A very early step in splice site recognition is exon definition, a process that is as yet poorly understood. Communication between the two ends of an exon is thought to be required for this step. We report genome-wide evidence for exons being defined through the combinatorial activity of motifs located in flanking intronic regions.Strongly co-occurring motifs were found to specifically reside in four intronic regions surrounding a large number of human exons. These paired motifs occur around constitutive and alternative exons but not pseudo exons. Most co-occurring motifs are limited to intronic regions within 100 nucleotides of the exon. They are preferentially associated with weaker exons. Their pairing is conserved in evolution and they exhibit a lower frequency of single nucleotide polymorphism when paired. Paired motifs display specificity with respect to distance from the exon borders and in constitutive versus alternative splicing. Many resemble binding sites for heterogeneous nuclear ribonucleoproteins. Specific pairs are associated with tissue-specific genes, the higher expression of which coincides with that of the pertinent RNA binding proteins. Tested pairs acted synergistically to enhance exon inclusion, and this enhancement was found to be exon-specific.The exon-flanking sequence pairs identified here by genomic analysis promote exon inclusion and may play a role in the exon definition step in pre-mRNA splicing. We propose a model in which multiple concerted interactions are required between exonic sequences and flanking intronic sequences to effect exon definition. Despite the wide acceptance of this idea, especially in metazoans where intron size is much greater than exon size, most biochemical investigations of splicing have focused on protein-protein interaction across introns, rather than on complexes that form across exons [All pre-mRNA splicing reactions involve the removal of an intron from between two exons and so require the pairing of the splice sites at the two ends of the intron; such pairing can be considered as a mandatory 'intron definition' step in splicing. However, it is likely that the initial recognition of most splice sites also involves 'exon definition,' the identification of two splice sites across an exon. This idea was first put forth to explain the observation that appending a 5' splice site downstream of the second exon in a two-exon pre-mRNA molecule greatly enhances splicing of the upstream intron in vitro . There hin vitro . One canss exons ,4.vice versa [It is possible that spliceosomal components themselves mediate this concurrent recognition of splice sites ,6. For ice versa ,10, implce versa  and coulce versa .A simple first step in the end-to-end recognition of an exon could be the binding of proteins at the two ends of the exon that are capable of specifically interacting with each other. If there is a limited repertoire of such proteins, then their existence should be signaled by the occurrence of specific combinations of sequences that serve as binding sites for these putative exon definition factors. Such pair-wise combinations can act to promote the intron definition step in splicing. The binding of the same heterogeneous nuclear ribonucleoprotein (hnRNP) at the two ends of a long intron can promote splicing ,14. A cocis-acting elements that act in combination at an earlier step in splicing, interacting from the two ends of an exon to mediate exon definition. Whereas most past computational searches for cis-acting splicing elements have focused on single motifs [Here we have sought evidence for e motifs -18, heree motifs ,19-21. Se motifs ,23.We found that more than 15% of exons harbor flanking motif pairs that are strongly associated with each other. These pairs are found around constitutive and alternative exons but not pseudo exons and their pairing is evolutionarily conserved. They are also associated most frequently with exons that appear relatively weak by other criteria. Specific pairs are also associated with tissue-specific genes. When tested in a heterologous context, these motif pairs were found to synergistically enhance exon inclusion. This enhancement proved to be context dependent, with specificity that was imparted by exonic sequences. Thus, the communication between exon ends may involve multiple interactions across the exon and its intronic flanks.We extracted intronic regions upstream of the polypyrimidine tract of exons (upstream of -14 relative to the 3' splice site) and downstream of the consensus 5' splice site (downstream of +6 relative to the 5' splice site). We limited our search to 100-nucleotide intronic regions, as these have been seen to harbor distinctive motifs ,19-21,245 × 45 = 410). If we set the P-value cutoff at 1/410 (referred to hereafter as 10-6), we expect to see around one pentamer pair having a P-value smaller than this cutoff if pentamers in one intronic region are independent of those in the other region. Examining about 80,000 human constitutive exons, we found more than 60,000 pentamer pairs (approximately 6% of 410) that passed this P-value cutoff. The top motif pairs detected all shared similar GC contents, being either GC-rich or AT-rich . A similar method was used by Friedman et al.  in a sea6 Figure , row 1; 6 Figure , row 2. s Figure . The strAs an additional control, we asked whether the co-occurrence of pentamer pairs could also be found around other genomic sequences of a similar size. We examined pseudo exons , definedsrc splicing [We next analyzed alternatively spliced exons using BBC-COOC and again found significantly co-occurring motifs. For three of the four regional classes, alternative exons gave rise to only about 40% of the number of motif pairs yielded by constitutive exons. This result might be attributable to the lower statistical power afforded by the smaller number of the former  compared to the latter . Interestingly, in the regional class UpDd, alternative exons yielded more co-occurring pairs than constitutive exons. This excess of alternative splicing motifs associated with a downstream distal region (more than +50 nucleotides) echoes the discovery of intronic elements regulating the alternative splicing of individual exons  as wellsplicing . For mosP-values for all 1,048,576 pairs for each set of regions can be found at [Table S2 in Additional file found at .We determined the distance limits for regions harboring co-occurring motif pairs by extending the BBC-COOC analysis to pairs of 50-nucleotide stretches symmetrically spaced at 50-nucleotide intervals away from the borders of constitutive and of alternatively spliced exons. For both types of exons the frequency of co-occurring motif pairs dropped off sharply beyond 100 nucleotides from the exon borders but could still be detected out to about 200 nucleotides, although not further Figure . These dP-values less than the P-value cutoff of 10-6 by definition; very few of these motif pairs have P-values less than the P-value cutoff when examined in any of the other three regional combinations  was comparable in almost all cases; for 98% of the co-occurring pairs, the lowest number of expected pairs (based on the null hypothesis) was within a factor of two of that for the defining region (UpDp in this case). The same was true for the other three regional combinations shown in Figure We asked whether the lower number of motifs pairs passing the cutoff of If these motifs are cooperating to enhance splicing, then this cooperation may be quite sensitive to the distance between a motif and its nearest splice site. For example, motifs A and B may be able to cooperate to enhance splicing of an exon between them, but if motif B is moved 50 nucleotides closer to the splice site, this pair is no longer effective. Such context dependence has previously been seen for exonic splicing enhancers  and repr-6 cutoff were not among those that passed the cutoff from alternative exons and vice versa . The fact that the pairing of these motifs has been conserved in primate evolution supports the idea that they are functional, perhaps working in concert to promote exon splicing through exon definition.If co-occurring motif pairs interact across exons to promote splicing, then their pairing should be evolutionarily conserved. We addressed this question by comparing human and macaque sequences. For each of the four regional classes, we identified human constitutive exons that harbor co-occurring pairs and then collected the macaque orthologs of those exons . ConservThe co-occurring pair hypothesis predicts that mutations that occur in these motifs should have a higher likelihood of disrupting exon splicing than those that occur in the same motifs when they are alone. Therefore, the former would be more likely to be eliminated by purifying selection. Thus, the motifs of co-occurring pairs should have a lower SNP density. Consistent with this prediction, for all four regional classes the SNP density was significantly lower when the motifs were paired than when they were unpaired for both human constitutive exons and alternative exons Figure . This obIf intronic co-occurring pairs act to promote splicing, then they might be expected to contribute more frequently to exons that are otherwise relatively deficient in splicing signals. We compared all constitutive exons that contain co-occurring motif pairs of a particular class  to the constitutive exons of a set that did not contain such pairs. The exons of the second set were exactly matched to the first set in the GC content of the relevant paired intronic regions so as to minimize the influence of base composition on any correlations seen. For instance, regions high in GC content will tend to be associated with splice sites that are high in GC content , which iCo-occurring motifs tended to have lower ESE coverage, higher ESS coverage and poorer 3' splice site scores compared to exons without co-occurring motifs  no greater than that seen among random pentamers with the same overall base composition . Thus, secondary structure does not seem to be playing a role in the selection of these motif pairs.If the intronic motifs discovered here function to promote splicing, they may overlap with previously reported motifs computationally predicted to do the same. We compared the 38 unique downstream motifs from the constitutive and alternative UpDp classes with pentamers located in downstream intronic flanks that were predicted to be ISEs based on their relative abundance and/or evolutionary conservation -21,24. TP-value threshold for detecting these co-occurring pairs, the actual proportion of human constitutive exons with functioning co-occurring pairs may be much higher. This abundance would allow co-occurring motif pairs to play a role in the splicing of many human constitutive exons. For constitutive exons, motif pairs that originate from proximal regions tend to be clustered at the proximal end of the 50-nucleotide region (closer to the splice site); on the contrary, motifs from distal regions are spread throughout the distal region . Because we set a stringent n Figure . Interesn Figure .Many of the motifs in the co-occurring pairs resemble the binding sites of hnRNPs or other RNA binding proteins, including hnRNPs A1/A2, C, D, F/H,G, I (PTB), K, L, M, and 9G8 , and significantly depleted in appendix, superior cervical ganglion and skeletal muscle. Interestingly, this pattern correlated  with the tissue-specific expression levels of hnRNP H  with the levels of both hnRNP K and C genes in these tissues , including the 3' and 5' splice sites, that is, the 223-nucleotide exon plus 14 nucleotides upstream and 6 nucleotides downstream. Deprived of its natural intronic flanks, the Hb2 exon undergoes very little inclusion (approximately 2%) in this context. Tandem pentamers from five co-occurring pairs (numbered 1 to 5) were then inserted in various combinations on either side of the Hb2 exon. Each of the first four pairs  were chosen because they are found in over 1,000 human constitutive