inserted anywhere in the genome with a TTAA tetranucleotide sequence. From this vantage point, both CRISPR/Cas9 and TALEN endonucleases offer the potential benefit of targeting the BAC into a specific location in the genome. To test this possibility, we designed a strategy for applying the same CRISPR/Cas9 and TALENs, that we previously usedRosa26 locus, to stimulate HDRhSIRPA BAC by retrofitting a cassette containing 900 base pairs of Rosa26 homology arm with sequences flanking the endonuclease cut site. In our laboratories, we have shown that 900 base pair homology arm length is sufficient for targeting circular KOMP vectors (approximately 9 kb) in the mouse genome at high efficiency (data not shown), and that 4.5 kb linearized constructs with similar homology arms can be targeted using CRISPRs and TALENs to Rosa26 and Hprt in mouse and rat zygotesRosa26 sgRNA or TALEN recognition sequence separating the 5′ and 3′ homology arms, and the other cassette that lacks the recognition sequence. By designing cassettes with or without the sgRNA and TALEN recognition sequences, the aim was to simultaneously linearize or keep the BAC circular in the injected embryo when the endonuclease cleaved the genomic DNA at the targeted Rosa26 locus. The hSIRPA-BAC that was retrofitted with the linearization site will be referred to as hSIRPA-BAC-Rosa26+, and the BAC without the linearization site will be referred to as hSIRPA-BAC-Rosa26−.We have thus far shown the efficiency with which functionally viable humanized hSIRPA-BAC-Rosa26+ did not show toxic effects on embryo survival immediately before microinjection (76.3% viable), yielded 60 day-15 fetuses (40.5% relative to the number of zygotes transferred), resulted in 1 founder PCR positive for the BAC (1.7% of fetuses) and 22 founders (36.7% of fetuses) with mutations around the cleavage site in the Rosa26 locus , yielded 28 day-15 fetuses (32.2% relative to the number of zygotes transferred), resulted in 1 founder PCR positive for the BAC (3.6% from fetuses) and in 12 founders (42.8% from the fetuses) with mutations around the cleavage site of the Rosa26 locus (hSIRPA-BAC-Rosa26+ did not show insertion in the Rosa26 locus as depicted by negative junction PCRs (data not shown). Therefore, despite that sgRNAs for the Rosa26 locus were highly active, as evidenced by high rates of mutations, indicating that the hSIRPA-BAC-Rosa26+ was likely linearized, the frequency of transgenic BAC rats was not increased as compared to the one observed using the circular hSIRPA-BAC-Rosa26−.The modified BACs were microinjected with the Rosa26 sgRNA and Cas9 protein because we previously demonstrated that Cas9 protein was more efficient than mRNA to obtain genome insertion of donor DNA26 locus . Microin26 locus . The fouhSIRPA-BAC-Rosa26 with TALE nuclease mRNA which we previously showed to efficiently and reproducibly target plasmid donor DNA integration by HDR in the Rosa26 locushSIRPA-BAC-Rosa26+ did not show toxic effects on embryo survival immediately before microinjection (68.9% viable), yielded 39 day-15 fetuses (32% relative to the number of zygotes transferred), resulted in no founders PCR positive for the BAC (0% of fetuses), and in 9 founders (23.1% of fetuses) with NHEJ mutations around the cleavage site of the Rosa26 locus , yielded 13 day-15 fetuses (19.7% relative to the number of zygotes transferred), resulted in 0 founder PCR BAC positive (0% from fetuses) and in 4 founders (30.8% from the fetuses) with mutations around the cleavage site of the Rosa26 locus  rats faithfully expressing functionally viable human SIRPα. While the procedures involved in the production of transgenic rats and mice are similar, the efficiency of creating transgenic rats has remained more difficult than mice64647In this study, we have generated for the first time: 1) highly efficient piggyBac and Sleeping Beauty, and engineered endonucleases such as ZFNs, TALENs and CRISPRs bursted onto the scene allowing manipulating and editing the genome with efficiency and precision never before possible. Because producing transgenic mice have traditionally been less difficult than rats, a large body of literature has accumulated demonstrating the ease with which the genomes of mice can be manipulated using these tools to, for example, reprogram somatic cells in into iPS cells, induce random mutagenesis, conduct precise genome editing through oligo replacement, and knock out gene function via indels333739494251525354As methods for generating transgenic rats improve over time, the last decade has also witnessed a rapid advancement in genome engineering technologies. Among them, tools based on transposable element systems such as SIRPA mice.In spite of the great potential engendered in these newly emerging genome engineering tools, BAC transgenesis continues to be a method of choice for many. The advantages imbued in BAC-based transgenics stem from the large genomic insert size, encompassing one or more genes with essential regulatory elements for proper expression of the transgene, and insulating elements that curb epigenetic silencing due to position effect variegation5760616263piggyBac TIR elements can be used to mobilize a fraction of BAC as large as 100 kb into the genome of mouse ES cells, and Suster and colleagues also showed in the same year BAC transposition in zebrafish using Tol2 transposable elementset al. reported that BACs modified with piggyBac TIRs can be injected directly into mouse zygotes for efficient generation of founders carrying the transgenepiggyBac, nor any other genome engineering tools, has yet to be reported for generating BAC transgenics.The major limitations of using BACs have been the cumbersome handling procedure and low insertion efficiency in the genome. A major breakthrough came in 2011 when Li and colleaguespiggyBac, CRISPR/Cas9 and TALEN mediated approaches may enhance hSIRPA-BAC transgenesis in the rat zygote. To do this, we strategically modified the BAC with elements compatible for recognition by these tools. Through this approach, we discovered that piggyBac provided an efficient approach for generating hSIRPA-BAC transgenic rats that are germline transmissible, and faithfully express functionally viable human SIRPα protein in the monocytes of F1 offspring. We have also demonstrated that monocytes from BAC transgenic rats express human SIRPα, as observed in human monocytes, indicating that regulation of expression by hSIRPA-BAC is conserved. Since rat monocytes from BAC transgenic rats express human SIRPα which