Application of exogenous ethylene or its m growth .cis-elements in their promoters [The ET perception and signal transmission cascade in Arabidopsis has been linked to the transcriptional activation of Ethylene Response Factors (ERFs) ,4. As trromoters . We hypoERF genes in the Populus trichocarpa genome versions 2.0 and 2.2 using regular expression method and pfam search for the ERF domain. These ERFs grouped into 11 distinct groups, similar to ERFs identified in Arabidopsis and rice [ERF transcripts were detectable in stem tissues of in vitro or greenhouse grown hybrid aspen. The responsiveness of all ERFs to short term ACC treatments  and to short or long term ethylene treatments , was assessed by qPCR. Most of the ERFs responded to at least one of the treatments, mostly by increased transcript accumulation. We identified ERFs that were specifically induced within the early ACC/ET- or the late ET-response. In addition, some ERFs showed prolonged induction up to 2 weeks of ET treatment. These different transcript patterns indicate that different ERFs may be involved in distinct, temporally distinguished processes during the ACC/ET- induced secondary xylem growth response.We identified 169 ERF candidates and investigated whether those were responsive to endogenous ET-signals in leaned stems during TW formation. We compared the induction of those 26 candidate ERFs in TW with ERF accumulation during long-term (2 weeks) ET treatment. Interestingly, a significant overlap of ERF induction in both conditions was found. From the 20 ERFs that were induced after long-term ET treatment, 16 had an increased transcript abundance during tension wood formation. This indicates that on an ERF transcript basis, tension wood formation is largely comparable to a long-term ET treatment.According to their expression and capacity to be induced by ET or ACC, we selected 26 pLMX5 promoter [ERFs in transgenic plants was confirmed and two to seven lines of each overexpressed ERF were phenotyped in a greenhouse trial. In general, ERF overexpression caused only mild alterations of overall plant stature . Only overexpression of one ERF candidate led to a severe dwarf phenotype with thin stems, reduced fiber and vessel size, reduced height growth and smaller leaves. The absence of any striking phenotypes in all other plants suggests that other regulators in addition to the overexpressed ERFs may be necessary to mimic the enhanced growth response observed during ET/ACC/TW mediated stimulation of secondary xylem growth.Based on the transcript data, twenty ERFs were selected for overexpression in hybrid aspen cambium/xylem under the promoter . SuccessERFs led to changes in cell wall composition in xylem tissues when overexpressed. This suggests that these ERFs have the ability to modify cell wall composition in wood forming tissues and may regulate the expression of cell-wall biosynthesis genes. This hypothesis is now under further investigation.A Fourier-Transformed Infrared spectroscopic and Pyrolysis GC-MS based screening of five lines for each of the 20 overexpressed ERFs revealed that five


CSN3, identified by screening our prior GWAS results in conjunction with our exome results, was found to contain an interesting coding polymorphism as well as containing excess rare variation as compared with the other genes evaluated. In conclusion, while rare coding variants may predispose to the risk of ischemic stroke, this fact has yet to be definitively proven. Our study demonstrates the complexities of such research and highlights that while exome data can be obtained, the optimal analytical methods have yet to be determined.The genetic architecture of ischemic stroke is complex and is likely to include rare or low frequency variants with high penetrance and large effect sizes. Such variants are likely to provide important insights into disease pathogenesis compared to common variants with small effect sizes. Because a significant portion of human functional variation may derive from the protein-coding portion of genes we undertook a pilot study to identify variation across the human exome  in 10 ischemic stroke cases. Our efforts focused on evaluating the feasibility and identifying the difficulties in this type of research as it applies to ischemic stroke. The cases included 8 African-Americans and 2 Caucasians selected on the basis of similar stroke subtypes and by implementing a case selection algorithm that emphasized the genetic contribution of stroke risk. Following construction of paired-end sequencing libraries, all predicted human exons in each sample were captured and sequenced. Sequencing generated an average of 25.5 million read pairs (75 bp×2) and 3.8 Gbp per sample. After passing quality filters, screening the exomes against dbSNP demonstrated an average of 2839 novel SNPs among African-Americans and 1105 among Caucasians. In an aggregate analysis, 48 genes were identified to have at least one rare variant across all stroke cases. One gene,  Stroke is the fourth leading cause of death and the leading cause of disability in the US MYH3 gene that cause the disease DHODH for Miller Syndrome (OMIM: #263750 - accessed 2010 July), a rare autosomal recessive Mendelian disease characterized by an under-sized jaw, droopy eyes, cleft lip or palate, and incomplete or unusual limb development Several studies have successfully employed exome sequencing to identify rare mutations that are responsible for Mendelian disease. In a proof-of-principle study, four affected individuals with Freeman-Sheldon syndrome (FSS - OMIM: #193700 - accessed 2010 July) and eight HapMap controls underwent exome were sequencing to demonstrate that that this technique could identify the known mutations in the #263800 - accessed 2010 July) and Bartter's (OMIM: #607364 - accessed 2010 July) syndromes, both of which are characterized by low blood pressures. The investigators demonstrated that heterozygote mutation carriers had significantly lower mean long-term values for systolic  and diastolic  BP and a 59%  lower risk of developing hypertension by age 60 years. In summary, individuals in the lower blood pressure ‘tail’ of the overall blood pressure distribution were found to have rare coding genetic variants influencing renal salt handling that contributed to blood pressure variation at the population level.While these examples demonstrate the usefulness of the exome approach in identifying rare variants associated with Mendelian disease, other studies are now revealing the role of rare variants in complex disease. In one such study, rare variants were determined to be associated with development of the well-established complex disease of hypertension parkin gene in the PARK2 locus that cause sporadic early-onset parkinsonism parkin variants that play a role in the common late-onset forms of Parkinson disease (age of onset >45 years) Various studies of complex diseases indicate that young onset disease is more likely due to multiple rare exonic variants with large effects as compared to late-onset disease. As an example, studies identified rare mutations in the Regarding stroke, twin studies and familial aggregation studies suggest a stronger genetic contribution to early onset than later onset stroke. Reports from the National Academy of Sciences-National Research Council Twin Registry showed a greater concordance in monozygotic twins relative to dizygotic twins when the twin cohort was younger In summary, this background information demonstrates the usefulness of the exome approach in identifying rare variants associated with Mendelian disease and also implies that by studying individuals in the extreme phenotypic tails in common complex disease, one can identify rare variants that have population-level effects on disease risk. While young-onset stroke is clearly not a Mendelian transmitted disease, rare variants of high penetrance could conceivably contribute to risk among the young-onset stroke phenotype. As such, a young age-of-onset can be considered as an extreme tail of the liability threshold distribution which is also often a characteristic of Mendelian disease. In this report, we describe our pilot study efforts to identify rare variants associated with ischemic stroke and the complexities of such research. We believe that sequencing will ultimately identify rare risk variants and that exome sequencing, given its ability to identify rare variants of high penetrance, is an excellent methodology to begin these efforts.All human subjects research presented herein has been reviewed and approved by the Institutional Review Boards of the University of Maryland and the Mayo Clinic. Written informed consent was obtained from all participants involved in the study.Ten ischemic stroke subjects were selected for exome sequencing and analyses; 8 African-American participants from the Genetics of Early Onset Stroke Study and 2 European-Caucasian participants from the Siblings with Ischemic Stroke Study.GEOS is a population-based study of ischemic stroke among Caucasian and African-American young adults in the Baltimore-Washington area that is composed of patients and controls recruited in 3 different time periods: Stroke Prevention in Young Women-1 (SPYW-1) conducted from 1992–1996, Stroke Prevention in Young Women-2 (SPYW-2) conducted from 2001–2003, and Stroke Prevention in Young Men (SPYM) conducted from 2003–2007. The study population see Table 1 Case participants were hospitalized with a first cerebral infarction identified by discharge surveillance from one of 59 hospitals in the greater Baltimore-Washington area and direct referral from regional neurologists. SPYW-1 included cases ages 15–44 years recruited within one year of stroke and was designed with a 1∶2 case-to-control ratio. SPYW-2 and SPYM included cases ages 15–49 recruited within three years of stroke and was designed with a 1∶1 case-to-control ratio. The abstracted hospital records of cases were reviewed and adjudicated for ischemic stroke subtype by a pair of neurologists according to previously published procedures Control participants without a history of stroke were identified by random-digit dialing. Controls were balanced to cases by age and region of residence in each study and were additionally balanced for ethnicity in SPYW-2 and SPYM.cases and controls risk factors including hypertension, diabetes mellitus, and myocardial infarction were determined by asking the study participant (or his/her proxy if the participant was unable to answer) if he/she had ever been told by a physician that he/she had the condition. Similarly, age, race, ethnicity, oral contraceptive use, and smoking history were determined by subject or proxy report. Less than 5% of participants had proxy interviews or assisted interviews. Cigarette smoking information included: ever smoker, current smoker, former smoker, number of years smoking, average number of cigarettes smoked during smoking years, and pack-years of smoking. , who have had brain-biopsy-proven central nervous system vasculitis at any time, who have Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL), Fabry disease, homocystinuria, mitochondrial encephalopathy with lactic acidosis and stroke-like episodes (MELAS), or sickle -cell anemia, or who had a mechanical aortic or mitral valve, or bacterial endocarditis at the time of stroke onset, were excluded from the Proband and Concordant Sibling groups. Patients who gave a positive answer to any of the 8 questions on the Questionnaire for Verifying Stroke-Free Status (QVSFS), or who deemed unreliable historians by the study interviewer due to moderate or severe impairment of speech, language, hearing, or memory, were be excluded from the Discordant Sibling group.Probands: Prior to enrollment, patients' medical records and test results were reviewed by a study neurologist to confirm the diagnosis of ischemic stroke. Eligible patients were classified by stroke subtype, according to the TOAST criteria Considerable thought was given as to which ten cases to include in our analyses, as the goal of this pilot was to not only to demonstrate the effective use of exome methodologies, but that these exomes would then also be combined with additional future exomes for further analyses. Additionally, future collaborative efforts between the GEOS and SWISS studies were also considered. Having two SWISS European-Caucasian samples run on the University of Maryland platform and through the bioinformatics pipeline would allow for future quality control evaluations on SWISS samples undergoing exome sequencing elsewhere.selecting only one subtype would have been the optimal methodology. As seen in As described in the Introduction, prior successes to identify causative disease variants implementing exome analyses have centered on the use of a highly defined phenotype. Given the heterogeneity of stroke and numerous stroke subtypes, each of which might be considered