exons. The fifth pair (CGCCG/CGCGC) has the most significant P-value of association (2.9 × 10-12).The abundance of co-occurring motif pairs flanking constitutive exons and their absence around pseudo exons, their exclusive residence within 200 nucleotides of the exon borders, their conservation as pairs in evolution, their association with weaker exons and the resemblance of many of them to known RNA binding protein binding sites suggested that the two motifs of a pair may cooperate across an exon to promote exon splicing. To test this idea we used a three-exon minigene with terminal exons and introns derived from the Chinese hamster et al. [Motif pairs 1 to 4 greatly enhanced the inclusion of the Hb2 exon when provided as flanking pairs, yielding 10- to 45-fold increases; motif 5 was without any substantial effect Figure . When onet al.  and Elenet al. , not to To explore the generality of this result, we repeated the experiment using a different central exon, a weakened version of exon 5 of the Wilm's tumor gene (WT1-5: the 51-nucleotide exon body plus 26 nucleotides upstream and 6 nucleotides downstream). Remarkably, the results of co-occurring pair inclusion were now the reciprocal of what was seen for Hb2: co-occurring pairs 1 to 4 were without effect but pair 5 yielded a robust and synergistic 20% exon inclusion Figure . A thirdSince alternative exons by definition often fail to be included, we explored the possibility that co-occurring pairs around alternative exons cooperate to prevent splicing, rather than enhance it. The WHW exon exhibits 70% inclusion in the absence of any added sequences and so provides a sensitive assay for silencing. Three such co-occurring pairs , chosen because they resemble hnRNP binding sites, were inserted in combinations on either side of the WHW exon. All three pairs demonstrated synergy in enhancing exon inclusion, lending no support to the silencing idea Figure . InteresThese results lead to two conclusions. The first is that the two motifs of a pair are acting not additively but synergistically, suggesting that they act in a concerted manner. The second is that these motifs act in a context-dependent manner, and the specificity of this context can be provided by exon body sequences.Exon definition can be thought about at several levels. Perhaps the most fundamental level is as an observable phenomenon: the competence of a splice site at one end of an exon is dependent on competence at the other end ,7,30,46.The specificity seen in the tested constructs suggest two exon definition models. In one model Figure , the twoHuman mRNA sequences and ESTs were downloaded from the UniGene database  and wereFor all exons that we used for BBC-COOC analysis, we first removed any repeats defined by RepeatMasker . If two Pseudo exons were defined as having lengths between 50 and 250 nucleotides and consensus values of ≥75 for 3' splice sites and ≥78 for 5' splice sites. The consensus values were based on a position-specific weight matrix and were calculated essentially according to Shapiro and Senapathy [Ui exons have a pentamer motif Ui in the upstream intronic region and nDj exons have a pentamer motif Dj in the downstream region. If the existence of motif Ui in the upstream region is independent of the existence of motif Dj in the downstream region, the number of exons with both motif Ui in the upstream region and motif Dj in the downstream region (k) follows the hypergeometric distribution with the probability given by:The total number of qualified exons for analysis is N. Among these exons we consider intronic pentamer pairs in which one motif occurs upstream and the other downstream of the exon. Of the N exons, nand where the expected value of k is Ui, and calculated the frequency of such exons as:Because the GC contents of the two intronic regions that flank the exon are likely to be highly correlated due to the existence of GC isochores in the human genome, we need to apply a strategy to remove this bias. Toward this end we grouped exons twice, first into 20 rows according the GC content in their upstream regions and then into 20 columns according to the GC content of their downstream regions. The GC content limits were adjusted so that each row contains the same number of exons; the same was done for each column. Thus, each row and each column contain N/20 exons, The result is a grid of 20 × 20 boxes where the exons in each box share a similar GC content both upstream and downstream . The final sum is:where MUi exons have motif Ui in the upstream intron and nDj exons have motif Dj in the downstream intron. We need to adjust nUi or nDj or both to make k' equal the theoretical mean of the hypergeometric distribution Ui the same and adjusted nDj to:which is the expected total number of exons with both motif Ui in the upstream intron and motif Dj in the downstream intron according to the null hypothesis. This carefully calculated expectation takes into account the GC content correlation between upstream and downstream intronic regions of an exon. Of all the N exons, nUi or adjusting both yielded similar results. It is this adjustment that takes the GC content correlation into account. We next counted the actual number of exons with both motif Ui in the upstream intron and motif Dj in the downstream intron (ka) across all 20 × 20 boxes:Adjusting na to follow the same hypergeometric distribution as k' with a probability:Under a null hypothesis, we would expect kP-values for all observed values of ka, where ka values greater than the mean indicate a positive correlation and vice versa. This method of dealing with the biased GC contents is similar to one used by the Burge laboratory to discover co-occurring pairs across introns [From this distribution we can calculate the  introns  except fP-value of >0.05 with its original partner. For example, for the non-co-occurring motif pair UpDpnon-pairing, Dpnon-pairing had the same GC content as the corresponding Dp but formed a non-co-occurring pair with Up; and for Upnon-pairingDp, Upnon-pairing had the same GC content as the corresponding Up but formed a non-co-occurring pair with Dp.Based on the coordinates of the human exons, orthologous macaque exons were extracted from a 17-genome multi-alignment . In thisHuman SNP data were downloaded from dbSNP build 130 . SNPs thE,Tissue, was calculated. As a control, we collected the same number of randomly chosen exons that did not contain the co-occurring pair . The number of exons in each control gene was matched to the same number of exons in the genes with co-occurring motifs. The fraction of control genes associated with each tissue/cell line was then calculated, FC,Tissue. The P-value for the significance of enrichment (or depletion) for each tissue/cell line was computed for the fraction of times that FE,Tissue was lower than or equal to FC,Tissue . The P-value cutoff for significance of tissue/cell lines bias was 0.001.From BioGPS  we obtaidhfr minigene modified from that previously described [To examine the cooperative activity of co-occurring pairs in splicing, we used a hamster escribed . The staescribed  and the escribed  as the kHEK 293 cells cultured in 35 mm dishes were transfected using Lipofectamine 2000  following the manufacturer's protocol. After cells were incubated for 24 hours, total RNA was extracted using illustra RNAspin Mini Kits . A sample of 400 ng of RNA was reverse transcribed (RT) using Omniscript  and a specific primer, AGAGTCTGAGATGGCCTGGCT, which pairs with a region in the third exon. One-tenth of the RT product was used as template in the following PCR amplification: forward primer, GTCAGATCCGCCTCCGCGTA; reverse primer, GTAAACGGAACTGCCTCCAA; initial denaturation, 94°C for 2 minutes; denaturation, 94°C for 45 s; annealing, 60°C, 1 minute; extension, 72°C, 1 minute; 20 cycles; final extension, 72°C, 5 minutes. Splicing products were separated in 1.8% agarose gels stained with ethidium bromide; the intensity of each splicing product was quantified with ImageJ. At least two independent transfections were performed for each construct. Proportion included (I) was defined as Included product/(Skipped product + Included product) in molar quantities.U = SU/Sno-insertion. For motif pair U:D, we evaluated the level of synergy by comparing the skipping fitness WUD of the pair insertion construct with the product of the skipping fitness values WU and WD of the corresponding single motif insertion constructs. The synergy index for promoting splicing inclusion is defined as SI = WUD - WU × WD. SI <0 indicates synergy, SI = 0 indicates no synergy, and SI >0 indicates anti-synergy [By analogy to previous synergy calculations ,45, we f-synergy ,45.BBC-COOC: base bias corrected co-occurrence; Dd: downstream distal; Dp: downstream proximal; ESE: exonic splicing enhancer; ESS: exonic splicing silencer; EST: expressed sequence tag; Hb2: human beta globin gene; hnRNP: heterogeneous nuclear ribonucleoprotein; ISE: intronic splicing enhancer; SI: synergy index; SNP: single-nucleotide polymorphism; Ud: upstream distal; Up: upstream proximal; WT1-5: exon 5 of the Wilm's tumor gene 1.SK and LC conceived and planned the research, analyzed the data and wrote the manuscript. SK carried out the experiments.Figures S1, S2, and S3, and Tables S1 and S3. Figure S1: distribution of pentamer pairs around constitutive exons. Figure S2: scheme for grouping exons according to the GC content of their flanks. Figure S3: tissue-specific expression of six hnRNPs. Table S1: results with no GC balancing. Table S3: overlap of UpDp downstream motifs with other reported intronic motif sets.Click here for fileTable S2. List of co-occurring pairs.Click here for fileTable S4. Tissue-specific expression of genes with co-occurring motif pairs.Click here for file


C. elegans, which have become a popular in vivo model for understanding the regulatory basis of lipid biosynthesis and deposition.Taurine plays an important role in reducing physiological stress. Recent studies indicated that taurine may serve as an anti-obesity agent at the cellular level. This study characterizes taurine’s potential anti-obesity function in C. elegans were raised on a normal or high-fat diet: N2  and RB1600, a mutant in tub-1 that serves as a tubby homologue and functions parallel to the 3-ketoacyl-CoA thiolase gene (kat-1) in regulating lipid accumulation. Taurine’s effect on lipid deposition was characterized according to assays of Sudan black B staining, triglyceride content measurement, food consumption, and mobility comparison.Two strains of When N2 was treated with taurine after the culture in the high-fat media, the worms showed lower lipid accumulation in the assays of the Sudan black B staining and the triglyceride quantification. The anti-obesity effect was less evident in the experiment for RB1600. When the amount of taurine was increased for the high-fat-diet-treated N2 strain, fat deposition decreased and mobility increased in a dose-dependent manner. In the food consumption assays, taurine did not cause a significant change in food intake.Taken together, these results strongly imply that taurine plays an important role in reducing fat deposition by modulating cellular pathways for lipid accumulation and stimulating mobility, but not the pathways for lipid biosynthesis and food intake. Taurine, or 2-aminoethanesulfonic acid, is the major component of bile and is present in many types of animal tissues, including in the gall bladder, blood vessels, eyes, and lower intestine. Although taurine is less known as an amino acid, it is commonly used as a functional component of various types of health drinks. It plays a vital role in adjusting physiological pathways in antioxidation and detoxification. Recent studies indicated that taurine deficiency is associated with persistent obesity, which can be prevented by dietary taurine supplementation ,2. This C. elegans is a very useful animal system in unearthing crucial biological mechanisms that are readily applicable to many higher-level