interacts with human CD47, it is likely that the transgenic rat monocytes will receive phagocytosis inhibitory signals from human SIRPα. Immune humanization of immunodeficient rats has been reported to be undetectableSIRPA transgenic rats will likely increase efficiency.Here, we aimed to examine whether piggyBac mediated hSIRPA-BAC transgenesis in rats, we provide an efficient tool in the hands of the research community, for expanding the existing platform for generating hSIRPA-BAC transgenic rats in various immune compromised strains13SIRPA transgenic rats will likely be useful in other biomedical and translational research arena as well, for example, a growing body of literature suggests the relevance of SIRPA in inflammationBy laying out a blueprint for generating Sirpa). The efficiency of BAC transgenesis by this approach was comparable to that of the same BAC using classical transgenesis . In contrast, the piggyBac-mediated approach increased the efficacy of BAC transgenesis compared to that of classical transgenesis . BAC insertion using ssODNS and guide RNAs occurred in the targeted locus whereas piggyBac-mediated BAC transgenesis resulted in random insertions in TTAA sites. Nevertheless, the insertion site of the piggyBac-mediated BAC transgene can be easily determined by splinkerette PCR, and thus the risk of insertional mutations can be evaluated. It is surprising that Yoshimi et al.’s CRISPR/Cas9 approach using ssODNs resulted in BAC insertion into the targeted locus, whereas the use of longer homology arms in our study did not allow for targeted integration. The different outcome may be attributed to the differences in mechanisms underlying insertions using ssODNs versus homology arms. In the case of ssODNs, it is likely that the insertion involved ssODN-mediated nonhomologous end-joining73Sirpa vs. rat Rosa26) may be differentially susceptible to insertion of BACs by the two repair pathways. Importantly, researchers now have the choice of performing BAC transgenesis either using Cas9/ssODNs- or piggyBac-mediated integration, with each system having advantages and disadvantages. The CRISPR/Cas9 approach may be particularly attractive once optimal parameters for this approach have been established, as it conceivably allows for precise editing of the genome including the creation of models where the human gene replaces the mouse or rat homolog.Concomitantly with the submission of our manuscript, the genomic insertion of a BAC using the CRISPR/Cas9 approach has been described in the ratet al.piggyBac mediated BAC transposition in mouse zygotes. Our manuscript shows that piggyBac transposition of BACs is also an efficient method in rats, resulting in increased transgenesis with typically a single copy insertion faithfully expressing the transgene. Moreover, the insertion sites are easily identifiable using simple PCR. Our results support the possibility that this approach may also be useful for generating transgenic animals in other mammalian species of biomedical or agricultural significance. We envision that in the near future, rapidly advancing technologies will allow for an efficient targeting of BACs into specific loci in the genome for the production of rat transgenics. Likewise, the emergence of newer, more advanced, technologies may no longer benefit from BAC-based transgenics for simple procedures such as knocking out genes or knocking in reporters. However, it is premature to disregard BACs as “obsolete” as recently claimedThe report by Rostovskaya piggyBac TIR elements. The RP11-887J4 BAC clone was acquired from BAC/PAC Resources (bacpac.chori.org), and retrofitted using the recombineering technology. The recombineering cassette carrying the TIR elements was generated by subcloning the 5′ TIR and 3′ TIR elements derived from pPB-CAG-EBNXN (Wellcome Trust Sanger Institute) into a pUC19 vector backbone with the spectinomycin resistance genes in between the TIR elements. The recombineered BAC clone was PCR and sequence verified.Human BAC, RP11-887J4, carrying 176,233 bps of genomic DNA segment covering the SIRPA transcript coding sequence, as well as the region spanning 52,081 bps up- and 78,424 bps down-stream of the transcript start and end points, was selected for retrofitting with the The RP11-887J4 BAC clone was retrofitted with a recombineering cassette carrying 1049 and 1038 bp length rat Rosa26 homology arms flandking the Rosa26 sgRNA recognition sequence. The cassette was generated by PCR amplifying the 5′ and 3′ homology arms from CHORI230-418P10, for which one of the primers carried the Rosa26 sgRNA recognition sequence. The PCR amplified fragments were subcloned into a pUC19 vector backbone with neomycin resistance gene driven by PGK and EM7 as a selection cassette using the Gibson Assembly kit (NEB).in vitro using the mMESSAGE mMACHINE Kit (Ambion) according to the manufacturer’s instructions. The transcribed mRNA was purified using the MEGAclear Kit (Ambion).To prepare the hyPBase mRNA transcript, pCMV-hyPBase (generous gift from the Wellcome Trust Sanger Institute) was digested with SalI restriction endonuclease, and cleaned up using the PCR Cleanup Kit (Macherey Nagel). 500 ng of the linearized plasmid was used as a template to transcribed Rosa26 and donor DNA as previously been described in detail2 at 37 °C for at least 1 h. Surviving embryos were then implanted on the same day of microinjection in the oviduct of pseudo-pregnant females (0.5 days post coitum) and allowed to develop until embryonic day 15 or until normal delivery. Offspring were obtained by crossing BAC positive founders with wild-type rats.The rats used in this work were from the Sprague-Dawley (SD/Crl) strain . The study was approved by the Ethics Committee on Animal Experimentation of the Pays de la Loire Region, France, in accordance with the guidelines from the French National Research Council for the Care and Use of Laboratory Animals (Permit Numbers: APAFIS#692), as such, the methods themselves were conducted in “accordance” with the approved guidelines. The BAC constructs and hyperactive transposase were injected at different concentrations indicated in the results section  and 3. TEmbryos or pups were identified by PCR using the following primers: 5′-CTCTACGCGCTTTCTTGTCC-3′ and 5′- AACGTCAGCCTCCAGGTATG-3′. The standard amplification profile consisted of 5 mn at 95 °C, 2 mn at 62 °C followed by 35 cycles of 30 s at 72 °C, 10 s at 95 °C, 10 s at 60 °C and 3 min at 72 °C. Amplicons were analyzed using microfluidic capillary electrophoresisGenomic DNA was isolated and digested with Sau3AI. The digested genomic DNA was ligated to 50 nM of splinkerette adaptors using T4 ligase and ligation buffer from NEB. Nested PCR was used to amplify the genomic DNA sequence adjacent to the 5′ and 3′ TIR ends. The PCR products were sequenced to determine the precise transposition sites.Using 20 ng of isolated genomic DNA, qRT-PCR reactions were performed with SYBR Green PCR Master Mix (Applied Biosystems) according to manufacturer’s instructions. 