as a separate complex disease, implementing this methodology in our stroke population was somewhat more difficult. In an effort to maximize the genetic contribution to stroke risk, we limited our case selection to only one gender , cases with an early age-of-onset, a positive family history of stroke, and the presence of no  standard stroke risk factors. We also worked to limit the stroke subtypes selected, although in retrospect FMNL2 (HGNC:18267), which encodes a formin-related protein, and rs1986743 (P = 2.65×10-7) in the intron of ARL6IP6 (HGNC:24048), which encodes ADP-ribosylation-like factor 6 interacting protein 6. As such, no SNP attained genome wide significance in our modestly-sized GWAS, supporting the notion that a few common variants do not account for the majority of stroke risk.Here we briefly describe our recently completed GWAS on the GEOS study population, as some of these results were utilized to inform on our exome analyses. Our group recently implemented the Illumina 1 M Quad GWAS chip on the GEOS population (including the 8 GEOS exome samples). The SNPs included on this chip were chosen based upon allele frequencies greater than 5% within HapMap datasets; therefore, rare variants were specifically excluded. Following extensive data cleaning procedures, GWAS analyses were performed with a manuscript detailing these results recently published Table S1). Also, as related to our future plans, GWAS data from these same study subjects can be utilized to select the optimal control subjects from other pre-existing publically available exome controls samples (e.g. NHLBI GO ESP available at dbGAP) to minimize confounding secondary to admixture.Having GWAS data on our exome cases allowed us to assess and compare genotype calls from a quality control standpoint .The GWAS data used in these analyses is available at database of Genotypes and Phenotypes (dbGaP)  within the Institute for Genome Sciences (IGS) at the University of Maryland School of Medicine. Construction of paired-end Illumina sequencing libraries were performed as prescribed by the manufacturer. These libraries will include unique 6 bp index sequences to enable multiplexed sequencing of more than one library per flowcell lane. Following library construction, Agilent's SureSelect technology was utilized to target capture of all predicted human exons in each sample for high-throughput sequencing using the Illumina Genome Analyzer IIx (GAIIx) platform. The method utilizes custom-designed, biotinylated RNA oligonucleotides for hybridization to the target sequence Following hybridization of library fragments to the RNA baits, the bound fragments were captured by streptavidin-coated magnetic beads, and unbound fragments were washed away. The RNA was then digested, leaving captured library fragments ready for sequencing. The captured library fragments were sequenced using an Illumina Genome Analyzer IIx. We sequenced 100 bp from both ends of each library fragment. Each lane of Illumina GAIIx sequencing generated 80–100 million high-quality, passed-filter sequence read pairs. Of these, approximately 75% were uniquely mappable reads with an estimated 70–90% of the uniquely mappable reads falling on or within 200 bp of a targeted exon. This sequence coverage yielded at least 20× sequence coverage for more than 75% of targeted exons, leading to high confidence SNP calls with low false positive rates In addition to performing the exome capture and sequencing, the Genomics Resource Center provided bioinformatics support for the project. Raw data from the sequencer was processed using Illumina's RTA and CASAVA pipeline software, which includes image analysis, base calling, and sequence quality scoring, as well as pre-existing GRC-developed pipelines for sequence assessment and quality control. Following basecalling by Illumina pipelines, the quality control pipeline assessed basecall quality, converted Illumina quality scores (ASCII+64) to Sanger-style/Phred-like scores (ASCII+32) and truncated reads where the median Phred-like quality score fell below Q20. The pipeline also plotted base composition by cycle/position to identify potential cycles with aberrant chemistry errors or sample bias. These processes were parallelized using a high-performance Sun Grid Engine computer grid, which contains more than 1000 cores and five terabytes of available RAM. Further analysis of the resulting sequence reads were performed using our human exome analysis pipeline based upon the Burrows-Wheeler Aligner (BWA), and the Genome Analysis Toolkit (GATK) Initial alignment to the human reference genome (hg18) using BWA was followed by statistical analyses that calculated the percent of reads uniquely mapping, the percent of properly paired reads, and statistics that evaluate the exome capture efficiency. These included percent of mapped reads within targeted regions and within a single library-fragment-length of targeted regions (+/−200 bp). We also calculated coverage of targeted regions and percent of targeted regions that met multiple coverage criteria . The resulting alignments were stored in BAM file format and we then used the Samtools package to identify and remove sequence reads that resulted from duplication of library fragments. These duplicated library fragments were artifacts of the necessary PCR step in library construction and are easily identified by having the same start and stop position in the alignment. Typical rates of duplication ranged between 10–20%. Removal of these duplicated reads prevented artificially high coverage and quality calculations and reduced false-positive variant calling.Because Illumina quality scores tend to be over-inflated, after removal of PCR duplicate reads, the Genome Analysis Toolkit Following these data quality control and pruning steps, variant detection (SNPs and indels) was performed using samtools. SNPs are called using a stringent set of quality criteria, including a minimum SNP quality score greater than 50, an allele balance less than 0.75, and a maximum of two SNPs within any 10 bp window. SNPs passing these quality criteria and falling within the targeted capture regions are stored in VCF format and annotated according to the RefSeq genes and isoforms in which they are found. The SNPs are further annotated according to classification, including coding-synonymous, missense, nonsense, intronic, UTR-5′ and UTR-3′. These classifications can be made at the isoform level to detect variants with differing potential functional impact on alternatively spliced gene products.http://www.broadinstitute.org/igv/ - accessed 2010 July), which enables display of tracks including read alignments, coverage levels, and SNP calls from multiple samples simultaneously alongside gene annotations. This provides a powerful tool for detailed comparative analysis between samples. Final data included raw sequence files, alignment files, SNP databases, and Integrated Genomics Viewer visualization.The analyzed exome data set, including both VCF and BAM files, can be visualized using the Broad Institute Integrated Genomics Viewer , additional information regarding the GEOS population is included here. The GEOS study population includes 1816 participants, consisting of 889 cases and 927 controls with the population characteristics demonstrated in The characteristics of the 10 selected cases for exome sequencing are listed in As described, we captured and sequenced ten stroke exomes. To evaluate the efficiency and success of our targeted capture and high-throughput sequencing of all human exons, we constructed paired-end Illumina sequencing libraries from whole human genomic DNA extracted from our ten clinical samples, using Agilent's SureSelect 37 Mb All Exon Kit to capture exonic DNA fragments Though the targeted capture method enables a high rate of enrichment for the targeted regions, non-specific capture also occurred. In our pilot samples, an average of 55% of aligned reads mapped to the targeted regions of the genome and 71% mapped onto or within 200 bp of the targeted regions. These rates of successful capture meet or exceed the expectations set by previous studies After sequencing, our data went through several data cleaning and alignment steps in preparation for SNP calling; these procedures were explained in the Methods. The application of these methods to our 10 exomes resulted in SNP calling as denoted in http://www.1000genomes.org/about - accessed 2011 July). Comparisons of all SNPs identified in our 10 exome study subjects and those within the same exomic regions from subjects within the 3 controls populations are seen in First, our data has significant overlap with these datasets, highlighting the quality of our exome data. Second, the overlap between our data and the various populations is incomplete, demonstrating that there are numerous previously unidentified variants in our exomic dataset. Third, the two differing African-American control populations have incomplete overlap, emphasizing the need to use well-matched controls (to the cases); implementing principal component and ancestry informative marker analyses will minimize confounding secondary to population admixture.To further evaluate the quality our data we sought publically-available exome (genome) data that could be used for comparison purposes. Here it is important to note that while one might consider utilizing only publically-available unaffected control reference data for comparison with our case-only exome data, such methods may introduce unbeknownst errors. While such methods may be appropriate when studying rare Mendelian diseases that express a highly-defined phenotype, complex diseases by nature have a less well-defined phenotype, with the genetic contribution to any specific complex disease being un-established across any potential publically-available control population. As such we propose that unaffected matched controls to the cases under evaluation be utilized along with the publically available control data, then statistically evaluating for differences in the controls population prior to case-control comparisons. In our pilot study no matched controls were utilized, however 3 appropriate publically-available control populations were identified in dbSNP. These included 61 African Ancestry individuals from the Southwest US (ASW), 100 Yoruba ) and 116 Caucasians of Western European ancestry (CEU), all of which were sequenced as part of the 1000 Genomes Project , with the results of several different analyses utilizing our 10 pilot case exomes now presented.first were the genes containing the “top" lacunar stroke coding variants as identified by our GWAS study and the second were a series of candidate genes selected from the literature Pursuant to our hypothesis that excess rare variation may play an important role in gene-specific stroke risk, we decided to evaluate specific genes among our exome data. Before we could embark on such analyses, control data was required and was obtained as described above. Two sets of genes were chosen for these analyses. The not believe that common variation is marking rare variation, but hypothesize that potentially a combination of common and rare variation in these genes may work together to increase risk. Utilizing our GWAS results nonsynonymous coding SNPs present. This was performed implementing SIFT http://sift.jcvi.org/www/SIFT_dbSNP.html - accessed 2010 July) which identified the nonsynonymous coding SNPs  and also rated the variants as deleterious or not. Among the pooled, African-American and Caucasian groups respectively, 17, 14 and 17 missense variants were identified; of these 4, 3, and 2 were considered to be deleterious, inducing amino acid changes purported to affect protein function. We then identified the genes containing these variants. Of note, there were no SNP (or gene) overlaps between the two ethnicities. The ‘top’ lacunar stroke coding variants from our GWAS results were identified by sorting our lacunar stroke specific results  by p-value smallest to largest. This was performed in the pooled sample and by ethnicity (African-Americans and Caucasians). Our hypothesis was that while none of these common variants reached genome-wide significance, maybe these gene-specific variants were marking stroke susceptibility genes and that potentially unknown excess rare-variation could also play a role. To be clear, we do MTHFR, F5, F2, ITGB3, ITGA2, LTA, SERPINE1, ACE, ApoE, NOS3, and NINJ2.The candidate genes selected for our second set of analyses were chosen on the basis that they harbor common genetic variants associated with ischemic stroke that have positively replicated in follow-up studies HLA-DPB1 and CSN3  had the greatest values. Given the expected high variability of HLA genes, only CSN3 (Kappa-Casein) SNPs were further evaluated. Among the ten CSN3 SNPs in the exome data, three were previously unidentified as per dbSNP, each