organisms, including humans ,6. IndeeLipid accumulation involves complex processes of lipid biosynthesis, transport, and storage -13. LipiIn C. elegans, the gene products of tub-1 are believed to manage lipid accumulation in cooperation with those of kat-1, which govern fatty acid beta-oxidation -17. The  tub-1 gene were tested under four classes of experiments. The worms were analyzed using Sudan black B staining, food intake assay, triglyceride measurement, and mobility assessment.This study characterized taurine as an anti-obesity agent in C. elegans that were fed a high-fat diet. The strains of N2, both the normal and RB1600, that mutated in theE. coli OP50 as a food source. The taurine-containing media were prepared by smearing an appropriate amount of taurine stock solution onto the media. The stock of E. coli OP50 was prepared by culturing it overnight in an LB medium until concentrated particles were formed. The quantity of OP50 in the media was adjusted depending on the initial worm input and the duration of the worm culture.C. elegans were grown on standard nematode-growth media that contained high or normal content of cholesterol as 25.0 or 12.5 μM, respectively. During each experiment, the worms were grown on the nematode growth media (NGM) that contained The anti-obesity effect of taurine was studied via microscopy on stained worms. Individual worms were fixed and stained with Sudan black, which stains lipid stores into opaque blue. For the staining, the worms were removed from the culture plates and were starved for 6 h. After they were washed in M9 media, they were fixed with 1% paraformamide in M9. Then they were subjected to three freeze-thaw cycles and dehydrated in 95% ethanol. The staining was performed in 70% ethanol saturated with Sudan black B.The lipid contents were quantitated by measuring the triglyceride content of the worms that were treated under taurine or taurine-free conditions. The worms were thoroughly washed in TBS and homogenized and sonicated in a Triglyceride Assay Buffer (TAB) and the triglyceride content was assessed using a BioVision triglyceride assay kit . After brief centrifugation, the samples were diluted to a final volume of 50μl/well. Following the dilution of the samples in two microliters of lipase, the samples were incubated for 20 min at room temperature to convert their triglycerides into glycerol and fatty acid. The optical density of each of the incubated samples was measured at 570 nm for the colorimetric assay in a microplate reader. The calculations were made by subtracting the background value derived from the 0 triglyceride standard from all the sample readings. The triglyceride concentration (nmol/μl) was calculated according to the formula [TC] = Ts (nmol) / Sv (μl) .9 cfu). The time taken for total intake of the food input was measured and compared among the treatment and genotypes.Worms were treated on taurine or taurine-free media for 12 hours; then, they were subjected either to fast or to copious feeding with OP50 for 6 h on NGM media. Twenty worms of 1-day-old were transferred onto NGM plates covered with lawn of identical amount of OP50 .The anti-obesity effect of taurine was studied under a microscope. Fig. tub-1 mutant positively responded to taurine by showing a lower triglyceride content under the taurine treatment. The inconsistency between the staining and the quantitation results in the tubby worms implies that taurine may work in the stage of fat biosynthesis rather than in the stage of fat accumulation.In addition to a visual comparison of the lipid droplet that was formed in the Sudan black B staining experiment, this experiment assessed the total amount of triglycerides in C. elegans. The measurement that was performed in this experiment genuinely referred to the lipid biosynthesis in the worms. The triglyceride content was shown to correspond to those in the experiment and to the dose-dependent anti-obesity effect of taurine Fig. . In the No considerable difference was seen between the worms that were treated with taurine and the worms in the taurine-free conditions. Fig. After the tunicamycin-treated worms were further grown on taurine-containing media, the extents of the worms’ movement after various treatment concentrations were visually compared . The visual comparison of the worms’ tracks revealed that the taurine-treated worms travelled longer distances than the non-taurine-treated control Table .. The poUsing C. elegans systems, this study tested taurine’s potential as an anti-obesity agent by helping reduce lipid accumulation in cells. Taurine significantly decreased fat storage depending on its concentration. Without negatively affecting food intake, taurine showed substantially reduced fat accumulation and increased activity, which matched the reduction of lipid storage under its treatment.Complex cellular mechanisms coordinate the equilibrium between lipid utilization and accumulation -23. BeinOne of the findings in this study is that taurine had only a minor anti-obesity effect on the tub-1 mutant, unlike on the normal strain. Unlike the wild type, the tubby mutants are known to store more fat due to their inability to control excessive transport to fat storage tissues or to react properly to metabolic signals from ciliated neurons ,29. ThusThe Sudan black B staining and triglyceride assays showed inconsistent results concerning the tub-1 mutants in response to taurine. That is, the Sudan black B staining did not show noticeable sensitivity to taurine application, but the triglyceride content measurements showed a significant difference between the taurine-free and the taurine-treated worms. These inconsistent results strongly indicate that the taurine had a more immediate impact on lipid deposition than on fat synthesis.As in mammals, C. elegans accumulates fat both from its diet and via de novo biosynthesis, and stores lipids in subcellular compartments. Not enough information is available, however, on how C. elegans acquires, synthesizes, uses, and stores lipids, but the consistent results of this study indicate that taurine diminishes lipid storage by enhancing physical activities, rather than by reducing food uptake. Despite these data, the mechanism behind the subcellular targeting and sequestration that dictate the magnitude and numbers of these deposits remains to be determined.tub-1 mutants. Unlike tub-1, N2 positively responded to taurine according to its triglyceride content assay. The increased mobility with the taurine treatment may contribute to reduced lipid deposition via enhanced lipid consumption. An additional study is necessary to clarify the potential mechanisms for the fat storage reduction. A new study may be rewarding in finding a definite agent that would modulate tub-1/kat-1 and sbp-1 molecules. Collective treatment of these molecules with taurine is a good way to generally cope with obesity.In summary, this study showed that taurine affects lipid deposition in a dose-dependent manner. Reduced lipid storage was evident in the wild-type worms but not in the SREBP: sterol regulatory element-binding protein; TAB: triglyceride assay buffer; kat-1: 3-ketoacyl-CoA thiolase-1; NGM: nematode growth mediaThe authors declare that they have no competing interests.HMK maintained C. elegans and carried out the various taurine treatments and data collection. CHD participated in the design of the study and performed the statistical analysis. DHL participated in obtaining worm strains and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.


Many smoking-cessation programs and pharmaceutical aids demonstrate substantial health gains for a relatively low allocation of resources. Genetic information represents a type of individualized or personal feedback regarding the risk of developing lung cancer, and hence the potential benefits from stopping smoking, may motivate the person to remain smoke-free. The purpose of this study was to explore what the impact of a genetic test needs to have within a typical smoking-cessation program aimed at heavy smokers in order to be cost-effective.Two strategies were modelled for a hypothetical cohort of heavy smokers aged 50 years; individuals either received or did not receive a genetic test within the course of a usual smoking-cessation intervention comprising nicotine replacement therapy (NRT) and counselling. A Markov model was constructed using evidence from published randomized controlled trials and meta-analyses for estimates on 12-month quit rates and long-term relapse rates. Epidemiological data were used for estimates on lung cancer risk stratified by time since quitting and smoking patterns. Extensive sensitivity analyses were used to explore parameter uncertainty.The discounted incremental cost per QALY was AU$34,687  over 35 years. At a willingness-to-pay of AU$20,000 per QALY gained, the genetic testing strategy needs to produce a 12-month quit rate of at least 12.4% or a relapse rate 12% lower than NRT and counselling alone for it to be equally cost-effective. The likelihood that adding a genetic test to the usual smoking-cessation intervention is cost-effective was 20.6% however cost-effectiveness ratios were favourable in certain situations .The findings were sensitive to small changes in critical variables such as the 12-month quit rates and relapse rates. As such, the cost-effectiveness of the genetic testing smoking cessation program is uncertain. Further clinical research on smoking-cessation quit and relapse rates following genetic testing is needed to inform its cost-effectiveness. Smoking remains a substantial health problem in many countries and is the largest modifiable risk factor for several cancers and a host of chronic diseases. Between 1980 and 2004, smoking prevalence in the Australian population dropped from 40% to 21% .et al. (1997) demonstrated the cost-effectiveness of smoking-cessation programs delivered by a general practitioner (GP)[The pivotal paper by Cromwell J oner (GP). Many suoner (GP). Howeveroner (GP). The extK-ras, GSTM1, CYP2D6, c-MET, NKX2-1, LKB1, BRAF) implicated in lung cancer onset [While tobacco smoking is the largest known risk factor for lung cancer occurring in 85-90% of cases, only 10-15% of smokers develop lung cancer. Recent The genetic link to lung cancer has implications for the design of smoking-cessation programs. Genetic information represents a type of individualized or personal feedback regarding the risk of developing lung cancer, and hence the potential benefits from stopping smoking, may motivate the person to remain smoke-free. Central to this is the potential to address the issue of optimistic bias, the underestimation of one's own risk of a harmful outcome relative to the average smoker. Recent developments in genetics suggests that some people respond well to genetic information about risk of lung cancer ,11, are Several companies now offer genetic testing for lung cancer susceptibility however they offer a single nucleotide polymorphism (SNP) test for lung cancer risk result and no other clinical data is used for their risk assessment. Our author (R.Young) heads a clinical research program at Auckland Hospital, New Zealand, offering patients a SNP-based test involving 20 SNPs and assessment of other clinical variables  within usual clinical practice for smoking-cessation. Early results show that intentions to quit smoking among 250 participants based on genetic testing for lung cancer risk were around 88% in those at elevated risk of lung cancer. The economic value of the adopting this new technology into practice is yet to be determined.To date, no smoking-cessation study has examined the cost-effectiveness of offering genetic tests in the context of disease prevention but other studies have investigated genetic testing to guide the choice of pharmacotherapy among individuals attempting to stop smoking ,14. GeneA Markov state transition model was constructed in TreeAge Pro 2009 software  Figure . The modet al. 2002 [We compared a usual smoking-cessation program