200 nM of primer pair used for genotyping the presence of the human SIRPA gene was used for qRT-PCR. The Ct values were calculated using Applied Biosystems’ SDS2.4 software. To determine the copy number of the transpositioned BACs, the Ct values derived from the primer pair used for amplifying the human SIRPA gene was normalized to the GAPDH gene in the rat genome.Rat peripheral blood leukocytes were isolated from heparinized blood by red blood cell lysis. hSIRPA was detected using an anti-human SIRPA monoclonal antibodies . Recombinant human CD47-Fc  was also used to detect human SIRPA expression since it does not bind to rat SIRPA. Rat SIRPA was detected using anti-rat SIRPA-FITC monoclonal antibody . Several isotype negative controls were used: APC-mouse IgG1, FITC-mouse IgG2a, PE-mouse IgG1 (BD Pharmingen). All monoclonal antibodies, hCD47-Fc and human IgGs were used at 10 ug/ml. Fluorescence was analyzed with a FACSVerse flow cytometer (BD Biosciences), and FlowJo software was used to analyze data.How to cite this article: Jung, C. J. et al. Comparative Analysis of piggyBac, CRISPR/Cas9 and TALEN Mediated BAC Transgenesis in the Zygote for the Generation of Humanized SIRPA Rats. Sci. Rep.6, 31455; doi: 10.1038/srep31455 (2016).


Clusterin (CLU) is a ubiquitous multifunctional factor involved in neoplastic transformation. The CLU transcript variants and protein forms play a crucial role in balancing cells proliferation and death.in vivo model system consisting of both neoplastic tissues and fine needle aspiration biopsy (FNAB) samples isolated from patients undergoing thyroidectomy.We investigated the regulation of CLU transcript variants expression in an The immunohistochemical analyses showed an overall CLU up-regulation in papillary carcinoma. A specific CLU2 transcript variant increase was registered using qPCR in papillary carcinomas while CLU1 decreased. In addition, the analysis of CLU transcripts expression level showed an increase of the CLU2 transcript in the TIR 3 patients with histologically confirmed thyroid cancer.indeterminate” thyroid nodules.Our results suggest the existence of a specific alteration of CLU2:CLU1 ratio towards CLU2, thus providing the first circumstantial evidence for the potential use of CLU transcript variants as effective biomarkers for a more accurate assessment of the so called “ Thyroid nodules are extremely common most of them are not cancerous, while malignant lesions derived from thyroid epithelial cells are relatively rare. The initial evaluation of thyroid nodules commonly involves thyroid function tests, ultrasound examination (USG) and fine needle aspiration biopsy (FNAB) of selected nodules. Clinically recognized thyroid carcinomas constitute 5-7% of all thyroid nodules and 1% of all human malignant tumours. The annual incidence of thyroid cancer varies worldwide from 0.5 to 10 per 100,000.Thyroid carcinoma usually originates from follicular cells, and medullary carcinoma, which is a form of thyroid cancer, originates from the parafollicular C cells. Distinct histological types of follicular cell-derived cancers (FCDC) are recognized: the majority of cases are papillary, including its major subtype follicular variant (FVPTC); the remaining are follicular, oxyphilic or Hurthle cell, poorly differentiated and anaplastic carcinomas. Such tumour subtypes substantially differ from each other in terms of propensity to recurrence, distant spread and metastatic involvement ,2.Over the past 15 years, the application of molecular technologies to the study of these neoplasms has revealed critical genetic pathways associated with the development of specific thyroid tumour types . The resFurthermore, signals from paracrine and/or autocrine pathways, alone or in combination, may regulate these processes, the final effects depending on the net balance between stimulatory and inhibitory factors. As the tumour progresses, neoplastic cells often lose the inhibitory growth factors in the regulation of cell proliferation, while the expression of positive autocrine growth factors increases. In this regard, the aberrant expression or function of regulatory genes, particularly of those encoding for adhesion factors, growth factors, apoptotic factors and their receptors, invariably occurs in several cancers, including thyroid carcinoma . Among tCLU is a glycoprotein with a slightly ubiquitous tissue distribution and an apparent involvement in biological processes ranging from neurodegeneration in Alzheimer’s disease to cancer initiation and progression ,6. IndeeRecent studies have demonstrated there is no relationship between CLU transcript variants and CLU protein forms, showing as CLU1 and CLU2 code for the same secreted protein (sCLU) also identical regarding the post-transcriptional modifications (i.e. glycosylation) ,12. HoweIncreased expression of CLU has been found in different diseases where either abnormal cell death or proliferation occurs . MoreoveCLU was identified by microsequencing in the culture medium of porcine thyrocytes . Treatmein vitro and in vivo, and that there was a complex mechanism of regulation of CLU expression in the normal and cancerous thyroid tissue , where E is the average amplification efficiency calculated by DART-PCR for each primers pair and Ct is the average Ct obtained for the calibrator  and for the target . Moreover, for each CLU transcript variants the relative expression ratio (rER) with respect to the analysed thyroid tissue was calculated by applying the following formula: [(1 + E(target))^target]/[(1 + E(EC)) EC], where EC is the endogenous control and CLU1 the calibrator. The average data from at least two independent experiments are reported.1 μl of each cDNA was used as template in qPCR assays, performed in triplicate with ABI PRISM 7900HT (Applied Biosystems) using the QuantiTect® SYBR Green PCR Master Mix (Qiagen). Amplification parameters have been previously listed . FluoresData are reported as the mean ± SEM. Statistical analysis was performed using the Student’s t-test.