with an allele count (AC) of 5% (i.e. 1 in 20). Notably, two of these three SNPs were classified as non-synonymous annotated using the VcfCodingSnps tool developed at University of Michigan (http://genome.sph.umich.edu/wiki/VcfCodingSnps - accessed 2010 July). Of the remaining seven established SNPs (i.e. in dbSNP), three were not seen in any of our control groups; corresponding ACs of 15%, 5%, and 5%. Of the remaining four SNPs seen in both the exome and control data, two are also non-synonymous SNPs both with AC = 5%; one is intronic with AC = 5%; the last is synonymous with AC = 20%. Overall, these allele counts are similar to the minor allele frequency as seen in the Control groups if present. We are currently having several International Stroke Genetics Consortium (ISGC) collaborators (http://www.strokegenetics.org/ - accessed 2011 July) evaluate their GWAS data with respect to this gene.Gene-based analysis of our 10-case exome data for both sets of genes (‘top’ GWAS lacunar hits and Candidate) as compared to the control populations were then initiated. Of note, we felt a combined analysis (8 African-American and 2 Caucasian) was appropriate given our limited number of exomes and pursuant to the hypothesis that shared risk mechanisms may exist across ethnicities. First, we screened the exome data of our cases to identify both the known (common and rare) and novel (rare) variants in the coding regions of each gene, followed by a screening comparing variant counts and overlap between our data and the control populations as shown in one rare variant, with this number dropping to 48 when the 2 Caucasian cases were also included. Taking this a step further by implementing a “double-hit hypothesis" (i.e. compound heterozygote) to evaluate for two novel variants in the same gene isoform, only 6 genes  satisfied this criterion. Three of the six genes identified are involved in various forms of metabolism , and of these, one was previously associated with diabetes (AQP7). Of the three remaining genes, one is involved in the inflammatory response (HLA-A), one is solely expressed in the CNS (OR4C3), and the function of the last is unknown (FRG2C). CTBP2 had three isoforms identified while ARDS had two. Counting the number of novel mutations (2 or more) per each gene isoform per sample is shown in Pursuing the hypothesis that stroke susceptibility genes may be enriched for novel variants across cases, we screened the exome data for genes in which every case had a least one rare variant. This methodology identified 70 gene isoforms where each African-American case had at least CTPB2 isoforms (NM_001329 and NM_ 001083914) were seen to have a non-sense codon occurring in all 10 samples.Our first impression of these numbers was that they seemed high. Nevertheless, we continued our analyses to further classify these variants by type as demonstrated in CTPB2 isoforms, the same variant in all 10 samples was identified occurring at basepair position 126717592 resulting in T to A substitution  and a LYS to nonsense codon in position 8 of the protein. This CTBP2 region is graphically demonstrated in http://www.broadinstitute.org/igv/ - accessed 2010 July), which displays the read alignments, coverage levels, and SNP calls from multiple samples simultaneously (3 in this case) alongside gene annotations. Pertinent to our CTBP2 data, we see that in the three sample tracks shown the aligned sequence coverage demonstrates the T to A substitution resulting in the nonsense codon as compared to the two bottom tracks showing the annotated RefSeq and codon position. While finding a novel stop codon among our cases would be a highly important finding, we believed that this finding is due to non-specific capture of a non-exome region that has a similar sequence to our exome region of interest, in other words this is a gene-duplication artifact. In explanation, all the cases were heterozygote at this location. Further, the A–C haplotype as seen in http://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi?chooseRs=coding&go=Go&locusId=1488; following links on the rs numbers one finds that “Lee" submitted both rs76555439 and rs78849795 - accessed 2011 January). Further evaluating Exploring these two CSN3 and HLA-DPB1, which appeared to have excess variation in our stroke cases. Further analyses of CSN3 are ongoing; however given the expected high variability of HLA genes, no further analyses of HLA-DPB1 has occurred. None of the previously identified candidate genes studied demonstrated ‘excess’ rare variation in our exome samples. A compound heterozygote analyses pursuant to the hypothesis that stroke susceptibility genes may be enriched for novel variants across cases was also performed, identifying several genes with rare variation across all 10 cases. In particular, CTPB2 appeared to contain a nonsense codon that appeared in all 10 cases, however after further analyses this was identified as a false positive. As a pilot study we identified several important methodological considerations and pitfalls that should be considered when implementing such methods. First, we emphasize the importance of selecting a highly defined phenotype among the study subjects, such as selecting on one stroke subtype, few established vascular risk factors, and positive a family history of stroke. Further selecting the study subjects in the extremes of the phenotypic tail (i.e. young-onset stroke) may also further maximize the genetic contribution to stroke risk. Second, as our results demonstrate, investigators must evaluate for the presence of potential false positive findings, a major pitfall regarding this type of research. Utilizing existing databases for non-diseased control samples may be fraught with difficulties as a control populations ascertained to study a different disease may contain an individual with the disease under study (in this case stroke). When studying rare variation, introducing such an error would be particularly troublesome.Our exome capture, sequencing, and analyses are among the first to examine for the presence and affect of rare variation in ischemic stroke. Given that the optimal methodologies regarding exome analyses are not fully-established in the study of complex disease, including stroke, we developed a variety of analyses to examine our exome data. First, pursuant to our hypothesis that excess rare variation may play an important role in gene-specific stroke risk, we evaluated specific genes chosen based upon our predominant stroke subtype, lacunar stroke, and also by selecting genes based upon prior candidate gene studies. The exomic analyses informed upon by our lacunar stroke GWAS results identified two genes, CSN3), which warrants additional discussion. As described, by screening our prior GWAS results for genes containing coding SNPs in lacunar stroke, and then evaluating the implicated genes in our exome data, we identified CSN3  as containing an interesting coding polymorphism as well as containing excess rare variation. CSN3 maps to chromosome 4 at 4q21.1 and contains 5 exons covering 8.85 kb on the direct strand from position 71108305 to 71117153 . The predicted protein has 182 aa . While the function and tissue expression pattern of this gene is are not fully established, Kappa-casein is known to stabilize micelle formation preventing casein precipitation in human breast milk (http://www.genecards.org/ - accessed 2011 July). Expression studies have demonstrated the presence of kappa-casein in a variety of soft tissue/muscle tissue cancers CSN3 was consistently highly expressed in all arterial segments analyzed.While our gene specific analyses failed to identify excess rare variation in any of the ischemic stroke candidate genes evaluated, our analyses evaluating our exome data in consideration of our prior GWAS results identified one gene, kappa-casein (CSN3 inhibited NF-kappaB activation triggered by tumor necrosis factor (TNF), but not interleukin-1 (IL-1). Moreover, over-expression of CSN3 also inhibited NF-kappaB activation triggered by proteins involved in TNF signaling, including TNF-R1, TRAF2, RIP, and NIK, but not by TRAF6, a protein involved in IL-1 signaling. These data suggest that CSN3 is a specific negative regulator of TNF- but not IL-1-induced NF-kappaB activation pathways.Other studies have indicated that kappa-casein may play a role in immune and inflammatory responses via regulation of the transcription factor nuclear factor kappaB (NF-kappaB) CSN3 was also identified as a novel candidate genes for type 2-diabetes mellitus (T2-DM) in a genome-wide association scan in the Old Order Amish CSN3 SNP rs3775745 was found to be associated with T2-DM in the Amish (p = 0.002), with this result replicating in a Mexican-American population (p = 0.003). Of note, none of our exome study subjects had diabetes.Exome based approaches offer different, yet complimentary, information to ongoing stroke genome wide association study (GWAS) efforts, including ongoing projects implementing substantially larger samples sizes as compared to our GWAS sample. While we are optimistic about the success of GWAS to identify stroke associated variants, we do not believe that GWAS results will account for all stroke risk. This point of view was recently supported by the results of a Cohorts for Heart and Aging Research in Genomic Epidemiology (CHARGE) Consortium GWAS study evaluating ischemic stroke risk in which only 2 intergeneic SNPs were found to achieve genome-wide significance While genome-wide association studies (GWAS) can identify common variants, they are not suited for situations where genetic architecture is such that multiple rare disease-causing variants contribute significantly to disease risk. This is because GWAS chips most often implement common variants as identified through the HapMap project and these variants do not serve as markers for rare variation Our pilot study has several limitations. Most notably the small sample size which was directly attributable to the costs associated with exome sequencing. Further, given that stroke is a prototypical complex disease with numerous risk factors and subtypes, the genetic mechanisms of each stroke (by subtype) included in our study is likely somewhat different. However, as described in the Methods section, we worked to limit the presence of risk factors in our young-stroke onset samples. Our study was also hampered by a lack of standardized analysis programs for exome analysis. At present the analytic methodologies regarding rare-variant analyses are an area of active methodological research http://www.cidr.jhmi.edu/ - accessed 2011 July). Quality metrics include missing genotype call rates by sample and SNP, Hardy-Weinberg analysis, and detailed global and local population substructure analysis using principle components analysis Our study also has several advantages. First, our study population is an ideal sample to evaluate since young age-of-onset is an extreme phenotype that is likely enriched with rare variants; further supported by the fact that numerous familial aggregation studies implicate a greater genetic risk at younger ages. Another strength is that our exome samples were also genotyped on the Illumina 1 M Quad [Omni Quad] as part of our groups' participation in the GENEVA consortium CSN3, identified by screening our prior GWAS results for genes containing coding SNPs in lacunar stroke, was found to both contain an interesting coding polymorphism as well as excess rare variation in our exome data as compared with the other genes evaluated. Additional research will be required to determine if CSN3 variants are truly associated with ischemic stroke risk. Lastly, while rare coding variants may predispose to the risk of ischemic stroke, this fact has yet to be definitively proven. Our study demonstrates the complexities of such research and highlights that while exome data can be obtained, the optimal analytical methods have yet to be determined.In closing, our study evaluated a highly prevalent complex disease for which the genetic architecture remains uncertain. Prior and ongoing studies implementing genome-wide association techniques are only capable of identifying common variants associated with disease. The extent to which ischemic stroke is determined by rare or low frequency variants has not yet been explored. Our study demonstrates the feasibility of utilizing exome based techniques to address this important research gap by implementing several different analyses at the variant and gene levels. Our study also highlights several of the important considerations in this type of research, such as attaining a highly specific phenotype and utilizing an extreme phenotype that is likely enriched with rare variants; in our case young-onset subtype-specific stroke. One gene, Table S1Call rate concordance between overlapping exome and GWAS SNPs.(DOC)Click here for additional data file.