with an alternative involving the usual smoking-cessation program and a genetic test some point after  completing the program . The beIn our model, we assumed our cohort were 50 year old heavy-smoking men and women (> 20 cigarettes per day) who presented to their GP, and were willing to participate in a smoking-cessation program. The usual smoking-cessation program comprised of GP advice, telephone counselling and nicotine replacement therapy (NRT) administered over 12 weeks  over 35 years. The number of quitters at 12 months is also presented to highlight the shorter-term impact. The level of effectiveness of smoking-cessation enhanced with a genetic test was based on a randomised clinical trial involving 557 participants-21 did nThe QALY is a generic outcome measure preferred for use in economic evaluations combining survival time adjusted for quality of life. A structured literature review was undertaken to locate recent preference-based quality of life scores (or utility weights) for lung cancer. Eleven studies from 1997-2008 were uncovered. The utility weights used in the present study were based on direct utility assessment using standard gamble interviews  and a seThe costs and outcomes for the two options were combined into incremental cost-effectiveness ratios (ICERs), that is, incremental cost per quitter and incremental cost per QALY gained. The ratios are calculated as follows:Where C = costs, E = effects (QALYs or quitters), GT = genetic testing arm and USC = usual smoking-cessation arm and represent the additional costs per health benefit of the genetic testing component. Our analysis took a payer perspective when measuring and valuing resources used for the two options. This included two payers; the consumers and health providers and the analysis aggregated the costs from both payers. Direct costs borne by the consumers (smokers) included over-the-counter NRT and the genetic test Table . Health Threshold analyses were undertaken to separately determine at what quit and relapse rates the genetic testing arm was cost-effective. To determine if any variables were primarily driving the cost-effectiveness results, one-way sensitivity analyses on all parameters were undertaken Table . Of partA probabilistic sensitivity analysis was also performed, re-sampling from nominated distributions of data inputs through 10,000 iterations. Beta distributions were assigned to probabilities  and gamma distributions were assigned to cost variables because these are often right-skewed. The simulated mean ICER (QALYs) with 95% confidence intervals (CI) was generated. Finally, to assess the structural uncertainty of our model, we re-examined the model for men and women separately because it is well known that men are heavier smokers and have higher risks of lung cancer compared to women. We also explored the model for all persons starting at age 30 and 60 years. During our analyses, we assumed a willingness-to-pay ICER threshold of $20,000 per QALY gained to guide the interpretation of the findings, a level in keeping with higher-end cost-effectiveness ratios found in previous evaluations of smoking-cessation programs[The cost-effectiveness results suggest that for smokers offered a smoking cessation program with a genetic test, an additional $300 on average is incurred compared with a usual smoking-cessation program  and higher for women $46,408 per QALY   ICERs for men because they have relatively higher numbers of life-years to gain from stopping smoking,25. HoweWhen our model was re-assessed for 30 year olds, the long-term effects were severely eroded due to discounting and relapse rates. Therefore, the overall effectiveness was very small, inflating the cost-effectiveness ratio. This finding would indicate that the genetic testing arm is potentially suitable only in older (at least 50 year olds), long-term smokers or that the NRT and counselling needs to be repeatedly offered in relapsed smokers [Our choice of relapse rates is an important variable in our model both in terms of the values used, which were taken from a meta-analysis, and the et al. (2002) for a critical estimate, quit rate at 12-months following the genetic test[et al.'s sample of mostly lower-socioeconomic smokers may be a difficult group to intervene in but likely to be relevant and generalisable to other settings like Australia where a higher proportion of disadvantaged people also smoke. Potentially adverse consequences of genetic testing include emotional distress, concerns about discrimination and implications for telling family members positive results. These issues were omitted from our analysis. Our results relate to QALYs gained from preventing lung cancer onset and we did not incorporate improved survival gains due to the potential avoidance of other major diseases linked to smoking . Again, the impact is that our effects may be underestimated and overall ICERs conservative. A further limitation of the study was the omission of the potential implications of interactions between the level of susceptibility, test properties and quit rates that may impact on the cost-effectiveness findings, introducing further uncertainty. Based on McBride's findings, 33% of the participants in the GT arm had a positive genetic test for the missing gene GSTM1 for elevated susceptibility to lung cancer. However, quit rates in these participants were similar to those with negative tests and therefore behavior change was not hindered by the GT results. This finding is supported by our own pilot work with further results on this issue forthcoming.While our model was responsive to an ageing cohort and other time-dependent variables, some limitations are apparent and a number of assumptions were necessary. Data estimates are based on those available in published randomized controlled trials and may not reflect real-world practice . It is acknowledged that many individuals permanently cease smoking on their own accord with no psychological or pharmacological assistance. The present study examines the relative effectiveness of a smoking cessation program compared with a smoking cessation program given in conjunction with a genetic test. Extensive sensitivity analyses explored parameter uncertainty and aspects of the structural uncertainty . We relied on a single, randomized clinical trial by McBride Lung cancer is the leading cause of cancer death in many developed countries and the prognosis is poor with a 1-year survival of 34% and 5-year survival of 12%. AlthougIn certain circumstances, specifically, if a smoking-cessation program delivering a genetic test, NRT and counselling produced a 12-month quit rate of at least 12.4% then it would represent a potentially sound health care investment for 50 year old heavy smokers. Overall, our findings showed that a genetic test option in addition to the use of NRT and counselling would produce very similar costs and effects than NRT and counselling alone. Further research on the quit rates at 12 months and beyond following a genetic testing strategy is required to strengthen our findings.CI: Confidence interval; COPD: Chronic obstructive pulmonary disease; GT: Genetic test; ICER: Incremental cost-effectiveness ratio; NRT: Nicotine replacement therapy; QALY: Quality adjusted life years; SNP: Single nucleotide polymorphisms; USC: Usual smoking cessationDr Robert Young is a Scientific Advisor to Synergenz BioSciences who sponsored separate, but related, projects in lung cancer genetics and risk assessment scores.Dr L Gordon, N Hirst and Dr P Brown declare that they have no competing interests.LGG: performed the systematic review, data analyses, interpretation and drafted the manuscript.NGH: assisted with systematic review, data analyses, interpretation and presentation of the findings and manuscript writing.RPY: provided clinical expertise, idea conception and intellectual input and interpretation of the overall findingsPMB: provided senior public health expertise, intellectual input and guidance during manuscript writing.All authors have contributed substantively to writing the manuscript and have approved the final version.Figure S1: Threshold analysis of quit rate required for the genetic test strategy to have equivalent net benefits as usual smoking-cessation, at a willingness to pay (WTP) of $20,000. Figure S2: Threshold analysis of proportion of relapse rate required for the genetic test strategy to have equivalent net benefits as usual smoking-cessation, at a WTP of $20,000 Figure S3: Proportion of cohort who are quitters or relapsers, by age and genetic test or usual smoking-cessation arms. Figure S4: Proportion of cohort who develop early or advanced lung cancers, by age and genetic test or usual smoking-cessation arms. Figure S5: Results of one-way sensitivity analyses on key parameter values showing change in base case incremental cost per QALY ratio Table S1 - Model assumptionsClick here for file


Further testing revealed the association to be focused on polymorphisms within the androgen-to-estrogen conversion sub-pathway, for both breast  and endometrial cancer . The sub-pathway association was validated in the Finnish sample of breast cancer . Further tumor subtype analysis demonstrated that the association of the androgen-to-estrogen conversion sub-pathway was confined to postmenopausal women with sporadic estrogen receptor positive tumors . Gene-based AML analysis suggested CYP19A1 and UGT2B4 to be the major players within the sub-pathway. Our study indicates that the composite genetic determinants related to the androgen–estrogen conversion are important for the induction of two hormone-associated cancers, particularly for the hormone-driven breast tumour subtypes.Despite the central role of estrogen exposure in breast and endometrial cancer development and numerous studies of genes in the estrogen metabolic pathway, polymorphisms within the pathway have not been consistently associated with these cancers. We posit that this is due to the complexity of multiple weak genetic effects within the metabolic pathway that can only be effectively detected through multi-variant analysis. We conducted a comprehensive association analysis of the estrogen metabolic pathway by interrogating 239 tagSNPs within 35 genes of the pathway in three tumor samples. The discovery sample consisted of 1,596 breast cancer cases, 719 endometrial cancer cases, and 1,730 controls from Sweden; and the validation sample included 2,245 breast cancer cases and 1,287 controls from Finland. We performed admixture maximum likelihood (AML)–based global tests to evaluate the cumulative effect from multiple SNPs within the whole metabolic pathway and three sub-pathways for androgen synthesis, androgen-to-estrogen conversion, and estrogen removal. In the discovery sample, although no single polymorphism was significant after correction for multiple testing, the pathway-based AML global test suggested association with both breast ( Estrogen exposure is the most important risk factor for breast and endometrial cancers. Genetic variation of the genes involved in estrogen metabolism has, however, not been consistently associated with these two cancers. We posited that the genetic risk associated with the estrogen metabolic genes is likely to be carried by multiple variants and is therefore most effectively detected by multi-variant analysis. We carried out a comprehensive association analysis of the estrogen metabolic pathway by interrogating SNPs within 35 genes of the pathway in three tumor samples from Sweden and Finland. Through pathway-based multi-variant association analysis, we showed that the genetic variation within the estrogen metabolic pathway is associated with risk for breast and endometrial cancers and that the genetic variation within the genes involved in androgen-to-estrogen conversion is particularly important for the development of ER–positive and sporadic breast tumors in postmenopausal women. Our study has demonstrated that the influence of genetic variation on hormone exposure has an impact