FGFR kinase amplification or mutation. There are several ongoing clinical trials of multitargeted agents in general NSCLC populations, including but not limited to patients with squamous disease. Other FGF/FGFR-targeted agents are in earlier clinical development. While results are awaited from these clinical investigations in squamous NSCLC and other disease settings, additional research is needed to elucidate the role of FGF/FGFR signaling in the biology of NSCLC of different histologies.With the introduction of targeted agents primarily applicable to non-small cell lung cancer (NSCLC) of adenocarcinoma histology, there is a heightened unmet need in the squamous cell carcinoma population. Targeting the angiogenic fibroblast growth factor (FGF)/FGF receptor (FGFR) signaling pathway is among the strategies being explored in squamous NSCLC; these efforts are supported by growth-promoting effects of FGF signaling in preclinical studies (including interactions with other pathways) and observations suggesting that FGF/FGFR-related aberrations may be more common in squamous versus adenocarcinoma and other histologies. A number of different anti-FGF/FGFR approaches have shown promise in preclinical studies. Clinical trials of two multitargeted tyrosine kinase inhibitors are restricting enrollment to patients with squamous NSCLC: a phase I/II trial of nintedanib added to first-line gemcitabine/cisplatin and a phase II trial of ponatinib for previously treated advanced disease, with the latter requiring not only squamous disease but also a confirmed  Histologic determination in advanced non-small cell lung cancer (NSCLC) has only recently become a fundamental consideration in guiding treatment decisions EGFR mutations and ALK gene rearrangements, the presence of which are to be confirmed by molecular analysis) are predominantly seen in adenocarcinomas NSCLC-directed targeted therapies introduced into clinical practice over the past decade are mainly applicable to the treatment of patients with adenocarcinomas. These include the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) gefitinib  With the lack of applicability of the newest agents for treating NSCLC, squamous NSCLC poses unique challenges in the clinic and is being recognized as a subset with particularly high need for new therapies. Among tumors classified as squamous NSCLC, heterogeneity in angiogenic and proliferative behavior has been described FGFs belong to a family of highly conserved polypeptide growth factors FGF genes have been identified in humans, of which the chromosomal locations have been established with one exception (FGF16) FGF3, FGF4, and FGF19, all on chromosome 11q13, and both FGF6 and FGF23 on chromosome 12p13) illustrates formation of the FGF family via gene and chromosomal duplication and translocation FGFR mutations have been associated with developmental disorders and identified across a number of malignancies, including lung cancer  levels have been shown to be increased in the NSCLC population (including both squamous cell and adenocarcinoma histologies) relative to healthy controls There is a substantial and growing body of literature to support that the FGF signaling pathway interacts with and influences other signaling pathways involved in the development and progression of NSCLC. For example, the VEGF and FGF/FGFR pathways have been shown to act synergistically in promoting tumor angiogenesis FGFR1 amplification specifically in squamous NSCLC, with amplification of a region of chromosome segment 8p11-12 (which includes the FGFR1 gene) in 21% of squamous tumors versus 3% of adenocarcinomas (P < 0.001) FGFR1 amplification in squamous NSCLC but not other histologic subtypes of lung cancer FGFR1 copy number in surgically resected squamous versus nonsquamous NSCLC  FGFR1 gene was numerically but not statistically higher for squamous versus adenocarcinoma histology  Interestingly, there appear to be some FGF/FGFR signaling pathway-related distinctions between NSCLC cases of squamous cell versus adenocarcinoma histology r = 0.17; P = 0.002), but even more strongly in the subset with nodal disease  Accumulating evidence points to a role for FGF signaling in the disease invasion and metastasis characteristic of NSCLC FGFR1 amplification as an independent negative prognostic factor (while exhibiting a dose-dependent association with cigarette smoking) in patients with squamous NSCLC r = 0.34; P = 0.01), with rates of 78% and 40% for patients with elevated and normal bFGF levels, respectively. Additionally, this study found a significant correlation between bFGF levels and VEGF levels  and that the combination of growth factors was a significant prognostic factor on univariate but not multivariate analysis, although conclusions were confounded by the presence of elevated levels of both bFGF and VEGF in only six patients. In a Japanese retrospective analysis of predictors of long-term survival among 71 patients with surgically resected NSCLC of adenocarcinoma or squamous histology, mean bFGF levels were significantly higher in cases of metastatic nodal involvement and high levels were most strongly correlated with poor prognosis in patients also exhibiting high VEGF levels (P < 0.0001) FGFs/FGFRs have been identified as potential predictive and prognostic markers in NSCLC. In a number of studies, pretreatment bFGF levels have been correlated with prognosis in the NSCLC population FGFR1 amplification, cell growth inhibition of an NSCLC line with focal FGFR1 amplification was demonstrated via FGFR1-specific small hairpin ribonucleic acids (shRNAs) or small molecule inhibitors A number of preclinical observations collectively suggest that FGF/FGFR signaling may be exploited as a therapeutic target in the NSCLC population. In the aforementioned DFCI study, in which 21% of squamous tumors exhibited α and β, fms-related tyrosine kinase 3 (FLT-3), and members of the src family N = 26, including three with squamous histology) of first-line nintedanib in combination with carboplatin/paclitaxel in advanced NSCLC, among seven patients with a confirmed partial response, two had squamous histology and one had mixed large cell/squamous histology FGFR kinase amplification or mutation per genotyping (NCT01761747). This trial has a target accrual of 40 patients and a primary endpoint of response. Orantinib , an oral TKI that targets VEGFR2, PDGFRβ, and FGFR1, was evaluated in a phase I trial  in combination with carboplatin/paclitaxel as first-line therapy for advanced NSCLC, with 13 partial responses observed among 33 evaluable patients Ongoing clinical trials of