The Riverine Barriers Hypothesis (RBH) posits that tropical rivers can be effective barriers to gene flow, based on observations that range boundaries often coincide with river barriers. Over the last 160 years, the RBH has received attention from various perspectives, with a particular focus on vertebrates in the Amazon Basin. To our knowledge, no molecular assessment of the RBH has been conducted on birds in the Afrotropics, despite its rich avifauna and many Afrotropical bird species being widely distributed across numerous watersheds and basins. Here, we provide the first genetic evidence that an Afrotropical river has served as a barrier for birds and for their lice, based on four understory bird species collected from sites north and south of the Congo River. Our results indicate near-contemporaneous, Pleistocene lineage diversification across the Congo River in these species. Our results further indicate differing levels of genetic variation in bird lice; the extent of this variation appears linked to the life-history of both the host and the louse. Extensive cryptic diversity likely is being harbored in Afrotropical forests, in both understory birds and their lice. Therefore, these forests may not be “museums” of old lineages. Rather, substantial evolutionary diversification may have occurred in Afrotropical forests throughout the Pleistocene, supporting the Pleistocene Forest Refuge Hypothesis. Strong genetic variation in birds and their lice within a small part of the Congo Basin forest indicates that we may have grossly underestimated diversity in the Afrotropics, making these forests home of substantial biodiversity in need of conservation. Alfred Russel Wallace was the first to hypothesize that tropical rivers are effective barriers to animal distributions Wallace . This hyIn contrast to the study of Amazonian taxa, molecular systematic assessments of the RBH for vertebrate taxa in the extensive tropical forests of west and central Africa has been largely limited to studies of primates and rodents distributed across Guineo-Congolean  watersheds and river basins . The Congo River flows 4372 km, 80% of which is between 567 and 293 m in elevation, and ranges up to 15 km in width Runge . It is tAlethe castanea (Fire-crested Alethe), Bleda syndactylus (Red-tailed Bristle-bill), Illadopsis rufipennis , Stiphrornis xanthogaster (Eastern Forest Robin), Neocossyphus poensis (White-tailed Rufous Thrush) and Bleda ugandae (Yellow-eyed Bristle-bill), and four that are forest habitat generalists: Terpsiphone rufiventer (Red-bellied Paradise Flycatcher), Criniger calurus (Red-tailed Greenbul), Myioceyx leconteii (African Dwarf-Kingfisher) and Smithornis rufolateralis (Rufous-sided Broadbill). Our results provide the first genetic evidence that an Afrotropical river has served as a barrier to gene flow for four understory specialist bird species and their lice based on samples collected from sites north and south of the Congo River.Here, we report results from a taxonomically diverse suite of bird species, six of which are forest understory specialists: Bleda syndactyla and Stiphrornis xanthogaster, we also included preserved tissues samples collected by the Field Museum of Natural History (FMNH) from areas north and well to the east of the Congo River  parasitizing Alethe castanea, Myrsidea sp. parasitizing Terpisphone rufiventer and Sturnidoecus sp. (Phthiraptera: Ischnocera) parasitizing Terpsiphone rufiventer.Birds were prepared in the field as standard museum skins, with associated tissue and blood samples. Prior to preparation, birds were exposed to ethyl acetate and brushed for louse ectoparasites. Birds were identified to species and lice to genus using morphology and both have been catalogued into the Texas Cooperative Wildlife Collection at Texas A&M University. Of the specimens brushed for ectoparasites, there were only three instances where lice were collected from individuals of the same bird species on opposite sides of the Congo River: Our strategy was to assess whether haplotypes on opposite sides of a river(s) were reciprocally monophyletic with respect to one another and whether the pattern (if any) across species was temporally similar, indicating a shared evolutionary history with respect to divergence time.b (cyt-b) gene for all avian samples; we also amplified the nuclear βact3 gene for Bleda syndactyla and the nuclear β-fibrinogen intron-5 gene (TGFβ2) for Stiphrornis xanthogaster. We amplified the cytochrome oxidase I (COI) gene for lice. PCR amplifications used published primers and protocols  have been deposited on GenBank under accession numbers KC349953-KC349959 (lice) and KC355099-KC355178 (birds).For birds and lice, whole genomic DNA was extracted from tissue using the DNeasy tissue extraction kit ; louse specific protocols were used for lice . We used MrModelTest . Sequence data for each understory species were analyzed using MRBAYES . We therefore performed analyses enforcing a strict clock as well as a relaxed, uncorrelated lognormal clock for the substitution rate for comparison. We also varied the speciation prior, using a Yule process as well as a speciation birth-death process speciation prior. Relative evolutionary rates were estimated among the three gene trees  by setting the prior of the rate for Alethe at 1.0. For each analysis, two separate MCMC runs were run for at least 60 million generations with parameters sampled every 1000 steps, and incorporating a 10% burn-in. Combining of MCMC runs, and assessment of parameters and trees were performed as above.*BEAST  using the hierarchical approximate Bayesian computation acceptance/rejection algorithm.MsBayes  and some of which are not  understory specialists, we found no evidence of across river genetic diversification; we did not analyze these species further. We found no variation in either the nuclear βact3 gene across Bleda syndactyla samples, or nuclear TGFβ2 gene for Stiphrornis xanthogaster.We generated mitochondrial sequence data for 10 species of birds based on our field sampling across the Congo River and its tributaries , Table 1Alethe castanea, Bleda syndactlya, Illadopsis rufipennis, and Stiphrornis xanthogaster), Bayesian analysis resulted in two highly supported clades. These clades consist almost entirely of haplotypes from birds collected from north of the Congo River versus haplotypes of birds collected from south of the river  or Pleistocene origins (other lineages). With the exception of southern bank Bleda syndactyla haplotypes, northern and southern clades across lineages have generally similar TMRCA (Estimates of time to most recent common ancestor (TMRCA) in each avian lineage , Table 2ar TMRCA , Table 2Bleda syndactyla being about twice the other species , consistent with the older estimated age for this species  parasitizing Alethe castanea individuals from north and south of the Congo River were 10% divergent in their DNA, whereas their hosts were just 2.2% divergent (uncorrected p-distances). We also found across-Congo divergences of 4% in a Myrsidea species taken from another bird species (Terpsiphone rufiventer). These across-river divergences contrasts with a lack of genetic divergence in another louse species , also parasitizing T. rufiventer. Notably, T. rufiventer occupies intermediate levels of a variety of forest types, and shows no genetic divergence across the Congo.With respect to lice, Bleda syndactyla. Nor did we find variation in the nuclear TGFβ2 gene for Stiphrornis xanthogaster; variation in this gene appears useful only at the interspecific level, where it supports a sister-relationship between xanthogaster and sanghensis, relative to other Stiphornis species . The divergence in mitochondrial data is in the realm of 2Ne, suggesting that no nuclear gene variation should be expected  showed no genetic variation across the Congo River. In addition to shared habitat, all understory specialists we surveyed are species that follow and prey on army ant swarms  feed on host body fluids (blood), whereas ischnoceran lice  feed on feathers. The feeding behavior of amblycerans increases exposure to the host's immune system  recently found to harbor extensive geographically structured genetic diversity across Afrotropical forests (Marks From our results, it is evident that potentially extensive cryptic diversity is being harbored in Afrotropical forests. With respect to birds, these results run counter to the assertion of Fjeldså and Lovett (Fjeldså and Lovett ts Marks . That eats Marks . And whits Marks .Our analyses provide the first intraspecific molecular assessments of the Riverine Barrier Hypothesis for birds and their lice distributed in the Afrotropics. Because our results are based on data from a very small area of the Congo Basin, extensive cryptic diversity is likely being harbored in Afrotropical forests. More broadly, the possibility of high levels of cryptic diversity in the Afrotropics is important when considering that macroecological studies use distributional information in range maps (Hansen et al.