on breast cancer development, especially on the development of hormone-driven breast tumor subtypes. Our study has also highlighted that future genetic studies of the estrogen metabolic genes should focus on the androgen-to-estrogen conversion process. Estrogen exposure is critical for the development of both breast and endometrial cancers and represents the most well-established risk factors for both diseases. Estrogen is a metabolic product whose circulating level is determined by de novo synthesis, conversion from other steroid hormones, and mechanisms of estrogen elimination. These metabolic processes are regulated by a network of enzymes encoded by different genes, suggesting that genetic variation within these metabolic genes may impact on breast and endometrial cancer risk. Genetic variation within the estrogen metabolic pathway has been intensively investigated, mostly by analyzing single variant effects in a limited number of candidate genes, SNPs and study subjects. The inadequacies of study design and analytical methodology have caused these studies to be underpowered for detecting moderate genetic effects which, not surprisingly, has led to inconsistent results Herein, we present a comprehensive analysis of genetic variation in the estrogen metabolism pathway and its association with breast and endometrial cancer risk using a pathway-based approach.CYP19A1, and for endometrial cancer, the smallest p-value was 0.00017 at rs12595627 in CYP19A1. The single-SNP associations were all moderate. Only rs12595627  survived the conservative Bonferroni correction for multiple testing at α = 0.05. Overall, however, the single-SNP p values appeared to deviate from their null distribution of no association . The single-SNP associations were suggestive, but instead of any single variant having a strong effect, there appeared to be multiple weak associations within the metabolic pathway.We performed single SNP association analysis in 1596 breast cancer cases, 719 endometrial cancer cases and 1730 population controls from Sweden. Of the 239 tagSNPs analyzed, 17 SNPs (7.1%) had p-values less than 0.05 for breast cancer, and 18 SNPs (7.5%) had p-values less than 0.05 for endometrial cancer . For brea priori defined sub-pathways, each performing specific metabolic function  and endometrial  cancers  and endometrial  cancer . In contrast, the other two sub-pathways showed no association with either form of cancer. For approximately half of the Swedish subjects in the breast cancer study (797 cases and 764 controls) we have genome wide association study (GWAS) data available. We used this to assess the possible influence of population stratification on our results. For the GWAS dataset, the genomic inflation factor, λgc, was1.015. Assuming an equal level of population stratification (in terms of the fixation index FST) in the current study and the GWAS sub-study, we estimated the genomic inflation factor, λgc, to be 1.030 in the current study, using the relationship between FST, sample size and λgc described in gc value of 1.030 for genomic control-based correction of population stratification, the corrected global AML p-values for breast cancer are 0.052 for the entire pathway and 0.011 for the androgen-estrogen conversion sub-pathway, leaving our results largely unchanged. Even if λgc was as large as 1.05 in the current study, the global test p-value for the androgen-estrogen conversion sub-pathway would still be as low as 0.014. To further ensure that the observed associations could not be due to the employment of 319 paraffin-embedded tissue samples in the analysis, we re-ran analyses excluding 319 paraffin-embedded tissue samples, and (at the same time) excluding 33 SNPs with call rates of less than 95%. Results were very similar. For example, for breast cancer, p-values were 0.028 and 0.009 for the entire pathway and for the androgen-estrogen conversion sub-pathway, respectively. To validate the association in the androgen-to-estrogen conversion sub-pathway, we genotyped the 120 SNPs of this sub-pathway in an additional 2245 breast cancer cases and 1287 controls from Finland and performed the same AML analysis by using the 118 successfully genotyped SNPs. The validation analysis in the Finnish sample revealed similar evidence of association between the androgen-to-estrogen conversion sub-pathway and breast cancer   cancer . The ass= 0.015) . The non= 0.015) . The SNPpglobal = 0.009 and 0.018 respectively), postmenopausal patients without family history , and postmenopausal patients with estrogen receptor positive (ER+) tumors   status and divided all the patients into subgroups. We then compared subgroups of patients, defined on values of these variables, with controls, to evaluate the role of the androgen-to-estrogen conversion sub-pathway in different patient subgroups. First, we compared patient subgroups against all the controls in the combined Swedish and Finnish samples. The subgroup results showed that in the combined samples, significant association was observed in postmenopausal patients (ctively) . No signThen, to rule out the possibility that the above subgroup results were caused by the mismatch between the patient subgroups and the controls in terms of the variables which defined patient subgroups, we performed the second subgroup analysis where the controls were also divided into subgroups according to family history and menopausal status . The secWe further investigated the impact of reproductive risk factors on the genetic association of the androgen-to-estrogen conversion sub-pathway with breast cancer. Because the risk factor information is not available for the Finnish controls, the analysis of the reproductive risk factors was performed in the Swedish samples where information on such factors is available. We performed the AML analysis of the androgen-to-estrogen conversion sub-pathway with adjustment for the reproductive risk factors  and HRT use. We investigated this primarily to assess whether any of the reproductive risk factors could be in the causal pathway. Since p-values remained almost unchanged in all analyses , it appeCYP19A1 with both breast  and endometrial  cancer and UGT2B4  with breast cancer only. The associations in breast cancer survived correction for multiple testing of 15 genes . We also observed suggestive association for UGT2B11 in breast and endometrial cancer as well as for HSD11B1, SULT2A1 and SULT2B1 in breast cancer. Consistent with the pathway-based associations, the gene-based associations are generally more significant in sporadic postmenopausal patient samples than in the whole breast cancer sample (except SULT2B1). Furthermore, the importance of CYP19A1 and UGT2B4 in breast cancer risk is supported by the fact that excluding either gene from the global test of the sub-pathway reduced the global significance of association for the sub-pathway, from 0.0015 to 0.011 for CYP19A1, and to 0.010 for UGT2B4. However, the fact that the association for the sub-pathway remained significant, after excluding either gene, suggests that, although CYP19A1 and UGT2B4 are the major players, genetic variation within other genes also contributes to the association within the sub-pathway.Attempting to refine the association within the androgen-to-estrogen conversion sub-pathway, we performed a gene-based AML analysis in the combined Swedish/Finnish breast cancer sample and the Swedish endometrial cancer sample. Among the 15 genes tested , strong Our pathway-based multi-SNP association analysis revealed a significant association between genetic variants in the androgen-to-estrogen conversion sub-pathway and the risk of two hormone dependent cancers. The association was particularly strong for ER+, sporadic breast cancer. Single SNP analysis did not reveal a similar association. We used the AML-based multi-SNP analysis, which has been shown to be more powerful than single SNP tests to yield significant and consistent association, when genetic risk is carried by multiple risk alleles each with moderate effect Pathway-based approaches are just beginning to be applied in association analysis Unless one enzyme is the rate limiting step for the entire metabolic pathway, it is not likely that small functional perturbations of individual variants would have a major impact on the overall effect of the metabolic pathway. To test the hypothesis that several genetic variants, each conferring weak to moderate effects, contribute to genetic risk, we adopted a systematic pathway-based approach for association analysis by testing the joint effect of multiple genetic variants in a progressive fashion from the whole metabolic pathway to biochemical sub-pathways and further down to individual genes. Such a progressive approach allows us to not only establish consistent association in three cancer samples from two different populations but also to refine the association of the androgen-to-estrogen conversion component of the metabolic pathway. Our study may therefore have advanced our understanding of the role of estrongen metabolism in breast and endometrial cancers by 1) accounting for the ambiguity surrounding the genetic association results and 2) indicating the androgen-to-estrogen conversion to be the important component of the metabolic pathway in modulating the risk and therefore to be a worthy focus for future studies.CYP19A1CYP19A1 polymorphisms and postmenopausal breast cancer. Moreover, pharmacological inhibition of aromatase prevents recurrences in postmenopausal women with estrogen-receptor-positive breast cancer and new contralateral primaries CYP19A1 by suggesting that the modulation of aromatase activity by either germ-line variation or pharmacological agents can influence the development of ER+ tumour in postmenopausal women. Furthermore, the convergence of genetic and pharmacological effects of CYP19A1 also raises therapeutic possibilities. For example, other genes implicated by our genetic study, such as UGT2B4, might also be pharmacological targets for treating breast cancer.After menopause, ovarian estrogen production dramatically declines and conversion of adrenal androgens to estrogens in peripheral tissues becomes the major source of circulating estrogens. The final step of this conversion is catalyzed by aromatase, encoded by BRCA1, BRCA2, p53, ATM, CHK2) appear to be more involved in predominantly breast cancers associated with family history of disease.Hormone exposure is a common risk factor for breast and endometrial cancer. Our employment of the three samples of two different hormone-related cancers from two different populations allowed us to apply a very stringent criterion for declaring an association. Furthermore, results of our breast cancer patient subgroup analysis indicate that the genetic determinants within the androgen-to-estrogen conversion sub-pathway may play a more prominent role in postmenopausal women with sporadic ER+ tumors, further suggesting that the modulation of hormone exposure by genetic variation may have a differential impact on breast tumor subtypes. Endogenous sex hormone level appears to be associated with breast cancer risk in postmenopausal women It is worth noting that the contribution of genetic polymorphisms to risk is a function of both their prevalence and penetrance and thus the relative importance of individual SNPs may vary from population to population. More studies in different populations are needed to fully understand the role of the androgen-to-estrogen conversion sub-pathway in breast cancer. We also want to highlight that our results are of genetic association in nature, and further studies are needed to confirm the findings and to identify functional variants causally linked to cancer risk.Swedish subjects were from a population-based case control study of breast and endometrial cancer as described Finnish breast cancer cases consist of two series of unselected breast cancer patients and additional familial cases ascertained at the Helsinki University Central Hospital. The first series