FGFR-inhibiting multitargeted tyrosine kinases in advanced squamous NSCLC or advanced NSCLC in general, including but not limited to squamous histology, are summarized in Table P = 0.0019) as well as in the histologic subsets with squamous disease (P = 0.02) or adenocarcinoma (P = 0.02) P = 0.0359). Cediranib primarily targets VEGFR2 but has demonstrated some inhibitory activity against FGF-induced proliferation, albeit 275-fold less selective than its inhibition of VEGF-induced proliferation As shown in Table P = 0.0082) The EGFR-directed monoclonal antibody cetuximab  FGFR1-amplified advanced squamous NSCLC (or FGFR1-amplified advanced breast cancer or FGFR2-amplified advanced esophagogastric cancer), with serial biopsies being performed to assess molecular effects (NCT01795768) Finally, there are ongoing clinical investigations of other FGF/FGFR-targeted agents in advanced malignancies, although not specific to NSCLC or squamous NSCLC. The pan-FGFR inhibitors AZD4547  and BGJ398  are being evaluated in a phase I trial for advanced solid tumors; for BGJ398, eligibility criteria include confirmed FGFR-related alterations. A nonrandomized phase II trial of AZD4547 monotherapy is enrolling previously treated patients with Future directions include studies to assess anti-FGF/FGFR agents in resectable disease , or even as a chemoprevention strategy FGFR1 gene amplification strengthen the rationale for this novel approach. Multitargeted small molecule inhibitors that inhibit FGFR along with other angiogenic pathways/receptors are the most advanced in clinical development, although none have yet to reach phase III evaluation in squamous-exclusive NSCLC study populations. Further research efforts are needed to more fully characterize the manner by and degree to which FGF signaling influences the underlying biology of specific NSCLC histologies.While there have been several molecularly targeted agents developed for the treatment of nonsquamous NSCLC, there appears to be a unique opportunity to develop anti-FGF/FGFR-based regimens for the treatment of NSCLC of squamous histology. Recent research findings supporting a propensity for squamous NSCLC to exhibit increased


Synovial fibroblasts in persistent inflammatory arthritis have been suggested to have parallels with cancer growth and wound healing, both of which involve a stereotypical serum response programme. We tested the hypothesis that a serum response programme can be used to classify diseased tissues, and investigated the serum response programme in fibroblasts from multiple anatomical sites and two diseases. To test our hypothesis we utilized a bioinformatics approach to explore a publicly available microarray dataset including rheumatoid arthritis (RA), osteoarthritis (OA) and normal synovial tissue, then extended those findings in a new microarray dataset representing matched synovial, bone marrow and skin fibroblasts cultured from RA and OA patients undergoing arthroplasty. The classical fibroblast serum response programme discretely classified RA, OA and normal synovial tissues. Analysis of low and high serum treated fibroblast microarray data revealed a hierarchy of control, with anatomical site the most powerful classifier followed by response to serum and then disease. In contrast to skin and bone marrow fibroblasts, exposure of synovial fibroblasts to serum led to convergence of RA and OA expression profiles. Pathway analysis revealed three inter-linked gene networks characterising OA synovial fibroblasts: Cell remodelling through insulin-like growth factors, differentiation and angiogenesis through _3 integrin, and regulation of apoptosis through CD44. We have demonstrated that Fibroblast serum response signatures define disease at the tissue level, and that an OA specific, serum dependent repression of genes involved in cell adhesion, extracellular matrix remodelling and apoptosis is a critical discriminator between cultured OA and RA synovial fibroblasts. The fibroblast-like synoviocyte (FLS) has become prominent as a mediator of disease persistence, supported by the persistent in vitro phenotype of these cells exemplified by the severe combined immunodeficiency (SCID) mouse model of cartilage invasion . Osteoartz et al . The fintz et al  illustrain vivo counterparts [RA FALS display an agressive phenotype, which involves increased invasiveness and resistance to apoptosis . Becauseterparts . We therterparts . We wereterparts . The hypSince the ‘core serum response’ signature (CSR) developed by Chang et al was based on fibroblasts derived from normal skin, lung and gum we reasoned that it may not fully represent the responses of synovial fibroblast populations in diseased joints. We therefore set out to experimentally define a serum response signature based on fibroblasts derived from both RA and OA patients. In order to take account of the documented effect of site on fibroblast transcriptional profiles and to address the possibility of multiple site involvement in systemic disease, we compared the serum response of fibroblasts of synovial origin to those of bone marrow and skin from the same individual. We set out to identify differentially expressed genes in at least one of the fibroblast groups. By using a SAM multiclass approach we could identify a signature of 3633 genes (FDR < 3% followed by a two-fold cut-off). Microarray gene expression was correlated with qPCR data for validation purposes . PrincipAnatomical location Serum Response (High vs. Low Serum)Disease type (RA vs. OA)While the PCA described above provides initial evidence for the existence of a molecular hierarchy of transcriptional control, it does not quantify the effect and it does not identify the specific genes linked to anatomical location, serum response and disease type. We therefore set out to directly quantify the differences in gene expression across all three levels of organisation and performed the following direct comparisons:Having defined the serum response signatures in each anatomical location and within each disease group we asked whether the serum response programs overlap significantly Samples derived from all three different anatomical locations of RA patients exhibited a greater overlap in serum response profile than OA samples, which exhibited more divergent, site-specific serum responses  and matrix components with signalling to differentiation and angiogenesis pathways. ITGB3 binds to multiple extracellular matrix components and growth factors including platelet-derived growth factor (PDGF), Collagens and Fibulin 2 [αvβ3 monoclonal antibody (Vitaxin) was withdrawn after