Talon cusp is a dental anomaly also known as an eagle’s talon. It is an extra cusp on an anterior tooth which arises as a result of evagination on the surface of a crown before calcification has occurred. The exact etiology is unknown. The inci-dence of talon cusp is less than 6%. Commonly involved teeth are maxillary incisors, usually unilateral but in some instanc-es bilateral. The classical radiographical feature of talon cusp is double teeth appearance. The anomaly has been reported to be unusual in the mandibular dentition. This article reports an unusual case of talon cusp of permanent mandibular central incisors. The majority of reports about talon cusp show that the permanent dentition has been involved three times more often than the primary dentition. The anomaly has been reported to be unusual in the mandible.A 14-year-old boy presented the Department of Oral Medicine and Radiology, Dental Hospital, Mangalore, India, with the complaint of mal-aligned lower anterior teeth. Patient’s medical history was non-contributory, and no abnormalities were noted on general examination. On intra-oral examination, two talon cusps were noted on the lingual surfaces of both mandibular central incisors. An over-retained mandibular primary left central incisor was also observed . On radi3 This abnormality is probably induced by trauma or other localized insults affecting the tooth germ.5 Genetic factors have also been suggested by some authors.7 Population-based phylogenic and genetic studies involving dental variations such as cusp of carabelli and talon cusp has been carried out recently in Hungary.8 It appears to be more prevalent in patients with Rubinstein-Taybi syndrome, Mohr syndrome , Sturge-Weber syndrome  or incontinentia pigmenti achromians.10 Our case was not associated with any known abnormal systemic developmental syndromes.Talon cusp affects both sexes but males have a higher incidence than females. Most of the cases are unilateral, but one fifth of the cases are bilateral. Studies have revealed that maxillary incisors are most commonly affected and mandibular incisors are least commonly affected.11Clinical problems noted with talon cusp cases include attrition, breast-feeding problems, compromised esthetics, occlusal interference, accidental cusp fracture, interference with tongue space, temporomandibular joint pain, displacement of the affected tooth, irritation of tongue during speech and mastication, periodontal problems because of excessive occlusal force, misinterpretation of radiographs of teeth with talon cusp before eruption and caries susceptibility because of developmental grooves on the talon.13Early diagnosis and definitive treatment is important for talon cusp. Caries in the deep developmental grooves on the lateral aspect of the cusp should be removed and the cavity filled with glass ionomer restorative material. Non-carious grooves are cleaned with slurry of pumice, acid etched and sealed with fissure sealant. If talon cusp causes premature contact and occlusal interference, the cusp should be reduced gradually on consecutive visits over 6-8 week intervals to allow time for deposition of reparative dentin for pulpal protection. After each grinding procedure, the tooth surface should be covered with a desensitizing agent. Conservative techniques such as complete reduction of the cusp followed by calcium hydroxide pulpotomy for an immature tooth or root-canal therapy have also been carried out.Talon cusp although unusually seen, needs to be diagnosed and kept under observation to prevent further complications. Our report highlights this unusual anomaly occurring in an unusual site.


High-throughput genomic technologies enable researchers to identify genes that are co-regulated with respect to specific experimental conditions. Numerous statistical approaches have been developed to identify differentially expressed genes. Because each approach can produce distinct gene sets, it is difficult for biologists to determine which statistical approach yields biologically relevant gene sets and is appropriate for their study. To address this issue, we implemented Latent Semantic Indexing (LSI) to determine the functional coherence of gene sets. An LSI model was built using over 1 million Medline abstracts for over 20,000 mouse and human genes annotated in Entrez Gene. The gene-to-gene LSI-derived similarities were used to calculate a literature cohesion p-value (LPv) for a given gene set using a Fisher's exact test. We tested this method against genes in more than 6,000 functional pathways annotated in Gene Ontology (GO) and found that approximately 75% of gene sets in GO biological process category and 90% of the gene sets in GO molecular function and cellular component categories were functionally cohesive (LPv<0.05). These results indicate that the LPv methodology is both robust and accurate. Application of this method to previously published microarray datasets demonstrated that LPv can be helpful in selecting the appropriate feature extraction methods. To enable real-time calculation of LPv for mouse or human gene sets, we developed a web tool called Gene-set Cohesion Analysis Tool (GCAT). GCAT can complement other gene set enrichment approaches by determining the overall functional cohesion of data sets, taking into account both explicit and implicit gene interactions reported in the biomedical literature.http://binf1.memphis.edu/gcatGCAT is freely available at  Microarray technologies are routinely used to examine gene expression profiles under different experimental conditions. However, statistical analysis of microarray experiments still remains challenging, due in part to sensitivity  of the method as well as technical, biological and multiple testing confounds. A large amount of effort has focused on developing mathematical models to normalize and identify differentially expressed genes To incorporate biological information into algorithms for identification of meaningful gene sets, most of the existing methods utilize functional category enrichment analysis based on Gene Ontology (GO) In recent years, a growing number of studies have focused on estimating the literature-based functional coherence of gene groups. In 2002, Raychaudhuri Previously, we developed a method which utilized Latent Semantic Indexing (LSI), a variant of the vector space model of information retrieval, to determine the conceptual relationships between genes from information in MEDLINE titles and abstracts ftp://ftp.ncbi.nlm.nih.gov/gene/DATA). PubMed IDs (PMIDs) corresponding to homologous genes were combined. Orthology data were downloaded from Mouse Genome Informatics site. To reduce false positives, PMIDs referring to more than 10 genes were removed as these citations usually described genomic experiments and contained no functional information. After filtering, there were 17,451 human and 21,903 mouse genes in our gene document collection . We focused on the GO categories that contained 5 to 1000 genes, which resulted in a total of 6681 GO categories across the human and mouse collections.The Gene Ontology data file (version 5.7.2) was downloaded from http://rpy.sourceforge.net/) were utilized to create the web front end and back end. Cytoscape (http://www.cytoscape.org/) was used to create and visualize the gene network graphs. The Scriptaculous (http://script.aculo.us/) and prototype.js javascript libraries were used to create limited visual effects within the web browser. The data for individual genes was obtained from the NCBI ftp site.The GCAT software was implemented using a Dell X-series server with quad-core 1.6 GHz Pentium Xeon processors and 8 GB of RAM. Python, MySQL, R and rpy  procedure is shown in The GO database contains greater than 22,000 functional classifications (GO terms) in three major categories: biological process, cellular component and molecular function. Genes are assigned to GO terms by human experts based on published experimental results. We assumed that genes assigned to each GO term share similar functions and treated them as gold standard to evaluate the robustness of the LPv methodology. We calculated LPv for 6,681 mouse and human GO terms that contained between 5 and 1,000 genes . The aveExamination of the LPv distributions of the three main GO categories  revealed−7, 7.1×10−8 and 0.282 for development, AD and cancer, respectively.To investigate the sensitivity of LPv methodology, we examined 50 manually selected genes related to three functional categories: development, Alzheimer disease (AD) and cancer The genes in both the development and Alzheimer disease groups were highly connected, whereas the genes in the cancer group were mostly disconnected, causing the LPv of the cancer gene set to be insignificant. The average number of abstracts associated with each group was 229, 243, and 439 for development, AD and Cancer, respectively. These results indicate that the LPv method can tolerate bias created by well-studied genes in even small subsets of genes, since inclusion of Trp53 (and others) in the cancer field still produced a significant LPv for the 50-gene set.http://binf1.memphis.edu/gcat). Users need to simply select the organism (human or mouse), the microarray platform (currently limited to Affymetrix), and input the list of genes by typing or pasting either gene symbols or Entrez Gene IDs. GCAT displays the LPv along with a network graph, where genes are represented as nodes and the edges represent cosine values >0.6. GCAT also provides the LSI similarity scores, which can be downloaded in tabular form, between all genes in the input set. In addition, a gene annotation table is provided, in which the gene IDs are hyperlinked to the Entrez Gene website and the abstract number associated with each gene is hyperlinked to PubMed website. Besides, GCAT displays the common log-entropy terms derived from the gene abstracts which can provide insights into biological functions of the gene set.A major goal of this study was to produce an automated and robust method to evaluate the functional coherence of (or literature support for) gene lists derived from genomic studies. Thus, we developed a web tool called Gene-set Cohesion Analysis Tool (GCAT) to enable calculation of LPv on the fly  can be very high for a number of reasons, including those associated with multiple hypothesis testing. To reduce false positives, biologist often select differentially expressed genes (DEGs) using a combination of arbitrary criteria such as the lowest expression p-value, highest magnitude of change, and/or a fold change cut off of 2. However, these selection criteria may not necessarily be biologically relevant and could result in loss of true positives. We postulate that by using a literature-based cohesion analysis approach, biologist should be able to have an objective criterion to evaluate various statistical feature selection methods and be able to arrive at a biologically relevant gene set more effectively.−32, suggesting that they are highly functionally related in the literature (−44). Moreover, genes that were changed greater than 2-fold and had an expression p-value<0.05 produced the smallest LPv (1.4×10−63). In contrast, genes that were changed greater than 2-fold and had an expression p-value>0.05 or genes that were changed less than 2-fold and had an expression p-value<0.05 did not show significant literature cohesion. These results demonstrate the utility of using an objective criteria based on functional information in the literature to fine tune statistical analysis of high-throughput experiments.To test the utility of GCAT for microarray analysis, we used a previously published microarray data set of interferon stimulated gene expression in mouse embryonic fibroblasts terature . Genes tNext, we explored whether GCAT could be used to compare the results of different feature extraction methods. We applied our method to a previously published acute lymphoblastic leukaemia (ALL) dataset including four different groups In a previous study, we developed a novel method to determine the conceptual relationships between genes directly from Medline abstracts using LSI In the present study, LSI-derived gene-gene similarities were used to determine a literature cohesion p-value (LPv) for gene sets. This work is significant because it provides a robust method for researchers to objectively evaluate the literature support for any given gene set derived from high-throughput technologies. The method can assist researchers in choosing appropriate feature selection methodologies or prioritizing multiple gene lists generated by different algorithms.The accuracy and sensitivity of LPv was evaluated using the gene sets associated with GO terms and a human-curated dataset. We found that most of the gene sets had statistically significant literature cohesion, indicating that our method correctly identifies functional coherence of gene sets. Greater than 90% of the gene sets in the molecular function and cellular component categories had significant LPv. In contrast, only 75% of groups in the biological process category were significant. These results are very similar to those obtained by Raychaudhuri et al Although the presence of well-studied genes can negatively impact LPv calculation, the method appears to be quite robust. The LPv of a human-curated set of 50 genes, which included well-studied genes such as Apoe, App, and Trp53 was statistically significant (LPv 0.007), whereas a subset of 31 cancer related genes had LPv of 0.282. This result suggests that cancer is generally well-studied and that genes involved in cancer are associated with many pathways and processes. Therefore, some caution is necessary when interpreting LPv for gene sets which contain a disproportionate amount of well-studied genes. To provide some feedback to the users of GCAT, we display the number of abstracts for each gene and provide a hyperlink to PubMed.A number of other groups have developed methods to determine literature-based functional coherence of gene/protein sets Microarray experiments are complex and subject to technical and biological variability that hinder inference analysis. Consequently, different statistical procedures must be carefully considered for each microarray experiment. Numerous statistical methods are available for identifying differentially expressed genes To our knowledge, one other web tool besides GCAT is available for estimation of statistical significance of protein-protein networks, called Studying Networks in the Omic World (SNOW) Another program, called Gene Set Enrichment Analysis (GSEA), determines enrichment p-values for the biological or functional categories cataloged in various databases −32) in the small gene set. The availability of GCAT will provide an objective measure to evaluate the appropriateness of various FDR adjustment methods. Ultimately, it would be very useful to incorporate LSI-derived literature similarities, perhaps as priors in a Bayesian model, to adjust the FDR for microarray datasets. Taken together, GCAT provides a unique tool for genomic researchers to objectively determine the most appropriate analytical approaches for their particular experimental system.Perhaps the biggest statistical challenge that faces microarray studies is the propensity for a high FDR previously defined as false discovery rate caused by multiple hypothesis testing. Current FDR adjustment strategies are dependent on the magnitude of differentially expressed genes, gene expression variability, and sample size Figure S1Venn Diagram of differentially expressed genes (DEGs) generated by three different statistical tests on four different microarray datasets: ALL1 (A), ALL2 (B), ALL3 (C) and ALL4 (D).(TIF)Click here for additional data file.