of 884 patients was collected in 1997–1998 and 2000 and covers 79% of all consecutive, newly diagnosed cases during the collection periods Risk factor information and tumour characteristics were available for all the Swedish samples and the Finnish cases, but were missing for the Finnish controls. The Finnish samples (mean age = 56 for the cases and 41 for the controls) were younger than the Swedish samples (mean age = 63 for both the cases and controls). All the risk factor and tumour characteristics information of the subjects are summarized in Written informed consent was obtained from all participating subjects, and the study was approved by the Institutional Review Boards in Sweden, Finland and at the National University of Singapore.DNA was extracted from 4 ml of whole blood using the QIAamp DNA Blood Maxi Kit (Qiagen)and non-malignant cells in paraffin-embedded tissue using a standard phenol/chloroform/isoamyl alcohol protocol tagSNPs program 2R coefficient, which quantifies how well the tagSNP haplotypes predict the genotype or haplotypes an individual carries. We chose tagSNPs so that common SNP genotypes and haplotypes (frequency ≥0.03) were predicted with 2R≥0.8 2R value of 0.85. We then estimated how well the tagSNP haplotypes of the remaining SNPs predicted the dropped SNP, an evaluation that can provide an unbiased and accurate estimate of tagSNP performance We selected 35 genes involved in estradiol or estrone metabolism and expressed in the breast (based on published literatures). We selected 1007 single nucleotide polymorphisms (SNPs) in these genes and their 30kb flanking sequences from the dbSNP (build 124) and Celera databases, aiming for a marker density of at least one SNP per 5kb . These SGenotyping was performed using the Sequenom system . All genotyping results were generated with positive and negative controls and checked by laboratory staff unaware of case-control status. Of the 302 tagSNPs, 42 SNPs failed in the development stage of Sequenom genotyping assays. SNPs with a call rate <85% (8 SNPs), minor allele frequency <1% (9 SNPs) or out of Hardy-Weinberg Equilibrium  were excluded from further analysis. Overall, 239 tagSNPs from the 35 genes were successfully genotyped . The genThe Cochran-Armitage trend test was performed for each of the 239 SNPs. One approach for assessing the departure of the distribution of the (Cochran-Armitage) test statistics from the  null distribution (no SNPs associated) has been described by Tyrer et. al. Table S1Selected characteristics of the subjects of the Swedish breast cancer sample.(0.03 MB DOC)Click here for additional data file.Table S2Tumour characteristics of the cases of Swedish and Finnish breast cancer samples.(0.03 MB DOC)Click here for additional data file.Table S3Genes and number of SNPs used in analyses.(0.08 MB DOC)Click here for additional data file.Table S4Twenty-five most significant SNPs for breast cancer in Swedish sample.(0.07 MB DOC)Click here for additional data file.Table S5Twenty-five most significant SNPs for endometrial cancer in Swedish sample.(0.07 MB DOC)Click here for additional data file.Table S6Twenty-five most significant SNPs for breast cancer in Finnish sample.(0.07 MB DOC)Click here for additional data file.


A phenomenon by which pre-irradiation of the abdomen of mice reduced the lung-colony-forming efficiency of i.v.-injected tumour cells is described. The extent of lung-colony inhibition was shown to depend on both the dose and timing of abdominal irradiation. The maximum inhibitory effect was obtained when mice received 1200 rad gamma-irradiation to the abdomen 5--7 days before tumour-cell challenge, but there was no effect when abdominal irradiation was given 1 or greater than or equal to 14 days before challenge, or when radiation doses were less than 600 rad. In mice less than 3 weeks old, the effect was much less marked than in adults. The target tissue which, when irradiated, exerted the inhibitory influence on lung-colony formation was located in the ventral half of the abdomen in all 4 quadrants, and was probably gut. Radioactively labelled tumour cells were arrested normally in the lungs of irradiated mice, but were cleared more rapidly without evidence of sequestration in the irradiated gut. The most plausible mechanism seems to be that irradiation of the gut induces the production of natrual killer cells with anti-tumour activity, though this has not been conclusively established.


Although aging is accompanied by diminished functioning, many elderly individuals preserve a sense of well-being. While the concept of "successful aging" has been popular for many decades, little is known about its psycho-physiologic and endocrine underpinnings. KORA-Age is a population-based, longitudinal study designed to determine the prevalence of successfully aged men and women between 65 and 94 years old in the MONICA/KORA Augsburg cohort of randomly selected inhabitants. Specifically, we aim to identify predictors of successful aging and to elucidate bio-psychosocial mechanisms that maintain mental health and successful adaptation despite adverse experiences of life and aging.Components of successful aging were assessed in a telephone survey of 4,127 participants (2008-2009) enrolled in the MONICA/KORA cohort, on average, 13 years earlier. Psychosocial, somatic and behavioural predictors are used to determine factors that contribute to successful aging. An age-stratified random sub-sample  participated in a personal interview where further psychological mechanisms that may underlie successful adaptation  were examined. The interactions among neuroendocrine systems in the aging process are investigated by studying the cortisol/dehydroepiandrosterone-sulfate ratio, the level of insulin-like growth factor I, and oxytocin.Longitudinal determinants of successful aging can be assessed based on a follow-up of an average of 13 years. A comprehensive analysis of biological as well as physio-psychological information provides a unique opportunity to investigate relevant outcomes such as resilience and frailty in the elderly population. The elderly constitute the fastest growing segment in the population. Increasing age is often associated with worsening physical and mental health, decreasing functional ability and cognitive impairment . MoreoveThere is no unanimous definition of "successful aging". To date, it remains unclear whether the construct refers simply to the absence of disability or to the combination of specific positive attributes among others, well-being. A recent literature search identified 28 studies with 29 different definitions . The HonHowever, successful aging has been also defined as more than the absence of disability and multi-morbidity. Even in the context of chronic physical impairment, aged individuals may experience successful aging, which points to a distinction between being chronically ill and regarding oneself as sick ,8. AccorRowe & Kahn  distinguSpecialised fields of study share only minimal agreement with the view of the elderly themselves. The Leiden85-plus Study  gained dThere are four objectives in this study: 1) to determine the prevalence of different definitions of successful aging in a representative sample of elderly individuals in the MONICA/KORA study; 2) to identify predictors of successful aging; 3) to phenotype the mental health of the elderly; 4) to identify biomarkers of successful aging. These aims are briefly described below.In the present investigation, different definitions of successful aging will be analysed Table :The purpose of our analysis is to assess the age- and sex-related prevalence of participants above 65 years who meet the criteria of successful mental and cognitive aging based on a random sample of the population recruited between 1984 and 2001 . In collSpecific aims are:• To assess the prevalence of successful aging according to the psycho-cognitive, biomedical, laymen's and combined approach.• To investigate the inter-relation of different domains of successful aging• To assess gender and age-related differences in the prevalence of successful aging• To characterize participants who meet the psycho-cognitive criteria of successful aging, despite somatic and functional impairments (multi-morbidity and disability).Successful aging may not occur by chance. However, knowledge about predictors and sequential events that facilitate successful aging is sparse. The main objective is to elucidate the predictive power of a wide range of midlife psychosocial, somatic and behavioural factors that contribute to successful aging. Elderly participants with a good mental health as defined in Objective 1, will be compared to those with impaired mental health on the following variables, which were assessed in their midlife:• Education and social class• Social network• Work conditions• Chronic psychological stress and job stress• Hyperactivity and/or hostile behaviour patterns (Type A/B Patterns)• Depression and/or mental exhaustion and/or Type D behaviour• Sleep disorders• Cardiovascular risk factors, in particular diabetes, hypertension, hypercholesterolemia and hypertriglyceridemia• Life style: physical activity, healthy nutrition, smoking• Self-perceived health and/or somatic complaintsThere is growing evidence that midlife cardiovascular risk factors are associated with vascular and even primary degenerative dementias, including Alzheimer's disease -18. HoweThe aim of this study is to investigate the influence of external object relations  and internal object relations (attachment) on the development of late-life depression. The absence of major depression is among the strongest early predictors of successful aging . Among tThe following hypothesis will be tested: Individuals with good social support and secure attachment have the lowest prevalence of late-life depression, followed by those with little social support but secure attachment, and finally the highest rates of depression will be observed in participants with little social support and no secure attachment.The novel aspect of the study is the systematic investigation of social support and attachment for the development of late-life depression in a large sample of elderly participants.The purpose of this study is to identify biomarkers that are individually, or in combination, associated with successful aging. With this aim, we will define quantitative traits of successful and unsuccessful aging (as characterised in Objective 1) in the random sub-sample of 1079 participants. We will analyse the circulating concentrations of pituitary and adrenal hormones at rest and will focus on cortisol and DHEA-S (dehydroepiandrosterone-sulfate) , IGF-I (insulin-like growth factor) (somatotropic axis) and oxytocin (neurohypophysis). Only a few studies thus far have paid particular attention to the aged, and those investigating individuals above the age of 65 have mainly used a purely somatic definition of disease. Furthermore, most of the studies are "treatment oriented", i.e. those attempting to prove the efficacy of hormone (replacement) therapy. Within the concept of "successful aging" outlined above, and especially with respect to the difference between "being chronically ill" and "self-perceived health", no studies are presently available that attempt to define a "hormonal profile" potentially associated with successful aging.In general, it is our hypothesis that overall "successful aging", well-being, indices of mental health, cognitive function and - as a consequence - overall health status are more appropriately reflected by ratios between stress hormones (cortisol) and protective hormones . This approach may identify disturbances in the balance between the activation of the hypothalamic-pituitary-adrenal (HPA) axis and the ability of an individual to cope with such challenges.The course of the KORA-Age project is depicted in Figure 1) A mortality follow-up in 2008 of all 65 - 94 year old participants of the KORA cohort. N = 4,564.2) A telephone interview of living participants