trials failed to show therapeutic benefit [β3 and αvβ3 integrin knockout mice exhibit enhanced pathological angiogenesis of implanted tumours while ITGB3 over-expressing models demonstrate small new vessels deficient in pericytes [αvβ3 integrins in angiogenesis therefore remains controversial. The CD44 pathway covers a broad spectrum of genes, including the MAPKinases and PI3Kinase that are all implicated in control of apoptosis and survival [β linked pathways, that form a part of this response, to sub-classify disease. These data also demonstrate at a transcriptome level that low serum conditions will increase the likelihood of identifying disease-specific signatures.We hypothesised that the CSR of Chang et al would differentiate persistent inflammatory RA from OA and normal synovium, and showed this to be the case at the tissue level. This observation places inflammatory arthritis at the centre of the long-standing debate regarding the relationship between wound healing, cancer and persistent inflammation . Compariibulin 2 –29. Treaibulin 2  as well ibulin 2 . ITGB3:Pibulin 2 . Angioge benefit . Furtherericytes , 34. Thesurvival , 36. Furhttp://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE12021) [We retrieved a dataset from the NCBI GEO database which contained synovial tissue biopsy samples from RA, OA and normal patients (SE12021) . The datSE12021) , 38. ExpFibroblasts were isolated as previously described  from synCells were seeded in duplicate at the required density using 10% FCS. Once attached, the medium was removed and the cells washed in serum free medium before replacing with fibroblast medium, which was identical but contained either 10% or 0.1% FCS . mRNA wa5 fibroblasts was reverse transcribed using Superscript Vilo (Life Technologies). 1 μl of the resulting cDNA was added to the real-time PCR master mix (Applied Biosystems) and TaqMan primers/probes were used to measure the relative quantities of SLIT3, PIAS1 and TNS3 (VIC labelled) with GAPDH (FAM labelled) as control using an ABI 7900HT machine (Applied Biosystems).Synovial fibroblasts from 3 OA and 3 RA patients were cultured as above. Cells were trypsinised and RNA extraction performed using an RNeasy kit (Qiagen). RNA from 1×10Out of the 108 samples retrieved six samples did not meet quality control and hence did not undergo further analysis. These included 1x Bone Marrow RA low serum, 1x Bone Marrow RA high serum, 1x Skin OA low serum, 1x Synovial OA low serum, 1x Synovial OA high serum and 1x Skin OA high serum sample. Gene expression data from the Affymetrix® arrays were processed and normalized using RMA methodology. Data were processed to remove genes with expression close to the detection level.Statistical analysis for microarrays (SAM)  as impleTo test whether the CSR signature is able to distinguish between normal, RA and OA samples retrieved from the public domain we applied a K-nearest neighbour algorithm as implemented by the knn.cv function in the ‘class’ library in the statistical environment R. This methodology uses a leave-one-out-crossvalidation. For each sample in the training set the k nearest neighbours are identified and the classification decided by majority vote. We have tested 2 < k < 10 and observed that the accuracy was independent of the value of k .Genes differentially expressed between all sample groups were visualised using Principal component analysis (PCA), a technique designed to represent highly multi-dimensional objects in a lower dimensionality space . The firhttp://www.ingenuity.com) that uses the Ingenuity Pathways Knowledge Base (IPKB) to explore biological interaction networks. Each gene identifier is mapped to a global molecular network within IPKB. An algorithm then generates networks based on the connectivity of focus genes, limited to a maximum of 35 genes for simpler visualisation. For the downstream effect analysis gene lists and calculated fold changes were uploaded to the IPKB. For each biological function within the IPKB an activation z-score is calculated by the software. Here the molecular network within IPKB is interrogated and experimentally observed causal relationships between genes and function extracted. These connections provide a literature-derived direction of effect which is used in combination with the foldchange to calculate the activation state of the given functional terms. As multiple genes may be connected to a function a statistical approach is necessary to calculate its significancy as compared to a randomly derived variable. This variable is obtained by randomly choosing 1 or -1 with equal probability representing an “increased” or “decreased” state respectively. For each number of genes affecting the functional term the number of “increased” (N+) and “decreased” (N−) predictions is summed up for the random. This however assumes that there is only 1 possible downstream effect to a given function. To correct for this the IPAKB uses a weighted z-score statistics in which:Mactivating and Minhibiting represent the number of findings underlying an edges “activation” or “inhibition” respectively. This is then used to correct for the direction of change within the z score algorithm. The z-score can therefore be calculated as:Gene sets derived from differential gene expression and Venn diagram analyses were analysed using the IPA application  procedure. The percentage of correct classification using a majority rule is indicated below the sample labels.(TIFF)Click here for additional data file.S2 FigA) Shows the Venn diagram for the response to serum in all tissues in OA. B) represents the serum response of all tissues in RA patients.(TIFF)Click here for additional data file.S1 TableRhF, rheumatoid factor; DMARD, Disease-modifying antirheumatic drug; MTX, methotrexate, weekly dose; D-PEN, D-penicillamine; Gold, intramuscular gold, monthly dose; AZA, azathioprine; SSA, sulphasalazine.(DOCX)Click here for additional data file.S2 TableData were obtained from 3 synovial fibroblast lines. qPCR, quantitative PCR; PIAS1, protein inhibitor of activated STAT-1 .(DOCX)Click here for additional data file.S3 TableOverview of the Functional associations in the serum response between RA and OA fibroblasts within each anatomical site. The numbers represent “number of genes increased”/“number of genes decreased” within each term.(DOCX)Click here for additional data file.S1 DatasetAn excel file containing all results from the SAM analysis for all comparisons performed, including up and down regulated genes with their associated Gene ID, Fold Change and q-value (%).(XLSX)Click here for additional data file.