A skewed assemblage of two epi-, meso- and bathypelagic fish families makes up the order Myctophiformes – the blackchins Neoscopelidae and the lanternfishes Myctophidae. The six rare neoscopelids show few morphological specializations whereas the divergent myctophids have evolved into about 250 species, of which many show massive abundances and wide distributions. In fact, Myctophidae is by far the most abundant fish family in the world, with plausible estimates of more than half of the oceans combined fish biomass. Myctophids possess a unique communication system of species-specific photophore patterns and traditional intrafamilial classification has been established to reflect arrangements of photophores. Myctophids present the most diverse array of larval body forms found in fishes although this attribute has both corroborated and confounded phylogenetic hypotheses based on adult morphology. No molecular phylogeny is available for Myctophiformes, despite their importance within all ocean trophic cycles, open-ocean speciation and as an important part of neoteleost divergence. This study attempts to resolve major myctophiform phylogenies from both mitogenomic sequences and corroborating evidence in the form of unique mitochondrial gene order rearrangements.Solivomer arenidens positioned as a sister taxon to the remaining neoscopelids. The enigmatic Notolychnus valdiviae is placed as a sister taxon to all other myctophids and exhibits an unusual second copy of the tRNA-Met gene – a gene order rearrangement reminiscent of that found in the tribe Diaphini although our analyses show it to be independently derived. Most tribes are resolved in accordance with adult morphology although Gonichthyini is found within a subclade of the tribe Myctophini consisting of ctenoid scaled species. Mitogenomic sequence data from this study recognize 10 reciprocally monophyletic lineages within Myctophidae, with five of these clades delimited from additional rearranged gene orders or intergenic non-coding sequences.Mitogenomic evidence from DNA sequences and unique gene orders are highly congruent concerning phylogenetic resolution on several myctophiform classification levels, corroborating evidence from osteology, larval ontogeny and photophore patterns, although the lack of larval morphological characters within the subfamily Lampanyctinae stands out. Neoscopelidae is resolved as the sister family to myctophids with Mitogenomic results from DNA sequences and unique gene orders corroborate morphology in phylogeny reconstruction and provide a likely scenario for the phylogenetic history of Myctophiformes. The extent of gene order rearrangements found within the mitochondrial genomes of myctophids is unique for phylogenetic purposes. Lanternfishes (Myctophidae) and blackchins (Neoscopelidae) comprise the only two families in the order Myctophiformes, superorder Scopelomorpha , a groupVibrio were found by Foran  (Table Genomic DNA was extracted using the Qiagen Puregene extraction kit following the manufacturer´s protocol and used directly for long and accurate amplification PCR (LA PCR) of the entire mitochondrial genome . DependiUUR in addition to alignment using previously determined myctophid RNA genes [Raw sequence data were processed and formatted with the software 4peaks Ver. 1.7.2 , TextwraNA genes .ARTn). Missing nucleotides, e.g. tRNA-Thr and Pro genes for some species, were coded as missing characters. Levels of mutational saturation were calculated from p-distances for 1st, 2nd and 3rd codon positions using PAUP* Ver. 4.0b10 [nRTn).Alignment of the 12 protein coding genes was done by eye whereas the 22 tRNA genes and 12S+16S rRNA genes were aligned with a probabilistic multiple alignment approach using ProAlign Ver. 0.5 . The ND6. 4.0b10   and the two tRNA-Met genes observed in Notolychnus. Pairwise sequence variation of the pseudogenes and detected tRNA-Met genes in Diaphini were compared in order to validate the INC-regions as actual tRNA-Met pseudogenes, the latter expected to show higher substitution rates.All 41 myctophiform mitogenomes were scrutinized for mt features such as rearranged gene orders and INC-regions. All INC-sequences were analyzed for sequence similarity in relation to possible duplicated regions in the mitochondrial genome by using NCBI Blast , tRNAscap-values.Topology comparison from morphological phylogenies and this study were tested by using the approximately unbiased log-likelihood test  implemenThe organization of the mitogenome is unique in all fishes of the family Myctophidae, showing rearranged gene orders for either the whole family, tribal or subtribal clades, whereas Neoscopelidae show the canonical vertebrate gene order. All these features are summarized in Figure L-region was much longer than the usual vertebrate sequence in this region of about 30–40 base pairs (56–321 in myctophids included in this study). We were not able to find any conserved sequence blocks or secondary cloverleaf structure typical of tRNA genes [1. WANYC-gene order found in all myctophids. The tRNA-Cys and Tyr have shifted positions and the putative ONA genes  for thisψ gene order found in the tribe Diaphini. The tRNA-Gln and Met have shifted positions with the typical L- and H-strand coding intact (tRNA-Gln is coded on the L-strand and tRNA-Met coded on the H-strand). An INC-region (tRNA-Met pseudogene denoted as Mψ) is present between tRNA-Gln and ND1 in all diaphinid taxa included. A similar gene order was also observed for Notolychnus valdiviae although the INC-region is a tRNA-Met gene for this taxon (IMQM gene order). This particular gene rearrangement was analyzed and is discussed in detail below.2. IMQMLampanyctus, Nannobrachium, Stenobrachius and Triphoturus all show tRNA-Glu pseudogenes in the canonical position between ND6 and Cytb, verified from sequence comparisons and partial secondary cloverleaf structures  showed differences related to lampanyctine tribal relationships  and Notolychnus valdiviae was recovered as the sister taxon to the remaining myctophids (high support). Myctophine and lampanyctine subfamilies were also recovered as monophyletic in all analyses. The tribe Diaphini was recovered as the sister lineage to the other lampanyctines, found in all analyses although with low support values, despite the unresolved tribal relationships within the remaining lampanyctines. The tribe Electronini was recovered as the sister lineage to the other myctophines with maximum levels of support and a clade consisting of Benthosema spp. and Diogenichthys atlanticus was resolved as the sister lineage to the tribes Gonichthyini and Myctophini. Ten myctophid lineages are recognized from this study and are noted according to current tribal classification as A-J in Figures Neoscopelidae and Myctophidae were both recovered as monophyletic with ψ gene order and putative tRNA-Met duplication in Diaphini, an interesting finding is observed in the monotypic basal branching taxon Notolychnus valdiviae, exhibiting two identified tRNA-Met genes resulting in an IMQM gene order. The two tRNA-Met genes both show the CAU-anticodon although they are highly divergent with respect to sequence similarity (~20% p-distance). Neigbour-joining distance method analyses of the tRNA-Met genes and their pseudogene counterparts showed clear phylogenetic structure in the diaphinid pseudogenes  in the analyses, in order to examine a possible connection to Diaphini (IMQMψ gene order) concerning this gene-order rearrangement, showed no phylogenetic relationship between these taxa  and the topology with Notolychnini constrained within Diaphini as indicated from the tRNA-Met duplication event was clearly not rejected (P = 0.407).Comparing constrained topologies according to morphology with the best scoring ML-tree from this study rejected a Notolychnini-Lampanyctinae relationship  in the canonical position of the putative origin of L-strand replication was found within all myctophids  resolved as sister taxon to the remaining family Myctophidae from mitogenome sequences. Paxton [Notolychnus as sister taxon to the subfamily Lampanyctinae  as sister group of the very speciose Diaphus (78 species) [Diaphus have been suggested from the presence of Dn-Vn, Ant, Suo and So photophores  was well supported from both sequences and a unique IMQ-gene order Figure , all in species) -91. Fourby Bolin  and mosttwo SAOs .Lampanyctodes hectoris is not included in this study, a myctophid showing ambiguous morphological characters concerning tribal placement. Paxton [Lampanyctodes as a possible early divergence based on several characters shared with the most plesiomorphic diaphinid genus Lobianchia. Conversely, Ahlstrom et al. [Lampanyctodes as being specialized within Gymnoscopelini from a similar larval form and development to that found within Lampadena, arguing that characters reflect habitat instead of true phylogeny.Gymnoscopelini (clade C) sensu Paxton  was foun. Paxton  noted Lam et al.  noted LaCeratoscopelus and Bolinichthys formed a clade (D) as either sister group to the remaining lampanyctines or included within them  comprising  Figures . In Nann Parvilux as well. Parvilux, and seeLampadena and Taaningichthys (F) is found monophyletic and completely congruent with morphology. Taaningichthys was previously a part of the genus Lampadena[Lampadena chavesi, L. dea and L. speculigera were more closely related to Taaningichthys than the remaining species of Lampadena based on expanded neural arches on the anterior vertebrae as found in Taaningichthys. In fact, Paxton [Lampadena and especially Taaningichthys are among the largest and deepest living myctophids, with the latter showing reduction of the lateral line and in the number of body photophores. This apparent reductional trend has resulted in one taxon, Taaningichthys paurolychnus, having lost all body photophores although retained