from the KORA cohort older than 65. N = 4,127.3) A medical examination of a sub-probe of the cohort 65 - 89 years old. N = 1,079These interviews and examination were carried out between September 2008 and November 2009 , who were older than 65 years at the end of December 2007, to partake. The surveys were carried out in 1984/85 (S1), 1989/90 (S2), 1994/95 (S3) and 1999/2001 (S4). All these surveys were based on representative samples of the population of Augsburg and two surrounding counties. Participants were 25 to 74 years old at the time of each survey. The participation rates ranged between 79% in S1 and 67% in S4 .The current research project is a part of the telephone interview survey and home or on-site visits which includes approximately 4,127 participants of the former MONICA/KORA surveys. The mental health module is a composite of instruments tailored to screen for important features of mental health indicators in participants in the age groups of 65 - 94 years.WHO-Five Well-being Index covers positive mood , vitality (being active and waking up fresh and rested), and general interests (being interested in things) [•  things) ,22. EachEuro-Qol (EQ-5D) describes the health-related quality of life states consisting of five dimensions  each of which can be reported as one of three responses. The responses record three levels of severity (no problems/some or moderate problems/extreme problems) within a particular EQ-5D dimension, and yields a range of scores from 5 to 15 [•  5 to 15 ,10.GDS-15  from Sheikh and Yesavage [• Yesavage  evaluateGeneralised Anxiety Disorder scale (GAD-7): A recently developed screening instrument to assess generalised anxiety disorder [• disorder  is availResilience: We will apply the German version of the 11-item short form Resilience scale [• ce scale  to assesce scale .The total interview time by the successful aging mental health requires approximately 7 to 13 min (out of 26 min. planned for the whole interview) depending on the cognitive status of participants.Cognitive Status: Participants will be screened by telephone using a short cognitive status test - the modified Telephone Interview for Cognitive Status (TICS-m) [• (TICS-m) . The TIC(TICS-m) .All participants included in the Successful Aging Predictor Study are derived from the ongoing MONICA/KORA Augsburg Cohort Studies. Extensive and highly quality controlled data sets have been taken from the participants including anthropometric, laboratory measures, and psychosocial data sets. These data provide the unique opportunity to screen for a wide range of predictors for successful aging covering a life span of up to 25 years .Beziehungsspezifische Bindungsskalen für Erwachsene: BBE). The BBE is a 14 items self-rating questionnaire to measure attachment styles of adults along the dimensions of "secure-fearful" and "dependent-independent" [Depressive symptomatology will be assessed with the Geriatric Depression Scale (GDS-15). Social network will be assessed with the "Social Network Index". The scores of this index are between 1 and 4 . The "sopendent" ,32.To define a "neuroendocrine profile" potentially associated with successful aging, we have selected specific hormones of hypothalamic-pituitary-adrenal axis origin, which decline substantially with age and may be involved in the maintenance of cognitive function and mental health. We expect to observe age- and sex related differences in hormone concentrations. To allow identification of differences between the groups related to "successful aging", the population size was chosen based on these assumptions on hormone level variability Table :The respective data for oxytocin are not available yet. In a smaller study, Forsling et al.  reportedregression and classification tree (CART) technique [For descriptive statistical analysis, associations between categorical variables will be carried out with the chi-square test. Group differences in continuous variables will be analysed by the t-test (variables with 2 categories) or the F test (more than 2 categories). To test for trends of different definitions of successful aging (Objective 1) in 5-year age intervals, groups will be coded with their respective median values. Characteristics associated with increased successful aging (Objectives 2-4) will be identified by age-adjusted and by multivariable logistic regression analysis with stepwise variable selection. To account for the possibility of effect modifications, second-order interaction terms for all factors fulfilling the entry criterion will be included in the initial regression model. All analyses will be performed separately for men and women. Moreover, to assess age-adjusted sex differences in prevalence of various successful aging definitions, logistic regression models with age, the respective characteristic, sex and the interaction term characteristic*sex will be included in the model equation. We recently gained experience  with theechnique  which isParticipants will be informed about research procedures and will be asked to sign an informed consent. The study protocol was submitted and approved by the Ethical Committee of the Bavarian medical association (Ethik-Kommission Nr. 08064).This study protocol illustrates a collaborative work useful for researchers interested in innovative and realistic investigation, which is aimed at understanding the psycho-bio-medical-social characteristics of successful aging in a population-based sample. A discussion of specific strengths and limitations of this study follows below.The first and obvious strength of this study is its feasibility, because the study design is based on the ongoing MONICA/KORA studies, which began in 1984 in Augsburg, Germany. All relevant methodological and logistical features of the study were successfully tested in previous studies, when, for example, a very low loss to follow-up rate was achieved . SpecifiAnother strong aspect of the design of this study is the large number of measurements. A great variety of psychological tests and biomarkers are used, making it possible to analyze the role of potential mediating variables as predictors of successful aging as well as the development of multiple symptoms over time.An additional advantage of this study concerns the large sample size, making it easy to draw firm conclusions about prevalence rates, and analytic results.This is a non-intervention, prospective cohort study with the limitations inherent to non-experimental research. The possibility of bias and residual confounding can never be entirely eliminated, and the ability to infer causation is correspondingly limited . NonetheIn conclusion, it is clear that society is faced with an ever growing elderly population with increasing medical demands. This study aims to contribute to the evidence-based research that may promote individual well-being in the elderly.(DHEA-S): dehydroepiandrosterone-sulfate; (IGF-I): insulin-like growth factor; (EQ-5D): EuroQol Instrument; (GDS-15): Geriatric Depression Scale; (GAD-7): Generalised Anxiety Disorder scale; (TICS-m): modified Telephone Interview for Cognitive Status; (MOPSY): MONICA Psychosocial Optional Study; Relation-specific Attachment Scales for Adults ; (CART): classification and regression tree.The authors declare that they have no competing interests.st objective. KHL planned the 2nd objective, initiated the study, formulated the research grant and drafted the manuscript. KHL, DH and GK planned the 3rd objective. KHL and MB planned the 4th objective. AP is coordinator of KORA-Age: All authors read and approved the final manuscript.MEL and RTE will carry out analysis and interpretation of data, and drafted the manuscript. KHL, BH, CB and AK planned the 1The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2288/10/36/prepub


Although microarray technology has become the most common method for studying global gene expression, a plethora of technical factors across the experiment contribute to the variable of genome gene expression profiling using peripheral whole blood. A practical platform needs to be established in order to obtain reliable and reproducible data to meet clinical requirements for biomarker study.We applied peripheral whole blood samples with globin reduction and performed genome-wide transcriptome analysis using Illumina BeadChips. Real-time PCR was subsequently used to evaluate the quality of array data and elucidate the mode in which hemoglobin interferes in gene expression profiling.We demonstrated that, when applied in the context of standard microarray processing procedures, globin reduction results in a consistent and significant increase in the quality of beadarray data. When compared to their pre-globin reduction counterparts, post-globin reduction samples show improved detection statistics, lowered variance and increased sensitivity. More importantly, gender gene separation is remarkably clearer in post-globin reduction samples than in pre-globin reduction samples. Our study suggests that the poor data obtained from pre-globin reduction samples is the result of the high concentration of hemoglobin derived from red blood cells either interfering with target mRNA binding or giving the pseudo binding background signal.We therefore recommend the combination of performing globin mRNA reduction in peripheral whole blood samples and hybridizing on Illumina BeadChips as the practical approach for biomarker study. Peripheral blood has recently become an attractive prime tissue for biomarker detection because of its critical role in immune response, metabolism, and communication with cells, and extracellular matrices in almost all tissues and organs in the human body, as well as its being less invasive and the simplicity of sample collectionMany different techniques are used to handle peripheral blood samples prior to RNA isolation based on the experimental design: PAXgene (whole blood), QIAamp (Platelets and White Blood Cells), and Ficoll and BD-CPT (Mononuclear cells). Several studies have been conducted to make a comparison between methods by examining their reproducibility, variance, and signal-to-noise ratiosThe Illumina Sentrix human-6 v2 array uses gene-specific 50-mer probes attached to 3 µm beads with an average of 30 redundant features for each transcript, allowing six samples to be profiled per BeadChip simultaneouslyNo difference was seen between pre- and post-globin reduction samples when comparing RNA quality using a bioanalyzer. Pre-globin reduction samples looked to be of similar quality to post-globin reduction samples, with two sharp peaks of 18 s and 28 s RNA around 2000 bp and 4000 bp. However, the cRNA from pre-globin reduction samples showed different signals from standard cRNA. Hemoglobin showed an additional sharp peak on the top of cRNA Fluorescence absorbance curve in pre-globin reduction samples and the sharp peak disappeared in post-globin reduction samples. In addition, most of the pre-globin reduction samples only showed a band at 800 bp in cRNA electrophoresis figure and post-globin reduction samples exhibited smeared bands (200 bp–6000 bp) as normal standard cRNA (Data not shown).A great deal of evidence shows that, when compared to other techniques, whole blood samples prepared by the PAXgene method typically result in Affymetrix microarray data with a low rate of genes detected as “present” with respect to the background noise on the chip, as well as large intra-group variance. The great abundance of hemoglobin mRNA is thought to account for this poor performanceThe BeadStudio software used to process Illumina's BeadChip data provides a “detection p-value” that can be used to determine whether a particular probe was detected against background noise. After correcting for multiple rounds of hypothesis testing, we considered adjusted p-values below 0.05 to be “detected” or “present.”Given the above definition, the average number of present calls per array in the post-globin reduction group was 11921.73±296.38, whereas the average number of present calls per array