We developed a pipeline for genome-wide association analysis and meta-analysis of CNV genotypes measured across multiple studies using SNP genotype array intensity data from different platform technologies. We then undertook cohort-level genome-wide association studies of CNV with lung function in a subset of 4 cohorts  with lung function measurements and meta-analysed the results. Follow-up was undertaken for CNVs which were well tagged by SNPs, in up to 146,871 individuals.Genome-wide association studies of Single Nucleotide Polymorphisms (SNPs) have identified 55 SNPs associated with lung function. However, little is known about the effect of copy number variants (CNVs) on lung function, although CNVs represent a significant proportion of human genetic polymorphism. To assess the effect of CNVs on lung function quantitative traits, we measured copy number at 2788 previously characterised, common copy number variable regions in 6 independent cohorts (P = 0.0007) with Forced Vital Capacity (FVC) downstream of BANP on chromosome 16 that shows evidence of replication by a tag SNP in two independent studies (replication P = 0.004). In addition, we provide suggestive evidence (discovery P = 0.0002) for a role of complex copy number variation at a previously reported lung function locus, containing the rootletin gene CROCC, that is not tagged by SNPs.We generated robust copy number calls for 1962 out of 2788 (70 %) known CNV regions genome-wide, with 1103 measured with compatible class frequencies in at least 2 cohorts. We report a novel CNV association  contains supplementary material, which is available to authorized users. Quantitative lung function traits are of major public health relevance and large GWAS have identified multiple common variants which collectively explain only a small proportion of the phenotypic variance. For lung function traits  are deletions or duplications of parts of a chromosome, ranging from a few hundred to a few million base pairs in length , leadingPlatforms primarily designed for SNP genotyping can additionally provide an estimate of copy number by looking for deviations in the intensity signal from each allelic SNP probe; “non-polymorphic” probes (probes at genomic positions where there is no known SNP) have been added to some GWAS platforms specifically for this purpose. The wide availability of these intensity data presents an opportunity to measure genome-wide copy number variation across multiple studies and bring together large sample sizes for association testing.Here we describe a pipeline for genome-wide CNV genotype measurement using SNP array intensity data. We first tested the pipeline and defined appropriate quality control filters using 6 cohorts comprising intensity data from 5 different genotyping platforms. We then undertook an analysis of genome-wide CNV associations with lung function for up to 12,403 individuals in a subset of 4 cohorts with lung function measurements at 1962 copy number variable loci. We show how this approach can identify multiallelic variants that are not well-tagged by SNPs and that potentially explain some of the missing heritability of lung function quantitative traits.n = 3496), Young Finns Study , British 1958 Birth Cohort , Study of Health in Pomerania , Raine Study (n = 1685) and the Avon Longitudinal Study of Parents and Children ). The aim of this first stage was to identify whether common CNVs could be measured with consistency across multiple cohorts with different intensity data. In the second stage we tested for association of copy number with lung function in 4 cohorts which had lung function measurements. Lung function association results were then meta-analysed across cohorts.There were 2 stages of the analysis. In the first stage we refined a pipeline for copy number calling to determine the quality control steps required to obtain consistent copy number measurement across multiple cohorts and genotyping platforms. In this stage we analysed intensity data from SNP genotyping platform experiments for 24,237 samples from 6 different cohorts  from a map of 11,700 copy number variants across the genome published by The Genome Structural Variation Consortium , which iThe Log R Ratio (LRR) of the intensity signals from the allelic probes used in the SNP genotyping was used to estimate copy number. LRR is the ratio of the intensity to the expected intensity of the relevant genotype cluster and is a transformation of the raw X and Y intensity signals that enhances the correlation with copy number. The LRR was available for all of the cohorts apart from ALSPAC where the sum of the X and Y intensities from each of the allelic probes was used instead. Replicate samples and samples which failed intensity noise filtering  were available for 3 adult cohorts  and 1 child cohort (ALSPAC). We tested for association of FEV1, FVC and FEV1/FVC with CNVR copy number within each cohort and results were then meta-analysed across up to 4 cohorts as the primary analysis and then across up to 3 cohorts of adults to look for age specific effects. Only CNVRs called with the same number of classes and consistent class frequencies across cohorts in stage 1  to account for correlation within families [For stage 2, lung function measurements (FEVfamilies . A detai2 > 0.7), we sought replication of suggestive signals of association (P < 0.001) for lung function in a large GWAS. Linkage disequilibrium (r2) between each CNVR and SNPs within 1 Mb of the CNVR start and end positions was measured using samples in the BHS cohort that had both copy number genotypes and imputed SNP genotypes (1000 Genomes Project Phase 1 imputation reference panel). For replication we used results from the SpiroMeta and CHARGE consortia meta-analysis of 48,201 individuals across 23 studies [n = 48,943, Additional file n = 49,727, Additional file 2, sex, height, pack years in smokers where available, and ancestry principal components where available. Residuals