the supra- and infracaudal glands. Lampadena yaquinae is found nested within the Lampadena species included and corroborates Paxton [Dorsadena[Lampadena. Likewise, the subgenera Lampadena and Lychnophora proposed by Fraser-Brunner [4[A clade consisting of Lampadena althoughLampadena based onLampadena noted th, Paxton  noted ths Paxton  in synonDorsadena with Lam-Brunner , supportrunner [4, are notrunner [4.The tribe Electronini (G), erected from low-leveled PVO and PLO photophores , composeBenthosema and Diogenichthys (clade H) were peculiar and the IQM-region and WANYC-region of both Electronini and Benthosema-Diogenichthys were highly differentiated compared to all other myctophids. Benthosema glaciale showed new positions of tRNA-Gln and -Met to the downstream region of the WANYC-region  included in this study renders Myctophini paraphyletic. We note that broader taxonomic representation of the genera Gonichthys, Centrobranchus, Tarletonbeania and Loweina is necessary concerning phylogenetic position of Gonichthyini within Myctophinae. However, evidence from mt sequences and a unique gene order in part of Myctophini  showed this particular gene arrangement whereas M. asperum and M. orientale showed the typical myctophid gene order also found in Centrobranchus choerocephalus and Symbolophorus californiensis, the latter four forming a well supported clade J  [Bathygadus antrodes, possessed all of the gadiform rearrangements although somewhat differently arranged from other subclades. This indicates early rearrangement events subsequently modified into presently observed synapomorphic gene orders for specific gadiform clades. The most basal branching in Myctophidae, Notolychnus, shows mt rearrangements such as two tRNA-Met genes and a comparatively large INC-sequence between ATP6 and CO3. These features are either modified or lost in other myctophids. On the other hand, several rearranged gene orders found in various myctophid tribes are not traceable in Notolychnus. Gene order rearrangements within Myctophidae provide a unique example of the phylogenetic utility of mt gene orders and the problems associated with this group of markers.Observations of unique gene rearrangements in Myctophidae, combined with their utility as clade defining synapomorphies, are quite numerous compared to similar findings within other vertebrate groups . It shouiformes)  and in viformes) . Birds (iformes) , althougiformes) . Plethodiformes) . The latiformes)  postulatiformes)  and mantiformes) , with thiformes)  and acroiformes) . As mentiformes) . The latiformes) , who fouiformes)  initiateMitogenomes from myctophiform fishes provide a unique empirical case of mt DNA sequences and gene order rearrangements that corroborate evolutionary history. Future mitogenomic determination of the 200+ remaining species within Myctophidae will most certaninly increase our knowledge concerning issues associated with gene order rearrangement events and their utility as phylogenetic markers. Multiple different gene orders found throughout the mt genome, utility as phylogenetic markers and the unknown causes and mechanisms associated with gene duplication and subsequent retention and/or removal of sequences, calls for the determination of complete mt genomes over single genes. Our results support the use of osteology and photophores in phylogeny of Myctophiformes, the latter being a very unique feature of any deep-sea group of organisms. Larval ontogeny is also informative albeit difficult to use on higher phylogenetic levels and particularly within the subfamily Lampanyctinae.The authors declare that they have no competing interests.All authors contributed in preparation of the study. JYP performed molecular lab work, initial analyses and drafted the original manuscript. DR, HT and TPS continually reviewed and discussed lab-procedures. JYP and EW finalized the analyses. JYP, IB, EW and MM finalized the manuscript. All authors read and approved the final manuscript.Table T2. Partitioning schemes used in the present study suggested by PartitionFinder.Click here for fileSaturation plots of pairwise distances of 1st, 2nd and 3rd codon positions in the 12 protein coding genes employed in the analyses. Vertical axis shows p-distance for each codon position against total p-distance . Saturation from RY-coding of the 3rd codon positions included  although not used in the present study from early onset of saturation.Click here for file Secondary structure of INC-regions. Sequence alignments and secondary tRNA structures of INC-regions are shown for synapomorphic spacers pertaining to gene order rearrangements 1, 3, 5 and 8 presented in Figure Click here for file Base pair details of INC-regions found in Myctophidae. Eight mt regions correspond to gene order and INC-region synapomorphies presented in Figure Click here for fileIMQMψ gene order in a clade of scarid parrotfishes. Taxa constituting a monophyletic clade in Scaridae all show similar patterns in the IMQ-region as observed within Diaphini. Grey areas show INC-regions with number of base pairs noted for each taxon and the arrow indicates possible gradual removal of INC-regions.Click here for file


The primary cilium is a microtubule-based organelle that projects from the surface of most vertebrate cells and plays a crucial role in a number of signaling processes. Even though an increasing number of cilia-associated proteins have been identified in the past decade, the molecular mechanisms controlling primary cilia formation remain poorly understood. Here, we performed a RNA interference (RNAi) based screen using telomerase-immortalized human epithelial retina (RPE1) cells to search for kinases and kinase-associated proteins with a role in ciliogenesis. In this cell line, ciliogenesis can be induced upon serum withdrawal. In order to identify both, positive and negative regulators of ciliogenesis, we performed the RNAi screen under reduced serum conditions instead of stringent starvation. This allowed us to detect not only proteins that reduced ciliogenesis upon depletion, but also those proteins that increased the number of ciliated cells upon depletion. To screen in high-throughput, we developed an automated microscope based readout using acetylated tubulin as a cilia marker. Our primary screen identified 103 potential regulators of ciliogenesis. To validate these, we used for a secondary screen independent siRNAs and confirmed 47 positive regulators and 4 negative regulators. The confirmed hits were associated with various biological processes as intracellular signaling, cell cycle control or cytoskeleton organization. Here, we will present the in-depth characterization of the microtubule-affinity regulating kinase 4 (MARK4), which has been identified as a novel positive regulator of primary cilia assembly.


We report a case with Non-Hodgkin Lymphoma with a focus of intense hypermetabolism in the sellar region in the primary staging and posttreatment whole body F-18 FDG PET. Further evaluation with magnetic resonance imaging after posttreatment FDG PET revealed a pituitary adenoma. Endocrinologic workup was normal consistent with nonfunctioning pituitary adenoma and endocrinologists decided to follow up the patient by yearly magnetic resonance imaging. This case demonstrates a nonfunctioning pituitary adenoma by whole body FDG PET and emphasizes the importance of pursuing incidental findings detected in the sella on PET imaging.Conflict of interest:None declared. Pituitary adenoma is a relatively common tumor, originating from the pituitary gland . It can A 49 year old man who presented with a mass located in the left axillary region enlarging during the last seven months. The patient underwent tru-cut biopsy and histopathological examination was consistent with non-Hodgkin’s lymphoma . A whole body FDG PET was performed for staging which revealed widespread malignant lymphadenopathy and involvement of the bone-marrow . Also thPituitary adenoma is a relatively common tumor, originating from the pituitary gland . PituitaFDG PET/CT is used in the evaluation of most types of malignancy, which has resulted in significant clinical impact on patient management. FDG PET/CT is more accurate than CT or other conventional imaging modalities for the diagnosis of previously unknown, recurrent or metastatic cancer foci, based in part on its whole-body imaging capabilities. PET scanning is based on the principle that metabolically active cells such as those in tumors are more likely than normal cells to take up the labeled glucose analogue, FDG, to become detectable. However, several benign or non-neoplastic conditions avidly accumulate FDG, causing ambiguity in interpretation of results. (3) Incidental foci of abnormal FDG uptake detected during whole-body PET/CT examinations may represent previously unknown malignant sites, physiological variants or benign lesions unrelated to cancer.Pituitary glands do not normally accumulate FDG and are not visualized on FDG PET imaging ,3,4. TheWe describe a patient with NHL who was noted to have a focus of intense hypermetabolism in the sellar region in the primary staging and posttreatment whole body FDG PET which might be due to involvement of pituitary gland with lymphoma or a benign pituitary adenoma. Campeau et al. reported on a patient with mucosa-associated lymphoid tissue lymphoma who had a non-functioning pituitary adenoma with FDG avidity . In our In conclusion incidental pituitary uptake on FDG PET is a very rare finding. Most cases of incidental pituitary FDG uptake were diagnosed as clinically non-functioning adenomas, and there were a few functioning adenomas. Further evaluations, including hormone assays and pituitary MRI are warranted when pituitary uptake is found on FDG PET/CT and it is strongly due to benign etiology when persistent pituitary uptake seen on both pre and posttreatment PET scans.