in the pre-globin reduction group was significantly lower and more variable, at 8987.75±1264.94. Hence, samples in the post-globin reduction group showed improved probe detection and reduced intra-group detection call variance with respect to those in the pre-globin reduction group . In addinot called in the pre-globin reduction group. Such probes might represent nucleotide sequence-specific susceptibility to globin-induced noise, or some other systemic effect.In order to determine whether the improvement in detection was a consistent phenomenon , we sought to identify probes that were consistently detected in the post-globin reduction group, and consistently 1876 probes showed a consistent pattern of improved detection, where at least 75% of the adjusted detection p-values were <0.05 for the post-globin reduction group and at least 75% were >0.05 in the pre-globin reduction group with a statistically significant difference between the distributions of pre- and post-globin reduction adjusted p-values . Here, sIn addition, no genes were found to pass the detection criteria described above  in the pre-globin reduction group, but failed to pass the criteria in the post-globin reduction group.In order to show that the 1876 probes with consistently improved p-values are not an artifact of random fluctuation of the p-values reported by the BeadStudio software, a randomized simulation was run to estimate the number of probes that would pass our selection criteria under random re-association of improved detection calls to probe identifiers. After 100 random trials, none of the simulations produced more than one probe that passed our criteria for being significantly improved by globin reduction. These results indicate that the 1876 probes identified here were not an artifact of the stochastic nature of microarray hybridization and instead represent a set of probes whose improved detection was strongly dependent on the removal of globin RNA from the whole blood samples.The sensitivity threshold of microarray measurements defines the concentration range in which accurate measurements can be achieved. One of the advantages of the BeadArray platform is that it requires less mRNA for hybridization. This makes BeadArray more sensitive than any other platforms.All of the 1876 genes with consistently improved detection p-values exhibited higher expression intensity in the post-globin reduction group than in the pre-globin reduction group . The BeaIn order to show that the post-globin reduction improves the practical usability of whole blood mRNA samples, the pre- and post-globin reduction samples were used to select gender marker genes. A similar approach was used by Debey et alThe result of the selection process shows a clear performance improvement when finding gender marker genes using post-globin reduction samples instead of pre-globin reduction samples. For example, when the top ten probes are selected in each of the ten iterations using the pre-globin reduction data, only one was selected in at least half of the iterations. In contrast, when using the post-globin reduction experiments four probes, representing three distinct genes on the Y chromosome, were selected. As well as improving the detection of low intensity and biologically informative transcripts, the globin reduction treatment also resulted in 298 genes  exhibiting apparent differential expression between the pre- and post-globin reduction groups. Among these, 75 genes were up-regulated with fold changes ≧2; 223 genes were found down-regulated with fold changes ≤0.5. We postulate first that hemoglobin may compete with the target mRNA in binding to the probe, thus contributing to a higher-than-normal pseudo binding background signal. Second, the overabundance of hemoglobin may interfere with target mRNA binding through an unknown mechanism.We used real-time PCR to show that the RNA levels of these genes were, in fact, no different in the pre- and post-globin reduction samples, and that the differential expression observed in the array data was an artifact of the noise induced by the overabundance of globin RNA in the pre-globin reduction samples. Four hemoglobin genes, HBA1, HBB, HBD, and HBE1, together with another down-regulated gene, as well as 2 up-regulated genes in bead array were randomly chosen to verify by real-time PCR. The two most abundant hemoglobin genes, HBA1 and HBB, were observed at lower levels in the post-globin reduction group with respect to the pre-globin reduction group in both the array data and in the real time PCR, indicating that the globin reduction protocol was effective. Two other hemoglobin genes, HBD and HBE, were also significantly down-regulated by globin reduction in both assays. However, AYTL2, the gene observed to be down-regulated in the array data, and the 2 genes that showed higher expression levels in the array data, ccl5 and DYRK2, all showed no significant change in expression level in the real-time PCR assay .Expression profiling using peripheral whole blood samples is an attractive method for biomarker detection. However, hemoglobin, which represents as much as 70% of the total mRNA population in peripheral whole blood samples isolated by the PAXgene tube, effectively dilutes the mRNA population and interrupts the gene expression profiles in the Affymetrix arrayhttp://www.ncbi.nlm.nih.gov/blast/Blast.cgi), respectively. However, the non-hemoglobin gene that was observed to be down-regulated after globin reduction in the microarray data, AYTL2, showed no significant change in the real-time PCR data. This suggests that globin reduction did not actually decrease the level of other genes' mRNA, but rather allowed for more accurate measurement of these levels when the samples were hybridized to microarrays. The decreased intensity observed for non-hemoglobin genes on the post-globin reduction microarrays might be due to high abundance hemoglobin providing a non-specific pseudo-binding signal. On further analysis, no significant sequence similarity was found between these down regulated genes and HBA1 and HBB. This again supports our hypothesis that the higher expression in pre-globin reduction samples of some genes might be due to a non-specific pseudo-binding signals from hemoglobin. In addition, the two up-regulated genes (ccl5 and DYR) exhibited no significant changes in expression level in the real time PCR data. This indicates that hemoglobin may interfere with target mRNA binding through an unknown mechanism and result in lower expression signals from the pre-globin reduction microarrays.Real-time PCR is commonly used to validate the mRNA expressions acquired from microarray experiments due to the greater specificity of the primer vs. microarray probesemonstrates that the combination of performing globin mRNA reduction in peripheral whole blood samples and hybridizing on Illumina BeadChips is a practical approach for biomarker research. Our future study will focus on the cancer biomarker detection by using this established platform.In summary, this study dThis study was conducted under protocols approved by the Children's Hospital Boston Institutional Review Board. Blood samples were obtained from 11 subjects who voluntarily agreed to participate and gave written informed consent. Peripheral blood was drawn with a BD safely Lok™ blood collection set  into PAXgene RNA collection tube  according to the standard procedure. Total RNA was prepared with the PAXgene Blood RNA Kit (Qiagen) according to the manufacturer's instructions with an on-column DNase digestion step. RNA quantity and quality were determined by a NanoDrop ND-1000 Spectrophotometer  and an Experion™.Since a large amount of hemoglobin exists in erythrocytes, several studies have shown decreased present calls, reduced accuracy, and increased variability among replicates in an Affymetrix GeneChip array when using PAXgene RNA collection technologyIn vitro transcription (IVT) technology, along with biotin UTP, was employed to generate multiple copies of biotinylated cRNA The labeled cRNA was purified via Filter Cartridge and quantified by NanoDrop and RiboGreen® . The integrity of cRNA was evaluated using an Experion™ (Bio-RAD).100 ng of total RNA was applied to generate cRNA by using a Illumina TotalPrep RNA Amplification Kit (Ambion). Reverse transcription with the T7 oligo (dT) primer was used to produce first strand cDNA. The cDNA then underwent second strand synthesis and RNA degradation by DNA Polymerase and RNase H, followed by clean up. The labeled cRNA target (1.5 µg) was used for hybridization to an array according to the Illumina Sentrix humanref-6 beadchip protocol. A maximum of 10 µl cRNA was mixed with a 20 µL GEX-HYB hybridization solution. The preheated 30 µl assay sample was dispensed onto the large sample port of each array and incubated for 18 hours at 58°C at a rocker speed of 5. Following hybridization, the samples were washed according to the protocol and scanned with a BeadArray Reader .Hemoglobin α (HBA1), β (HBB), δ (HBD), and ε(HBE1) genes together with 3 other randomly chosen genes from a differential expression list were picked for evaluating the array data by using real-time PCR. Primers were designed using primer 3 and shown in Detection p-values produced by the BeadStudio software were corrected for multiple hypothesis testingIn order to show that the probes with consistently improved detection p-values were not an artifact of random chance, a randomized simulation was run. Each trial in the simulation consisted of identifying, for each blood sample (a pre- and post-globin reduction pair of microarray experiments), those probes with p-values greater than 0.05 in the pre-globin reduction data and less than 0.05 in the post-globin reduction data. Those p-values were then randomly reassigned among the probes whose p-values were not below 0.05 in both the pre- and post-globin reduction data. After performing this random re-association for each blood sample, we applied the same criteria for selecting significantly improved probes as was applied to the observed data, and recorded the number of probes that passed the selection criteria. Thus, our simulation generates a distribution according to the null hypothesis that an improved p-value pair is equally likely to be associated with any probe whose p-values are not already below 0.05 in both the pre- and post-globin reduction samples.To verify the efficacy of the globin reduction treatment in the context of gene selection, the following normalization and selection process was run. Analysis of the raw intensity data revealed that, between pairs of arrays, a non-linear relationship existed between corresponding pairs of probes. To correct this, we used a Loess adjustment as implemented in the BioConductor package for R e, the ReliefF algorithm finds e's nearest neighbor with the same class (gender) using Euclidian distance over all genes. The nearest neighbor of the other class (opposite gender) is also found in the same manner. These selections are called the hit and miss, respectively. The importance of each gene is then computed by taking a normalized sum of differences between the distance from e to the hit and the distance from e to the miss. Thus, the greater the difference between the hit and miss, the greater the importance of the gene in distinguishing class. This method has the advantage that it makes no assumptions about the distribution of expression intensities. Marker genes were identified by picking out genes that were consistently ranked highly across at least half of the repeats. Heatmaps, as well as Student's t-test p-values describing the ability of the genes identified by the above method to distinguish gender were generated in R.After normalization, the data was split into pre- and post-globin reduction groups, each containing eight female and three male samples. We then performed gene selection independently on these two groups of 11 experiments using the ReliefF algorithmTable S1(0.52 MB XLS)Click here for additional data file.