were inverse rank inverse-normal transformed after adjustment apart from FVC in SpiroMeta and CHARGE, which was untransformed [1 and FEV1/FVC we used inverse-variance weighted meta-analysis across the replication cohorts and for FVC we used Z-score meta-analysis. All replication resources were comprised of European ancestry individuals. For replication, we used a Bonferroni corrected 5 % threshold for the number of CNVRs with a tag SNP available. Replication was not available for CNVRs not well-tagged by SNPs (r2 > 0.7).For CNVRs which were well-tagged by SNPs  terms using DAVID [used GTEx to perfo2 test described in Additional file For the first stage of the analysis, aimed at developing a pipeline for copy number calling across different genotyping platforms, intensity data was available for 24,259 samples from 6 cohorts. After filtering, 19,308 samples were taken forward for CNVR clustering and copy number calling. The total number of CNVRs called and the proportions of deletions, amplifications and multiallelic CNVRs called in each cohort varied across genotyping platforms (Table P < 2.5 × 10-5); the full set of association results are provided in Additional file P < 0.001) which included 12 CNVR-trait combinations (11 distinct CNVRs) in the adult cohorts analysis , 7 . A SNP tagging CNVR6854.1  showed replication of association with FVC in UK BiLEVE and in the meta-analysis of the 3 replication cohorts (Table P < 0.006). The direction of effect of this CNVR was consistent across the 3 discovery CNVR cohorts and was in the same direction in SpiroMeta, CHARGE and UK BiLEVE for the SNP allele positively correlated with increased copy number, but in the opposite direction in the non-significant UK Biobank association . The tag SNP rs7501378 is 420 bp further downstream of BANP than CNVR6854.1  were for 3 overlapping and correlated CNVRs within or near to CROCC, a gene that encodes rootletin, a component of cilia  with lung function in this study, no enrichment of gene ontology terms was seen (P < 0.05).Across the 45 genes implicated by CNVRs showing nominal evidence of association -mediated deactylation pathway [We developed a quality-control pipeline for genome-wide association analysis and meta-analysis of CNV genotypes and generated robust copy number calls across cohorts for 1962 CNVs genome-wide across 6 cohorts comprising 5 different genotyping platforms. Of these, 1103 CNVs could be clustered consistently in at least 2 cohorts, 777 in 3 cohorts, 371 in 4 cohorts and 70 in 5 cohorts. No CNVRs could be clustered across all 6 cohorts .1/FVC (P = 2.0-2.7 × 10-4) and which involved the CROCC gene in a region that has previously been shown to be associated with lung function [2 = 0.005) by the previously reported SNP showing strong evidence of association with FEV1/FVC , which is within 25 kb [1/FVC in addition to the 3.2 % of variance in FEV1/FVC already accounted for by genome-wide significantly associated SNPs [Crocc knockout mice that loss of rootletin (the protein encoded by Crocc) prevents the formation of the ciliary rootlet in airway epithelial cells leading to a reduction in motile cilium function with pathological changes consistent with insufficient mucociliary clearance [CROCC is in a region which is annotated as containing several overlapping CNVRs [CROCC warrant further verification. Determining the precise genomic architecture of the region is needed to understand the nature of the associations observed in this study.An additional five CNVRs which showed suggestive evidence of association with lung function in our study were complex (i.e. did not have 3 copy number classes) and hence not well tagged by bi-allellic 1000G SNPs. Amongst these were 3 correlated CNVRs which had the strongest evidence of association for FEVlearance . CROCC ing CNVRs  . We were able to generate compatible copy number calls across at least 2 cohorts for over one thousand CNVRs genome-wide. Differences in concordance of CNV class frequencies between pairs of cohorts with SNP genotyping data from the same or similar versions of Illumina platforms could be due to differences in intensity data quality. It was evident that older versions of the platforms had poorer probe coverage of the CNVRs than the more recent platforms, some of which had content designed specifically for the larger set of CNVRs from which the subset analysed here were selected. We chose to use the number of copy number classes and class frequencies from one cohort as the best estimate of the true number of classes to which other cohorts were compared with validation of the genotypes measured in this reference cohort against CNV genotypes in an independent cohort. As new cohorts emerge and existing cohorts update their genotype data using newer arrays, compatibility across cohorts for increasing numbers of CNVRs is likely to increase.BANP on chromosome 16 and present evidence that copy number variation may play a role at a locus previously reported as being associated with lung function (CROCC).We demonstrate how common CNV regions can be reliably and consistently called across cohorts, using an existing calling algorithm and rigorous quality control steps, using SNP genotyping array intensity data. Using this approach, we describe a novel signal of association with FVC for a copy number variable locus downstream of 1, forced expiratory volume in 1 second; FVC, forced vital capacity; GWAS, genome-wide association study; LRR, Log R ratio; SHIP, study of health in pomerania; SNP, single nucleotide polymorphism; UK BiLEVE, UK biobank lung exome Variant Evaluation; YFS, young finns study1000G, 1000 genomes project; ALSPAC, avon longitudinal study of parents and children; B58C, British 1958 birth cohort; BHS, busselton health study; CHARGE, cohorts for heart and aging research in genomic epidemiology; CNV, copy number variant; CNVR, copy number variable region; ERS/ATS, european respiratory society / american thoracic society; FEV