Cancer cells are characterized by massive dysegulation of physiological cell functions with considerable disruption of transcriptional regulation. Genome-wide transcriptome profiling can be utilized for early detection and molecular classification of cancers. Accurate discrimination of functionally different tumor types may help to guide selection of targeted therapy in translational research. Concise grouping of tumor types in cancer maps according to their molecular profile may further be helpful for the development of new therapeutic modalities or open new avenues for already established therapies.Complete available human tumor data of the Stanford Microarray Database was downloaded and filtered for relevance, adequacy and reliability. A total of 649 tumor samples from more than 1400 experiments and 58 different tissues were analyzed. Next, a method to score deregulation of KEGG pathway maps in different tumor entities was established, which was then used to convert hundreds of gene expression profiles into corresponding tumor-specific pathway activity profiles. Based on the latter, we defined a measure for functional similarity between tumor entities, which yielded to phylogeny of tumors.We provide a comprehensive, easy-to-interpret functional cancer map that characterizes tumor types with respect to their biological and functional behavior. Consistently, multiple pathways commonly associated with tumor progression were revealed as common features in the majority of the tumors. However, several pathways previously not linked to carcinogenesis were identified in multiple cancers suggesting an essential role of these pathways in cancer biology. Among these pathways were 'ECM-receptor interaction', 'Complement and Coagulation cascades', and 'PPAR signaling pathway'.The functional cancer map provides a systematic view on molecular similarities across different cancers by comparing tumors on the level of pathway activity. This work resulted in identification of novel superimposed functional pathways potentially linked to cancer biology. Therefore, our work may serve as a starting point for rationalizing combination of tumor therapeutics as well as for expanding the application of well-established targeted tumor therapies. Cancer is one of the leading causes of death . If segrHere, we employed an extensive functional genomics approach to analyze gene expression profiles of the complete human cancer related microarray datasets from the Stanford Microarray Database . Our aimGene expression data were obtained from microarray experiments, downloaded from the Stanford Microarray Database . IgnorinInitially the raw data were loaded into R, invalid cDNA probes removed (e.g. flag 'contaminated' or 'failed') and a background correction was applied. After the calculation of the log2 fold changes relative to the control RNA the quantile normalization was applied to each experiment. In all these initial steps we made use of the 'limma' R package . Next, wA gene was considered upregulated (respectively downregulated) if it had a M-value > 1.0 (resp. < -1.0) and a p-value smaller than 0.01 in a one sided one-sample t-test. This procedure defined between 391 and 1433 up- and between 424 and 2150 downregulated tumor associated genes figure . Note: Aftp://ftp.genome.jp/pub/. The KEGG pathway data were parsed by a Perl [Metabolic reaction and signaling event information were acquired from the PATHWAY database of the Kyoto Encyclopedia of Genes and Genomes (KEGG) . These py a Perl  script uFor each tumor entity - KEGG pathway pair, we tested whether the set of genes that are differentially expressed in the given tumor entity is enriched in the set of genes belonging to the given KEGG pathway. Testing was performed using an one-sided exact Fisher test. This resulted in a 28 tumor entities × 208 KEGG pathways matrix containing the p-values of each test.To get a better overview, we implemented a Python  script, Using the web-based tool PubMatrix  for liteIn a second step, we used the binarized functional cancer map (1 respectively 0 representing significant resp. non-significant entries) to calculate a similarity score between tumor entities. The similarity between two tumor entities T1 and T2, was quantified by calculating the Cohen's Kappa Coefficient  of correWe calculated the quadratic agreement matrix among the tumor entities. The result was visualized with the R function heatmap . EuclideAPOH), previously not mentioned in context with cancer, T cell receptor beta constant 1 (TRBC1) a widely undescribed gene and also not associated in context with cancer and alpha-fetoprotein (AFP), a major plasma protein reported to be associated with several carcinomas [ORM2) another plasma protein whose specific function has not yet been determined and not linked to carcinogenesis was found to be regulated in 25 tumor tissues. Furthermore, two genes were identified to be regulated in 24 tumor tissues, inter-alpha (globulin) inhibitor H2 (ITIH2), a gene involved in extracellular matrix stabilization and related to prevention of tumor metastasis [KIAA0101 which is associated with hepatocellular and thyroid carcinoma [In order to investigate individual and conserved signaling and metabolic events in tumor progression, we analyzed a dataset of 649 human two-color cDNA microarray tumor samples obtained from the Stanford Microarray Database . These srcinomas -26. Furttastasis -29 and tarcinoma ,31.Focusing on deregulated genetic signaling events across multiple tumors as well as metabolic events in tumor progression, we analyzed the differentially expressed genes in context of the functional categories as well as signaling and metabolic pathway maps provided by the Kyoto Encyclopedia of Genes and Genomes (KEGG) . After aTo evaluate the significantly enriched KEGG pathway maps in context of published literature to tumor progression already described and mostly unknown we utilized the web-based tool PubMatrix . The 'CeFurther use of the functional cancer map was made to calculate tumor phylogeny by laying down new starting points for the efficient combination of tumor therapeutics as well as for expanding the application area of well-established tumor therapies. We postulated that tumor entities with comparable significantly enriched KEGG pathway maps demonstrate comparable activation of biochemical processes and, thus, biochemical behavior. As a measure of closer or distant relationship between two tumor entities we applied the Cohen's Kappa Coefficient  to the bDespite comprehensive gene expression sets available on multiple human tumor entities, only few studies have attempted to assess and compare biological behavior of the tumors on the basis of functional microarray analysis and evaluation of commonly deregulated and conserved genetic signaling and metabolic events. Moreover, previous analyses focused on individual mechanisms or did not compose tumor-specific gene expression information into detailed, structured maps, impeding a systems view on cancer. However, in particular this kind of information could potentially guide medical research in the context of new applications and novel combinations of existing chemotherapeutics. Therefore, we here present gene expression information of tumors according to pathway activity profiles deemed to better reflect biological behavior. Our analysis of multiple tumor entities using these activity profiles resulted in a clearly structured functional cancer map figure . Note: DAs a proof of principle and supporting our approach, we found well documented biological events such as cell cycle and apoptosis to be highly conserved among the diverse tumor entities. These findings confirmed the reliability of the generated functional cancer map. Deregulated balance between proliferation and cell death represents a pro-tumorigenic mechansim in human carcinogenesis . Both acHowever, besides extensively studied and repeatedly documented signaling pathways highlighted in our analysis several pathways were found to be commonly enriched among differentially regulated genes which have not been clearly attributed to tumorigenesis. Thus, our work identified key roles of additional signaling pathways in cancer biology. Deregulations or mutations in the molecules involved in the 'Cell Cycle' and 'ECM-receptor interaction' pathway, preserving the physical link between extracellular matrix and actin cytoskeleton, are also closely connected to tumor progression . BesidesTAFIa), by inactivating the anaphylatoxins C3a, C4a and C5a and dysregulation of those genes has been implicated in tumor growth [Almost no studies were published on the functional pathway of 'Complement and Coagulation cascades'. Few studies demonstrated a potential role of individual genes within this pathway in cancer development. Some members of the complement system can be modulated by the antifibrinolytic protein, activated thrombin-activatable fibrinolysis inhibitor  signaling pathway regulates a multitude of genes important for diverse cellular functions including cell proliferation, cell differentiation, immune response and apoptosis. Once activated, PPARgamma will preferentially bind with retinoid X receptor alpha and signal antiproliferative, antiangiogenic, and prodifferentiation pathways in several tissue types. Since ligands and other agents influencing this pathway have revealed anticancer effects in a variety of human cancers ,50 efforFinally, we noticed an enrichment of genes invovlved in the functional pathways "Diabetes type I" and "gluycolysis/gluconeogesis". Besides our data several clinical reports support a synergism between Diabetes mellitus and cancer development. Konishi I et al. reported a correlation between a diabetic pattern of the 75 g oral glucose tolerance test OGTT and hepatocarcinogenesis and considered it a significant risk factor for hepatocellular carcinoma (HCC) in patients with hepatitis C virus . Also HuIn summary, there is accumulating evidence for a synergy between diabetes mellitus and an increased risk for and decreased survival of patients with HCC. Our data suggests that important interactions may be identified by analyzing the enriched functional pathways of "Diabetes type I" and "gluycolysis/gluconeogesis" previously not extensively studied in context of cancer.We introduced a measure of profile similarity by which tumor entities were structured based on semantic aggregated profiles, which means significant, individual tumor mechanisms calculated out of gene expression data, into a dendrogram figure . In ordeNotably, examining downregulated genes and clustering of the tumor entities by means of KEGG pathways, we found our sample to almost perfectly cluster according to the germ layer of the tissue of tumor origin. However, this finding needs to be further analyzed and applied to robust statistical testing ,55.Multiple tumor entities have been analyzed for signaling and metabolic events in tumor progression and composed into a functional cancer map. The resulting detailed, structured map should lead to a more systematic view on common and individual tumor mechanisms. This work lead to the identification of novel superimposed functional pathways closely linked to cancer biology. Among these pathways were 'ECM-receptor interaction', 'Complement and Coagulation cascades', and 'PPAR signaling pathway'. Furthermore tumor phylogeny was calculated with respect to their biological behavior. Both, the functional cancer map paired with the indication for tumor phylogeny could serve as a starting point for the efficient combination of targeted tumor therapeutics as well as for expanding the application area of well-established tumor therapies.The authors declare that they have no competing interests.Primary data preparation and normalization: TB, RK; Data analysis: MK, TM, JM, PRG, AT; Statistics and pathway analsis: FS, SB, ATr; Manuscript preparation: MK, JM, ATr, AT. All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1755-8794/4/53/prepubList of regulated tumor genes with respect to tumor entities and corresponding p-values / q-values.Click here for fileFunctional cancer map with respect to upregulated genes.Click here for fileFunctional cancer map with respect to downregulated genes.Click here for fileFunctional cancer map without any filtering for outlier genes.Click here for filePubMatrix result for the literature mining of the enriched KEGG pathway maps in context of published literature to tumor progression.Click here for fileTumor phylogeny: Calculation of the tumor phylogeny was done by applying the Cohen's Kappa Coefficient to the binarized upregulated functional cancer map.Click here for fileTumor phylogeny: Calculation of the tumor phylogeny was done by applying the Cohen's Kappa Coefficient to the binarized downregulated functional cancer map.Click here for file